Medical Ethnobiology and Ethnopharmacology in Latin America 2013

Evidence-Based Complementary and Alternative Medicine

Guest Editors: Ulysses Paulino de Albuquerque, Edwin L. Cooper, Maria Franco Trindade Medeiros, Rômulo Romeu da Nóbrega Alves, and Ana H. Ladio Medical Ethnobiology and Ethnopharmacology in Latin America 2013 Evidence-Based Complementary and Alternative Medicine

Medical Ethnobiology and Ethnopharmacology in Latin America 2013

Guest Editors: Ulysses Paulino de Albuquerque, Edwin L. Cooper, Maria Franco Trindade Medeiros, Romuloˆ Romeu da Nobrega´ Alves, and Ana H. Ladio Copyright © 2014 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in “Evidence-Based Complementary and Alternative Medicine.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Editorial Board

Mahmood Abdulla, Malaysia Jen-Hwey Chiu, Taiwan Ching-Liang Hsieh, Taiwan Jon Adams, Australia William C. S. Cho, Hong Kong Jing Hu, China Zuraini Ahmad, Malaysia Jae Youl Cho, Korea Gan Siew Hua, Malaysia Ulysses Albuquerque, Brazil Seung-Hun Cho, Republic of Korea Sheng-Teng Huang, Taiwan Gianni Allais, Italy Chee Yan Choo, Malaysia BennyTanKwongHuat,Singapore Terje Alraek, Norway Ryowon Choue, Republic of Korea Roman Huber, Germany Souliman Amrani, Morocco Shuang-En Chuang, Taiwan Angelo Antonio Izzo, Italy Akshay Anand, India Joo-Ho Chung, Republic of Korea Kong J., USA Shrikant Anant, USA Edwin L. Cooper, USA Suresh Jadhav, India Manuel Arroyo-Morales, Spain Gregory D. Cramer, USA Kanokwan Jarukamjorn, Thailand Syed Asdaq, Saudi Arabia Meng Cui, China Yong Jiang , China Seddigheh Asgary, Iran Roberto Cuman, Brazil Zheng L. Jiang, China Hyunsu Bae, Republic of Korea Vincenzo De Feo, Italy Stefanie Joos, Germany Lijun Bai, China Roc´ıo Vazquez,´ Spain Sirajudeen K.N.S., Malaysia Sandip K. Bandyopadhyay, India Martin Descarreaux, USA Z. Kain, USA Sarang Bani, India Alexandra Deters, Germany Osamu Kanauchi, Japan Vassya Bankova, Bulgaria Siva Durairajan, Hong Kong Wenyi Kang, China Winfried Banzer, Germany Mohamed Eddouks, Morocco Dae Gill Kang, Republic of Korea Vernon A. Barnes, USA Thomas Efferth, Germany Shao-Hsuan Kao, Taiwan Samra Bashir, Pakistan Tobias Esch, Germany Krishna Kaphle, Nepal Jairo Kenupp Bastos, Brazil Saeed Esmaeili-Mahani, Iran Kenji Kawakita, Japan Sujit Basu, USA Nianping Feng, China Jong Yeol Kim, Republic of Korea David Baxter, New Zealand Yibin Feng, Hong Kong Cheorl-Ho Kim, Republic of Korea Andre-Michael Beer, Germany Josue Fernandez-Carnero, Spain Youn Chul Kim, Republic of Korea Alvin J. Beitz, USA Juliano Ferreira, Brazil Yoshiyuki Kimura, Japan Yong Boo, Republic of Korea Fabio Firenzuoli, Italy Joshua K. Ko, China Francesca Borrelli, Italy Peter Fisher, UK Toshiaki Kogure, Japan Gloria Brusotti, Italy W. F. Fong, Hong Kong Nandakumar Krishnadas, India Ishfaq A. Bukhari, Pakistan Romain Forestier, France Yiu Wa Kwan, Hong Kong Arndt Bussing,¨ Germany Joel J. Gagnier, Canada Kuang Chi Lai, Taiwan Rainer W. Bussmann, USA Jian-Li Gao, China Ching Lan, Taiwan Raffaele Capasso, Italy Gabino Garrido, Chile Alfred Langler,¨ Germany Opher Caspi, Israel Muhammad Ghayur, Pakistan Lixing Lao, Hong Kong Han Chae, Korea Anwarul Hassan Gilani, Pakistan Clara Bik-San Lau, Hong Kong Shun-Wan Chan, Hong Kong Michael Goldstein, USA Jang-Hern Lee, Republic of Korea Il-Moo Chang, Republic of Korea Mahabir P. Gupta, Panama Tat leang Lee, Singapore Rajnish Chaturvedi, India Mitchell Haas, USA Myeong S. Lee, UK Chun Tao Che, USA Svein Haavik, Norway Christian Lehmann, Canada Hubiao Chen, Hong Kong Abid Hamid, India Marco Leonti, Italy Jian-Guo Chen, China N. Hanazaki, Brazil Ping-Chung Leung, Hong Kong Kevin Chen, USA K. B. Harikumar, India Lawrence Leung, Canada Tzeng-Ji Chen, Taiwan Cory S. Harris, Canada Kwok Nam Leung, Hong Kong Yunfei Chen, China Thierry Hennebelle, France Ping Li, China Juei-Tang Cheng, Taiwan Seung-Heon Hong, Korea Min Li, China Evan Paul Cherniack, USA Markus Horneber, Germany Man Li, China ChunGuang Li, Australia Andrea Pieroni, Italy Mei Tian, China Xiu-Min Li, USA Richard Pietras, USA Evelin Tiralongo, Australia Shao Li, China Waris Qidwai, Pakistan S. C. Tjen-A-Looi, USA Yong Hong Liao, China Xianqin Qu, Australia MichaThl Tomczyk, Poland Sabina Lim, Korea CassandraL.Quave,USA Yao Tong, Hong Kong Bi-Fong Lin, Taiwan Roja Rahimi, Iran K. V. Trinh, Canada Wen Chuan Lin, China Khalid Rahman, UK Karl Wah-Keung Tsim, Hong Kong Christopher G. Lis, USA Cheppail Ramachandran, USA Volkan Tugcu, Turkey Gerhard Litscher, Austria Gamal Ramadan, Egypt Yew-Min Tzeng, Taiwan Ke Liu, China Ke Ren, USA Dawn M. Upchurch, USA I-Min Liu, Taiwan Man Hee Rhee, Republic of Korea MarynaVandeVenter,SouthAfrica Gaofeng Liu, China Mee-Ra Rhyu, Republic of Korea Sandy van Vuuren, South Africa Yijun Liu, USA JoseLuisR´ ´ıos, Spain Alfredo Vannacci, Italy Cun-Zhi Liu, China Paolo Roberti di Sarsina, Italy Mani Vasudevan, Malaysia Gail B. Mahady, USA Bashar Saad, Palestinian Authority Carlo Ventura, Italy Juraj Majtan, Slovakia Sumaira Sahreen, Pakistan Wagner Vilegas, Brazil Subhash C. Mandal, India Omar Said, Israel Pradeep Visen, Canada Jeanine Marnewick, South Africa Luis A. Salazar-Olivo, Mexico Aristo Vojdani, USA Virginia S. Martino, Argentina Mohd. Zaki Salleh, Malaysia Y. Wang, USA James H. McAuley, Australia Andreas Sandner-Kiesling, Austria Shu-Ming Wang, USA Karin Meissner, USA Adair Santos, Brazil Chenchen Wang, USA Andreas Michalsen, Germany G. Schmeda-Hirschmann, Chile Chong-Zhi Wang, USA David Mischoulon, USA Andrew Scholey, Australia Kenji Watanabe, Japan Syam Mohan, Malaysia Veronique Seidel, UK Jintanaporn Wattanathorn, Thailand J. Molnar, Hungary Senthamil R. Selvan, USA Wolfgang Weidenhammer, Germany Valerio´ Monteiro-Neto, Brazil Tuhinadri Sen, India Jenny M. Wilkinson, Australia H.-I. Moon, Republic of Korea Hongcai Shang, China Darren R. Williams, Republic of Korea Albert Moraska, USA Karen J. Sherman, USA Haruki Yamada, Japan Mark Moss, UK Ronald Sherman, USA Nobuo Yamaguchi, Japan Yoshiharu Motoo, Japan Kuniyoshi Shimizu, Japan Yong-Qing Yang, China Frauke Musial, Germany Kan Shimpo, Japan Junqing Yang, China MinKyun Na, Republic of Korea Byung-Cheul Shin, Korea Ling Yang, China Richard L. Nahin, USA Yukihiro Shoyama, Japan Eun Jin Yang, Republic of Korea Vitaly Napadow, USA Chang Gue Son, Korea Xiufen Yang, China F. R. F. Nascimento, Brazil Rachid Soulimani, France Ken Yasukawa, Japan S. Nayak, Trinidad And Tobago Didier Stien, France Min H. Ye, China Isabella Neri, Italy Shan-Yu Su, Taiwan M. Yoon, Republic of Korea Telesphore´ Nguelefack, Cameroon Mohd Roslan Sulaiman, Malaysia Jie Yu, China Martin Offenbacher, Germany Venil N. Sumantran, India Jin-Lan Zhang, China Ki-Wan Oh, Republic of Korea John R. S. Tabuti, Uganda Zunjian Zhang, China Y. Ohta, Japan Toku Takahashi, USA Wei-bo Zhang, China Olumayokun A. Olajide, UK Rabih Talhouk, Lebanon Hong Q. Zhang, Hong Kong Thomas Ostermann, Germany Wen-Fu Tang, China Boli Zhang, China Stacey A. Page, Canada Yuping Tang, China Ruixin Zhang, USA Tai-Long Pan, Taiwan Lay Kek Teh, Malaysia Hong Zhang, Sweden Bhushan Patwardhan, India Mayank Thakur, India Haibo Zhu, China Berit Smestad Paulsen, Norway Menaka C. Thounaojam, India MedicalEthnobiologyandEthnopharmacologyinLatinAmerica2013, Ulysses Paulino de Albuquerque, Edwin L. Cooper, Maria Franco Trindade Medeiros, Romuloˆ Romeu da Nobrega´ Alves, and Ana H. Ladio Volume 2014, Article ID 576382, 1 page

What the Iberian Conquest Bequeathed to Us: The Fruit Trees Introduced in Argentine Subtropic—Their History and Importance in Present Traditional Medicine, Pablo C. Stampella, Daniela Alejandra Lambare,´ Norma I. Hilgert, and Mar´ıa Lelia Pochettino Volume 2013, Article ID 868394, 17 pages

Chemical Composition and Validation of the Ethnopharmacological Reported Antimicrobial Activity oftheBodyFatofPhrynops geoffroanus Used in Traditional Medicine,Diogenes´ de Queiroz Dias, Mario Eduardo Santos Cabral, Debora´ Lima Sales, Olga Paiva Oliveira, JoaoAntoniodeAraujoFilho,˜ Diego Alves Teles, JoseGuilhermeGonc´ ¸alves de Sousa, Henrique Douglas Melo Coutinho, JoseGalbertoMartinsdaCosta,MartaReginaKerntopf,R´ omuloˆ Romeu da Nobrega´ Alves, and Waltecio´ de Oliveira Almeida Volume 2013, Article ID 715040, 4 pages

The “Hidden Diversity” of Medicinal Plants in Northeastern Brazil: Diagnosis and Prospects for Conservation and Biological Prospecting, Deyvson Rodrigues Cavalcanti and Ulysses Paulino Albuquerque Volume 2013, Article ID 102714, 29 pages

The Key Role of Cultural Preservation in Maize Diversity Conservation in the Argentine Yungas, NormaI.Hilgert,FernandoZamudio,VioletaFurlan,andLuc´ıa Cariola Volume 2013, Article ID 732760, 10 pages

Wild Animals Used as Food Medicine in Brazil,Romuloˆ Romeu Nobrega´ Alves, Tacyana Pereira Ribeiro Oliveira, and IereceLucenaRosaˆ Volume 2013, Article ID 670352, 12 pages

Acute and Chronic Toxicity of an Aqueous Fraction of the Stem Bark of Stryphnodendron adstringens (Barbatimao)˜ in Rodents,MarcoAntonioCosta,Joao˜ Carlos Palazzo de Mello, Ed´ılson Nobuyoshi Kaneshima, Taniaˆ Ueda-Nakamura, Benedito Prado Dias Filho, Elisabeth Aparecida Audi, and Celso Vataru Nakamura Volume 2013, Article ID 841580, 9 pages

Comparison of Brazilian Plants Used to Treat Gastritis on the Oxidative Burst of Helicobacter pylori-Stimulated Neutrophil, Cibele Bonacorsi, Luiz Marcos da Fonseca, Maria Stella Gonc¸alves Raddi, Rodrigo Rezende Kitagawa, and Wagner Vilegas Volume 2013, Article ID 851621, 8 pages

In Vitro Antileishmanial Activity of Essential Oil of Vanillosmopsis arborea (Asteraceae) Baker,Aracelio´ Viana Colares, Fernando Almeida-Souza, Noemi Nosomi Taniwaki, Celeste da Silva Freitas Souza, JoseGalbertoMartinsdaCosta,K´ atia´ da Silva Calabrese, and Ana Lucia´ Abreu-Silva Volume 2013, Article ID 727042, 7 pages Contents

Which Approach Is More Effective in the Selection of Plants with Antimicrobial Activity?, Ana Carolina Oliveira Silva, Elidiane Fonseca Santana, Antonio Marcos Saraiva, Felipe Neves Coutinho, RicardoHenriqueAcreCastro,MariaNellyCaetanoPisciottano,ElbaLucia´ Cavalcanti Amorim, and Ulysses Paulino Albuquerque Volume 2013, Article ID 308980, 9 pages

Antiulcerogenic Activity of the Hydroalcoholic Extract of Leaves of Croton campestris A. St.-Hill in Rodents,FranciscoE.B.Junior,´ Dayanne R. de Oliveira, Elizangelaˆ B. Bento, Laura H. I. Leite, Daniele O. Souza, Ana Luiza A. Siebra, Renata S. Sampaio, Anita O. P. B. Martins, Andreza G. B. Ramos, Saulo R. Tintino, Luiz J. Lacerda-Neto, Patricia R. L. Figueiredo, Larissa R. Oliveira, Cristina K. S. Rodrigues, Valterlucio´ S. Sales, Francisco R. S. D. N. Figueiredo, Emmily P. Nascimento, Alefe B. Monteiro, Erika´ N. Amaro, JoseG.M.Costa,HenriqueDouglasMeloCoutinho,´ Irwin R. A. de Menezes, and Marta R. Kerntopf Volume 2013, Article ID 579346, 10 pages

Bonellia albiflora: A Mayan Medicinal Plant That Induces Apoptosis in Cancer Cells,RosaMoo-Puc, Juan Chale-Dzul, and Edgar Caamal-Fuentes Volume 2013, Article ID 823453, 8 pages

Biological Activities and Chemical Characterization of Cordia verbenacea DC. as Tool to Validate the Ethnobiological Usage, Edinardo Fagner Ferreira Matias, Erivania˜ Ferreira Alves, Beatriz Sousa Santos, CelestinaElbaSobraldeSouza,Joao˜ Victor de Alencar Ferreira, Anne Karyzia Lima Santos de Lavor, Fernando Gomes Figueredo, Luciene Ferreira de Lima, Francisco Antonioˆ Vieira dos Santos, Florido´ Sampaio Neves Peixoto, Aracelio´ Viana Colares, Aline Augusti Boligon, Rogerio´ de Aquino Saraiva, Margareth Linde Athayde, Joao˜ Batista Teixeira da Rocha, Irwin Rose Alencar Menezes, Henrique Douglas Melo Coutinho, and JoseGalbertoMartinsdaCosta´ Volume 2013, Article ID 164215, 7 pages

Evaluations of the Antimicrobial Activities and Chemical Compositions of Body Fat from the Amphibians Leptodactylus macrosternum Miranda-Ribeiro (1926) and Leptodactylus vastus Adolf Lutz (1930) in Northeastern Brazil, Mario Eduardo Santos Cabral, Diogenes´ de Queiroz Dias, Debora´ Lima Sales, Olga Paiva Oliveira, Diego Alves Teles, JoaoAntoniodeAraujoFilho,˜ JoseGuilhermeGonc´ ¸alves de Sousa, Henrique Douglas Melo Coutinho, JoseGalbertoMartinsdaCosta,´ Marta Regina Kerntopf, Romuloˆ Romeu da Nobrega´ Alves, and Waltecio´ de Oliveira Almeida Volume 2013, Article ID 913671, 7 pages

Efficacy of Plectranthus amboinicus (Lour.) Spreng in a Murine Model of Methicillin-Resistant Staphylococcus aureus Skin Abscesses,FranciscoFabio´ Martins de Oliveira, Alba Fabiola Torres, Thially Braga Gonc¸alves, Gilvandete Maria Pinheiro Santiago, Cibele Barreto Mano de Carvalho, Milena Braga Aguiar, Lilia Maria Carneiro Camara, Silvia Helena Rabenhorst, Alice Maria Costa Martins, JoseTelmoValenc´ ¸a Junior, and Aparecida Tiemi Nagao-Dias Volume 2013, Article ID 291592, 9 pages Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2014, Article ID 576382, 1 page http://dx.doi.org/10.1155/2014/576382

Editorial Medical Ethnobiology and Ethnopharmacology in Latin America 2013

Ulysses Paulino Albuquerque,1 Edwin L. Cooper,2 Maria Franco Trindade Medeiros,3 Rômulo Romeu da Nóbrega Alves,4 and Ana H. Ladio5

1 Biology Department, Federal Rural University of Pernambuco, Rua Dom Manoel de Medeiros, Recife, PE, Brazil 2 Laboratory of Comparative Neuroimmunology, Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA 3 Campus Cuite,´ Centro de Educac¸ao˜ e Saude´ (CES), Unidade de Educac¸ao˜ (UAE), Curso de Cienciasˆ Biologicas,´ Universidade Federal de Campina Grande (UFCG), Olho D’Agua´ da Bica S/N, Cuite,´ PB, Brazil 4 Departamento de Biologia, Universidade Estadual da Para´ıba, Monteiro, PB, Brazil 5 Laboratorio Ecotono, Universidad de Comahue, Bariloche, Argentina

Correspondence should be addressed to Ulysses Paulino Albuquerque; [email protected]

Received 23 December 2013; Accepted 23 December 2013; Published 22 January 2014

Copyright © 2014 Ulysses Paulino Albuquerque et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ethnobiology is a rapidly growing science, especially in Latin Ulysses Paulino Albuquerque America. Undoubtedly, growth in this field has many expla- Edwin L. Cooper nations, including the diversity in the lines of research, the Maria Franco Trindade Medeiros continuous training of researchers, and the great biological Romuloˆ Romeu da Nobrega´ Alves and cultural diversity in Latin America. This special issue Ana H. Ladio contains 14 manuscripts covering different approaches in ethnobiology. We should highlight the fact that most of the papers are focused on the pharmacological evaluation of plants or animals used in traditional medical practices. Of these 14 manuscripts, eight address the evaluation of the biological activities of medicinal plants, while two evaluate the biological activity of a number of animals used for medicinal purposes in Northeastern Brazil. Additionally, at least three papers in this special issue explore the relationship between healthcare and both cultural practices and foods. In addition, one paper employs a relatively simple approach to bring attention to the wide variety of medicinal plants that are used in Brazil, the diversity of which is possibly overestimated by taxonomic misconceptions. Thus, we hope that this collection of papers, accom- panying the annual issue of “Medical Ethnobiology and Ethnopharmacology in Latin America,” will give our readers valuable insights into diverse areas of ethnobiology research. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 868394, 17 pages http://dx.doi.org/10.1155/2013/868394

Research Article What the Iberian Conquest Bequeathed to Us: The Fruit Trees Introduced in Argentine Subtropic—Their History and Importance in Present Traditional Medicine

Pablo C. Stampella,1,2 Daniela Alejandra Lambaré,2,3 Norma I. Hilgert,2,4 and María Lelia Pochettino1,2

1 Laboratorio de Etnobotanica´ y Botanica´ Aplicada, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, 1900 La Plata, Buenos Aires, Argentina 2 CONICET, Argentina 3 Laboratorio de Botanica´ Sistematica´ y Etnobotanica,´ Facultad de Ciencias Agrarias, Universidad Nacional de Jujuy, San Salvador de Jujuy, Argentina 4 Instituto de Biolog´ıa Subtropical, CONICET, Facultad de Ciencias Forestales, Universidad Nacional de Misiones, CeIBA, 3370 Puerto Iguazu,´ Misiones, Argentina

Correspondence should be addressed to Mar´ıa Lelia Pochettino; [email protected]

Received 4 May 2013; Accepted 4 July 2013

Academic Editor: Ana H. Ladio

Copyright © 2013 Pablo C. Stampella et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

This contribution presents information about the history of introduction, establishment, and local appropriation of Eurasian fruit trees—species and varieties of the genera Prunus and Citrus—from 15th century in two rural areas of Northern Argentina. By means of an ethnobotanical and ethnohistorical approach, our study was aimed at analysing how this process influenced local medicine and the design of cultural landscape that they are still part of. As a first step, local diversity, knowledge, and management practices of these fruit tree species were surveyed. In a second moment, medicinal properties attributed to them were documented. A historical literature was consulted referring to different aspects on introduction of peaches and citric species into America and their uses in the past. The appropriation of these fruit-trees gave place to new applications and a particular status for introduced species that are seen as identitary and contribute to the definition of the communities and daily life landscapes. Besides, these plants, introduced in a relatively short period and with written record, allow the researcher to understand and to design landscape domestication, as a multidimensional result of physical, social, and symbolic environment.

1. Introduction the construction of a cultural landscape that includes the dimension of biocultural phenomenon, in the sense that it The native human groups—diverse in patterns of sub- is a physically, culturally, and historically determined envi- sistence and settlement strategies—that, since prehispanic ronment [1, 3]. times, inhabit different ecological territories of our coun- As of the process of European settling and peopling, try, constructed particular cosmovisions. Those cosmovi- like the Jesuit reductions and the founding of colonies in sions together with local knowledge shaped the practices of Argentine territory were, those cultural landscapes changed, management and use of those plant resources that defined as well as the characteristic ways of life of those ethnic the local landscape while designing a dynamic physical group. So, the breakup of natives spaces occurred along space where the own meanings and values of each people with the establishment of a new regime of exploitation were manifested themselves [1, 2]. This process includes and production, either politically or culturally imposed by the features that define domestication (cultural selection the Europeans, by means of the introduction of stuffs and and the emergence of a different reality), and its result is products from the Old World, for instance, cattle and several 2 Evidence-Based Complementary and Alternative Medicine crops that modified the configuration and representation of To Panama, landscape [1, 4–7]. Mexico, and Hawaii routes The settling of Spanish colonies in America led to the 10∘ beginning of plant species exchange between the New and the Old World that would affect multiple aspects of daily To Spain and Portugal life in both continents. Studies seldom approach the changes 0∘ produced since 1492 in the particular features of each group in agriculture, food, and even in their habits and customs— Empire of Brazil as medicine, rituals, and religious celebration. Instead, there 10∘ are many examples that refer to the expansion of plants since times much older than the arrival of Spaniards to America, and those cases show similar situations in other parts of the 20∘ world, in particular the movements of goods and species from Chaco the East and the South (Africa) to the classical Mediterranean. Among them, “sugar cane” (Saccharum × officinarum L.) 30∘ N can be mentioned which was rapidly adopted in the South of Europe through the advance of Islamic Empire [8]. As well, the European contact with Africa in the middle 15th 40∘ century was partially promoted for the desire of acquiring Patagonia spices and condiments. The silk route, established by the 1st and 2nd Century, constituted a very important commercial ∘ way for the exchange of cultivated spices. Plant species from 50 theOldWorldarrivedintoAmericaaspartofcultureand 80∘ 70∘ 60∘ 50∘ 40∘ idiosyncrasy of Iberian people, for example, “wheat” (several species of Triticum), “barley” (Hordeum vulgare L.) and “rye” (Secale cereale (L.) M. Bieb.), “broad bean” (Vicia faba L.) and City Spain-Portugal border otherpulses,seedand/orstonefruittreesofthegenerPrunus, Aquatic route Aboriginal border Cydonia, Malus,andPyrus (“plum,” “peach,” “quince,” “apple,” Land route and “pear”), and other temperate fruits, along with aromatic Figure 1: The Iberian conquest and colonization of America species and flowers that were included as exotic elements continent: an routes to terra firma of the 16th Century. within American native communities [5, 9–11]. All kinds of vessels were constantly exchanging plant germplasm between both worlds, and besides seeds, fruits, and adult plants, the transportation of branches, shoots, and grafts was also frequentramas (“ , retonos˜ , varetas y mugrones by Sepp [9]andPaucke[13]. Furthermore, several volumes (ingertos) de algunos arboles´ ...,” A n g l e r´ıa (1516) in de la about medicine and medicinal plants were found among Puente y Olea [12]), in barrels for wine. The main destination Jesuits goods in the moment of their expulsion from America of these trips was Antilles and later the continent (which was (Galeno de Pergamo´ (the 2nd Century), Rivero, Johann named “tierra firme,” that is, terra firma), Mexico and Peru Dolaeus (the 17th Century), and Suarez´ de Rivera, y Fuente the (16th Century). Then, according to chronicles, different Pierola´ (the 18th Century)), and it is supposed that they access routes were followed. This route, known as Caribbean- used also the books of Andrea Mattioli, Andres´ de Laguna, Andean, was accompanied by the one of Asuncion-La´ Plata Dioscorides´ and the Materia Medica of Father Joseph of River and the Brazilian one. Each of them had its own Montenegro, and Father Segismund Asperger [14]. On the particularities related to interethnic relationships, the origin other hand, it is worth mentioning that all around the world, of the introduced germplasm, and the associated knowledge different human groups attribute medicinal value to their with each plant and its management (Figure 1). food, and in this way, the species considered in this contribution, which all have been originated in Asia or Eastern Europe, were added to the Mediterranean record of useful 1.1. Fruit Trees and Local Medicine. Many cultivated plant plants by their biodynamic properties as well: citric as insect species with medicinal properties arrived to America, along repellentorantidoteinpoisoningandfordiversediseasesand with associated knowledge that was propagated by Span- symptoms, while the fruit trees of rose family were valued ish people. Their incorporation and appropriation by local as astringents, febrifuges, diuretics, and laxatives [15, 16]. In people involved a process of assignment of properties, the theparticularcaseofpeaches,theywereappreciatedasa progressive perception, and the search among the alternatives luxurious food, and in so much, they appear also in funebrial of obtaining, producing and applying of their medicinal contexts in the Mediterranean [17]. These therapeutical uses, use. even if they do not remain unaltered through the time, reflect De la Puente y Olea [12] gave the first references of these a minor incidence of commercial aspects than their use as medicinal species that were also mentioned in the description food and also show the local appropriation of these plants of the orchards of the priests of two Jesuit missions given through the generation of novel applications. Evidence-Based Complementary and Alternative Medicine 3

Based on what is mentioned earlier, this contribution important economic activities in Misiones are silviculture provides information about the medicinal use of two plant andagriculture,complementedwithcattleraising.Thefirst groups of Eurasian fruit trees in America, considering of them is based on monoculture of “pine” (Pinus spp.) and that their history of introduction, appropriation, and local “eucalyptus” (Eucalyptus spp.) destined to paper and wood recognition allows to interrelate them and to define them industriessettledinthearea,whichareinturnjobsource as representative elements of the landscape that they are for many inhabitants. The principal crops are “tobacco” currently part of. The two plant groups are part of larger scale (Nicotiana tabacum L.), “yerba mate” (Ilex paraguariensis A. studies and reflect the research performed by the authors on St.-Hil.), “tea” (Thea sinensis L.), and fruit trees, mainly citric different periods and regions of Argentine subtropics: (a)— (C. reticulata, C. × aurantium,andC. × latifolia). In summary, the case of citric, Citrus L., from northeastern Argentina the economy of the province is based in extractivism or and (b)—“peaches,” Prunus persica (L.) Batsch., from north- production of raw material with low industrial development western Argentina. From the ethnobotanical approach, the [26, 27]. In accordance with this scope, all the families inter- description, analysis, and comparison of the entry routes are viewed in rural contexts are characterized by their traditional presented, as well as the medicinal use of citric and fruit trees systems of subsistence production, with the occasional sale of of rose family in the past and in the present. This kind of surplus in urban tax-free fairs. study allows to make a time projection with the historical data and ethnobotanical information to reach their appropriation significance. 1.2.2. Northwestern Argentina (NWA): Dry Valleys and Yun- gas (Cloud Forests). Field works have been carried out in 1.2. Description of Local Landscape peasant communities of northwestern Argentina, settled in both western and eastern slopes of Andean foothills. In the 1.2.1. Northeastern Argentina (NEA): Phytogeographic Dis- west, the research was conducted in intermountain valleys tricts of “Campos” and “Urunday” in the South of Misiones. in the localities of Juella and Yacoraite, in Humahuaca Field works have been performed in two departments of Ravine (province of Jujuy), and in the East in two areas in thesouthoftheprovince,SanIgnacio(intheParanariver´ the province of Salta, in Los Toldos valley (Santa Victoria bank) and Concepcion´ de la Sierra (near the Uruguay river). Department) as well as in the villages that are settled in the This zone is a transition between the District of Campos and old Farm San Andres´ (in Oran´ department) (Figure 2). the District of Selvas Mixtas [18], where Mart´ınez-Crovetto Western intermountain valleys correspond, from the [19] places the district of Urunday. In this region, gallery phytogeographic point of view, to the Prepunena˜ or Bolivian- forests form corridors along Parana and Uruguay River and Tucuman province, characterized by a xerophytic vegetation, also along the course of the main streams, and they also while the eastern slopes belong to Yungas or cloud forest, cover the slopes of low hills as Santa Ana, San Juan, or which are determined by the presence of orographic rainfall San Jose.´ This is an impoverished forest, in transition to that define an exuberant vegetation, which varies accord- savanna, where forest islands within a grass matrix can be ing to the elevation shaping three different environments: observed, which is locally known as “capones” [20]. The Pedemontane Forest, Montana Forest, and Montane Cloud populations settled there are expressive of the history of the Forest [18, 28]. In spite of the marked differences resulting region. Diverse ethnic groups inhabited the area before the from the features of vegetation and general environment, European arrived there, and since the beginnings of the 20th in both regions agriculture is performed, destined not only Century, several migratory waves have arrived. At present, to family consumption but also to the sale of products in a mosaic of different cultures can be found in the area fairs and regional markets. This subsistence strategy has a shaping mixed communities: the “Criollos,” Mbya Guarani long history in the zone, where in addition to the typical people and the descendents of Polish, Ukrainians, Italians, American triada (Z. mays—Phaseolus spp.—Cucurbita spp.), Spaniards, Brazilians, and Paraguayans, among others. The several crops characteristic of Southern Andean Area were studied communities of TeyuCuar´ eandCerroM´ artires´ are grown, for example, Chenopodium quinoa Willd., Solanum a good example of people diversity, and they can be defined tuberosum L., and other species of Andean tubers like Oxalis as peasant communities [21]. This diversity is also reflected in tuberosa Molina and Ullucus tuberosus Caldas, among others thelanguageasbothSpanishandGuaraniarespokenandalso [29–31]. a local variation of Portuguese (in the Uruguay river area). As The inhabitants of these locations are descendents of for the economy, local groups derived their subsistence from native people; most of them with different degrees of mis- the “monte” (local name for the forest, [3]) through hunting, cegenation with Europeans arrived to the zone in different fishing, and gathering, and they also practiced a swidden moments. The language there spoken is a local variant of horticulture of Manihot esculenta Crantz, Ipomoea batatas Spanish, with the persistence of some words and grammatical (L.) Lam., Zea mays L., and different species of Phaseolus structures of Kechwa and Aymara [32–35]. and Cucurbita, along with other crops of low lands [22–25]. Native people living in the zone still carry out these activities, andtheyhaveaddedculturesintroducedintheareabythe 2. Materials and Methods Europeans, for instance, Oryza sativa L. and recently Glycine max (L.) Merr. They have also introduced livestock raising The present study is the result of a major ethnobotanical (cows,sheep,andminoranimals).Atpresent,themost research conducted in different stages. In the first moment, 4 Evidence-Based Complementary and Alternative Medicine

∘ 65∘ 55

Bolivia N

Paraguay Jujuy Yungas province settlements Paraguay 27∘ Chile Misiones ∘ 23 Dry valleys province settlements Northeastern

Argentina settlements Salta province Brazil

Figure 2: Localization of study areas in northwestern and northeastern Argentina.

the issue of agrobiodiversity richness and ways of man- 3. Results and Discussion agement were approached, and then the aspects related to local uses of the fruit trees were studied in depth to obtain 3.1. History of Entry Arrival and Use of Citrus in NEA. Citrus generalizations, in particular the therapeutic applications is native of east, south, and southeast of Asia, Australia, and that are the basis of this contribution. In Yungas, field southwest of Pacific Islands [44]. Most of citric species were work was performed between the years 1994 and 2000, introduced in Europe by Muslims during the 10th and 11th with the purpose to enquire about traditional medicine and [46] with the only exception of “citron” (Citrus medica L.) phytotherapy in general. In dry valleys and northeastern that was already known by Greeks and Romans, and those Argentina, the researches started in 2010 and still continue. fruits that were later introduced like “sweet orange” (Citrus In both cases, the object of the study is the local knowledge × aurantium L.), “tangerine” (Citrus reticulata Blanco), and and management of species and varieties of the genera “grapefruit” (C. × aurantium). Citric were brought to America Prunus and Citrus, respectively. Qualitative techniques have duringthesecondtripofColumbusin1493.Thechroniclers been used. The first settlers that took part in the research of that period mentioned that seeds of “oranges,” “lemons,” were randomly selected, and in some cases, the strategy of and “citron”, as well as “melons” and all sort of vegetables snow ball was applied [36, 37]. In NWA, the number of (pepitas y simientes de naranjas, limones y cidras, (ademas´ participants in the research reached in Yungas 59 people, in de) melones y de toda hortaliza [47]), have been taken to dry valleys 20 people, and in NEA 36 people. The interviews Central America from the Gomera Island (that belongs to the were oriented to the enquiry about local knowledge on Canarias archipelago). These references would correspond to the name or names by which the species are recognized, C. × aurantium L., C. × limon (L.) Osbeck,C.medica,and useful parts and allotted uses, with special reference to Cucumis melo L., respectively. To these “species” (in strict the therapeutical ones, including way of preparation and sense, hybrid taxa or cultivated varieties [44]), de la Puente administration. Likewise, by means of the review of a total of yOlea[12] added “toronjas” that would belong to C. maxima 38 bibliographic sources and historic documents of different (Burm.) Merr., “limas” of the group of C. × aurantifolia nature, ranging from the beginning of the 16th century to (Christm.) Swingle, and C. × limettioides Tanaka. These the present, the search for medicinal uses has been widened, authors also pointed out that the first routes of introduction and information about the entry of these crops in America of cultivated plants had been Andaluc´ıa and Castilla, in Spain; was obtained. The uses of these ethnovarieties in the past Guinea in Africa; and in Asia “orange” trees with large fruits and in the present were compared by means of chi-square which had been introduced from Filipinas. By the last quarter test. Voucher and other support materials (like branches and of the 16th century, several representatives of genus Citrus, leaves, as well as fruits) have been collected in the field as “oranges” and “lemons,” were already naturalized in the in the company of local inhabitants. This plant material Americas. Father Joseph de Acosta (who arrived to Peru in has been botanically identified [38–43], its taxonomy was 1572) mentioned that citric were the trees that had more controlled and updated [44, 45], and then it was deposited and better developed, and he was surprised because of the in the Herbary of Useful Plants and Collection of Fruits presence of orange groves, as well as other citric including and Seeds (CFS) of the Laboratorio de Etnobotanica´ y “limes,” “citron,” and similar (“limas,cidra,andyfrutade Botanica´ Aplicada (LEBA) of the Facultad de Ciencias Nat- este linage”[48]). Those cultivars of Citrus that had been urales y Museo, Universidad Nacional de La Plata, and in managed or domesticated in lesser extent, particularly those the Herbary of Museo de La Plata (LP). They have been used as rootstocks for grafted plants, show a special easiness assignedtheinitialandnumberofpersonalrecordsofeach to naturalization, related to the environments where they collector. have been planted, local people, fauna that eats their fruits, Evidence-Based Complementary and Alternative Medicine 5

“Orange”flowerwaterwasused(anditstillis)inconfec- × 10∘ tionery and the “bergamota” water (C. limon) in perfumery Orinoco [9, 52, 53]. After the Jesuits expulsion (1767), other uses are mentioned as, for instance, the use of bitter “orange” peel (C. × aurantium) to calm the ache of dysentery and as a tonic 0∘ Maynas too; the juice of “lima sut´ı” (C. × aurantifolia)tostoppaludic fever (“chucho”) and against the rush called “sudamina” o

Moxos “zarpullido”.Nevertheless, some of these properties have been 10∘ Chiquitos alreadydiscussedbyPisointhe16thcentury[54]. As from the contribution of Paucke [13] in the inventories 20∘ of the Jesuits expulsion made by Brabo [55], a record of the cultivars grown in Jesuitical missions can be approached. There were cultivated “lemons,” “sweet lemons,” “limones Guaranies N 30∘ ceut´ıes,” “sweet limes,” “citrons,” “toronjas,” and “sweet and sour oranges,” including cultivars like “Chinese orange” and others “oranges with fine peel.” Selected varieties were often 40∘ cultivated in orchards and gardens of Jesuits, where only the priests and a few chosen Guarani people, particularly skilled for horticulture, were allowed to enter. They were also grown in the colleges of the order placed in Buenos Aires, Cordoba,´ ∘ 50 Santa Fe, and Asuncion´ del Paraguay. The cultivars considered that commons or vulgar were frequently cultivated 80∘ 70∘ 60∘ 50∘ 40∘ in the Tupambae andoccasionallyinseveralAbambae— plots assigned to each family for subsistence production. Additionally, those varieties locally considered as “wild” grew City Spain-Portugal border Aquatic route Jesuit missions spontaneously, in process of naturalization, in different spaces Land route more or less modified by agroforestal management. With the estrangement of the religious order from Figure 3: The result of the Iberian conquest and colonization of Spanish colonies in 1768, missions were abandoned and America continent: Latin American Jesuit Missions (the 17th and during the following year, multiple migrations occurred to 16th Centuries). the neighbouring forest, to Corrientes, Santa Fe, Buenos Aires, and Asuncion,´ among other places [50]. Gardens were abandoned and a process of forest regeneration took place in several patches, formerly cultivated. It had to be expected that and their own process of hybridization and apogamy that are these anthropogenic landscapes were managed, especially complementary of sexual reproduction [1]. It seems that the those closer to buildings—some of them inhabited for a short entry (as well as the exit) of plant germplasm was continuous period, despite the Portuguese and Paraguayan invasions in and copious and more diversity appeared at the same time 1817 and 1818, in particular, fruits, yerba mate,andcotton when the different areas of the world met in touch. (Gossypium sp.) groves, no longer functional. Orange groves During the Jesuitical period, citric were cultivated in still remain by the interviewed settlers as present a few years different plots within the Guarani missions (Figure 3). The ago,andtheyhavebeendescribedandpraisedbythetravelers major diversity of fruits was found in the gardens of the in Misiones during the end of the 19th Century and the priests and in the colleges [1]. In addition, there were beginnings of the 20th [56–60]. “orange” groves belonging to the Tupambae (that were those community gardens for the production in large scale) destined not only for local consumption but also for the sale 3.2.HistoryofArrivalandUseof“Peaches”inNWA.Prunus [49, 50]. The fruits were used in medicinal preparations persica (Prunoideae) is a species belonging to the botani- against putrid fever (typhus), intestinal parasites, and stom- cal family Rosaceae. The genus is subdivided into several ach diseases, as appetizers and as antidotes against snake subgenera according to the morphology of the fruit. Based bites and other poisonous animals. The juice of the acidic on bibliographic record, its origin can be established in the varieties was considered to have cold properties in the frame mountainous zones of Tibet and southwest of China. Peaches of humoral medicine, and they were also uses as excipients couldbefoundintheMediterraneanbytheprinciples and sweeteners, for instance, the syrup and bark of “citron” of Christian area; nevertheless their taxonomy and history or “lemons” (C. × limon). Orange wine is cited as the most of evolution under domestication are still not completely healthyofallliquors,anditisusedasotherspiritsdistilled elucidated as for origin and dispersal centers [61, 62]. from fruits, as a remedy for coldness in native people. This The first galleons and other vessels coming from Sevilla wine was made with orange juice fermented in big glass arrived at the Antilles early in the 16th Century. They brought bottles with sugar, added with wine yeast fifteen days later and among other fruits (as mentioned previously) the fruits from left to stand for a whole year [51]. the Castillas or stone fruits among which a special reference 6 Evidence-Based Complementary and Alternative Medicine to “peaches” is made. Later, this germplasm entered Mexico leaves are used to heal grains and furuncles and mashed seeds and Peru (mainly in Lima), and from these points, the crop with egg white to stop hemorrhages [71]. was expanded to the rest of the continent [12]. In the specific case of the populations of NWA, historical sources and records show that these exotic fruit trees were 3.3. The Current Situation of Citrus. By the end of the introduced from Chile—following the route of the Pacific— 19th Century, Burmeister [71] referred to the presence of by the decade of 1550 by the expedition of Nunez˜ de Prado. “naranjos mandarinos” (tangerines, C. reticulata)inthe They thrived and by December 1552 a wealthy fructification province of Corrientes, and he recommended their intensive was described (“ya surg´ıan exuberantes los frutos de las cultivation together with “common oranges” and “chirimoyo” primeras siembras de los espanoles˜ ”[63]). (Annona cherimola Mill.) to export the fruits to large cities. The first seeds possibly arrived to Santiago del Estero, A few years early, Hieronymus [72]includedtheminhis which was equidistant in the route of communication publication about medicinal plants of Argentina under the between Peru and the Atlantic Ocean and from there dis- name of “naranjo fino” o “mandarino” (before Citrus deli- tributed to other provinces that were part of the colonial ciosa Tenore, now C. reticulata)withthesameusesthan government of Tucuman, created in 1563 and constituted by “sweet orange” (against scurvy, antitussive, and against bile the present Argentine provinces of Jujuy, Salta, Tucuman,´ diseases). “Pomelos” or “grapefruit” (C. × aurantium)entered Catamarca, Santiago del Estero, and the center of Cordoba.´ possibly by the same time or shortly before than “tangerine.” During this period, the influence of Chile on this government It is still discussed if “grapefruit” is a result of mutation or stopped and the district remained under the authority of hybridization, but there is agreement to consider Barbados Peru viceroy. The political space of Tucumanwasbornasa´ as the place of geographic origin by the principles of the 18th consequence of the discovery of R´ıo de la Plata Litoral and the Century, from where it disperses to the other Antilles islands exploration of the route that connect it with Peru. Its occu- not before a Century later [73, 74]. Maybe “grapefruits” were pation was guided by the need to expand the territory and mistaken for “pampelmuse” or “toronjas” (C. maxima). These to find new lands suitable for cultivation. Unlike the native new introductions were coincident with the reconfiguration groups that inhabited the south of Argentine territory, the of local people through the migratory process that had place Andean partialities that were comprised in this government in the first years of the 20th Century. The first immigrants have been soon subjected to the new administration after the were brought to Misiones by official colonization that started arrival of the Spanish founders. Such was the case that by the in 1898 with the arrival of Polish and Ukrainians from second half of the 16th and beginning of the 17th Century, Galitzia (one of the poorest zones of rural Europe) to the native people managed European crops, although these plants village of Apostoles.´ The state was also responsible for the did not integrate their daily diet [4, 5, 63–65]. peopling of those lands of dorsal central ridge (Alem,´ Obera,´ By that moment, in Argentina, the subsistence economy and Cainguas).´ On the other hand, private colonization was based on the exploitation of native workforce to labor incorporated mainly Deutsch immigrants from Brazil or in the country with plants and animals introduced from directly from Europe, and it covers mainly the zone of Upper Europe, with some local exceptions like “maize.” In 1590, a Parana.´ Later, Creole and Paraguayan immigrants arrived process of internalization of commercial circuit occurred, looking for job [75]. which became an integrated market from Potosi to Buenos New wisdoms and practices on the environment arrived Aires passing through Tucuman.´ Thus, the influence of Chile with the immigrants, as well as a different valoration of the on NWA region was relegated [66]. In this frame, Jujuy was plants with which they interacted; for example, the “oranges,” considered during colonial period a pathway employed by that in the new land were very common, in the origin natives and Spaniards to arrive to Peru, and it constituted a countries were such a precious good that they were given as a border of Tawantinsuyu (Inca Empire) and later a pathway to present for Christmas. Another source of variability appeared Bolivia (Potos´ı) [67]. with the industrialization of citric cultures. After the first Alongwiththefooduseofpeaches,therearearchae- quarter of the 20th Century, coincident with the start of ological evidence of their ceremonial use in ritual contexts citriculture in NEA, diverse cultivars were introduced from by native people during Spanish-Indigenous period by the different parts of the world (Africa, India, USA, China, and year 1600 [68]. Regarding their medicinal applications in Japan). The model of citric industry is the one developed the zone, the earliest mentions date from colonial period in the states of Florida and California (USA) that consisted and referred to the use of peaches as natural laxatives. In of new varieties grafted on rootstocks of “sour orange,” fact, in different publications referring to European uses of which was later replaced—because of CTV (Citrus tristeza “peaches,” the flowers and leaves are mentioned as to be used virus, a kind of Closterovirus) epidemy—by rootstocks of in infusion and/or syrup as child laxatives. Nevertheless, it is “sweet orange,” “Cleopatra orange,” “Rangpur lime,” an d warned that flowers could be toxic because of the presence of “sweet lime”, among others [76–79]. By the decade of 1970, a amygdaline, more abundant in some varieties than in others homogenization took place because of the increasing agrarian [43, 69, 70]. Poultices with the leaves on the abdomen are globalization and market request for uniform fruits as for recommended against parasites, as anti-inflammatory, and to size, shape, colour, and taste. At present, 78% of culti- heal wounds and herpes. The infusion of leaves with milk is vated citric belong to “tangerines” (“Satsuma,” “Clementina,” also mentioned as antiparasitic. The distilled water of flowers and “Murcott”), 17% to “oranges” (“Valencia Late,” “Salus- is used to remove specks from the skin. Dry and powdered tiana,” “Lanelate,” and “Newhall”), and the remaining 5% to Evidence-Based Complementary and Alternative Medicine 7

Table 1: The citric of NEA: local varieties and their botanical identity. The ∗ indicates the presumable local ethnovarieties.

Local name Local variety Botanical species Voucher Cidra Cidra Citrus maxima (Burm.) Merr. Stampella 34 (LP) Pomelo cidra C. maxima × Citrus × aurantium L. Not collected ∗ Pomelo Pomelo blanco C. × aurantium (grupo pomelo) Stampella 19 (LP) Pomelo rosado C. × aurantium (grupo pomelo) Stampella 78 (CFS) ∗ C. × aurantium (grupo naranja Apepusilvestre´ Stampella and Hilgert 10 (LP) amarga) ∗ C. × aurantium (grupo naranja Apepu´ Apepucasera´ Stampella 85 (CFS) amarga) ∗ C. × aurantium (grupo naranja Apepudulce´ Stampella 53 (CFS) amarga) ∗ Naranja silvestre C. × aurantium (grupo naranja dulce) Stampella 128 (LP) Naranja silvestre de fruto C. × aurantium (grupo naranja dulce) Stampella 162 (CFS) grande ∗ Naranja casera C. × aurantium (grupo naranja dulce) Stampella 112 (CFS) Naranja Naranja injertada C. × aurantium + Citrus trifoliata L. Commercial cultivar Naranja de ombligo C. × aurantium “Ombligo”+ Commercial cultivar (injertada) C. trifoliata Naranja calderon´ C. × aurantium “Calderon”´ Commercial cultivar Naranja que gu´ıa C. × aurantium Not collected ∗ Citrus reticulata Blanco “Comun´ de Mandarina silvestre Stampella 103 (LP) Concordia” ∗ Mandarina casera C. reticulata “Comun´ de Concordia” Stampella 121 (CFS) Mandarina injertada (con Citrus reticulata “Okitsu” (=C. Commercial cultivar lima) unshiu)+Citrus × limettioides Tanaka Mandarina Citrus reticulata “Encore”+ Citrus × Mandarina injertada (con limon (L.) Osbeck (=C. jambhiri Stampella 116 (LP) limon´ cidra) Lush.) Mandarina injerto con apepu´ C. reticulata “Encore” Stampella, Cabanillas, and Hilgert 172 (CFS) Mandarinacoloradao C. reticulata “Cleopatra” (=C. reshni Stampella, Cabanillas, and Hilgert 170 (CFS) mandarinola Tanaka) Mandarinacoloradajaponesa C. reticulata “Okitsu” Stampella, Cabanillas, and Hilgert 175 (CFS) Mandarina bergamota C. reticulata “Encore” Stampella 86 (CFS) ∗ Limon´ o limon´ comun´ C. × taitensis Risso (=C. limonia Osb.) Stampella 80 (LP) Limon´ amarillo Citrus × limon “Verna” (=C. limon)Commercialcultivar Limon´ lima Citrus × latifolia Tanaka Stampella, Keller, Nu´nez,˜ and Dutra 60 (CFS) Limon´ Citrus × limon (L.) Osbeck (=C. Limon´ cidra o limon´ rugoso Stampella, Hilgert, and Furlan 132 (CFS) jambhiri) Citrus × aurantifolia (Christm.) Limon´ sut´ıl o lima acida´ Not collected Swingle Citrus × limon (L.) Osbeck (C. × Limon´ real o limon´ aromatico´ Stampella, Cabanillas, and Hilgert 177 (CFS) latifolia × C. maxima?) Lima Lima, lima dulce Citrus × limettioides Tanaka Stampella 79 (CFS) Citrus japonica Thunb. (=Fortunella Quinoto Quinoto Stampella, Hilgert, and Furlan 150 (CFS) japonica [Thunb.] Swingle) Citrus trifoliata L. (=Poncirus Trifoliata Trifoliata Not collected trifoliata Raf.)

“lemons” (“Tahiti” and “Eureka”) [80]. In spite of this, several (from the “monte”). All the informants agree in considering cultivars—some of them in a naturalized status [81, 82]—are “apepu” as native and wild, belief strengthened by the fact conceived by most of the interviewed people as native fruit that this plant has a Guarani name. “Apepu” has different trees. “Apepu,”´ “naranja comun,”´ “mandarina comun,”´ “lima interpretations: some authors consider it formed by the prefix dulce,” and “limon´ mandarina” are considered wild fruits 𝑎 meaning fruit, pe that is peel, and pu meaning noise 8 Evidence-Based Complementary and Alternative Medicine

Table 2: Ethnovarieties (locally recognized varieties) of peaches (Prunus persica)fromdryvalleysinNOA.

Group of ethnovarieties of durazno Ethnovariety Voucher (Prunus persica) Amarillo entero 22 Lambare(CFS)´ Amarillo corazon´ rojo 31 Lambare(CFS)´ Durazno comun´ (endocarp adhered to Blanco 52 Lambare(CFS)´ mesocarp) Rosado 45 Lambare(CFS)´ Duraznojorge(=CholoCholito) 30Lambare(CFS)´ Durazno alancate (=Olancate) 60 Lambare(CFS)´ Durazno prisco (=Frisco that can be Amarillo 46 Lambare(CFS)´ opened) (endocarp not adhered to Blanco 50 Lambare(CFS)´ mesocarp) Rosado 20 Lambare(CFS)´ and breakup [83]. Others [84] instead consider that “apepu” drunk three times a day, and if discomfort still persists, it means flaccid, but ape would be dorsum or surface and pu, canbetakenfortwoconsecutivedays.Theshootsareused hollow sound, in reference to the noise made by the epicarp in humoral medicine and soaked in vinegar, in frictions to when knocked with the fingers, a distinctive feature of this remove heat from the body, or in infusion that has to be drunk fruit. three times to stop “chucho” (very high fever with sweat) [87]. As a result of this research, 8 ethnospecies of Citrus The pulp of the fruit with apple pulp is mixed and mashed (including 27 ethnovarieties) have been recorded which to use as a thickener element in the preparation of “yista”: a aresummarizedinTable 1. Local ethnovarieties are those solid mixture of plant components with basic properties used generally named as “communes,” “caseras,” and “silvestres” to chew “coca” (Erythroxylum coca Lam.) [88, 89]. (that mean common, domestic, and wild, resp.) and are The information referred to the use of the previously marked in Table 1. They are coincident with those historically mentioned ethnovarieties in the past, according to the lit- cultivated by the “Criollos” and can be found in the monte erature, as well as the recorded uses in the study area at andalsoinoldhousesmadeofperishablematerial,knownas present, is detailed in Table 3 and compared in Figure 4.By “tapera” (that means abandoned house). comparing the number of ethnovarieties used for each body system either in the past and in the present, higly significative differences (𝜒210 g.l., 𝑃 < 0.005 = 41.57) appeared (Figure 5). 3.4. The Current Situation of “Peaches”. Early introductions In Figure 5, it can be observed that the uses for affections of of seed and stone fruit trees of the rose family resulted in the digestive system are the most frequent in both periods and establishment of local cultures, which have been maintained that nowadays the usage of these resources becomes more by means of both seed or vegetative reproduction, but without importantinthetreatmentofdiseasesofrespiratorysystemas theintroductionofnewgermplasm.Forthatreason,those well as the circulatory, osteo-artro-muscle ones and humoral fruittreespopulationsareconstitutedbythesamevarieties medicine. introduced in times of the Colony, but they reflect local criteria of cultural selection as well as plant adaptations to a particular environment. In the case of “peaches,” those 500 4. Final Considerations years of history in the new settlement shaped a complex of 9 ethnovarieties comprised within 2 groups of ethnovarieties Characteristics of native people and of colonization streams (Table 2) locally identified by diverse attributes that diverge shaped cultural landscapes with particular traits that reflect fromthoseprizedbythemarket[85]. In this way, and on the identified plant species and their uses. Among the fruit although in the last years commercial “peaches” cultures trees introduced in the northern Argentina, peaches had a wereintended,incontrasttowhathappenswiththecitric long history of use in Europe (although their centre of origin in NEA, here the industrial cultivation of “peaches” has not was Asia). When peaches were brought to the American been established, and the commercial varieties introduced continent, during the early conquest and colonization, they did not represent an instance of diversification or entry of were introduced as luxurious foods, reproducing their history novel germplasm to local crop. The permanence is observed in Europe, where they had a reduced number of therapeutic tooinfoodandtherapeuticalusesgiventotheseplants.Many applications. The citric, instead, by that time had a short inhabitants of dry valleys make a preserve (“compota”) with historysharedwithEuropeanpeople,andtheyweremuch dry “peaches” (“pelones”) boiled with sugar, and the juice valued not only as food but as medicines too. In NWA, of the preparation is used to treat kidney diseases [86]. In thecolonizersintroducedcultivatedplantsfromSpainand Yungas, settlers cultivate “peaches” in the middle stage of the entered from Peru or Chile, though some contacts with the forest. There, a decoction made of dry flowers and powdered introductions performed through Buenos Aires port (over seeds is made to treat diseases related to intestines (diarrhea, R´ıo de la Plata) could be mentioned. From the ecological flatulence, and intestine inflammation). The preparation is point of view, Humahuaca Ravine and other dry valleys Evidence-Based Complementary and Alternative Medicine 9 ] ] 92 , 91 ] ] , 47 47 94 47 , ] 93 , 47 ] 47 90 ] 47 ] 47 Validation Antibacterial [ Immunomodulatory effect [ Antiulcer [ Fever [ Tonic, antioxidant, hypoglycemic, and anticholinesterase [ Fever, anthelmintic [ Antimicrobial, antimycobacterial and spasmolytic [ Anticancer activity [ To low blood pressure, sedative,fortheheart,andfor diabetes To low blood pressure, for diabetes, for the liver, sedative, digestive, for influenza, and for colds Digestive, for stomach ache, for flatulence, and for gastrointestinal diseases To chew coca (dietary supplement) Medicinal use For the stomach, stomach ache,fordiarrhea,against as“empacho,” tonic, and for the fever Refreshing (against heath, in humoral medicine) Leaf Leaf used Juice Juice Fruit To low blood pressure Epicarp Flowers For the heart Part of the plant ] 73 , 71 – 69 , 52 , 47 , Uses in the past Present uses in the study area 43 , 16 , 15 Medicinal Use [ To clean liver and stomach, tostomach alleviate inflammations, to alleviate fevers,improve blood, to and to cure wounds,and throat, uvula abscess. Sedative For halitosis, for dysentery, to comfort stomach, diuretic, and mild antidote For the stomach, tonic, stimulant, and expectorant AntispasmodicAfter the labour Appetizer, for stomach ache, and anthelmintic Antidote against poisons Appetizer, to invigorate stomach, and antidote against poison Leaf Toavoidevilspirits,antidoteagainstpoisons (emetic), insect repellent, to freshen upremedy breath, against plague, to invigorateand stomach against vomiting and fainting after labour Effectivepurgativetoremovepoisonfromthe body Part of the plant used Juice Epicarp Leaf Shoots FlowersSeedsFruit Medicinal Anthelmintic Fruit Juice Table 3: Summary and comparison of uses of the studied fruit trees. Past uses in Europe and present ones in study area. L. aurantifolia limettioides × × (Christm.) Sw. Citrus Tanaka Scientificname Citrus medica Citrus Common name Cidra Lima 10 Evidence-Based Complementary and Alternative Medicine ] 96 , ] 47 98 , 97 ] 47 ] 95 , 47 Validation Antiseptic, carminative, diuretic, eupeptic, vascular stimulant, protective vitamin, and antimicrobial [ Analgesic, antianemic, antiemetic, antiesclerotic, antipyretic, antiseptic, demulcent, moisturizing, remineraliser, antitoxic, and vulnerary [ Peel antiperoxidative, antithyroidal, antihyperglycemic, cardioprotective, anthelmintic, and antimicrobial [ Antidiarrhoeic, diuretic, intestinal mucosa protector, local haemostatic, vascular stimulant and protector, vitaminic, antioxidant, sedative, and anxiolytic [ Medicinal use For influenza, sudorific, for colds, and for sore throat For influenza, for cough, and to low blood pressure Dietary supplement, refreshing, sedative, digestive, and for respiratory diseases Refreshing, for liver, stomach ache, against indigestion, for fever, for blood, for cough, expectorant, for influenza, for pneumonia, bronchitis, colds, angina,purgative,as anti-inflammatory insectin bites, aphthas, for hangover, and for lose weight Leaf used Juice Juice Flower Part of the plant ] Table 3: Continued. 73 , 71 – 69 , 52 , 47 , Uses in the past Present uses in the study area 43 , 16 , 15 Medicinal Use [ Against sickness and vomits To prevent drunkenness To eliminate pimples and blackheads Anthelmintic and to eliminate kidney stones and sand To relieve thirst and febrifuge Antidote against poisons To invigorate stomach, appetitive, digestive, to improve breath, to strengthen heart, antidote, to improve hygiene, and to removefrom phlegm the palate To smooth the face and toof remove jaundice yellow color Part of the plant used Seed Epicarp Fruit To cure the plague and digestiveJuice Fruit (L.) limon × Scientificname Citrus Osbeck ´ on Common name Lim Evidence-Based Complementary and Alternative Medicine 11 ] ] 101 47 ] 100 , 99 , 47 Antimicrobial [ Validation Appetizer, cholagogue, demulcent, eupeptic, reduces cholesterol, tonic, vascular stimulant, it aids in digestion and relieves flatulence, cardiovascular health, anticancer, sedative, anxiolytic, and antiviral [ Antispasmodic, sedative and tranquillizer [ To low blood pressure, for influenza, digestive and for “empacho,” forcolumn “pasmo” “pasmo,” bone pain, and hemorrhoids, and angina Refreshing, vitamin supplement, for influenza, for respiratory diseases, cough and bronchospasm, for “pasmo,” and angina Refreshing, digestive, and sedative Fever, for influenza, digestive, and for hangover Sedative specially for children, fevers, influenza, for cough, emetic, antiseptic, tooth ache, and waist ache Medicinal use For flatulence, refreshing (humoral medicine), for hemorrhages, to low blood pressure, for influenza and fever, for cough, sedative, digestive, hepatic, laxative, angina, antiseptic, bone pain, and low back pain For influenza,cough, and for colds Leaf Leaf Leaf used Juice Fruit For influenza and for fever Fruit Sudorific and for influenza Sprout For influenza mesocarp mesocarp Epicarp and Epicarp and Part of the plant ] Table 3: Continued. 73 , 71 – 69 , 52 , 47 , Uses in the past Present uses in the study area 43 , 16 , 15 Medicinal Use [ To invigorate stomach, heart, and stomach tonic and fainting after labour Juice For liver, cough, and against scurvy Juice Part of the plant used Flowers To strengthen heart and stomach Epicarp To comfort stomach Flowers L. L. aurantium aurantium × × Scientificname Citrus Citrus reticulata Blanco Citrus Common name Naranja Naranja agria, apepu Mandarina 12 Evidence-Based Complementary and Alternative Medicine ] 104 ] – 105 102 ] 107 ] 106 Validation Laxative [ Skin diseases [ Hypotensive, antistress, against anxiety, hypoglycemic, and hypolipidemic [ Medicinal use Intestine diseases (diarrhea, flatulence, and intestine inflammation) Vitamin supplement, refreshing, for cough, catarrh and asthma, influenza, digestive, and to low blood pressure For fever Vitamin supplement and for influenza Against influenza, fever, for cough, and to low high blood pressure used Juice Fruit seeds Shoots Refreshing and febrifuge mesocarp Epicarp and Flowers and Part of the plant ] Table 3: Continued. 73 , 71 – 69 , 52 , 47 , Uses in the past Present uses in the study area 43 , 16 , 15 Medicinal Use [ Laxative, antiparasitic, anti-inflammatory, and healing Part of the plant used FruitLeaf LaxanteFlowers Laxative and to remove freckles Fruit Kidneys and sight Anti-inflammatory [ Seeds Against hemorrhage Leaf For liver and antidote Leaf L. L. aurantium × Scientificname Prunus persica Batsch Citrus Common name Pomelo Durazno Evidence-Based Complementary and Alternative Medicine 13

8 7 6 5 4 3 2 1 0 Past Past Past Past Past Past Past Past Past Past Past Present Present Present Present Present Present Present Present Present Present Present Humoral medicine Symbolic medicine Integral health Circulatory system Dermal system Digestive system Nervous system Reproductive system Respiratory system Urinary system Arthroscopic -osteomuscular system

Citrus × aurantifolia (Christm.) Sw., Citrus × limettioides Tanaka Citrus medica L. Citrus × aurantium L. Citrus reticulata Blanco Citrus × limon (L.) Osbeck Prunus persica L. Batsch

Figure 4: Number of therapeutic properties assigned per species per system.

50 Portugal, Africa, India, and the southeastern of Asia. Such 40 citric, through the agency of Jesuits, became conspicuous 30 20 elements in local landscape and turned into characteristic 10 cultures of the zone. Furthermore, several taxa have passed 0 to a naturalized status, and now they characterize also the spontaneous flora and are considered by the inhabitants as native from the zone. In both areas, NWA and NEA, in parallel to the establishment of these crops as an agricultural and economic resource, Integral health Integral Dermal system Urinary system Urinary Nervous system Nervous

Digestive system Digestive diverse use has been developed, for example, the therapeutic Circulatory system Circulatory Humoral medicine Humoral Respiratory system Respiratory Symbolic medicine Symbolic applications referred to throughout the text, and many of Reproductive system Reproductive

Arthroscopic system -osteomuscular them are novel with respect to those uses recorded in other Past parts of the world, as a result of local experimentation. Either Present past or present uses, although diverse, seem to be founded on Figure 5: Number of ethnovarieties used for each body system in the antioxidant properties of the fruits here considered, tak- the past and present. ingintoaccountthatoxidativedamage,causedbytheaction of free radicals, may initiate and promote the progression of a number of diseases such as the ones treated with citric and peaches. were good recipients of “peaches,” “apples” (Malus domestica Despite the differences, the examples here posed allow to Borkh.), “pears” (Pyrus communis L.), and “quinces” (Cydonia understand the processes of local appropriation of introduced oblonga Mill.). Consequently, both for cultural and environ- plants. The decision to incorporate foreign elements has mental reasons, the local agricultural communities adopted to be understood in the frame of the own cosmovisions rapidly these plants to cultivate them. But, along this process, of native groups from different zones of Argentina. Those new varieties consistent with local values had been selected, groups were going through a moment of strong trans- as well as those that showed more tolerance for the conditions formations, but they did not resign their prominence in in the new space. Thereby, varieties have been configured decision making which contributed to cultural reproduction. which are considered as defining a particular agricultural Indeed, they took an active part, either incorporating as context and recognized as an identity factor by the settlers: rejecting, redefining, or abandoning customs, objects, and “duraznos de la Quebrada” (in reference to Humahuaca products within the unceasing social and historic dynamics. Ravine). In NEA, on the other hand, the introductionof citric In this cultural frame, the appropriation of “peaches” and was the result of the confluence of two colonization streams: citrus turns these originally exotic fruits into identifying the Spanish one known as R´ıo de la Plata-Asuncion´ and elements that contribute to the definition of the communities theBrazilianonethatsummarizedgoodapportationsfrom and the landscape where their daily life has place. Given 14 Evidence-Based Complementary and Alternative Medicine

that those environments can undergo changes and so their Ministerio de Agricultura, Pesca y Alimentacion,´ Madrid, material nature frequently varies through time, these plants Spain, 1991. introduced in a relatively short period and with written [11] J. A. Gonzalez,´ M. Garc´ıa-Barriusoy, and F. Amich, “Medicina record allow the researcher to understand and to design tradicional en Espana˜ y Argentina: comparacion´ preliminar de landscape domestication, as a multidimensional result of algunas especies originarias de Europa,” in Avances Sobre Plan- either physical, social, and symbolic environment [108, 109]. tas Medicinales Andinas,N.D.VignaleandM.L.Pochettino, Eds., chapter 7, pp. 245–268, RISAPRET/CYTED, San Salvador de Jujuy, Argentina, 2009. Acknowledgments [12] M. de la Puente y Olea, LosTrabajosGeograficos´ de la Casa de la Contratacion´ ,EscuelaTipografica´ y Librer´ıa Salesianas, Sevilla, The authors especially thank the inhabitants of the areas Spain, 1900. in NEA and NWA for kindly sharing their knowledge and [13] F. Paucke, Hacia allayparaac´ a(Unaestadaentrelos´ time. The authors are grateful to the Laboratorio de Botanica´ indios mocob´ıes 1749–1767, Universidad Nacional del Tucuman,´ Sistematica´ y Etnobotanica´ (LABOCyE) FCA, UNJu, Labora- Tucuman,´ Argentina, 1944, Traduccion´ castellana por E. Wer- torio de Etnobotanica´ y Botanica´ Aplicada (LEBA) FCNyM, nicke. Tomo III, segunda parte. UNLP,theInstitutodeBiolog´ıa Subtropical (IBS) FacFor, [14] M. C. Vera de Flachs and C. Page, “Textos clasicos´ de medicina UNaM, and CONICET for material and financial support. en la botica jesu´ıtica del Paraguay,” Cuadernos del Instituto Our gratitude goes also to Guillermo Gil for advise in statis- Antonio de Nebrija,vol.13,pp.117–135,2010. tical analyses and to anonymous reviewers for improving the [15] J. Morton, Fruits of Warm Climates, Florida Flair Books, Miami, paper. Fla, USA, 1987. [16] J. Dr. Duke’s Phytochemical and Ethnobotanical Databases, References http://www.ars-grin.gov/duke/. [17] L. Sadori, E. Allevato, G. Bosi et al., “The introduction and [1] A. Capparelli, N. Hilgert, A. Ladio et al., “Paisajes cultur- diffusion of peach in ancient Italy,” in Plants and Culture: Seeds ales de Argentina: pasado y presente desde las perspecti- of the Cultural Heritage of Europe,J.P.MorelandA.M.Mercuri, vas etnobotanicas´ y paleobotanicas,”´ Revista de la Asociacion´ Eds.,pp.45–61,CentroEuropeoperiBeniCulturali,Ravello, Argentina de Ecolog´ıa del Paisaje,vol.2,no.2,pp.67–79,2011. Italy, 2009. [2] J. Ochoa and A. Ladio, “Pasado y presente del uso de plan- [18] A. L. Cabrera, Regiones Fitogeograf´ıcas Argentinas, Enciclopedia tassilvestresconorganos´ de almacenamiento subterraneos´ Agricultura y Jardiner´ıa.TomoII.,ACME,1976. comestibles en la Patagonia,” Bonplandia,vol.20,no.2,pp.265– [19] R. Mart´ınez-Crovetto, “Esquema fitogeografico´ de la provincia 284, 2011. de Misiones (Republica´ Argentina),” Bonplandia,vol.1,no.3, [3] M. L. Pochettino, M. R. Mart´ınez, and M. Crivos, “Land- pp.171–223,1963. scape domestication among two mbya-guaran´ ´ı communities in [20] J. C. Chebez, Gu´ıa de las reservas naturales de la Argentina. Misiones, Argentina,”in Ethnobiology and Biocultural Diversity, Nordeste, Editorial Albatros, Buenos Aires, Argentina, 2005. J.R.Stepp,F.S.Wyndham,andR.K.Zarger,Eds.,pp.696–704, University of Georgia Press, Athens, Ga, USA, 2002. [21] L. J. BartolomeandG.Schiavoni,´ Desarrollo y estudios rurales en Misiones, Ediciones CICCUS, Buenos Aires, Argentina, 2008. [4] A. Capparelli and R. A. Raffino, “Arqueoetnobotanica´ de El Shincal I: tallos finos, frutos y semillas,” Tawantinsuyu,vol.3, [22] L. Cadogan, Cadogan, segun´ Cadogan. Entrevista Diario pp.40–57,1997. La Tribuna, 9 de febrero de 1969, http://www.portalguarani .com/obras autores detalles.php?id obras=17830. [5] M. Giovannetti, “La conquista del noroeste argentino y los cultivos europeos,” Fronteras de la Historia,no.10,pp.253–283, [23] B. Susnik, Los abor´ıgenes del Paraguay: Tomo II. Etnohistoria de 2005. los Guaran´ıes: epoca´ colonial,MuseoEtnografico´ “A. Barbero” , Escuela Tecnica´ Salesiana, Asuncion,´ Paraguay, 1979-1980. [6] M. Giovannetti and V. Lema, “Cultivos introducidos por los europeos en el Shinkal de Quimivil: La presencia de los Hispano [24] M. Chase-Sardi, “El tekoha. Su organizacion´ social y los efectos en la supervivencia ritual,” in Entre Pasado Y Presente,A.Cetti, negativos de la deforestacion´ entre los Mbya-guaran´ ´ı,” Suple- A. Re, D. Rindel, and P. Valeri, Eds., pp. 410–429, VI Jornadas mento Antropologico´ ,vol.24,no.2,pp.33–41,1989. de jovenes´ investigadores en ciencias antropologicas,´ Instituto [25] E. Schaden, Aspectos Fundamentales de la Cultura Guaran´ı, Nacional de Antropolog´ıa y Pensamiento Latinoamericano, Centro de Estudios Antropologicos´ (CEADUC), Universidad Buenos Aires, Argentina, 2005. Catolica,´ “N.S. de la Asuncion”,´ Asuncion,´ Paraguay, 1998. [7] V. S. Lema, Domesticacion´ vegetal y grado de dependencia [26] G. Schiavoni, “Organizacion´ domestica´ y apropiacion´ de tierras ser humano-planta en el desarrollo cultural Prehispanico´ del fiscales en la Provincia de Misiones (Argentina),” Desarrollo Noroeste argentino [Tesis doctoral (inedita)]´ , Facultad de Cien- Economico´ ,vol.34,pp.595–608,1995. cias Naturales y Museo, Universidad Nacional de La Plata, La [27] G. Schiavoni, Colonos y ocupantes: parentesco, reciprocidad y Plata, Argentina, 2009. diferenciacion´ social en la frontera agraria de Misiones,Editorial [8] J. H. Galloway, “Sugar,” in The Cambridge World History of Universitaria, Universidad Nacional de Misiones, Posadas, Foods,K.F.KipleandK.C.Ornelas,Eds.,vol.1,chapterII,pp. Argentine, 1998. 437–449, Cambridge University Press, Cambridge, UK, 2000. [28] G. Navarro and M. Maldonado, Geograf´ıa Ecologica´ de Bolivia, [9] A. Sepp, Relacion´ de Viaje a las Misiones Jesu´ıticas, Edicion´ Vegetacion´ y Ambientes Acuaticos´ , Editorial Centro de Ecolog´ıa cr´ıtica de W. Hoffmann. Tomo I. Ed. EUDEBA, 1971. Difusion´ Simon´ I. Patino,˜ Santa Cruz, Calif, USA, 2002. [10] J. Garc´ıa Paris, Intercambio y difusion´ de plantas de consumo [29] G. Karasik, “Intercambio tradicional en la Puna Jujena,”˜ Runa, entre el Nuevo y el Viejo mundo, Servicio de Extension´ Agraria, vol. 14, pp. 51–91, 1984. Evidence-Based Complementary and Alternative Medicine 15

[30] G. F. Scarpa and P. Arenas, “Especias y colorantes en la cocina [48] B. de las Casas, Historia de las Indias, Tomo II. Edicion´ del tradicional de la Puna jujena˜ (Argentina),” Candollea,vol.51, Marques´ de la Fuensanta del Valle y D. JoseSanchoRayon´ pp.483–514,1996. Imprenta de Miguel Ginesta, Madrid, Spain, 1875. [31] N. I. Hilgert and G. E. Gil, “Traditional andean agriculture and [49] J. de Acosta, Historia natural y moral de las Indias,Impresoen changing processes in the Zenta river basin, Salta, Northwestern Casa de Juan Leon,´ Sevilla, Spain, 1590. Argentina,” Darwiniana,vol.43,no.1–4,pp.30–43,2005. [50] R. Carbonell, Estrategias de desarrollo rural en los pueblos [32]G.F.Torres,M.C.Bianchetti,andM.E.Santoni,“Ladieta guaran´ıes (1609–176), Antoni Bosch, Sociedad Estatal Quinto de los campesinos del Valle Calchaqu´ı y de la Puna y sus Centenario, Instituto de Estudios Fiscales & Instituto de Coop- determinantes culturales,” Kallawaya Serie Monografica´ ,no.1, eracion´ Iberoamericana, Barcelona, Spain, 1992. p. 46, 1985. [51] L. Galvez,´ Guaran´ıes y jesuitas. De la Tierra sin Mal al Para´ıso, [33] M. A. Albeck, Taller de Costa a Selva. Produccion´ e Intercambio Editorial Sudamericana, Buenos Aires, Argentina, 1995. entre los pueblos agroalfareros de los Andes Centro Sur,Instituto [52] P. de Montenegro, Materia Medica Misionera, Edicion´ digital Interdisciplinario Tilcara, Tilcara, Argentina, 1994. Biblioteca Virtual del Paraguay, 1945. [34] C. Troncoso, “El diseno˜ de las estrategias de vida de los [53] J. Cardiel, Breve relacion´ de las misiones del Paraguay,Secretar´ıa campesinos en Juella,” in La Quebrada. Geograf´ıa, Historia y de Cultura de la Nacion´ y Ed. Teoria, Buenos Aires, Argentina, Ecolog´ıa de la Quebrada de Humahuaca,C.Reboratti,Ed.,pp. 1994, Prologo´ de E. Maeder. 139–160, Editorial La Colmena, Buenos Aires, Argentina, 2003. [54] M. Bertoni, La civilizacion´ guaran´ı, Parte III. Etnograf´ıa. [35] R. A. Raffino, J. D. Gobbo, and A. Iacona,´ “De Potos´ıyTarijaa Conocimiento, 1927, Imprenta y Edicion´ “Ex Sylvis”, Puerto la frontera Chiriguana,” Folia Historica´ del Nordeste,vol.16,pp. Bertoni. 83–129, 2006. [55] F. J. Brabo, Coleccion´ de documentos relaticos alaexpulsi´ on´ de [36] R. H. Bernard, Social Research Methods. Qualitative and Quan- los jesuitas de la Republica´ Argentina y del Paraguay,Establec- titative Approaches, Sage, Thousand Oaks, Calif, USA, 2000. imiento Tipografico´ de J. M. Perez,´ Madrid, Spain, 1872. [37] U. P. Albuquerque, R. F. Paiva de Lucena, and L. V. F. Cruz [56] R. Lista, El territorio de las Misiones, Imprenta “La Universidad” da Cunha, Metodos´ e tecnicas´ na pesquisa Etnobiologica´ y de J. N. Klingelfuss, Buenos Aires, Argentina, 1883. Etnoecologica´ , Nuppea, Recife, Brazil, 1st edition, 2010. [57] E. L. Holmberg, “Viaje a misiones,” Bolet´ın de la Academia [38] W. Swingle, “The botany of Citrus and its wild relatives of the Nacional de Ciencias en Cordoba´ , vol. 10, pp. 252–288, 1887. orange subfamily (Family Rutaceae, subfamily Aurantioideae),” [58] V.Gambon,´ Atraves´ de las misiones guaran´ıticas, Angel´ Estrada in The Citrus Industry. History, Botany, and Breeding,H.Webber yC´ıa, Buenos Aires, Argentina, 1904. and L. Batchelor, Eds., pp. 129–474, University of California [59] J. B. Ambrosetti, Primer y segundo viaje a Misiones por Juan Press, Berkeley, Calif, USA, 1943. Bautista Ambrosetti. Comentado por J. C. Chebez y B. Gasparri, [39] H. Webber, “Cultivated varieties of Citrus,” in The Citrus Editorial Albatros, Fundacion´ de Historia Natural Felix´ de Industry. History, Botany, and Breeding,H.WebberandL. Azara,, Buenos Aires, Argentina, 2008. Batchelor, Eds., pp. 475–668, University of California Press, [60] J. B. Ambrosetti, Tercer viaje a Misiones por Juan Bautista Berkeley, Calif, USA, 1943. Ambrosetti,EditorialAlbatros,Fundacion´ de Historia Natural [40] J. Palacios, Citricultura Moderna, Hemisferio Sur, Buenos Aires, Felix´ de Azara, Buenos Aires, Argentina, 2008, Comentado por Argentina, 1st edition, 1978. J. C. Chebez y B. Gasparri. [41] C. Anderson, “Variedades cultivadas en el area´ del R´ıo [61] L. Depypere, P.Chaerle, K. V.Mijnsbrugge, and P.Goetghebeur, Uruguay,” in Manual para productores de naranja y mandarina “Stony endocarp dimension and shape variation in Prunus de la region´ del R´ıo Uruguay,A.Fabiani,R.Mika,L.Larocca,and section Prunus,” Annals of Botany,vol.100,no.7,pp.1585–1597, C. Anderson, Eds., Diversificacion´ Productiva, Manual Serie A, 2007. Nro.2,pp.63–92,INTA,Concordia,Canada,1996. [62]P.Burger,J.-F.Terral,M.-P.Ruas,S.Ivorra,andS.Picq, “Assessing past agrobiodiversity of Prunus avium L. (Rosaceae): [42] G. Delucchi, “Sinopsis de las especies de Rosaceae adventicias: a morphometric approach focussed on the stones from the subfamilia Prunoideae,” Bonplandia,vol.20,pp.73–94,2011. archaeological site Hotel-Dieuˆ (16th century, Tours, France),” [43] D. Rivera Nu´nez,˜ C. Obon´ de Castro, S. R´ıos Ruiz et al., Las Vegetation History and Archaeobotany,vol.20,no.5,pp.447– variedades tradicionales de frutales de la Cuenca del R´ıo Segura. 458, 2011. Catalogo´ Etnobotanico´ (1): frutos secos, oleaginosos, frutales de [63] J. R. Baez,´ “Breves apuntes sobre la migracion´ de las plantas hueso, almendros y frutales de pepita, Servicio de Publicaciones, agr´ıcolas Euro-Indianas en el momento del descubrimiento,” Universidad de Murcia, Murcia, Spain, 1997. Lilloa,vol.18,pp.311–360,1949. [44] D. X. Zhang and D. J. Mabberley, “Citrus,” in Flora of China,Z. [64] J. R. Baez,´ “La primera colonia agrohispana en el Tucuman´ Y.Wu,P.H.Raven,andD.Y.Hong,Eds.,pp.90–96,Chinaand (Siglo XVI),” Revista Argentina de Agronom´ıa,vol.14,no.2,pp. Missouri Botanical Garden Press, St. Louis, Mo, USA, 2008. 85–93, 1947. [45] D. Mabberley, “A classification for edible Citrus (Rutaceae),” [65] A. Capparelli, V. Lema, and M. Giovanetti, “Introduccion´ y Telopea,vol.7,pp.167–172,1997. dispersion´ de bienes de Viejo mundo: posibles rutas de ingreso [46] D. Mabberley, Plant-Book. A Portable Dictionary of Plants, a la provincia de Catamarca,” Memorias del Tercer Congreso Their Classification and Uses, Cambridge University Press, de Historia de Catamarca: arqueolog´ıa, cultura, educacion´ y Cambridge, UK, 3rd edition, 2008. geograf´ıa humana.pp.85–105,Bazan,´ 2007. [47] B. A.´ Arias and L. Ramon-Laca,´ “Pharmacological properties of [66] E. Tandeter, “Conquista y colonizacion,”´ in Historia Economica´ citrus and their ancient and medieval uses in the Mediterranean de America´ Latina, problemas y procesos,E.TandeterandJ.C. region,” Journal of Ethnopharmacology,vol.97,no.1,pp.89–95, Korol, Eds., pp. 21–57, Fondo de Cultura Economica,´ Buenos 2005. Aires, Argentina, 1998. 16 Evidence-Based Complementary and Alternative Medicine

[67] A. M. Lorandi, “El servicio personal como agente de desestruc- [85] L. Cadogan,´ “Diccionarios Mbya-Guaran´ı—castellano,” Bib- turacion´ en el Tucuman´ colonial,” Revista Andina,vol.6,no.1, lioteca Paraguaya de Antropolog´ıa vol. XVII. Fundacion´ L. pp. 135–173, 1988. Cadogan.´ CEADUC-CEPAG, Asuncion,´ Paraguay, 1992. [68] A, Capparelli, M. Giovannetti, and V.Lema, “Primera evidencia [86] D. A. Lambare´ and M. L. Pochettino, “Diversidad local y de arqueologica´ de cultivos europeos (trigo, cebada y duraznos) practicas´ agr´ıcolas asociadas al cultivo tradicional de duraznos y de semillas de algodon´ en el NOA: su significacion´ a traves´ Prunus persica (ROSACEAE), en el Noroeste de Argentina,” del registro del Shincal de Quimivil,” in Paleoetnobotanica´ Darwiniana, vol. 50, no. 2, pp. 174–186, 2012. del Cono Sur: estudios de casos y propuestas metodologicas,´ , [87] N. I. Hilgert, “Plants used in home medicine in the Zenta River B.Marconetto,N.Oliszewsky,andP.Babot,Eds.,pp.25–48, basin, Northwest Argentina,” Journal of Ethnopharmacology, Museo de antropolog´ıa-Facultad de Filosof´ıa y Humanidades, vol.76,no.1,pp.11–34,2001. Universidad Nacional de Cordoba,´ Cordoba,´ Spain, 2007. [88] N. I. Hilgert, S. Reyes, and G. Schmeda-Hirschmann, “Alkaline [69] P. Font Quer, Plantasmedicinales,elDioscorides´ renovado, substances used with coca (Erythroxylum coca,Erythroxy- Editorial LABOR, Barcelona, Spain, 11th edition, 1988. laceae) leaf insalivation in northwestern Argentina,” Economic [70] S. Castroviejo, Flora Iberica.´ Plantas vasculares de la Pen´ınsula Botany,vol.55,no.2,pp.325–329,2001. Iberica,´ e Islas Baleares, vol. VI ROSACEAE of F. Munoz˜ [89] N. I. Hilgert, “Plant species used during insalivation of “coca” Garmendia and C. Navarro, Eds.,RealJard´ın Botanico,´ C.S.I.C., (Erythroxylum coca var. coca, Erythroxylaceae) leaves,” Dar- Madrid, Spain, 1998. winiana,vol.38,no.3-4,pp.241–252,2000. [71] L. Manfred, Siete mil recetas botanicas´ a base de mil trescientas [90]B.Nagaraju,S.C.Anand,N.Ahmed,J.N.NarendraSharath plantas medicinales, Editorial Kier, Buenos Aires, Argentina, Chandra, F. Ahmed, and G. V. Padmavathi, “Antiulcer activity 1947. of aqueous extract of Citrus medica linn. fruit against ethanol- induced ulcer in rats,” Advances in Biological Research,vol.6,no. [72] C. Burmeister, Memoria sobre el territorio de Misiones, Minis- 1,pp.24–29,2012. terio de Agricultura de la Republica´ Argentina. Impr., Litogr. y Encuad. De J. Peuser, Buenos Aires, Argentina, 1899. [91]F.Conforti,G.A.Statti,R.Tundis,M.R.Loizzo,andF. Menichini, “In vitro activities of Citrus medica L. cv. Dia- [73] J. Hieronymus, “Plantae diaphoricae florae argentinae orevista´ mante (Diamante citron) relevant to treatment of diabetes and sistematica´ de las plantas medicinales, alimenticias o´ de alguna Alzheimer’s disease,” Phytotherapy Research,vol.21,no.5,pp. otra utilidad y de las venenosas, que son ind´ıgenas de la 427–433, 2007. Republica´ Argentina o´ que, originarias de otros pa´ıses se [92] G. B. Bairagi, A. O. Kabra, and R. J. Mandade, “Anthelmintic cultivan osecr´ ´ıan espontaneamente´ en ella,” Bolet´ın de la activity of Citrus medica L. leaves in indian adult earthworm,” Academia Nacional de Ciencias en Cordoba´ ,vol.4,no.3,pp. International Journal of PharmTech Research,vol.3,no.2,pp. 199–598, 1882. 664–667, 2011. [74]J.Kumamoto,R.W.Scora,H.W.Lawton,andW.A.Clerx, [93] N. E. Sandoval-Montemayor, A. Garc´ıa, E. Elizondo-Trevino,˜ “Mystery of the forbidden fruit: Historical epilogue on the E. Garza-Gonzalez,´ L. Alvarez, and M. del Rayo Camacho- origin of the grapefruit, Citrus paradisi (Rutaceae),” Economic Corona, “Chemical composition of hexane extract of Citrus Botany,vol.41,no.1,pp.97–107,1987. aurantifolia and anti-Mycobacterium tuberculosis activity of [75] Citrus Pages, http://users.kymp.net/citruspages/home.html. some of its constituents,” Molecules,vol.17,no.9,pp.11173–11184, [76] M. C. Gallero and E. M. Krautstofl, “Proceso de poblamiento 2012. y migraciones en la provincia de Misiones, Argentina (1881– [94] F. Spadaro, R. Costa, C. Circosta, and F. Occhiuto, “Volatile 1970),” Ava´,vol.16,pp.245–264. composition and biological activity of key lime Citrus auran- [77] H. Webber, “History and development of the Citrus industry”,” tifolia essential oil,” Natural Product Communication,vol.7,no. in TheCitrusIndustry.History,Botany,andBreeding,,H.Webber 11, pp. 1523–1526, 2012. andL.Batchelor,Eds.,pp.1–40,UniversityofCaliforniaPress, [95] L. M. Lopes Campelo,ˆ C. Gonc¸alves E Sa,´ A. A. C. de Almeida Berkeley, Calif, USA, 1st edition, 1943. et al., “Sedative, anxiolytic and antidepressant activities of Citrus [78] J. Palacios, Citricultura Moderna, Hemisferio Sur, Buenos Aires, limon (Burn) essential oil in mice,” Pharmazie,vol.66,no.8,pp. Argentina, 1978. 623–627, 2011. [79] A. Banfi, “Difusion´ de buenas variedades c´ıtricas,” IDIA,vol. [96] M. J. Dhanavade, C. B. Jalkute, J. S. Ghosh, and K. D. Sonawane, 90–92, p. 102, 1954. “Study Antimicrobial Activity of Lemon (Citrus lemon L.) Peel Extract,” British Journal of Pharmacology and Toxicology,vol.2, [80] A. Banfi and H. Benatena,˜ “Nuevas variedades en la coleccion´ no. 3, pp. 119–122, 2011. de citrus,” IDIA,vol.90–92,pp.101–102,1954. [97] H. S. Parmar and A. Kar, “Antiperoxidative, antithyroidal, [81] Compan˜´ıa Tabacalera de Misiones, http://www.cooptabmis antihyperglycemic and cardioprotective role of Citrus sinensis .com/citrusespanol.html. peel extract in male mice,” Phytotherapy Research,vol.22,no.6, [82] M. N. Seo and C. Xifreda, “Rutaceae,” in Flora Fanerogamica´ pp.791–795,2008. Argentina,A.M.AntonandF.O.Zuloaga,Eds.,vol.106,pp. [98] A. T. Rajarajan, V. G. Vijayasree, W. Kenichi, S. Vijaya Kumar, 1–22, 2008. G. Narasimman, and S. Sadish Kumar, “Anthelmintic and [83] P. C. Stampella, G. Delucchi, and M. L. Pochettino, “Natural- antimicrobial properties of peels of Citrus sinensis,” Pharmacol- izacion´ e identidad del “limon´ mandarina”, 𝐶𝑖𝑡𝑟𝑢𝑠 × 𝑡𝑎𝑖𝑡𝑒𝑛𝑠𝑖𝑠 ogyonline, vol. 1, pp. 363–368, 2009. (Rutaceae, Aurantioideae) en la Argentina,” Bolet´ın de la [99] J. A. Saonere Suryawanshi, “An overview of Citrus aurantium Sociedad Argentina de Botanica´ ,vol.48,no.1,pp.161–169,2013. used in treatment of various diseases,” African Journal of Plant [84] J. S. Storni, Hortus Guaranenesis. Flora, Universidad Nacional Science,vol.5,no.7,pp.390–395,2011. de Tucuman,´ Imprenta y Litograf´ıa de Miguel Violetto, [100] M.I.R.Carvalho-FreitasandM.Costa,“Anxiolyticandsedative Tucuman,´ Argentina, 1944. effects of extracts and essential oil from Citrus aurantium L,” Evidence-Based Complementary and Alternative Medicine 17

Biological and Pharmaceutical Bulletin,vol.25,no.12,pp.1629– 1633, 2002. [101] S. Husain Shahnaz, M. Ali, and B. Prasad Panda, “Influence of volatile constituents of fruit peels of Citrus reticulata Blanco on clinically isolated pathogenic microorganisms under In-vitro,” Asian Pacific Journal of Tropical Biomedicine,vol.2,no.3,pp. 1299–1302, 2012. [102] J. A. D´ıaz-Juarez,F.A.Tenorio-L´ 𝑎pez, G. Zarco-Olvera, L. D. Valle-Mondrag𝑎n, J. C. Torres-Narvaez,´ and G. Pastel´ın- Hernandez,´ “Effect of Citrus paradisi extract and juice on arterial pressure both in vitro and in vivo,” Phytotherapy Research, vol.23,no.7,pp.948–954,2009. [103] V. Gupta, P. Bansal, J. Niazi, and G. Kaur, “Anti-anxiety activity of Citrus paradisi var. star ruby extracts,” International Journal of PharmTech Research, vol. 2, no. 3, pp. 1655–1657, 2010. [104] A. A. Adeneye, “Hypoglycemic and hypolipidemic effects of methanol seed extract of Citrus paradisi Macfad (Rutaceae) in alloxan-induced diabetic Wistar rats,” Nigerian Quarterly Journal of Hospital Medicine,vol.18,no.4,pp.211–215,2008. [105] T.-Y.Shin, S.-B. Park, J.-S. Yoo et al., “Anti-allergic inflammatory activity of the fruit of Prunus persica: Role of calcium and NF- 𝜅B,” Food and Chemical Toxicology,vol.48,no.10,pp.2797– 2802, 2010. [106] A. H. Gilani, N. Aziz, S. M. Ali, and M. Saeed, “Pharmacological basis for the use of peach leaves in constipation,” Journal of Ethnopharmacology,vol.73,no.1-2,pp.87–93,2000. [107]Y.H.Kim,H.E.Yang,B.K.Park,M.Y.Heo,B.K.Jo,andH. P. Kim, “The extract of the flowers of Prunus persica,anew cosmetic ingredient, protects against solar ultraviolet-induced skin damage in vivo,” Journal of Cosmetic Science,vol.53,no.1, pp.27–34,2002. [108] K. Seeland, Ed., Nature is culture: indigenous knowledge and socio-cultural aspects of trees and forests in non-European cultures, Intermediate Technology Publications, London, UK, 1997. [109] C. Vitri, “La ruta de Diego de Almagro en el territorio argentino: un aporte desde la perspectiva de los caminos prehispanicos,”´ Revista escuela de historia, Facultad de Humanidades Universi- dad Nacional de Salta,vol.1,ano˜ 6, no. 6, 2007. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 715040, 4 pages http://dx.doi.org/10.1155/2013/715040

Research Article Chemical Composition and Validation of the Ethnopharmacological Reported Antimicrobial Activity of the Body Fat of Phrynops geoffroanus Used in Traditional Medicine

Diógenes de Queiroz Dias,1 Mario Eduardo Santos Cabral,2 Débora Lima Sales,3 Olga Paiva Oliveira,1 João Antonio de Araujo Filho,4 Diego Alves Teles,1 José Guilherme Gonçalves de Sousa,1 Henrique Douglas Melo Coutinho,1 José Galberto Martins da Costa,1 Marta Regina Kerntopf,1 Rômulo Romeu da Nóbrega Alves,5 and Waltécio de Oliveira Almeida1

1 Programa de Pos-Graduac´ ¸ao˜ em Bioprospecc¸ao˜ Molecular, Universidade Regional do Cariri, 63105-000 Crato, CE, Brazil 2 Programa de Pos-Graduac´ ¸ao˜ em Biotecnologia de Recursos Naturais, BioMol-Lab, Departamento de Bioqu´ımica e Biologia Molecular, P.O. Box 6043, Universidade Federal do Ceara,´ Campus do Pici, 60455-970 Fortaleza, CE, Brazil 3 Programa de Pos-Graduac´ ¸ao˜ em Etnobiologia e Conservac¸ao˜ da Natureza, Universidade Federal Rural de Pernambuco—UFRPE, 52171-900 Dois Irmaos,˜ Recife, PE, Brazil 4 Departamento de Qu´ımica Biologica,´ Universidade Regional do Cariri, 63105-000 Crato, CE, Brazil 5 Departamento de Biologia, Universidade Estadual da Para´ıba, 58429-500 Campina Grande, PB, Brazil

Correspondence should be addressed to Diogenes´ de Queiroz Dias; [email protected]

Received 22 February 2013; Revised 9 September 2013; Accepted 23 September 2013

Academic Editor: Ana H. Ladio

Copyright © 2013 Diogenes´ de Queiroz Dias et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. Phrynops geoffroanus is a small turtle that inhabits lakes, rivers, and streams throughout South America. The body fat of this animal is used as a folk medicine in Brazil for treating illnesses such as sore throats, ear aches, mumps, rheumatism, and arthritis. The present study evaluated the antimicrobial activity of oil extracted from Phrynops geoffroanus (OPG), determined its chemical composition, and discussed the implications of its use in traditional medicine. The OPG was obtained from the ventral region of this turtle using hexane as a solvent. The antimicrobial activity of OPG was tested against standard and multiresistance strains of bacteria and fungi and its composition was determined indirectly by analyzing the methyl esters of the component fatty acids. The OPG presented a clinically relevant antifungal activity against Candida krusei ATCC 6258 (MIC 128 𝜇g/mL). When the OPG was associated with the antibacterial and antifungal drugs, was observed a synergistic effect when associated the OPG with the gentamicin against the strain Pseudomonas aeruginosa 22. Our results indicated that OPG has clinically relevant antifungal activity against C. krusei, and demonstrated synergetic antibacterial activity in combination with commercial antibiotics against Pseudomonas aeruginosa.

1. Background practices at least since the first colonization of Brazil [5], and studies by Alves et al. [6]havedocumentedtheuse Reptiles are frequently used in making traditional medicines of natural products derived from reptiles (including leather, and their roles in folk practices for curing and/or preventing teeth, fat, meat, and bones) as foods and for ornamental and illnesses have been recorded in many different social-cultural medicinal purposes in rural and urban areas of that country. contexts throughout the world [1–4]. Historical documents Freshwater turtles are included among the many animals used showthatanimalshavebeenusedintraditionalmedicinal in traditional folk medicine and their shells, blood, eggs, 2 Evidence-Based Complementary and Alternative Medicine and body fat are sought after as zootherapeutic elements in 2.4. Analysis of OPG by Gas Chromatography Coupled to a northeastern Brazil [7, 8]. Mass Spectrometer (GC/MS). The analysis of volatile con- Among the 278 species of turtles in the world, approxi- stituentswascarriedoutinaHewlett-PackardGC/MS,model mate 20% occur in South America in 20 families; the most 5971, using the nonpolar DB-1 fused silica capillary column species rich family is Chelidae, with a total of 23 species (19 (30 m × 0.25 mm i.d., 0.25 𝜇m film), eluted with helium of which occur in Brazil, where they are popularly known as gas at 8 mL/min with split mode. Injector and detector ∘ ∘ “cagados”´ [9]. temperatures were set to 250 Cand200C, respectively. The ∘ ∘ Phrynops geoffroanus isasmallturtlewithapredomi- column temperatures was programmed from 35 Cto180C ∘ ∘ ∘ ∘ nantly carnivorous diet [10]. It is popularly known as “cagado-´ at 4 C/min and then from 180 Cto250Cat10C/min. Mass de-barbela” and is widely distributed in South American spectra were recorded from 30 to 450 m/z, with an electron countries [11, 12] in lakes, rivers, and streams with relatively beam energy of 70 eV. The individual components were large volumes of water [13]. identified by computer MS library searches, using retention P. g e off ro anu s is used by many traditional communities indices as a preselection routine and visual inspection of the in Brazil as a zootherapeutic [14, 15]—principally its fixed spectra from the literature for confirmation [20], as well as by oilfrombodyfat—totreatillnessessuchassorethroats,ear visually comparing standard fragmentation to that reported aches, mumps, rheumatism, and arthritis [16]. Many of these intheliterature[21, 22]. maladies (inflammations and dermatitis) are associated with pathogenic organisms, including bacteria and fungi, which 2.5. Microorganisms. Experiments were undertaken using suggest the existence of antimicrobial activity associated with clinical isolates of the bacteria Escherichia coli (EC27), Staphy- this turtle species, although no laboratory studies have yet lococcus aureus 358 (SA358), and Pseudomonas aeruginosa examined the efficiency of this popular folk remedy. (PA22). The strains Escherichia coli ATCC-10536, Staphylo- As such, the present work identified the chemical compo- coccus aureus ATCC-25923, Pseudomonas aeruginosa ATCC- nents of the body fat of Phrynops geoffroanus and evaluated 15442, and Klebsiella pneumoniae ATCC-4362wereusedas its antimicrobial activity (either alone or in association positive controls. Isolates of Candida albicans ICB12 and with antibiotics and antifungal drugs) and discusses the Candida krusei 6258 were used to evaluate antifungal activity, implications of its use as a traditional remedy. as well as in the modulation tests [23]. All of the lineages were maintained in heartinfusionagarslants(HIA, Difco). The ∘ cells were cultivated during the night before the trials at 37 C 2. Methods in a Brain Heart Infusion medium (BHI, Difco). 2.1. Zoological Material. Specimens of Phrynops geoffroanus ∘ 󸀠 2.6. Drugs. The antibiotics gentamicin, amikacin, and werecollectedinthemunicipalityofAiuaba(0636 S × ∘ 󸀠 neomycin were obtained from Sigma Chemical Corp., 40 07 W) in Ceara´ State, Brazil, in September/2011 using St. Louis, MO, USA. The antifungal drugs used were active collection techniques [17]. The specimens were sub- amphotericin B (Sigma Co., St. Louis, USA), mebendazol sequently anesthetized using ketamine (60 mg/Kg) and (Lasa—Pharmaceutical Industries LTDA., Brazil), nystatin xylazine (6 mg/Kg) [18] and sacrificed, and their body fat (Laboratorio´ Teuto Brasileiro S/A, Brazil), and metronidazole was removed. Reference specimens were fixed in 70% ethyl benzoate (Prati, Donaduzzi and Cia LTDA., Brazil). All of alcoholanddepositedinthezoologicalcollectionatthe these compounds were dissolved in sterile water before use. Cariri Regional University—URCA (collection numbers LZ- URCA 1328 and LZ-URCA 1329). This work was approved by 2.7. Tests of Drug Susceptibility. AtestsolutionofOPG the Animal Ethics Committee of the Universidade Regional was prepared using 20 mg of the oil dissolved in 1 mL of do Cariri—URCA under the Reference no.: 04/2012. dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany), generating an initial concentration of 20 mg/mL. This solu- 2.2. Preparation of the Oil from Phrynops geoffroanus (OPG). tion was subsequently diluted to 1024 𝜇g/mL with sterile The fixed oils present in body fat located in the ventral regions water. The minimum inhibitory concentrations (MICs) of ∘ the oil were determined in BHI using microdilution series of the turtles were extracted with hexane (60 C) for 6 h in 5 a Soxhlet apparatus. The hexane was subsequently decanted with suspensions of 10 CFU/mL and drug concentrations andfilteredandthesolventwasremovedbyheatinginawater of 1024 𝜇g/mL to 1 𝜇g/mL (double dilution series) [24]. The ∘ bath at 70 C for 2 h; the extracted oil was subsequently stored MIC was defined as the lowest concentration of a compound in a freezer for future analysis. that inhibited microbial growth. To investigate the potential of the oil as a modulator of antibacterial and antifungal drug activities, we determined the antibacterial (128 𝜇g/mL) and 2.3. Determination of the Fatty Acids. The fatty acids in antifungal (16 𝜇g/mL) MICs in subinhibitory concentrations; the OPG were analyzed indirectly by identifying their ∘ theplateswereincubatedfor24hoursat37C. corresponding methyl esters. The extracted oil (0.2 g) was saponified by refluxing for 30 min. in a solution of potas- 3. Results sium hydroxide and methanol, following the methodology described by Hartman and Lago [19]. The pH of the extract The body fat of Phrynops geoffroanus was found to be was adjusted, and the free fatty acids were methylated by acid composed of 84.63% and 13.38% of unsaturated and saturated catalysis to generate their methyl esters. methyl esters, respectively (Table 1). Evidence-Based Complementary and Alternative Medicine 3

Table 1: GC/MS characterization of the methyl esters of the fatty the synthesis of the endogenous microbial fatty acids. The acids in oil from the body fat of P. g eoff ro anu s . OPG is composed mainly of unsaturated fatty acids (84,63%),

a being the main component the palmitoleic and oleic acids Name T𝑟 (min) (%) (58,39% and 15,7%, respectively). The high concentration of Perlagonic acid 11,43 2,04 unsaturated FAs is different when compared with the FAs Pentadecylic acid 19,09 3,68 compositionobservedintheworkofScarlatoandGaspar Palmitoleic acid 22,31 58,39 [28],butsimilartotheworkofGasparandSilva[29], both Caprylic acid 23,98 0,84 studying different parts of Podocnemis expansa. Linoleic acid 24,63 4,50 The OPG when assayed alone against bacterial and fungal Linolenic acid 24,72 2,28 strains demonstrated an clinically nonrelevant antimicrobial = Oleic acid 24,83 15,70 activity against all strains, except against C. krusei,(MIC 128 𝜇 Erucic acid 24,90 3,76 g/mL). When the OPG was associated with antibiotics and antifungal drugs, was observed a synergistic effect when Palmitic acid 25,13 6,82 associated the OPG with the gentamicin against the strain Saturated esters — 13,38 Pseudomonas aeruginosa (PA 22) (Table 2). In studies with Unsaturated esters — 84,63 the body fat of the lizard Tupinambis merianae, Ferreira Total — 98,01 et al. [30] demonstrated that besides the usage of this body aRetention time. fat in the folk medicine against infectious diseases, this product demonstrated non-antimicrobial activity when used Table 2: MIC values (𝜇g/mL) of aminoglycosides against Escheri- alone or associated with antibiotics. The body fat of T. chia coli 27, Staphylococcus aureus 358, and Pseudomonas aeruginosa merianae presented 57 and 43% of unsaturated and saturated 𝜇 22 in the absence and presence of 128 g/mL of oil derived from the FAs respectively. Possibly, the antimicrobial and modulatory body fat of P. g eoff ro anu s . activity demonstrated by OPG can be due to the high percent EC 27 SA 358 PA 22 of unsaturated FAs detected in this work. Antibiotics Our results demonstrated that the body fat of P. g e of - MIC MIC MIC MIC MIC MIC Combined Combined Combined froanus has antimicrobial and modulatory activities, this work being the first report about a biological activity by this Amikacin 4,9 4,9 19,5 19,5 312,5 312,5 product, validating its usage by the folk medicine and indi- Neomycin 4,9 4,9 19,5 19,5 312,5 312,5 cating a clear relationship between the percent of unsaturated Gentamicin 2,44 2,44 9,8 9,8 39,1 9,8 fatty acids and this antimicrobial and modulatory activities.

OPG alone did not demonstrate any clinically relevant 5. Conclusion antibacterial activity, with a MIC ≥ 1024 𝜇g/mL for all of the bacterial strains tested—indicating that the body fat of The results of our work validate the antimicrobial and this turtle is inefficient when used alone in treating bacterial modulatory activities of OPG and possibly, of other body infections. When tested against fungal strains, the OPG fats with high level of unsaturated fatty acids. Due this demonstrated a MIC ≥ 1024 𝜇g/mL against Candida albicans fact, we recommend more studies to evaluate the use of the ICB12 and MIC 128 𝜇g/mL against C. krusei 6258, thus body fat of P. g e off ro anu s against other diseases informed by demonstrating clinically relevant antifungal activity against ethnopharmacological surveys. infections caused by the latter fungus. The OPG was also tested for possible antibacterial and antifungal activity when combined with commonly used antibiotics or antifungal Conflict of Interests drugs. Tests to confirm possible synergisms between OPG The authors declare that they have no competing interests and aminoglycosides (Table 2) were negative against multiresistant strains of E. coli (EC27) and Staphylococcus aureus (SA358). A synergistic effect was noted, however, against Acknowledgments Pseudomonas aeruginosa (PA22) when OPG was combined with gentamicin. Tests of the modulation of antifungal drugs The authors would like to thank the Coordenaçao˜ de when associated with OPG, likewise do not demonstrate Aperfeiçoamento de Pessoal de N´ıvel Superior—CAPES positive effects against Candida albicans ICB12 or Candida forthestudygrantsawardedtoMarioE.S.Cabraland ≥ 𝜇 krusei 6258 (both having MIC 1024 g/mL). Diogenes´ Q. Dias; the Fundaçao˜ Cearense de Apoio ao Desenvolvimento Cient´ıfico e Tecnologico—FUNCAP´ for 4. Discussion the study grants awarded to Debora´ L. Sales and Olga P. Oliveira, and for support provided to Waltecio´ O. Almeida The antibacterial and antifungal properties of several fatty (process BPI-0112-2.05/08); IBAMA for the collecting per- acids (FAs) were reported [25, 26] and according to Zheng mits (Autorizaçao˜ de coleta SISBIO—IBAMA: no. 30223-1, ∘ et al. [27], the mechanism of the antimicrobial activity is process n 76197587); and Robson W. Avila´ for identifying related to the action of the unsaturated fatty acids affecting the reptiles. We are grateful to the Conselho Nacional de 4 Evidence-Based Complementary and Alternative Medicine

Desenvolvimento Cient´ıfico e Tecnologico—CNPQ´ (PQ- [16] R. R. N. Alves, N. A. LeoNeto,G.G.Santana,W.L.S.Vieira,and´ 311713-2012-2) for the research grant awarded to W. O. W.O. Almeida, “Reptiles used for medicinal and magic religious Almeida and for the grant awarded to R. R. N. Alves and J. purposes in Brazil,” Applied Herpetology,vol.6,no.3,pp.257– G. M. da Costa. 274, 2009. [17] P.AuricchioandM.G.Salomao,˜ Tecnicas´ de Coleta e Preparaçao˜ de Vertebrados Para Fins Cient´ıficos e Didaticos´ ,InstitutoPau References Brasil de Historia´ Natural, Sao˜ Paulo, Brazil, 2002. [1] Z. Zhou and Z. Jiang, “International trade status and crisis for [18] F. A. B. Viana, Guia Terapeuticoˆ Veterinario´ ,Grafica´ e editora snake species in China,” Conservation Biology,vol.18,no.5,pp. CEM Ltda, Belo Horizonte, Brazil, 2003. 1386–1394, 2004. [19] L. Hartman and R. Lago, “Rapid preparation of fatty acid methyl [2] M. M. Mahawar and D. P. Jaroli, “Animals and their prod- esters from lipids,” Laboratory Practice,vol.22,no.7,pp.475– ucts utilized as medicines by the inhabitants surrounding the 476, 1973. Ranthambhore National Park, India,” Journal of Ethnobiology [20] J. W.Alencar, A. A. Craveiro, and F. J. A. Matos, “Kovats’ indices and Ethnomedicine,vol.2,article46,pp.1–5,2006. as a preselection routine in mass spectra library searches of [3]P.E.Vazquez,´ R. M. Mendez,´ O. G. R. Guiascon,´ and E. J. N. volatiles,” JournalofNaturalProducts,vol.47,no.5,pp.890– Pinera,˜ “Uso medicinal de la fauna silvestre em los Altos de 892, 1984. Chiapas, Mexico,”´ Interciencia,vol.31,no.7,pp.491–499,2006. [21] E. Stenhagen, S. Abrahamson, and F. W. Mclafferty, Registry of [4] J. Fretey, G. H. Segniagbeto, and M. Soumah, “Presence of sea Mass Spectra Data Base, Government Printing Office, Washing- turtle in traditional pharmacopoeia and beliefs of West Africa,” ton, DC, USA, 1974. Marine Turtle Newsletter,vol.116,pp.23–225,2007. [22] R. P. Adams, Identification of Essential Oil Components by [5] M. L. V. Silva, A. G. C. Alves, and A. V. Almeida, “A zooterapia Gas Chromatography/Quadrupole Mass Spectroscopy,Allured no Recife (Pernambuco): uma articulaçao˜ entre as praticas´ e a Publishing Corporation, Carol Stream, Ill, USA, 2001. historia,”´ Biotemas,vol.17,pp.11–95,2004. [23] H.D.M.Coutinho,L.N.Cordeiro,andK.P.Bringel,“Antibiotic [6]R.R.N.Alves,G.A.Pereira-Filho,K.S.Vieira,G.G.Santana, resistance of pathogenic bacteria isolated from the population W. L . S . Vi e i r a , a n d W. O. A l m e i d a , “R epteis´ e as populaçoes˜ of Juazeiro do Norte-Ceara,”´ Revista Brasileira de Cienciasˆ da humanas no Brasil: uma abordagem ethnoherpetologica,”´ in A Saude´ ,vol.9,pp.127–138,2005. Etnozoologia No Brasil: Importancia,ˆ Status Atual e Perspectivas, [24] M. M. Javadpour, M. M. Juban, W.-C. J. Lo et al., “De novo R. R. N. Alves, W.M. S. Souto, and J. S. Mourao,Eds.,pp.121–147,˜ antimicrobial peptides with low mammalian cell toxicity,” Nupeea, Recife, Brazil, 2010. Journal of Medicinal Chemistry,vol.39,no.16,pp.3107–3113, [7] F. S. Ferreira, S. V. Brito, S. C. Ribeiro, W. O. Almeida, 1996. and R. R. N. Alves, “Zootherapeutics utilized by residents [25] M. O. Nobre, P. S. Nascente, M. C. Meireles, and L. Ferreiro, of the community Poço Dantas, Crato-CE, Brazil,” Journal of “Drogas antifungicas´ para pequenos e grandes animais,” Cienciaˆ Ethnobiology and Ethnomedicine,vol.5,article21,pp.21–31, Rural,vol.32,no.1,pp.175–184,2002. 2009. [26] G. Agoramoorthy, M. Chandrasekaran, V. Venkatesalu, and [8] F. S. Ferreira, A. V. Brito, S. C. Ribeiro, A. A. F. Saraiva, W. O. M. J. Hsu, “Antibacterial and antifungal activities of fatty acid Almeida, and R. R. N. Alves, “Animal-based folk remedies sold methyl esters of the blind-your-eye mangrove from India,” in public markets in Crato and Juazeiro do Norte, Ceara,´ Brazil,” Brazilian Journal of Microbiology,vol.38,no.4,pp.739–742, BMC Complementary and Alternative Medicine, vol. 9, article 17, 2007. pp.17–24,2009. [27] C. J. Zheng, J.-S. Yoo, T.-G.Lee, H.-Y.Cho, Y.-H. Kim, and W.-G. [9]F.L.Souza,“Umarevisao˜ sobre padroes˜ de atividade, Kim, “Fatty acid synthesis is a target for antibacterial activity of reproduçao˜ e alimentaçao˜ de cagados´ brasileiros (Testudines, unsaturated fatty acids,” FEBS Letters,vol.579,no.23,pp.5157– Chelidae),” Phyllomedusa,vol.3,no.1,pp.15–27,2004. 5162, 2005. [10] F. B. Molina, “Observaçoes˜ sobre os habitos´ e o comporta- [28] R. C. Scarlato and A. Gaspar, “Composiçao˜ nutricional do casco mento alimentar de Phrynops geoffroanus (Schweigger, 1812) em da tartaruga (Podocnemis expansa) criada em cativeiro e em cativeiro (Reptilia, Testudines, Chelidae),” Revista Brasileira de idadedeabate,”Cienciaˆ e Tecnologia de Alimentos,vol.27,pp. Zoologia,vol.7,no.3,pp.319–326,1991. 41–44, 2007. [11] J. B. Iverson, A Revised Checklist with Distribution Maps of the [29] A. Gaspar and T. J. P. Silva, “Composiçao˜ nutricional da carne Turtles of the World,RichardPrivatelyPrinted,1992. da tartaruga-da-Amazoniaˆ (Podocnemis expansa) criada em [12] F. L. Souza, “Geographical distribution patterns of South Amer- cativeiro e em idade de abate,” Revista do Instituto Adolfo Lutz, ica side-necked turtles (Chelidae), with emphasis on Brazilian vol. 68, no. 3, pp. 419–425, 2009. species,” Revista Espanola˜ de Herpetolog´ıa,vol.19,pp.33–46, [30]F.S.Ferreira,S.V.Brito,J.G.M.Costa,R.R.N.Alves,H.D.M. 2005. Coutinho, and W. D. O. Almeida, “Is the body fat of the lizard [13]P.C.H.PritchardandP.Trebbau,The Turtles of Venezuela, Tupinambis merianae effective against bacterial infections?” Society for the Study of Amphibians and Reptiles, Oxford, UK, Journal of Ethnopharmacology,vol.126,no.2,pp.233–237,2009. 1984. [14] E. M. Costa-Neto, “Faunistic resources used as medicine by an Afro-Brazilian community from Chapada Diamantina National Park, State of Bahia. Brazil,” Sitientibus,vol.15,pp.211–219,1996. [15]F.D.B.P.MouraandJ.G.W.Marques,“Folkmedicineusing animals in the Chapada Diamantina: incidental medicine?” Ciencia & Saude Coletiva,vol.13,no.2,pp.2179–2188,2008. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 102714, 29 pages http://dx.doi.org/10.1155/2013/102714

Research Article The ‘‘Hidden Diversity’’ of Medicinal Plants in Northeastern Brazil: Diagnosis and Prospects for Conservation and Biological Prospecting

Deyvson Rodrigues Cavalcanti1,2,3 and Ulysses Paulino Albuquerque1

1 Laboratory of Applied Ethnobotany, Department of Biology, Federal Rural University of Pernambuco, Avenida Dom Manoel de Medeiros s/n, Dois Irmaos,˜ 52171-900 Recife, PE, Brazil 2 State University of Alagoas, AL 115 Km 3, 57601-000 Palmeira dos Indios,´ AL, Brazil 3 Federal Institute of Education, Science and Tecnology of Alagoas, Avenida das Alagoas s/n, Palmeira de Fora, 57601-220 Palmeira dos Indios,´ AL, Brazil

Correspondence should be addressed to Deyvson Rodrigues Cavalcanti; [email protected] and Ulysses Paulino Albuquerque; [email protected]

Received 25 April 2013; Accepted 8 June 2013

Academic Editor: Romuloˆ Romeu Nobrega´ Alves

Copyright © 2013 D. R. Cavalcanti and U. P. Albuquerque. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Increases in ethnobotanical studies and knowledge in recent decades have led to a greater and more accurate interpretation of the overall patterns related to the use of medicinal plants, allowing for a clear identification of some ecological and cultural phenomena. “Hidden diversity” of medicinal plants refers in the present study to the existence of several species of medicinal plants known by the same vernacular name in a given region. Although this phenomenon has previously been observed in a localized and sporadic manner, its full dimensions have not yet been established. In the present study, we sought to assess the hidden diversity of medicinal plants in northeastern Brazil based on the ethnospecies catalogued by local studies. The results indicate that there are an average of at least 2.78 different species per cataloged ethnospecies in the region. Phylogenetic proximity and its attendant morphological similarity favor the interchangeable use of these species, resulting in serious ecological and sanitary implications as well as a wide range of options for conservation and bioprospecting.

1. Introduction the empirical knowledge retained in different areas and with different origins is aggregated, conserved, and spread. Thus, Medicinal plants are freely circulated in Brazil, particularly the regional public markets are the pillars of a complex, open, in informal trade settings where several types of plants are and dynamic system of knowledge [1]. marketed for a wide range of illnesses (see [1]). Limited access Although promising for the biological prospecting of to specialty medicine and an increasing interest in the so- novel drugs and pharmaceutical products, actual research at called natural treatments account for the rapid increase of the such markets has some limitations, as the identity of the vast tradeinsuchproductsinBrazil[2]. majority of the plant species traded there cannot be safely The most important vendors of medicinal plants are established by means of conventional methods [1, 5–7]. located in urban centers, namely, in fairs and public markets, In contrast with community-based ethnobotanical sur- where consumers have easy access to a wide variety of medici- veys, where the investigated resources are directly accessible nal plant species together with the corresponding therapeutic in loco [8–11],researchatmarketsandfairsismuchmore indications [3]. More specifically, the regional public markets complex, as a significant proportion of the plant prod- act as spaces representative of the cultural production and ucts offered to the consumers are uncharacteristic or lack biological diversity of a given area [1, 4]andascenterswhere the elements required for accurate taxonomic identification 2 Evidence-Based Complementary and Alternative Medicine

(see [1, 5, 6]).Asarule,onlypartsoftheplantsaresold, It is safe to assume that in Brazil, as a function of the to wit, the ones allegedly containing the active therapeutic plant biodiversity, environmental diversity, and multicultural components, such as barks, roots, seeds, flowers, and leaves, and ethnic composition of the country [33], the number which are sometimes dehydrated, chopped, and/or ground. of homonym ethnospecies and consequently also the phe- As a result, it becomes quite easy to mix or mistake a similar nomenon of hidden diversity of medicinal plants is much species with or for another. more comprehensive and significant than suggested from the Several authors have previously expressed such concerns few occurrences recorded in the scientific literature. In the andproposedsomemethodologicalsolutionstotheproblem present study, we sought (1) to measure the hidden diversity, [1, 12]. Various palliative techniques have been suggested for that is, the number of medicinal plant species subsumed cataloging all medicinal plants available to the consumers underthesamecommonnameintheBraziliannortheast at public markets, some of which are quite specialized and region; (2) to establish the different types of underdiffer- expensive [13–15], whereas others are feasible but not always entiation of homonym ethnospecies; and (3) to assess the viable [16, 17]. influence of biological diversity on the number of homonym In addition to morphological similarities between ethnospecies.Finally,wesoughttoindicatesomeofthepossi- species, another factor that makes it difficult to interpret the ble implications for conservation and biological prospecting. ethnobotanical data collected at public markets is the fact Assuming that the variety of homonym ethnospecies in a that multiple plants species are frequently known by the same given region depends on the region’s biodiversity, one might vernacular name. Such events of semantic correspondence expect the following: (1) for the variation in the number of in ethnobotanical studies were initially detected by several homonym ethnospecies to be directly proportional to the size authors [18–21] and then properly systematized by Berlin of the sampled area, as larger areas theoretically include a [22] in a study that sought to determine the relationships wider variety of environments, and consequently, also greater between the biological and traditional classification systems, biological diversity and (2) that a significant number of the thus establishing the grounds of ethnotaxonomy. homonymethnospeciesshouldberepresentativeofthenative Within that ethnotaxonomic approach, Berlin [22, 23] flora compared to the group of species with one-to-one established the notion of underdifferentiation to define the correspondence. semantic correspondence between different species that share a vernacular name, of which two types were described. Underdifferentiation type 1 occurs when the species involved 2. Materials and Methods belongtothesamegenus,andtype2occurswhentheybelong to different genera. When only one species corresponds toa 2.1. Characterization of the Study Area. The northeast region given ethnospecies, correspondence is defined as one-to-one of Brazil includes nine federal units and represents a total area 2 or biunivocal [22, 23]. Several studies of local communities of 1,558,196 km ,whichcorrespondsto18%ofthecountry’s have employed these notions to identify similar patterns territory. It is located in an intertropical zone limited by the of semantic correspondence between different species24 [ – AtlanticOceantotheeastandnorth,theAmazonianrain- 28]. The species subjected to underdifferentiation have been forest to the northwest, and the Cerrado (Brazilian savan- termed ethnohomonyms. nah) domain to the west and southwest [34]. The vegeta- Althoughquitewelladjustedtothelocalsystems,such tion is mainly xerophytic, being the Caatinga (Brazilian xeric correspondences tend to overlap and become complex when shrubland), a highly peculiar biome with a high degree of different cultural origins become somehow intertwined [29, endemism [35–37]. Atlantic ombrophilous forest predomi- 30]. The overlapping of homonym ethnospecies makes the nates in the coastal area. Currently, this forest is one of the understanding of ethnobotanical data originating in environ- most seriously threatened biomes in the world, and only 5% mentswherecomplexculturalnetworksareestablishedeven of its original area remains [38, 39]. Enclaves of Cerrado and more difficult, as is the case with ethnobotanical studies at rainforest are widely present as areas of disjunct vegetation regional public markets (see [6]). [40–43], making the Brazilian northeast region a strategic We define here the “hidden diversity” of medicinal plants area from the perspective of global richness and biological as the set of different homonym ethnospecies “hidden” under diversity [44, 45]. the same vernacular name. We coined the term “hidden From the demographic point of view, the total population diversity” based on the analogy with the notion of a “hidden of the northeast region comprises approximately 49 million harvest,” which denotes the progressive and unofficially inhabitants, primarily distributed along the coastal area documented appropriation of the plant biodiversity in a given wheremoststatecapitalsandmajorcitiesarelocated,which area [31, 32]. together host approximately 40% of the population [34]. According to Krog et al. [6]theimpossibilityofdistin- The cultural diversity of the northeast region is high due to guishing among homonym ethnospecies is one of the major the ethnic miscegenation resulting from the colonization limitations to the advancement of ethnobotanical research of Brazil [46, 47], and the population includes Europeans, in public markets, particularly in the case of ethnopharma- mostly Portuguese and Dutch, black slaves from Africa, cological studies of plant conservation and bioprospection. and the various indigenous peoples. In addition, it is worth Although that phenomenon has previously been detected in observing that in the last ten years, the economic growth of a localized and sporadic manner, its full dimensions have not the region was significantly higher than the national average yet been established. [34]. Evidence-Based Complementary and Alternative Medicine 3

2.2. Data Survey. Six of the nine northeastern states were used the generic term “herbalist” (locally known as “erveiro”) included in the analysis based on the need to survey the to allude to any type of vendor of medicinal plants. The term widest possible diversity of cultural representations and ethnospecies is used in the present study to allude to the environments and the need to take into account the logistics common or vernacular names given to the medicinal plants. of access and permanence at the study sites. For the purposes Using a field notebook, we made records of the catalogs of the present study, we assumed that the expression of of plants traded by the herbalists as mentioned in semistruc- the regional culture is more diversified at the state capitals tured interviews [48].Forthepurposesofthestudy,theplants because they exhibit the largest population density, including availableinstockatthetimeofthestudyaswellasthose immigrants from other states and/or the inland cities. traded in the previous 12 months were taken into consid- The states and corresponding capitals sampled were as eration. The common names of the plants were recorded as follows: Maranhao/S˜ ao˜ Luiz, Ceara/Fortaleza,´ Para´ıba/Joao˜ spelled by the respondents. Pessoa, Pernambuco/Recife, Alagoas/Maceio,´ and Sergipe/ Aracaju. The primary site of medicinal plant trade in each 2.3. Data Analysis. The ethnobotanical data supplied by the state capital was identified, and thus the following markets herbalists in the various studied northeastern states were were selected: the Mercado Central (Central Market) in Sao˜ transcribed and entered in digital spreadsheets using MS Luiz/MA, Mercado de Sao˜ Sebastiao˜ (St. Sebastian Market) in Excel 2003 software, thus creating a Market Relational Fortaleza/CE, Mercado Central in Joao˜ Pessoa/PB, Mercado Database (MRD). The MRD was used to map the geograph- Sao˜ Jose´ (St. Joseph Market) in Recife/PE, Mercado da ical distributions of the ethnospecies across the Brazilian Produc¸ao˜ (Production Market) in Maceio/AL,´ and Mercado northeast region and identify the most frequently occurring Albano Franco (Albano Franco Market) in Aracaju/SE. ones. Following an initial exploratory visit, an appointment was In parallel, an Ethnobotanical Survey Database (ESD) was made for data collection. The plant vendors at each selected created and populated. For that purpose, 55 ethnobotanical marketwereinformedastothenatureofthestudyandinvited surveys of the northeastern states were identified, and the to participate. Some vendors refused immediately, and others listed species and ethnospecies were entered in the ESD. The initially agreed and then went back on their original agree- plants not identified at the species level were not included. ment. As a result, a total of 22 respondents were interviewed Only relevant studies were selected: most (45) were published and provided a representative sample of the vernacular names in major scientific journals, seven were Master’s dissertations, oftheplantstradedintheregion.InthestateofPernambuco, one was a doctoral thesis, one a book, and one the Develop- the ethnobotanical studies in public markets are already more ment Plan of a major Brazilian university (Federal University advanced. Albuquerque and colleagues [1] previously found of Bahia—Universidade Federal da Bahia, UFBA). a significant decrease in the availability of plant vendors in The data entered in both databases (MRD and ESD) this state based on only two samples obtained over an eight- were then crosschecked to produce a detailed list of the year period. The in situ observations and data collected for ethnospecies mentioned both in the ethnobotanical surveys the present study suggest that this decrease in availability may and by the respondents in our study, with the correspond- represent a general trend that can be explained by several ing species. This step allowed for the identification of the factors. For instance, the lack of regulation and control of homonym species and their clustering around the corre- the sector in regards to health and ecological aspects may sponding ethnospecies. generate mistrust and insecurity among vendors. The vendors We selected a sample corresponding to 40% of the eth- may also experience a lack of return research or “benefits” nospecies included in both databases (MRD and ESD) based that would otherwise entice them to be informants. In on their frequency in the ethnobotanical surveys. Thus, only addition, the harsh economic conditions of the country have the 165 most frequent ethnospecies out of a total of 406 listed removed a significant number of vendors from the market, in the ethnobotanical surveys were selected for analysis. and unrelenting derogatory campaigns have undermined the Thesamplingcriteriausedwerebasedontwoassump- informal trade markets in the media. Vendors in the informal tions: (1) ethnobotanical research is still incipient in most trade markets also experience increasing competition with of the northeast region, and thus, infrequent ethnospecies food stores, which are common in large urban centers and might suggest a merely temporary pattern of semantic cor- usually have better infrastructure, availability, and sanitary respondence, consequently masking the results, the number conditions. There is also a lack of interest in new generations of one-to-one correspondences in particular and (2) the to continue the family traditions of using and trading medic- ethnospecies most frequently mentioned in the regional eth- inal plants. nobotanical surveys might represent the patterns of semantic After the study was explained, the respondents freely correspondence in a more unequivocal and reliable manner. signed an informed consent form. The study was approved The corresponding species were allocated to three groups: by the Research Ethics Committee of the Federal University of one comprised the species with one-to-one correspondences, Pernambuco (Universidade Federal de Pernambuco—UFPE) the second, the homonym ethnospecies with type 1 under- no. 0039.0.172.0000-10, FR (Folha de Rosto—Title Page) differentiation, and the third, the homonym ethnospecies 3139660. with type 2 underdifferentiation, according to Berlin’s23 [ ] Although some authors [1] have reported that several classification. The corresponding species were subjected to terms are used to describe vendors of medicinal plants, even- synonym analysis; the names that are currently valid were tually including hierarchical criteria, in the present study, we duly recorded based on the List of Species of the Brazilian 4 Evidence-Based Complementary and Alternative Medicine

Flora 2012 [49]andthedatabaseoftheMissouriBotanical 100 Garden [50],whichwerealsousedtoestablishthebiogeo- 90 graphicstatusofeachspeciestoclassifythemasnativeor 80 exotic. 70 To assess whether underdifferentiationsensu ( Berlin 60 [23]), expressed as the number of homonym ethnospecies, 50 varies as a function of the biological diversity of a given 40 area, we compared the results corresponding to the northeast (%) Species 30 region with a geographically narrower sample, based on the 20 assumption that the larger the area, the wider the environ- 10 mental variety, and thus, the more diversified the flora. 0 Thatnarrowersamplewasrepresentedbythestateof PE NE Pernambuco, which is the northeastern state most thoroughly studiedfromanethnobotanicalperspective.Thenumbersof One-to-one ethnoespecies homonym ethnospecies and one-to-one correspondences of Under-differentiated ethnoespecies the northeast region were compared to those of Pernambuco. Figure 1: Percentages of ethnospecies that exhibited one-to-one cor- The frequency of species in the respective categories of respondence and underdifferentiation marketing in the northeast semantic correspondence (i.e., one-to-one and underdiffer- region and the state of Pernambuco, Brazil. entiation) was analyzed by means of 𝐺 tests [51]aswerethe percentages of native and exotic species in each group. 100 90 80 3. Results 70 The ethnospecies (𝑛 = 165)sampledbasedonthedatacol- 60 lected at the visited markets exhibited correspondence with 50 459 species, corresponding to 228 genera and 90 families 40 Species (%) Species (Table 1). The ratio of species to ethnospecies was 2.78. From 30 the total number of analyzed ethnospecies, only 41 (25%) 20 exhibited one-to-one correspondence, whereas 124 (75%) 10 exhibited underdifferentiation and correspondence to 418 0 One to one Under-differentiated species. Approximately 62% of the homonym ethnospecies exhibited two or three corresponding species, although in Native some cases, a single ethnospecies included up to nine corre- Exotic sponding homonym species, as, for example, “quebra-pedra” (stonebreaker) (Table 1). Figure 2: Percentages of native and exotic species in the group Analysis of the data corresponding to the state of Pernam- of ethnospecies that exhibited one-to-one correspondence and buco alone identified 138 out of the 165 ethnospecies found in underdifferentiation marketing in the northeast region and the state the northeast region, which exhibited correspondence with of Pernambuco, Brazil. 203 species. The ratio of species to ethnospecies was 1.46. The pattern of correspondence included 89 (64%) instances of one-to-one correspondence and 49 (36%) of underdiffer- under-differentiated ethnospecies compared to the one-to- entiation; the homonym ethnospecies represented a total of one ethnospecies (𝐺 = 22.52;df=1; 𝑃 < 0.00001). 114 species. Among the 418 homonym ethnospecies, 256 (61.3%) Comparison of the data from the state of Pernambuco were congeneric (type 1 underdifferentiation), and 77 (18.4%) and the northeast region showed variation in the number of exhibited correspondence at the genus level only (type 2 one-to-one correspondences that was inversely proportional underdifferentiation). That is to say, 61% of the species bear tothesizeofthesampledarea,whereasthenumberof correspondence to at least one other species of the same homonym ethnospecies varied in proportion to the size of genus with the same vernacular name, whereas 18.4% of the the sampled area, as shown in Figure 1. Consequently, the homonym ethnospecies exhibited correspondence with one homonym ethnospecies predominated in the northeast (NE) or more species belonging to other genera in the same family. sample (𝐺 = 48.41;df=1; 𝑃 < 0.00001). In some cases (20.3%), the homonym ethnospecies belonged In the group of homonym ethnospecies, 309 (74%) to different families, such as the ethnospecies “fedegoso” were representative of the native flora, and 109 (26%) were (coffee senna) and “capeba” (cow-foot leaf)Table ( 1). exotic species. In the group of ethnospecies with one-to- one correspondence, 15 (37%) were representative of the 4. Discussion native flora and 26 (63%) were exotic species (Figure 2). The proportion of native species relative to the proportion 4.1. Hidden Diversity in Regional Markets. Knowledge of the of exotic species was therefore significantly greater for the hidden diversity of medicinal plant species represents an Evidence-Based Complementary and Alternative Medicine 5 PE,PB,SE,CE,PI, MA, RN, BA PE, PB, PI, BA PE,PB,CE,RN PE,PB,RN,BA PE,PB,CE,PI,RN, BA – – – ] , 62 85 , 76 88 , 62 , , , 60 82 , 74 80 , 60 ] – ] , 59 , ]PE,RN,BA , 76 72 94 77 ]PB,CE,MA,BA 76 , ] 59 , , – 84 , 56 , , 79 – 67 78 70 76 92 , 73 , 55 – , , , , 54 79 , 78 , – 63 – 52 72 69 71 88 , ]MA ]PE,PB ]PE , 53 ]MA ]PB,CE , , , – , 65 52 , ]PE ]PE 62 9 , 70 , 68 65 89 57 91 68 , , 70 67 85 70 , ]CE ]RN – , , , ]PB ]PB ]BA ]BA ]PB ]SE ]BA , ]PB ]PI ]BA 52 11 , ]RN 11 3 3 65 65 , , , , ] , , , , , , , 95 79 57 79 83 60 82 1 1 61 1 89 78 88 66 56 77 68 3 56 90 85 3 56 1 1 81 [ 64 64 87 67 [ [ 80 [ N[ N[ N[ N N[ N N ˜ ao N [ (Vell.) H. ˜ ˜ ao) A. C. ao) A. C. (Less.) H. (Mart.) J. B. (L.) H. Rob. N [ Pdre N)Pedersen [ (Vell.) Allem . (L.) Kuntze N [ (L.) Benth. N [ Raddi N [ (Mull.) S. F. Mull. Arg. N [ (Allem (Allem DC. N [ Sw. N [ (Bong.) Cabrera N [ Mart. N [ Mart. ex Mull. Arg. N [ Mart. N [ Mart. N [ Sw. N [ L. N Mart. N [ (Spreng (Aubl.) Willd. N [ Mart. N [ Mart. N [ Vernonanthura phosphorica Rob. Luehea grandiflora Verbesina macrophyllaBlake ennnhr ferruginea Vernonanthura Rob. Myracrodruon urundeuva Commiphora leptophloeos Gillett Luehea candicans Vernonanthura brasiliana Spondias mombin Amburana cearensis Sm. Amburana cearensis Sm. Anadenanthera colubrina Brenan Schinus terebinthifolius Acalypha multicaulis Gochnatia velutina Luehea divaricata Annona coriacea Guettarda platypoda Guettarda angelica Annona crassiflora Luehea ochrophylla Pfaffiaglomerata Centrosema brasilianum Alternanthera brasiliana Lygodium venustum Lygodium volubile Dipteryx odorata ˜ ao )Less. . (Vell.) ˜ ˜ ao) A. C. ao) A. C. (Mart.) J. B. )Pedersen (Vell.) Allem . (L.) Kuntze (L.) Benth. ˜ ao Raddi (Mull.) S. F. (Vell.) Benth. Mull. Arg. (Spreng (Allem (Allem DC. Mart. Sw. Less. (Bong.) Cabrera Mart. Mart. ex Mull. Arg. Mart. Mart. Sw. L. Allem Mart. (Spreng (Aubl.) Willd. Mart. Mart. Willd. Pers. L. Schinus terebinthifolius Luehea speciosa Luehea candicans Vernonia scabra Vernonia ferruginea Gochnatia velutina Guettarda angelica Acalypha multicaulis Luehea grandiflora Verbesina macrophyllaBlake Luehea ochrophylla Centrosema brasilianum Luehea divaricata Pfaffiaglomerata Amburana cearensis Annona coriacea Vernonia polyanthes Myracrodruon urundeuva Amburana cearensis Guettarda platypoda Annona crassiflora Anadenanthera colubrina Alternanthera brasiliana Brenan Gillett Commiphora leptophloeos Brenan Piptadenia colubrina Torresea cearensisDipteryx odorata Spondias mombin Spondias lutea Sm. Sm. Lygodium venustum Bursera leptophloeos Lygodium volubile Anadenanthera macrocarpa Table 1: Ethnospecies marketed in the Northeast Brazil and the corresponding species cataloged in the scientificliterature. Euphorbiaceae Fabaceae Tiliaceae Fabaceae Asteraceae Burseraceae Schizaeaceae ´ a Anacardiaceae ˆ onito Amaranthaceae ¸oita Vernacular name FamilyAroeiraCaj Scientificname Anacardiaceae in theAbre original sourceCaminho Valid scientificAc cavalo Ac name Literature Origin Amburana Cumaru State Angelica Fabaceae Araticum Rubiaceae Angico Annonaceae Fabaceae Assa-peixe 6 Evidence-Based Complementary and Alternative Medicine PE,PB,SE,CE,RN PE,PB,CE,PI,BA PE,PB,SE,CE,PI, RN – , ] ] PE, PB, CE, RN 71 ] , 89 97 90 97 , , , 67 , , 87 94 77 94 , , , 63 , ]PE , 86 93 75 ]PE,CE,MA 66 , , 94 , 60 , , , 92 82 89 70 ]SE , 59 , , 65 , 59 – , 96 , 66 63 88 67 , ]PB ]PB,PI ]PE,MA , , 57 , 56 – , 56 , , 61 95 88 85 , 56 56 86 75 ]SE ]BA ]MA ]PB ]CE ]CE ]BA , 11 , ]BA ]BA ]CE ]PB ]PI , , ]PE 60 ]PE ]PE 3 11 , , ] , , , , , , 73 58 78 75 82 53 84 67 96 11 86 85 11 1 82 3 1 9 98 56 72 98 57 74 68 98 [ [ 73 77 N[ N[ N[ N[ N[ N[ E[ Cass. N [ (DC.) R. M. Kunth N [ (L.) DC. N [ (L.) Urb. N [ A. Juss. N [ (G.Don)D.F. (Spreng.) Taub. N [ A. Juss. N [ L. N [ (Benth.) Killip N [ (Benth.) Barneby Dryand. N [ Willd. N Jacq. N [ L. N [ DC. N (Benth.) L. O. (Mart.) Barneby and (DC.) H. S. Irwin (Jacq.) Merr. E [ Urb. N [ (Benth.) H. S. Irwin L. N [ Mart. and Zucc. N [ Table 1: Continued. Albizia inundata J. W. Grimes Conocliniopsis prasiifolia King and H. Rob. Senna martiana and Barneby Begonia reniformis Operculina macrocarpa Samanea tubulosa andJ.W.Grimes Senna spectabilis and Barneby Peltophorum dubium Piper marginatum Piper umbellatum Ageratum conyzoides Operculina alata Byrsonima sericea Centratherum punctatum Operculina hamiltonii Austin and Staples Byrsonima verbascifolia Hirtella ciliata Albizia polycephala Ipomoea dumosa Williams Byrsonima coccolobifolia Samanea saman Byrsonima correifolia Erythrina velutina Byrsonima gardneriana Urb. Miq. Piper peltatum Cass. (DC.) R. M. )Hayne Kunth . (L.) DC. (L.) A. Juss. Silva Manso (G. Don) D. F. (Jacq.) Benth. (Kunth) R. M. (Spreng.) Taub. A. Juss. L. (L.) (Benth.) Killip (Benth.) Barneby Dryand. Willd. Jacq. L. DC. DC. (Mart.) Barneby and (DC.) H. S. Irwin Urb. (Benth.) H. S. Irwin Schott (Wender Mart. and Zucc. Peltophorum dubium Albizia inundata J. W. Grimes Piper umbellatum Begonia reniformis Begonia huberi C. Pothomorphe peltata Piper marginatum Lourteigia ballotifolia King and H. Rob. Centratherum punctatum Albizia polycephala Byrsonima verbascifolia Conocliniopsis prasiifolia King and H. Rob. Ageratum conyzoides Operculina alata Senna martiana Begonia vitifolia and Barneby Byrsonima sericea Operculina convolvulus Hirtella ciliata Samanea tubulosa andJ.W.Grimes Byrsonima coccolobifolia Pithecellobium saman Senna spectabilis and Barneby Operculina macrocarpa Byrsonima gardneriana Byrsonima correifolia Erythrina velutina Ipomoea purga Operculina hamiltonii Austin and Staples Piperaceae Chrysobalanaceae Asteraceae Convolvulaceae Begoniaceae Fabaceae ıstula Fabaceae ´ ˜ ao de Vernacular name FamilyBalaio de veio Scientificname in the original source Valid scientificBatata de Purga nameBurd Literature Origin Canaf State Capeba Murici Malpighiaceae Mulungu Fabaceae velho Evidence-Based Complementary and Alternative Medicine 7 PE, PB, CE PE,PB,CE,RN,BA PE,PB,CE,PI,RN PE, PB, PI, MA, RN, BA PE,PB,SE,CE,RN ]PE,CE,RN,BA , , , ] 93 99 66 , 72 , , , , 99 72 , ] 84 92 – 71 65 , , , , , 71 95 99 63 , , , 83 79 65 62 , , , , , 64 90 90 56 ]PE,MA,RN 81 , 76 , – 61 59 , , , , , 93 85 62 ]CE 84 ]MA 55 , ] 78 , ] 67 59 , 57 , , , , , , 81 98 68 94 78 ] 93 75 , , ]BA 53 ]PE,SE 60 , 56 – , 57 – 65 , 56 , , , , , 83 , 92 68 95 66 100 71 92 92 75 ]RN ]PE ]BA ]BA ]BA ]PE , 52 11 ]SE , ]CE ]CE , ]PI ]PE 9 , 58 ]CE , 11 11 , , , , , , , , , , , 1 79 56 75 69 3 1 9 75 94 78 3 61 78 58 69 9 57 74 80 90 78 55 [ [ [ 76 75 68 [ 94 [ 73 N E[ N[ ex Benth. N [ and Schltdl. N . (K. Schum. and L. N [ (Chodat) Iltis N [ L. N [ L. E [ (Bong.) Steud. N L. E [ Benth. N [ Cambess. N [ Wittm. N [ Aiton N [ (Aubl.) L. P. Schumach. and Roxb. N [ Moric. N [ Cham L. N [ L. N Link N [ (Jacq.) Raf. N Mart. ex Zucc. N [ L. E [ Table 1: Continued. Phyllanthus tenellus Euphorbia hyssopifolia Sambucus nigra Phyllanthus amarus Thonn. Tarenaya spinosa Euphorbia thymifolia Euphorbia prostrata Sambucus racemosa Phyllanthus niruri Phyllanthus flaviflorus Lauterb.) Airy Shaw Caryocar coriaceum Tarenaya hassleriana Phyllanthus urinaria Sambucus australis Bauhinia cheilantha Caryocar brasiliense Oxalis divaricata Bauhinia forficata Phanera outimouta Queiroz Bauhinia acuruana Bauhinia ungulata Bauhinia subclavata Bauhinia smilacifolia Burch. ex Benth. and Schltdl. Mull. Arg. . (L.) Small (K. Schum. and L. L. L. (Bong.) Steud. L. Benth. Cambess. Aubl. Wittm. Aiton Schumach. and Roxb. Moric. Chodat Cham L. L. Link Mart. ex Zucc. L. Jacq. aesyce hyssopifolia Phyllanthus flaviflorus Euphorbia prostrata Sambucus nigra Cleome spinosa Phyllanthus tenellus Phyllanthus corcovadensis Euphorbia hyssopifolia Cham Euphorbia thymifolia Sambucus racemosa Phyllanthus amarus Thonn. Caryocar coriaceum Cleome hassleriana Bauhinia cheilantha Phyllanthus niruri Caryocar brasiliense Sambucus australis Phyllanthus urinaria Oxalis divaricata Lauterb.) Airy Shaw Bauhinia forficata Bauhinia ungulata Bauhinia outimouta Bauhinia acuruana Bauhinia smilacifolia Burch. Bauhinia subclavata Euphorbiaceae Oxalidaceae ˆ e Cleomaceae ´ o Fabaceae ¸amb Vernacular name FamilyMuc Scientificname in the original sourceMoror Valid scientificPiqui name Literature Origin Caryocaraceae Quebra pedra State Sabugueiro Adoxaceae 8 Evidence-Based Complementary and Alternative Medicine PE,PB,SE,PI,RN, BA PB,SE,CE,PI,RN PE,PB,CE,PI,MA PB, SE, PI, BA PE,PB,CE,RN , , , ]PB,SE,CE,RN ] , ]PE,CE ] 71 88 , 65 95 94 73 , 76 88 , , , ] , , , 63 61 84 , 89 92 , 95 73 70 , 77 , ]PB,CE,PI,BA , , , , , 62 ]PB,CE,PI 60 83 , 88 92 90 94 , , 74 64 64 , 88 , ]PE,PB,MA ] , – , , 56 – , ]PE,PB,RN 57 80 , 78 78 72 71 85 , 89 88 , 63 ]PE,PB,BA , 62 74 72 , , , , , , , 55 , , , 56 , 69 – 89 62 74 68 71 70 , 80 85 71 ]PB , , 60 , 59 59 , ]PE,MA , , , , , , 52 58 , , , 11 , 71 , 86 67 59 80 93 , 54 68 62 76 76 ]MA ]CE ]MA , ]PE 60 52 ]PB ]SE ]SE ]SE , ]PI , 9 57 ]PE 53 , , , 9 11 , , , ] , , , , , , , , , 69 57 61 9 3 3 95 58 55 75 11 53 1 77 68 56 1 66 95 3 60 57 85 9 75 [ [ 60 [ [ 66 [ 75 [ 95 N[ N[ N[ N[ N[ and Mill. E [ and Bonpl. Pic. Serm. Britton (Vell.) Taub. N [ Hoffm. ex Pohl N Cambess. N [ Kunth N L. N ) Lundell N [ (L.) (L.) (L.) Link E [ Mull. Arg. N [ (Willd.) DC. N [ Aubl. N [ (L.) Link N A. St.-Hil. N [ L. E (Vell.) Reitz N [ L. N [ Sm. N [ Netto Baill. N [ (Mill.) H. S. Irwin and ( Table 1: Continued. Croton campestris Periandra mediterranea Cnidoscolus quercifolius Croton heliotropiifolius Croton sonderianus Croton betaceus Mimosa somnians Humb. Senna uniflora Barneby ex Willd. Heliotropium elongatum Roem. and Schult. Mimosa sensitiva Turnera subulata Turnera guianensis Senegalia tenuifolia Turnera ulmifolia Guapira noxia Heliotropium indicum Uncaria tomentosa Echinochloa colona Rose Turnera chamaedrifolia Senna occidentalis Heliotropium procumbens Guapira opposita Lycopodiella cernua (Mull. Arg.) Mill. and Bonpl. (Vell.) Taub. Hoffm. ex Pohl Cambess. Kunth L. ) Lundell (L.) Pic. Serm. Willd. (L.) Link Baill. Aubl. Mull. Arg. (Willd.) DC. (L.) Link A. St.-Hil. L. Pax and K. Hoffm. Willd. (Vell.) Reitz L. Sm. Mart. ex Benth. Netto (Mill.) H. S. Irwin and ( Croton campestris Croton tenuifolius Croton heliotropiifolius Croton moritibensis Croton sonderianus Cnidoscolus phyllacanthus Pax and L. Hoffm. Turnera subulata Turnera guianensis Periandra mediterranea Turnera chamaedrifolia Heliotropium indicum Cnidoscolus quercifolius Croton rhamnifolius Periandra dulcis Turnera ulmifolia Senna occidentalis Mimosa sensitiva Uncaria tomentosa Acacia paniculata Echinochloa colona Guapira noxia Mimosa somnians Humb. ex Willd. Guapira opposita Lycopodiella cernua Senna uniflora Barneby Heliotropium procumbens Heliotropium elongatum Roem. and Schult. Rubiaceae Alismataceae Boraginaceae Lycopodiaceae Fabaceae Fabaceae ˜ ao Mole Nyctaginaceae Vernacular name FamilyFedegoso Scientificname in the original source Valid scientificFavela name Euphorbiaceae Velame Literature Origin Acansu Euphorbiaceae State Chanana Fabaceae Jo Turneraceae Unha de gato Evidence-Based Complementary and Alternative Medicine 9 PE, PB, PI, BA PE, PB, CE PE, PB, BA , , – 89 73 74 , , , ] 86 72 67 , , , 94 , 78 63 63 ]PB,MA,RN,BA , , , 93 ]PB,SE,CE,BA , 65 56 56 100 , , , 88 88 , ]PB,SE,RN ]PE,PB,BA , ]PE,MA , ] 53 56 53 ]PE 99 93 , , , 90 83 94 86 85 , , , , , 65 , , 53 52 52 , , , , 84 86 89 78 63 80 84 ]MA ]MA , ]CE ]RN ]BA , ]PE ]SE ]BA ]RN ]SE ]PI ]PI ]PI , ]SE , ]BA ]BA ]CE ]PB ]PI ]PI ]CE , 11 11 56 11 ]PE ] , , , , , , 97 78 58 85 55 79 66 87 82 11 74 61 74 87 82 59 87 69 66 58 90 1 1 95 58 56 60 1 80 1 66 77 [ 94 [ [ 76 N[ E[ N[ N[ . ˜ ao) Dunal N [ Murray N [ Vell. N [ Spreng (L.) Benth. N [ (Allem (Forssk.) (Mart.) Griseb. N [ Scop. N [ Moric. N [ L. N [ L. N L. E [ Agra N [ A. St.-Hil. N [ L. E [ Moric. N [ Kunth E [ L. N [ um Mart. N [ (Aubl.) Pers. N Choisy N [ M. Nee N [ Benth. N [ Dunal E [ Mart. ex Benth. N [ L. E [ L. N ich Vell. N [ Mart.exBenth.N[ Schweinf. ex Table 1: Continued. Solanum paludosum Solanum mauritianum Solanum granuloso-leprosum Solanum absconditum Ducke Luetzelburgia auriculata Solanum scuticum Solanum polyrtr Solanum albidum Solanum lycocarpum Talinum portulacifolium Asch. Andira paniculata Pityrocarpa obliqua subsp. brasiliensis (G. P. Lewis) Luckow and R. W. Jobson Vismia brasiliensis Luehea grandiflora Heliotropium angiospermum Heliotropium indicum Solanum paniculatum Cedrela fissilis Celosia argentea Plumbago scandens Vismia guianensis Ocimum americanum Andira nitida Andira vermifuga Cedrela odorata Centrosema brasilianum Elephantopus mollis Origanum majorana .Echinodorussubalatus . Chrysophyllum splendens ˜ ao) . Heliotropium transalpinum Murray Spreng Cham (L.) Benth. (Allem (Forssk.) (Mart.) Griseb L. L. L. Agra Vell. Aiton Moric. A. St.-Hil. L. D. Don Moric. Kunth L. (Pers.) J. F. Macbr. Mart. (Aubl.) Pers. Choisy Benth. um Dunal Mart. ex Benth. L. L. ich Vell. Mart. ex Benth. Schweinf. ex Solanum absconditum Solanum polytr Solanum paludosum Solanum auriculatum Solanum erianthum Solanum tabacifolium Solanum albidum Solanum lycocarpum Heliotropium angiospermum Heliotropium tiaridioides Andira paniculata Ducke Luetzelburgia auriculata Vismia brasiliensis Luehea grandiflora Centrosema brasilianum Heliotropium indicum Plumbago scandens Solanum paniculatum Cedrela fissilis Vismia guianensis Ocimum americanum Andira nitida Piptadenia obliqua Andira vermifuga Echinodorus subalatus Cedrela odorata Chrysophyllum splendens Talinum portulacifolium Asch. Elephantopus mollis Celosia cristata Origanum majorana Plumbaginaceae Alismataceae Sapotaceae Tiliaceae Fabaceae Boraginaceae Asteraceae Solanaceae Meliaceae Amaranthaceae Portulacaceae Vernacular name FamilyLingua de Vaca Scientificname in the original source ValidLacre scientific Clusiaceae Jurubeba name Literature Origin Cedro State Crista de galo Manjerona Lamiaceae Angelim Fabaceae 10 Evidence-Based Complementary and Alternative Medicine PB, CE, PI, MA, RN, BA PE, PB, BA PE,PB,SE,CE,RN, BA , , , 78 , 83 67 , 74 , , 79 64 , ]PB,CE,PI,BA 71 , – , 72 88 63 88 , , 67 ] , , , 69 74 98 58 86 , , 66 , , ] , , 71 ]PE 67 96 56 88 ]MA,BA , 82 61 ]PE , ] , , , , 53 , , 69 67 93 54 53 95 94 83 76 , ]CE , ]RN ]BA ]MA ]PB ]CE ]PB ]BA ]BA ]BA ]PB ]PB ]PB 59 ]SE , ]CE ]CE ]CE ]PE ]PE ]PE ]PB ]PI ]PI ]PI ]PI ]PE , 11 ]PE , , , , , , , 66 89 60 93 78 67 57 76 11 93 1 67 74 78 68 85 9 93 98 53 54 78 74 84 88 88 11 67 73 74 86 58 90 60 80 [ [ 90 84 [ 73 N[ N[ N[ N[ N[ N[ N[ N[ N[ N[ and . L. N [ Mart. N [ (Mart.) O. Tul. N [ L. N [ (Cham G. Mey. Griseb. (Vell.) Stellfeld L. N [ . W. T. A i t o n E [ DC. N [ Mart. N [ Cham. and (L.) L. N [ L. N [ L. E [ L. N [ L. E [ Kunth N [ Kunth N [ (L.) DC. N [ (Sw.) Steud. N [ L.L. N N [ L. N [ Kunth N [ Mart. ex L. E L. N Hook. f. E [ L.) Micheli Table 1: Continued. Erythroxylum vacciniifolium Borreria scabiosoides Schldl. Erythroxylum amplifolium E. Schulz Borreria verticillata Echinodorus grandiflorus Schltd Anemopaegma arvense and J. F. Souza Polygala paniculata Capraria biflora Smilax rotundifolia Emilia sonchifolia Equisetum arvense Nasturtium officinale Polygala gracilis Equisetum giganteum Cenostigma macrophyllum Cybianthus detergens Scoparia dulcis Equisetum hyemale Illicium verum Miconia albicans Plantago major Polygala paniculata Lantana camara Lantana camara Zornia latifolia Sm. Oxalis psoraleoides Cardiospermum corindum Cardiospermum halicacabum Verbesina diversifolia Lantana canescens and . L. Mart. Tul. (Mart.) O. L. (Cham Griseb. Smilax cissoides (Vell.) Stellfeld L. L. W. T. A i t o n DC. Mart. Cham. and ianum L. L. L. Benth. Wedelia calycina Rich L. L. Kunth (L.) DC. St.-Hill. (Sw.) Steud. L. L. L. Kunth Mart. ex L. L. Sm. Hook. f. Gardner L.) Micheli Erythroxylum vacciniifolium Spermacoce verticillata Borreria scabiosoides Schldl. Polygala paniculata Nasturtium officinale Smilax rotundifolia Emilia sonchifolia Cenostigma Erythroxylum amplifolium E. Schulz Echinodorus grandiflorus Schltd Capraria biflora Equisetum arvense Cybianthus detergens Polygala gracilis Equisetum giganteum Anemopaegma arvense and J. F. Souza Smilax japecanga Griseb.Smilax cissoides Smilax japecanga Griseb. Equisetum hyemale Smilax campestris Griseb. Smilax campestris Griseb. Lantana camara Lantana camara Miconia albicans Scoparia dulcis Plantago major Polygala paniculata Oxalis insipida A. Cardiospermum corindum Cardiospermum halicacabum Verbesina diversifolia Lantana canescens Wedelia scaberrima Illicium verum Zornia latifolia Polygalaceae Brassicaceae Asteraceae Rubiaceae Scrophulariaceae Fabaceae Primulaceae Erythroxylaceae Alismataceae Polygalaceae Verbenaceae Melastomataceae Bignoniaceae Plantaginaceae Plantaginaceae Oxalidaceae Verbenaceae Sapindaceae Asteraceae Fabaceae ´ a Vernacular name FamilyArrozinho Anil estrelado Scientificname Cavalinha Schisandraceae in the original source Valid scientific Equisetaceae Chumbinho nameCamar Literature Origin Canela de velho Catuaba State Japecanga Smilacaceae Vassourinha Transagem Evidence-Based Complementary and Alternative Medicine 11 PE,PB,SE,CE,RN PE,PB,SE,CE,RN, BA PB, CE, RN ] PE, PB, CE, PI, RN ] PE, SE, CE, RN, BA , 90 , ]PE 99 , 59 , , 73 94 81 , – , , 58 94 , , 75 70 75 76 ] – , , , , ]PE,CE,MA 83 56 99 71 , , 70 ]PE 74 64 67 78 , , ] ] , , ]PB,CE ]PE,CE,BA , , , 53 78 75 95 , , 70 95 72 99 , 83 ]PE,BA 88 60 63 61 63 , , , , , , , , , , , 52 69 73 84 71 , 90 , 67 80 85 , ]CE,PI ]PB,BA 85 59 , 60 59 ]MA,RN ]MA,BA , 56 56 , , , , , 11 , , , , , 60 81 , 64 60 84 63 62 67 63 88 78 77 ]SE ]SE ]SE , ]PB ]BA ]CE 53 55 77 , ]SE – ]PE ]CE ]PB , ]PI , 9 52 , ]CE , , 53 55 ]PE , , , , , , , , , , , 11 57 80 52 1 3 68 96 95 9 55 55 78 85 58 3 9 96 96 57 72 61 9 1 63 98 76 86 [ 62 76 77 [ [ 72 N[ N[ N[ N[ N N[ (Tul.) L. P. (Mart. ex G. Mill. N [ Mull. Arg. N [ DC. N [ Kunth N [ (Tul.) L. P. L. E [ L. N [ Baill. N (Benth.) Harley N [ L. E [ Mull. Arg. N [ (L.) Philipson N [ Barb. Rodr. N [ L. E [ (Baker)R.M.King Lam. N [ (Kuntze) E. Santos N [ Noblick N [ Aubl. N [ (Mart.) Becc. N [ L. E [ (Kuntze) K. Schum. N [ Benth. N L. E [ Table 1: Continued. Gymnanthemum amygdalinum (Delile) Sch. Bip. ex Walp. Poincianella bracteosa Queiroz Poincianella microphylla Don) L. P. Queiroz Cynara cardunculus Queiroz Gymnanthemum amygdalinum (Delile) Sch. Bip. ex Walp. Poincianella pyramidalis Prolobus nitidulus and H. Rob. Ocimum basilicum Ocimum campechianum Croton sonderianus Syagrus picrophylla Syagrus cearensis Ageratum conyzoides Stilpnopappus scaposus Croton blanchetianus Blainvillea acmella Polygala violacea Ocimum gratissimum Curcuma longa Bidens pilosa Licania rigida Ceiba glaziovii Hypenia salzmannii Croton heliotropiifolius Croton urticifolius Croton argyrophylloides Chorisia glaziovii . . Becc. Syagrus oleracea (Tul.) L. P. (Mart. ex G. Mill. Mull. Arg. ) K. Schum. DC. Cass. L. Tul. L. Baker (Benth.) Harley Willd. Mull. Arg. Barb. Rodr. L. (Baker)R.M.King Lam. (Kuntze) E. Santos L. Noblick Aubl. (Mart.) Kuntze L. ( Benth. L. Gymnanthemum amygdalinum (Delile) Sch. Bip. ex Walp. Cynara scolymus Poincianella microphylla Don) L. P. Queiroz Queiroz Caesalpinia bracteosa Poincianella pyramidalis Ocimum campechianum Croton sonderianus Syagrus cearensis Syagrus picrophylla Stilpnopappus scaposus Syagrus oleracea Ageratum conyzoides Vernonia condensata Ocimum basilicum Caesalpinia pyramidalis Tul Croton blanchetianus Baill Blainvillea rhomboidea Prolobus nitidulus and H. Rob. Polygala violacea Ocimum gratissimum Curcuma longa Ceiba glaziovii Licania rigida Bidens pilosa Hypenia salzmannii Croton rhamnifolius Croton urticifolius Chorisia glaziovii Croton argyrophylloides Polygalaceae Asteraceae Malvaceae Lamiaceae Bombacaceae ˜ ao Zingiberaceae ´ e Arecaceae ˜ ao Asteraceae ¸afr Vernacular name FamilyAlcachofra Asteraceae ScientificAc name in the original source ValidAlfavaca scientificCatol Lamiaceae nameMentrasto Literature Origin Catingueira State Fabaceae Marmeleiro Euphorbiaceae OiticicaPic Barriguda Chrysobalanaceae 12 Evidence-Based Complementary and Alternative Medicine PE,PB,SE,CE,PI, MA, RN, BA PE,PB,SE,CE,PI, RN ]PE,CE,RN,BA – ] 99 , , 67 97 ]PE,MA,BA , 81 ] , , 94 , 65 84 101 90 ]PE,PB,BA – , , 79 – , 93 , , 57 83 63 94 99 78 , , , 78 , ]PE,CE 81 , , , , 55 75 57 84 94 74 , , 94 76 – , ]PE,CE,BA ]PE,CE,PI , 69 , , , , 53 69 52 83 78 93 63 , 70 93 75 ]MA , , ]RN,BA , ]BA , ]BA , ]PB,CE , 56 , , , 11 , 11 68 68 69 69 79 90 67 , 78 78 55 ]PB , 62 72 84 , , ]RN ]MA ]MA ]CE , , , ]BA , ]PE , ]PE , ]PI , ]PB 9 , 53 ]PE ]MA ]RN 9 , , , , , , 72 67 67 100 53 52 67 66 66 61 68 1 56 67 90 72 57 56 54 57 3 9 98 68 81 65 [ 69 83 [ 60 N[ E[ N[ ˜ ao N [ ) Link . Allem (L.) Kuntze N [ L. E [ L. E [ Andrews E [ L. E [ W. T. A i t o n E [ L. E [ L. N L. N [ (Spreng (Hook. and Arn) L. E [ L. E [ L. E [ L. E [ (Spreng.) Pedersen N [ (L.) Skeels E [ (L.) R. K. Jansen N [ A.St.-Hil. and Moq. N [ Jacq. N [ L. E (Camb.) A. Lima E [ L. E [ (Kunth) Cass. N [ ex Benth. Otto Table 1: Continued. Jodina rhombifolia Reissek Justicia gendarussa Burm. f. Artemisia vulgaris Pfaffiaglomerata Ocimum carnosum and Justicia pectoralis Alternanthera brasiliana Mentha pulegium Mentha spicata Gossypium arboreum Gossypium herbaceum Chrysophyllum arenarium Gossypium barbadense Polygala paniculata Gossypium hirsutum Plectranthus barbatus Ruta graveolens Acmella oleracea Ximenia americana Polygala bryoides Nasturtium officinale Artemisia vulgaris Prunus domestica Acmella ciliata Syzygium cumini Rorippa pumila ˜ ao Allem (L.) Kuntze L. L. Andrews L. W. T. A i t o n L. L. L. Hook. and Arn. L. L. L. L. (Spreng.) Pedersen (L.) R. K. Jansen A. St.-Hil. and Moq. Jacq. (L.) Druce L. (Camb.) A. Lima L. (Kunth) Cass. Benth. Pfaffiaglomerata Alternanthera brasiliana Iodina rhombifolia Justicia pectoralis Artemisia vulgaris Justicia gendarussa Burm. f. Mentha pulegium Gossypium arboreum Gossypium herbaceum Ocimum selloi Mentha spicata Gossypium barbadense Polygala bryoides Polygala paniculata Nasturtium officinale Plectranthus barbatus Acmella oleracea Gossypium hirsutum Ximenia americana Ruta graveolens Prunus domestica Chrysophyllum arenarium Acmella ciliata Artemisia vulgaris Eugenia cumini Rorippa pumila Asteraceae Amaranthaceae Acanthaceae Lamiaceae Polygalaceae Brassicaceae Olacaceae Lamiaceae Sapotaceae Myrtaceae Rosaceae Asteraceae ısia Asteraceae ´ ˜ ao Malvaceae ˜ ao Vernacular name FamilyVique Scientificname in theAgri original source Valid scientificAlgod nameAmeixa Literature Origin Anador State ArrudaArtem Rutaceae Evidence-Based Complementary and Alternative Medicine 13 PE,PB,CE,BA PE,PB,SE,CE PE, RN, BA PE,PB,SE,CE,PI, MA, RN, BA , – ] , , 79 66 , , 99 71 83 , , ] , 77 63 – , , 94 67 97 81 81 ]PB,CE,PI , – , – 61 , ]PE,CE,RN,BA 75 , , 98 63 92 76 95 79 99 , , ]PE,BA , , 58 , 71 , , – , 87 89 69 69 60 88 78 , 94 – , , , , , ] 56 60 , , , , 78 52 87 58 56 63 77 93 75 ]CE , 92 ] PI, MA , ]PE , , , , ] , 54 53 – , , 9 , , 71 98 74 , 53 56 53 86 93 71 56 88 75 ]MA ]PE ]BA ]BA ]BA ]BA , ]PE ]BA , ]CE 11 , 11 79 3 , , , , , , , , , , , , , 84 66 78 69 78 84 1 82 11 80 11 54 1 1 61 9 11 56 94 [ 54 73 83 [ 68 [ [ 84 84 E[ N[ N[ N[ N[ E[ N[ (Mart.) DC.) Benth. N [ Wall. ex A. ( L. N Mart. N [ G. B. Ownbey E (Gardner) (Gomes) Benth. N [ (Mart.) Plumel N [ (Spruce ex J. Presl E Nees and Mart. N [ Nees and Mart. N [ Gaert. E [ (L.) Voss E [ Schrad. ex J. C. Mart. N Table 1: Continued. Bambusa vulgaris Wendl. Himatanthus sucuuba Mull. Arg.) Woodson Himatanthus bracteatus Woodson Abarema cochliacarpos Barneby and J. W. Argemone subfusiformis Gochnatia oligocephala Cabrera Dendrocalamus giganteus Munro Himatanthus drasticus Stryphnodendron adstringens Coville Bambusa bambos Maytenus distichophylla Carduus benedictus Maytenus rigida Stryphnodendron coriaceum Plathymenia reticulata Nectandra cuspidata Anacardium occidentale Cinnamomum verum Nectandra leucantha Mart. (Mart.) DC.) Benth. Blume Wall. ex A. ( L. Mart. G. B. Ownbey (Gardner) (Gomes) (Retz.) Willd. Benth. (Mart.) Plumel (Spruce ex Nees and Mart. L. Nees and Mart. Gaert. Schrad. ex J. C. Mart. Himatanthus drasticus Bambusa vulgaris Wendl. Argemone subfusiformis Himatanthus sucuuba Mull. Arg.) Woodson Bambusa arundinacea Maytenus distichophylla Carduus benedictus Woodson Stryphnodendron barbatimam Abarema cochliacarpos Barneby and J. W. Pithecellobium cochliacarpum (Gomes) J. F. Macbr. Stryphnodendron coriaceum Argemone mexicana Plathymenia reticulata Gochnatia oligocephala Cabrera Dendrocalamus giganteus Himatanthus bracteatus Maytenus rigida Munro Nectandra cuspidata Stryphnodendron adstringens Coville Cinnamomum zeylanicum Anacardium occidentale Nectandra leucantha Asteraceae Asteraceae Papaveraceae Fabaceae Lamiaceae Lauraceae ˜ ao Fabaceae Vernacular name FamilyBambu Scientificname in the original source Poaceae Janauba Valid scientific Apocynacaee Barbatim name Literature Origin Bom nome State Celastraceae CajuCardo santo Anacardiaceae Candeia Canela 14 Evidence-Based Complementary and Alternative Medicine PE,PB,SE,CE,RN, BA PE,PB,SE,CE,PI, MA, RN, BA , ] 58 , , , 100 76 80 57 , , , ] PB, CE, PI, MA, BA , – 75 99 71 78 ]PE,CE,MA,RN,BA 55 , , , , ]PE,MA,RN,BA ]PE,PB,RN,BA , 58 93 , 72 95 68 ]PE,PB 76 , ]PE,CE 53 ]PB,MA,BA 99 99 , – , , , , , 56 89 92 65 85 , 69 93 67 75 , , 52 , 83 , 84 ] , – , , , , , 53 86 84 56 82 , 11 95 71 83 67 , 60 ]MA , , , 79 72 , , – , , , , 52 , 9 81 85 55 , 66 67 , 63 93 79 63 ]MA , ]PE ]BA ]MA ]PB ]BA , ]SE 57 ]PB ]PB ]PI ]CE ]PI ]PI ]PE , , , ]RN 9 65 68 3 , , , ]CE , – , , , , 1 1 94 1 56 66 87 68 84 81 74 75 77 74 78 61 53 86 61 9 69 1 9 97 66 77 60 88 [ [ 61 78 N N[ Pirani N [ L. N [ Vogel N [ ´ ecul N [ Poelln. E [ . Tr Desf. N [ Cav. E [ (Jacq.) R. Br. E [ (L.) Mill. E [ (L.) R. Br.a E [ (Jacq.) L. N [ Ducke N [ (Lam.) Pers. E [ (Andrews) Haw. E [ (Andrews) Haw. E [ A. St.-Hil. N [ Mart. N [ (Cambess.) Miers N [ L. E [ Palau E [ Willd. N [ DC. N Dwyer N [ L. N [ Spreng (Mill.) N. E. Br. ex Britton Table 1: Continued. Kalanchoe pinnata Lippia alba and P. Wilson Leptolobium dasycarpum Cecropia peltata Cereus jamacaru Cecropia pachystachya Opuntia ficus-indica Copaifera coriacea Copaifera lucens Copaifera reticulata Copaifera officinalis Cecropia palmata Copaifera langsdorffii Kalanchoe blossfeldiana Malvaviscus arboreus Kalanchoe crenata Cardiospermum halicacabum Zanthoxylum hamadryadicum Leonotis nepetifolia Guatteria australis Eschweilera ovata Aloysia citriodora Baccharis crispa Kalanchoe crenata Melissa officinalis Leucas martinicensis Pirani L. ´ ecul (Vogel) (Lam.) Oken Poelln. Salisb. Tr Cambess. Cambess. Desf. Cav. (Jacq.) R. Br. (L.) Mill. (L.) R. Br. (Jacq.) L. Ducke (Lam.) Pers. A. St.-Hil. Mart. (Cambess.) Miers (Less.) DC. L. Willd. DC. Kunth Dwyer L. (Mill.) N. E. Br. ex Britton Bryophyllum calycinum Kalanchoe pinnata Bryophyllum pinnatum Cecropia peltata Opuntia ficus-indica Copaifera coriacea Copaifera lucens Lippia alba and P. Wilson Copaifera reticulata Copaifera officinalis Kalanchoe brasiliensis Cecropia palmata Cecropia pachystachya Cereus jamacaru Copaifera langsdorffii Zanthoxylum hamadryadicum Acosmium dasycarpum Yakovlev Kalanchoe blossfeldiana Kalanchoe brasiliensis Malvaviscus arboreus Leonotis nepetifolia Guatteria australis Cardiospermum halicacabum Eschweilera ovata Leucas martinicensis Baccharis trimera Lippia citriodora Melissa officinalis Crassulaceae Verbenaceae Crassulaceae Malvaceae Annonaceae Sapindaceae Rutaceae Fabaceae Lecythidaceae Lamiaceae Lamiaceae ´ uba Urticaceae ˜ ao de ˜ ao Francisco S Vernacular name FamilyMandacaruCarqueja Cactaceae Scientificname in the original source ValidCidreira scientific Asteraceae Pra tudo name Literature Origin Copaiba Fabaceae State Courama Cord Emba Imbiriba Evidence-Based Complementary and Alternative Medicine 15 PE,PB,CE,MA,RN, BA PE,PB,CE,RN,BA PE,PB,CE,MA,RN, BA PE, PB, PI, MA, BA PE,PB,SE,CE,PI, MA, RN, BA , , , , ] , 68 , 83 , 76 – 93 ]PE,PB,PI , 84 , , 88 100 – 65 , 62 , , 75 , , 68 84 100 74 , ]PE,BA 79 , , ]PE,PB,CE 86 93 , , 63 , , 60 , 69 , 94 72 63 78 , 93 72 , 78 ] , , , , ]PE 85 ]PE,BA 92 , – ] , 56 , 59 , 63 , 84 56 63 , 68 76 72 94 70 , 100 81 95 76 ]PB,MA,BA , , , , , 83 , , ]PE,RN,BA , ] , , 53 , , 57 , 56 , 52 52 , 79 57 69 76 69 66 93 68 , 99 93 75 81 99 80 , , , ]PB , , , , , , , 52 , , , , , , 11 56 11 11 , 72 88 , , 63 63 67 57 54 ]PB 84 , , 67 72 92 93 78 79 94 ]CE ]BA , ]BA ]BA ]PE ]PB , 9 9 11 , , ]PE , , , , 3 3 , ] , , , , , , , , , , , 68 100 80 53 89 60 67 84 3 53 53 3 1 81 56 52 1 3 77 53 52 [ 78 [ 94 [ [ [ 69 92 93 64 76 89 Benth. N [ L. E [ L. E L. E [ Mill. E [ Mill. E [ Mill. E [ Reissek N [ L. E H. Rainer N [ Mart. ex Reissek N [ (Brongn.) Vogel N [ (Nees) Mez N [ Roscoe E L. E Mart. N Mart. and Schauer N [ (Mart.) Kausel N [ (Seub.) Franco E [ L. E [ Vattimo N [ Table 1: Continued. Laurus azorica Annona cherimola Ocotea duckei Calliandra depauperata Ziziphus joazeiro Laurus nobilis Annona muricata Annona neosericea Foeniculum vulgare Anethum graveolens Lippia thymoides Zollernia ilicifolia Maytenus ilicifolia Pimpinella anisum Foeniculum vulgare Ocotea glomerata Ocimum gratisssimum Ziziphus cotinifolia Rosmarinus officinalis Zingiber officinale Plinia cauliflora Benth. L. L. (Mart.) O. Berg L. Mill. Mill. Mill. Reissek L. Mart. ex Reissek (Brongn.) Vogel (Nees) Mez Roscoe L. Mart. Mart. and Schauer (R.E.Fr.)R.E.Fr. (Seub.) Franco L. Vattimo Laurus azorica Annona cherimola Calliandra depauperata Lippia thymoides Ocotea glomerata Ocotea duckei Rollinia sericea Foeniculum vulgare Anethum graveolens Maytenus ilicifolia Zollernia ilicifolia Annona muricata Ziziphus joazeiro Laurus nobilis Pimpinella anisum Foeniculum vulgare Ziziphus cotinifolia Ocimum gratisssimum Rosmarinus officinalis Zingiber officinale Myrciaria cauliflora Verbenaceae Fabaceae Celastraceae Fabaceae Lamiaceae ´ a Rhamnaceae Vernacular nameEspinheira Familysanta Gengibre Scientificname in theGraviola original source Zingiberaceae Valid scientificJaboticaba Annonaceae Ju Myrtaceae name Literature Origin LouroErva doce Lauraceae State Endro Apiaceae Alecrim Apiaceae 16 Evidence-Based Complementary and Alternative Medicine PE, PB, RN PE,PB,MA,RN,BA PE,PB,CE,MA,RN, BA PE, PI, MA, RN, BA PE,PB,CE,MA,RN, BA PE,PB,SE,CE,RN, BA PE,PB,SE,CE,PI, MA, RN, BA , , , , ] 94 , , , 99 68 76 99 78 , , ] , 56 , 93 , – – , 81 , 66 69 ]SE,BA 101 , 94 76 , 92 55 55 – – ] , , 79 , , , 76 , 101 62 67 , – 93 75 81 99 89 ] PI, MA, BA ] PI, MA , 53 53 , , , , , , , 66 ] , , 63 67 74 79 , 84 59 67 , 72 , 88 79 84 94 , , 93 52 52 , , ] , , , , , , 57 , , , ]PB,CE,BA 53 53 , 53 69 68 83 11 11 , 101 , 66 78 69 83 84 93 ]PE,BA , , , , ]PB 84 ]BA 89 , , , , – , , , , , 56 , , 11 ]PE,CE ]PE 9 52 11 9 , 94 83 79 77 , 66 66 , , , , 63 99 76 81 83 67 77 79 66 ]CE ] ]RN ]PE ]BA , , , , ]BA 82 ]CE ]PE , , ]BA 9 ]PE 3 3 3 78 93 3 11 , ] , , , , , , , , , , , , , , , , , 57 79 67 93 99 52 69 67 60 1 1 3 67 1 1 94 11 1 57 9 1 1 70 78 83 [ 100 [ 57 [ [ 79 [ 63 76 69 69 99 [ [ [ 78 94 N[ N[ N . L. E . Schltr. E [ Andrews E (DC.) Stapf E Hogg ex Sweet E [ Chaix E [ L. E L. N [ Mill. E (L.) Poit. N [ L. E [ L. N [ (L.) Poit. N [ (L.) Cogn Cham Cav. E [ L. N [ (Jacq.) Sw. N [ Molina E (L.) Hitchc. N [ (Jacq.) Roscoe N [ Schult. and Schult. f. E [ (L.) Burm. f. E Table 1: Continued. Plectranthus barbatus Costus arabicus Gymnanthemum amygdalinum (Delile) Sch.Bip. ex Walp. Aloe vera Costus spiralis Plectranthus neochilus Hyptis pectinata Lavandula officinalis Hyptis suaveolens Lavandula spica Matricaria chamomila Costus spicatus Physalis angulata Chiococca alba Peumus boldus Coreopsis grandiflora Persea americana Passiflora foetida Polygala paniculata Averrhoa carambola Luffa operculata Aloe socotrina Cymbopogon citratus L. . Aloysia lycioides Schltr. Andrews (DC.) Stapf Hogg ex Sweet Baker Chaix L. L. Mill. (L.) Poit. L. L. Mill. (L.) Poit. (L.) Cogn. (Andrews) Benth. [ Cham Cav. L. (Jacq.) Sw. Molina (L.) Hitchc. (Jacq.) Roscoe Schult. and Schult. f. (L.) Burm. f. Coleus barbatus Plectranthus barbatus Costus spiralis Costus arabicus Plectranthus neochilus Hyptis pectinata Lavandula officinalis Hyptis suaveolens Lavandula spica Aloe vera Aloe barbadensis Matricaria chamomila Costus spicatus Aloysia lycioides Coreopsis grandiflora Vernonia condensataVernonia bahiensis Toledo Peumus boldus Physalis angulata Chiococca alba Aloe socotrina Passiflora foetida Polygala paniculata Persea americana Luffa operculata Cymbopogon citratus Averrhoa carambola Lamiaceae Verbenaceae Lamiaceae Monimiaceae Solanaceae Rubiaceae Passifloraceae Polygalaceae ˜ a Asteraceae Vernacular name FamilyAbacateAlfazema Lauraceae Scientificname in the original source Valid scientificAlum Babosa name Literature Origin Xanthorrhoeaceae Boldo Cabacinha State Camomila Cucurbitaceae Cana de Asteraceae macaco Costaceae Canapum Caninana Capim santoCarambola Poaceae Oxalidaceae Evidence-Based Complementary and Alternative Medicine 17 PE,PB,CE,BA PE, RN, BA PE, PB PE,PB,CE,MA,BA PE,PB,CE,PI PE,PB,CE,PI,RN, BA , , ]PE,BA , 72 , 76 , , , , 94 75 93 ] PE, PB, CE, RN , , 93 72 , 69 ]PE,PB ] PE, PB, CE, MA, BA , 60 , , 92 93 72 ]PE , 84 93 , 71 , , 94 84 , 68 , , 57 65 , , , 81 63 , , 84 76 , ] 89 , ]PE 69 , ]PE,PB 78 92 ]PE,CE,BA ]PE,CE , 63 , ] , 56 63 , , , 67 83 56 , , 93 94 76 75 71 94 100 , 92 , , , 67 , 83 76 , , , , , 56 , 53 53 , , , 53 , 52 , , ]PB,CE 76 69 65 69 63 63 , 56 94 ] , , ]PE,PB 84 63 , , 65 68 , , , 53 , , ] 52 52 , , 92 11 , , 11 , , , 72 , , 56 55 86 67 , 56 55 56 100 81 93 88 ]CE ]CE , ]PB 53 57 11 ]PE 55 9 9 , , , , 11 11 3 , , , ] , ] , , , , , , , , , , , , 98 98 76 11 9 53 11 9 1 9 3 11 3 53 1 67 1 53 53 3 89 99 94 92 [ [ 76 [ [ 88 [ [ 79 .) A. Gray E [ DC. E L. E [ Mill. E L. E [ Hemsl Hook. E [ ( Labill. E R. E. Fr. N [ L. E [ L. E L. E L. E [ Aubl. N [ (Mart.)R.E.Fr. N [ Jacq. N [ Mart. N [ (Blume) D. Dietr. E L. E [ L. E [ L. E [ Table 1: Continued. Annona tomentosa Annona coriacea Alpinia speciosa Allium cepa Annona squamosa Sesamum orientale Helianthus annuus Tithonia diversifolia Justicia pectoralis Tagetes erecta Eucalyptus globulus Xylopia frutescens Eucalyptus citriodora Dianthus caryophyllus Carica papaya Acanthospermum hispidum Ricinus communis Symphytum officinale Solanum americanum Xylopia laevigata )A.Gray DC. L. Mill. L. Hemsl. Hook. ( Labill. R. E. Fr. L. L. L. L. L. (Pers.)B.L.Burtt Aubl. (Mart.)R.E.Fr. Jacq. Mart. (Blume) D. Dietr. L. L. L. Annona tomentosa Annona coriacea Tithonia diversifolia Allium cepa Alpinia speciosa Alpinia zerumbet andR.M.Sm. Eucalyptus globulus Annona squamosa Sesamum orientale Helianthus annuus Xylopia frutescens Allium ascalonicum L. Allium ascalonicum L. E [ Eucalyptus citriodora Justicia pectoralis Dianthus caryophyllus Acanthospermum hispidum Xylopia laevigata Tagetes erecta Ricinus communis Symphytum officinale Solanum americanum Carica papaya Sesamum indicum Liliaceae Caryophyllaceae Asteraceae Asteraceae ´ a Acanthaceae ˆ onia Zingiberaceae Vernacular nameCarrapateira FamilyCebola branca Euphorbiaceae Chamb Scientificname Col in the original source Valid scientificConfreiCravo branco Erva moura Boraginaceae Espinho de cigano Solanaceae nameEucalipto Literature Origin Pinha Myrtaceae Mamoeiro State Gergelim Annonaceae Caricaceae Girassol Pedaliaceae Imbira Asteraceae Annonaceae 18 Evidence-Based Complementary and Alternative Medicine PE,PB,CE,RN PE,PB,CE,PI,MA, BA PE, PB, SE, CE, PI, BA , , , 71 77 , 65 , , ] ]PE,PB,BA 68 75 , , 60 84 , 100 ] , 71 66 , , , 58 76 , 89 100 , 59 63 ] , , – , , 56 65 , 94 85 93 85 ]PE,RN,BA , 56 61 , , , , ]PE,PB,CE , , 54 63 80 ]PE,RN , 93 74 92 80 ]CE,BA 93 ]PB,PI ]PE,CE , , 55 55 , , , , 81 , 11 , , ]PE , 98 , 92 86 62 56 70 90 89 76 , ]PE 54 , 53 53 ]BA ]BA ]BA ]BA ]PE ]PI ]PI , 9 , , 11 65 , , , , , , , , , , 63 74 82 82 74 69 82 1 56 74 1 82 3 9 53 93 9 9 53 88 75 87 [ 69 [ [ N[ N[ N[ N[ N[ N[ N[ N[ N[ N N[ N[ E[ (Mart. (Mart. (Mart. (Mart. (Rizzini) (Cham.) (Brot.) L. Mart. Mart. (L.) Oken N L. E [ Hayne N [ L. N (Ridl.) Sandwith N [ L. E [ Y. T. Lee and (Silva Manso) Benth. (Silva Manso) Benth. . Table 1: Continued. Tabebuia aureaand Hook. f. ex S. Moore Hymenaea aurea Langenh. Handroanthus chrysotrichus ex DC.) Mattos Handroanthus spongiosus S.Grose Hymenaea stigonocarpa ex Hayne Handroanthus impetiginosus ex DC.) Mattos Carapichea ipecacuanha Andersson Handroanthus serratifolius (A.H.Gentry) S. Grose Tabebuia roseoalba Hymenaea martiana Handroanthus impetiginosus ex DC.) Mattos Tabebuia aureaand Hook. f. ex S. Moore Hybanthus calceolaria Handroanthus impetiginosus ex DC.) Mattos Handroanthus ochraceus Mattos Artemisia vulgaris Aeollanthus suaveolens ex Spreng Artemisia absinthium Hymenaea courbaril A. (L.) Baill. Mart. (Brot.) Stokes (Brot.) A. Rich. (Mart. ex DC.) (Mart. ex DC.) Mart. (L.) Oken (Mart. ex Lorentz Lorentz ex L. Lorentz (Vahl) G. Hayne L. (Ridl.) Sandwith Rizzini (Cham.) Standl. L. (Mart.) Bureau Y. T. Lee and (Silva Manso) Benth. (Silva Manso) Benth. . . Tabebuia caraiba Tabebuia roseo-alba and Hook. f. ex S. Moore Tabebuia aurea Hymenaea aurea Langenh. Tabebuia chrysotricha Psychotria ipecacuanha Hymenaea martiana Cephaelis ipecacuanha DC.) Standl. Tabebuia spongiosa Hymenaea stigonocarpa ex Hayne Tabebuia avellanedae ex Griseb. Tabebuia serratifolia Nicholson Artemisia vulgaris Griseb. Tabebuia impetiginosa Standl Hybanthus ipecacuanha Hybanthus calceolaria Hymenaea courbaril Tabebuia aureaand Hook. f. ex S. Moore Tabebuia avellanedae Tabebuia avellanedae Artemisia absinthium Aeollanthus suaveolens ex Griseb. ex Spreng Tabebuia impetiginosa Standl. Tabebuia ochracea Rubiaceae Violaceae ´ a Fabaceae Vernacular name FamilyIpe Scientificname in the original source Valid scientific Bignoniaceae Pau d’arco roxo Bignoniaceae name Literature Origin Pau d’arco Bignoniaceae State Pepaconha LosnaMacassa Asteraceae Lamiaceae Jatob Evidence-Based Complementary and Alternative Medicine 19 PE,PB,SE,CE,RN, BA PE, CE, MA, RN, BA , , 84 ]PE,PB,MA 67 , ] , ] PE, PB, CE, PI, BA 81 100 , 101 60 88 ]PB,RN , , , , 79 77 93 , 88 59 69 , , , , ]PE,PB,BA , 78 71 76 , 53 84 , ]PE 69 67 , , , , , 69 ]PE 62 76 11 68 , 78 , , 56 ]RN ]RN , 60 , ] , 93 , , 9 , 59 66 69 ]PB 99 99 , 56 99 75 ]RN ]BA ]BA , ]PE , ]PE ]PE ]PB ]PB ]SE ]SE ]CE ]CE ]CE ]PB 53 ]PE ]CE , ]PE , , 3 57 11 , , , ]CE ]PB , ]PB ]PB , , , 81 78 95 55 89 3 67 81 78 3 56 1 53 3 69 9 56 75 1 85 80 100 1 11 95 52 54 3 56 90 78 77 [ 92 [ 72 E[ N[ E[ . Codd E [ (Lam.) DC. N [ Hort.exL.H. L. E [ Schrank N [ Andrews E [ L. N [ Mart. N [ N. E. Br. N [ L. N [ L. N [ (Jacq.) Sweet N [ Humb. and Bonpl. L. E [ R. Grether N [ L. E [ Ulbr. N [ (Willd.) Briq L. E [ Benth. N [ L. N [ L. E [ L. E [ Benth. N [ Mart. ex Colla N [ L. N [ (L.) Less. E L. E (L.) Cav. E [ L. N [ J. Presl and C. Presl E [ Cav. N [ Table 1: Continued. Sida galheirensis Plectranthus unguentarius Citrus medica Malva erecta Sida linifolia Cucurbita argyrosperma Bailey Piriqueta guianensis Mentha piperita Waltheria americana Mimosa somnians ex Willd. Urena lobata Althaea rosea Mentha spicata Plectranthus barbatus Melochia tomentosa Geranium erodifolium Cucurbita pepo Mimosa invisa Sida cordifolia Mimosa pudica Melochia tomentosa Hyptis martiusii Piriqueta racemosa Citrus aurantium Egletes viscosa Mangifera indica Mimosa candollei Mimosa misera Achyrocline satureioides Gomphrena demissa Waltheria rotundifolia . Coleus forskohlii (Lour.) (Lam.) DC. Hort.exL.H. L. Schrank Andrews DC. Mart. N. E. Br. L. L. (Jacq.) Sweet (Christm.) Swingle Humb. and Bonpl. L. L. Ulbr. (Willd.) Briq L. Benth. L. Risso L. L. Benth. (L.) L. (L.) Osbeck Mart. ex Colla L. (L.) Less. L. (L.) Osbeck Cav. L. ia leptocarpa . Sida linifolia Piriqueta guianensis Mentha piperita Malva sylvestris Spreng Plectranthus barbatus Geranium erodifolium Coleus forskohlii Mentha viridis Plectranthus amboinicus Citrus aurantiifolia Citrus limon Citrus limonum Piriqueta racemosa Cucurbita pepo Mimosa invisa Citrus limonia Waltheria indica Schrank Mimosa pudica Urena lobata Achyrocline satureioides Hyptis martiusii Alcea rosea Cucurbita argyrosperma Bailey Egletes viscosa Mangifera indica Melochia tomentosa Waltheria rotundifolia Sida cordifolia Mimosa somnians Melochia tomentosa Sida galheirensis ex Willd. Mimosa misera Gomphrena demissa Lamiaceae Geraniaceae Malvaceae Asteraceae Lamiaceae Lamiaceae Lamiaceae Arecaceae Sterculiaceae Anacardiaceae Sterculiaceae Sterculiaceae Malvaceae Malvaceae ˜ ˜ a a ˜ aozinho Gomphrenaceae ˜ ao Rutaceae miuda grauda Vernacular name FamilyJerimumHortel Cucurbitaceae Scientificname in the original sourceHortel Valid scientificLim Macela name Literature Origin Malicia State Fabaceae Malva Manga espada Capit Malva rosa Malva branca 20 Evidence-Based Complementary and Alternative Medicine PE,PB,SE,CE,PI, MA, RN PE,PB,SE,CE,RN, BA PE,PB,MA,BA PE, CE, BA PE,PB,CE,PI,RN, BA PE,PB,SE,PI,RN, BA PE,PB,CE,PI,MA, RN, BA PE,PB,CE,PI,MA, RN, BA , , 61 , , 85 ] , , , , , 78 75 95 79 72 63 59 , ] ] , ] 82 , ] – , – , , , 76 99 95 69 76 93 94 101 ] 55 69 57 62 , , , , , 69 , – – , , , , , , , 75 94 88 75 67 79 93 100 90 66 53 94 53 ]PE,BA ]PE,CE,PI,BA , 67 59 , , , , 66 , , , , , , , , , , , 71 93 93 72 92 63 80 81 83 76 78 ]CE,BA 52 63 , 94 52 , , ]PB,RN 65 58 , , , , , , , , 56 , , – – , , , 69 69 83 83 90 11 11 56 77 66 90 75 79 79 61 76 , , ]PB,PI , , 92 53 55 – , , , ] , , , , , , , – , 54 , , , 9 9 , 57 71 78 70 60 55 67 , , 52 85 94 72 73 75 78 78 59 60 ]CE ]PE ]BA ]BA ]MA ]BA , ]PE , , , 83 ]PI 53 52 , ]PE , , ]MA 11 3 3 , ] , , , , , , , , – ]CE , , , , , , 55 84 68 57 3 1 1 74 1 57 62 94 84 9 53 60 53 3 11 56 53 56 61 80 63 76 [ [ [ [ 57 86 69 99 [ 57 [ [ 76 [ 70 81 83 L. E A. DC. N [ Mart. N (Loefl.) Stuntz N [ Eichler N [ L. E [ Kunth N [ L. E Mart. N [ L. E [ L. N Kunth N [ L. E [ Lam. N Mill. E [ Mart. N [ L. E Baill. N [ DC. N [ (L.) Blume N [ L. E [ L. E (Pohl) Baill. N [ L. N Table 1: Continued. Boerhavia coccinea Ocimum tenuiflorum Ocimum minimum Ocumum americanum Ocimum basilicum Boerhavia diffusa Combretum leprosum Guazuma ulmifolia Eugenia uniflora Croton conduplicatus Croton heliotropiifolius Croton cordiifolius Trema micrantha Combretum fruticosum Aspidosperma pyrifolium Eugenia pluriflora Mentha pulegium Aspidosperma parvifolium Jatropha gossypiifolia Combretum mellifluum Jatropha ribifolia Chenopodium ambrosioides Momordica charantia Luehea ochrophylla L. A. DC. Mart. (Loefl.) Stuntz L. Eichler L. Mart. L. Kunth Willd. L. Lam. Mill. Mart. L. Baill. L. (L.) Blume L. L. Jacq. (Pohl) Baill. L. (O. Berg) Kiaersk. Boerhavia hirsuta Boerhavia coccinea Ocimum minimum Ocimum sanctum Ociumum americanum Combretum leprosum Ocimum basilicum Aspidosperma parvifolium Aspidosperma pyrifolium Eugenia pitanga Combretum fruticosum Boerhavia diffusa Eugenia uniflora Croton conduplicatus Croton rhamnifolius Croton cordiifolius Combretum mellifluum Jatropha ribifolia Trema micrantha Guazuma ulmifolia Jatropha gossypiifolia Mentha pulegium Chenopodium ambrosioides Momordica charantia Luehea ochrophylla Ulmaceae Sterculiaceae Apocynaccae Cucurbitaceae Tiliaceae ˜ ao ˜ ao Lamiaceae ˜ ao roxo Euphorbiaceae ˜ ao de S Vernacular name FamilyManjeric Scientificname in the original sourceMastruz Valid scientificMel Chenopodiaceae Mufumbo name LiteratureMutamba Origin Combretaceae Pereiro State Pega pinto Nyctaginaceae PitangaPinh Myrtaceae PoejoQuebra faca Lamiaceae Euphorbiaceae Caetano Evidence-Based Complementary and Alternative Medicine 21 PE,PB,CE,PI,RN PE, CE, MA, BA PE, CE, MA, RN, BA PE,PB,CE,PI,MA, BA PE,PB,CE,PI,MA, RN, BA , ] PE, PB, SE, CE, BA , , , 81 , 83 94 , , 65 ]PE,PB,CE , , 78 , 78 81 , 78 , , 98 72 86 , 63 , , , 76 , 65 79 , 69 , , 76 89 63 61 , , , , 67 , 60 78 ] , 66 71 61 , , 86 57 , , , , , 66 59 76 100 ]PE,SE,BA ]PE,PB,BA 61 , 69 57 , , ] ] , 76 55 , ]PE,PB , , , 93 – 84 56 57 72 93 92 99 , 56 , 72 , 58 , 56 , , , ] , 56 53 , , , , , , 80 72 55 56 92 69 89 84 93 ]MA ]PE ]PE ]BA ]BA ]MA ]MA ]BA ]BA ]BA ]BA 11 ]PB ]SE ]SE ]SE ]SE ]SE , ]PE 55 53 9 53 ]PE , , 11 , ] , , , , , , , , , , , 84 67 77 95 95 95 11 94 69 79 95 95 53 1 68 53 9 53 3 9 58 94 76 9 84 3 66 68 80 [ 85 84 83 [ [ [ [ 84 68 83 N[ N N[ E[ N[ N[ . Schum . . ) Roem. and (L.) Moench E (L.) Vahl N [ )Briq . Cambess. E [ Kunth N [ Rottb. N [ (Mill.) Fuss E [ Hoffm. E [ G. Gaertn., B. (L.) Vahl N [ (L.) R. Br. N [ L. N [ Desr. (Jacq.) K (L.) Pers. N [ L. E ( Mill. N [ B. A. Gomes N [ (L.) Poit. N [ L. E (Lam.) H. S. Irwin L. N [ L. N [ Spruce ex Benth. N [ (Rich (L.) Poit. N [ (Benth.) H. S. Irwin L. N [ (L.) Hitchc. N [ L. N Table 1: Continued. Senna martiana and Barneby Petroselinum sativum Ipomoea asarifolia Schult. Senna corymbosa and Barneby Abelmoschus esculentus Petroselinum crispum Tephrosia purpurea Ipomoea pes-caprae Cyperus ligularis Cyperus surinamensis Fimbristylis dichotoma Senna alexandrina Bowdichia virgilioides Bowdichia nitida Petiveria alliacea Petiveria tetrandra Armoracia rusticana Mey., and Scherb. Hyptis pectinata Hyptis suaveolens Bixa orellana Coutarea hexandra Punica granatum Tamarindus indica Kalanchoe brasiliensis Cinchona officinalis Quassia amara Chiococca alba .Fimbristylismiliacea .Hyptismutabilis ) Roem. and (L.) Moench (L.) Vahl )Briq . Cambess. Kunth Rottb. (Mill.) Fuss Hoffm. G. Gaertn., B. Gaudich (L.) R. Br. Ruiz and Pav. Desr. (Jacq.) K. Schum. L. (L.) Pers. L. ( B. A. Gomes (L.) Poit. Wedd. L. (Lam.) H. S. Irwin L. L. Spruce ex Benth. (Rich (L.) Poit. (Benth.) H. S. Irwin Link L. L. Petroselinum sativum Tephrosia purpurea Petroselinum crispum Fimbristylis littoralis Senna martiana and Barneby Bowdichia nitida Ipomoea pes-caprae Cyperus surinamensis Fimbristylis dichotoma Abelmoschus esculentus Senna corymbosa and Barneby Coutarea hexandra Ipomoea asarifolia Schult. Petiveria alliacea Petiveria tetrandra Hibiscus esculentus Senna acutifolia Bowdichia virgilioides Cyperus ligularis Hyptis mutabilis Hyptis pectinata Hyptis suaveolens Punica granatum Kalanchoe brasiliensis Tamarindus indica Bixa orellana Armoracia rusticana Mey., and Scherb. Cinchona calisaya Quassia amara Chiococca brachiata Apiaceae Rubiaceae Convolvulaceae Punicaceae Brassicaceae Simaroubaceae Rubiaceae ´ a Lamiaceae ´ e Phytolacacceae ˜ a ˜ ao Crassulaceae Vernacular name FamilyQuiaboQuina Malvaceae Scientificname in the original source Valid scientificRom Sai nameSalsa Literature Origin Sambacait Sena State Sucupira Fabaceae Tamarino Fabaceae Guin Fabaceae UrucumTiririca Bixaceae Cyperaceae 22 Evidence-Based Complementary and Alternative Medicine PE,PB,SE,CE,RN, BA PE,PB,CE,PI ] PE, PB, PI, MA , 75 , 72 – – , 73 ]PE,PB,RN 79 ]PE,PI,RN , 58 67 , 71 , , , 99 70 75 53 , , 63 , 59 , ] , ] PE, PB, CE, RN , 93 66 73 52 , ]PB,SE 95 , 60 90 – ] , 56 , , , , 88 67 63 11 70 92 , , ]PE 93 , , 57 , 60 , 53 , 9 , , , 57 73 77 57 ]PB , 63 77 ]CE ]CE ]RN ]MA ]PB ]PB ]PB ]PE , ]PB ]PB , ]SE ]RN ]SE , ]BA ]PE ]PB 85 11 ]CE , ]PE 3 11 52 , , , , , , , 95 63 85 58 90 60 76 3 1 56 59 54 54 53 1 53 77 58 1 60 68 56 100 82 55 77 54 81 [ 60 76 [ N[ N[ N[ N[ N[ N[ N N[ N[ ˜ ao N [ ˜ ao) A. C. (Mull. Arg.) Mart. ex Allem (Vahl) Roem. L. E [ (Benth.) Ducke N [ Moric. N [ Lam. E [ (Benth.) Seigler Roem. and (Mart.) Benth. N [ (Allem (L.) Wight and ı. ´ Pax and K. Hoffm. N [ L. N [ L. N [ (Willd.) Poir. N [ (Willd.) Poir. N Ducke N [ (L.) Arthur N [ (Kunth) Roem. and um Dunal E [ L. E [ L. E [ (Gardner) Weigend N [ rich L. E [ ˜ ao, PI: Piau Table 1: Continued. Vachellia farnesiana Arn. Benth. Mimosa ophthalmocentra Mimosa tenuiflora Senegalia piauhiensis and Ebinger Eleocharis elegans Schult. Cnidoscolus phyllacanthus Pax and L. Hoffm. Calliandra spinosa Myracrodruon urundeuva Eleocharis interstincta and Schult. Cyperus articulatus Amburana cearensis Sm. Solanum stipulaceum Cyperus esculentus Schult. Mimosa acutistipula Cnidoscolus urens Piptadenia stipulacea Mimosa tenuiflora Cnidoscolus infestus Solanum albidum Physalis philadelphica Crataeva tapia Solanum lycopersicum Lamium album Solanum polyt Urtica urens Aosa rupestris ˜ ao ˜ ao) A. C. (Mull. Arg.) Allem Mart. ex MilL. (Vahl) Roem. (Benth.) Ducke Moric. (Benth.) Seigler Roem. and (Mart.) Benth. (Allem Pax and K. Hoffm. L. L. (Willd.) Poir. (Willd.) Poir. Ducke (L.) Arthur (Kunth) Roem. and (L.) Willd. um Dunal Brot. ex Hornem. L. L. ich (Gardner) Weigend L. ´ a. RN: Rio Grande do Norte, BA: Bahia, MA: Maranh Acacia farnesiana Mimosa tenuiflora Mimosa ophthalmocentra Benth. Calliandra spinosa Cyperus articulatus Mimosa acutistipula Senegalia piauhiensis and Ebinger Cnidoscolus infestus Eleocharis elegans Schult. Cyperus esculentus Physalis ixocarpa Cnidoscolus urens Cnidoscolus phyllacanthus Pax and L. Hoffm. Mimosa tenuiflora Solanum rhytidoandrum Sendtn. Solanum rhytidoandrum Sendtn. Amburana cearensis Eleocharis interstincta Lycopersicon esculentum Piptadenia stipulacea and Schult. Solanum polytr Solanum albidum Myracrodruon urundeuva Sm. Lamium album Solanum stipulaceum Schult. Aosa rupestris Crataeva tapia Urtica urens Anacardiaceae Euphorbiaceae Fabaceae Solanaceae Fabaceae Lamiaceae Loasaceae Urticaceae ´ a Capparidaceae Vernacular name FamilyJunco Scientificname in the original sourceTomate Cyperaceae Valid scientificTrapi Solanaceae Urtiga branca name Literature Origin Jurema branca Fabaceae State Jurema preta Jurubeba branca Imburana de cheiro PE:Pernambuco,PB:Paraiba.SE:Sergipe,CE:Cear Evidence-Based Complementary and Alternative Medicine 23 important tool because it might point to the possible patterns their side effects, contraindications, toxicity, and effective of substitution of homonym ethnospecies in a given area. therapeutic action. In the case of northeast Brazil, 75% of the plants traded in Because the only plant material available for species regional public markets exhibit correspondence with more identification is that sold at the markets, whereas the har- than one plant species. vesting sites are usually inaccessible due to their distance or As most (74%) such species are representative of the nat- the unavailability or mistrust of the harvesters—as a large ive flora, we might infer that the regional markets are largely part of harvesting is indiscriminate—the resolution of this supplied by natural stocks. Because the demand for medicinal impasse necessarily demands more specialized taxonomic plants is continuously increasing [2], the gradual exhaustion procedures, such as micrography and molecular taxonomy. or scarcity of resources might make the substitution of In this regard, several techniques have been widely appl- homonym ethnospecies unavoidable and increasingly more ied to the resolution of this type of taxonomic problem [14, frequent, particularly in the large cities where 70% of the 15, 104–106], to support scientific research and as a tool for population resides [34] and where access to medicinal plants the surveillance and control of commercial plant and animal is primarily mediated by commerce. products. Barcoding is one of the most promising among such Precisely for that reason, it is safe to assert that semantic techniques and has already been applied to the identification plurality is manifested most frequently at the public markets of plant species in public markets [107]. This technique of large cities, which are privileged spaces where significant consists of the identification of species based on the differ- amounts of people, products, and knowledge circulate on a entiation of genetic sequences in specific DNA regions108 [ ]. daily basis. Thus, such markets afford an extremely favorable The use of molecular taxonomy might in time become a scenario for comparative ethnobotanical studies at a regional very important and practical tool for cataloging the hidden level. diversity in public markets and thus contribute to a better understanding of the biodiversity flow in a given area and, In recent years, ethnobotanical research in regional public consequently, the frequency with which homonym eth- markets has provided an important platform for conservation nospecies are being interchangeably used in public markets. studies and biological prospecting. However, the limitations A reliable cataloging of this biodiversity affords multiple to species identification represent the major hindrance to the possibilities for further biological and cultural studies and growth of research in such locations [6]aswellastothe must be considered as crucial for the advancement of ethnob- assessment of hidden diversity and events of homonym eth- otanical research. nospecies substitution, as most of the plants are sold in parts or pieces that are sometimes completely uncharacteristic. 4.2. Implications for Conservation. From the perspective of Forthatreason,homonymethnospeciesgoeasilyunno- conservation at the regional level, one should not ignore ticed when commercial medicinal species are cataloged, the the hidden diversity of medicinal plants, as this diversity moresothemoreremarkablethemorphologicalsimilarities represents the possible variations in the range of species that are. In this regard, 61.3% of the hidden diversity of the are effectively used relative to the multiplicity of homonym medicinal plants of the northeast region is congeneric, that ethnospecies and the biological diversity of a given area. On is, exhibits type 1 underdifferentiation, which denotes phylo- such grounds, one might infer that the larger the number of genetic proximity and consequently morphological similarity homonym ethnospecies, the higher the odds that the pressure [102]. This similarity makes the understanding of the ethnob- of use is, or might eventually become, distributed among otanical data collected at public markets even more difficult. more than one plant population, as in our study where a Toprevent this situation, the criteria adopted for the iden- significant number of native homonym ethnospecies (74%) tification of species by some studies conducted in regional was found. public markets are based on the vernacular nomenclature, When, conversely, the frequency of use predominantly sometimes as a complementary identifier12 [ , 103]andother affects one species, the risks are patently greater for the species times as the primary criterion [6]. In places where the catalog affected but also for others with the same vernacular name, of medicinal plants and the data relative to their biodiversity as due to substitution, those others might become subjected are comprehensive, common names might possibly be used to an intense and fast extractivist pressure that compromises quite safely. However, this is definitely not the case in Brazil, their resilience, particularly in the case of the most vulnerable where the repertoire of medicinal plants in these marketing populations, leading to their collapse. spaces is largely a hidden diversity. The species Myracrodruon urundeuva provides a good Additionally, due to the explicit difficulty of recognizing example of the possible impact of the extractivist pressure on and identifying the plant species in public markets and more than one plant population. In this case, another species, the progressive increase in the substitution of homonym Schinus terebinthifolius,whichisalsonativeandbelongsto ethnospecies, the vulnerability of consumers tends to become the same family, is currently traded under the same generic more serious, and possible risks related to safety and efficacy name (“aroeira”—Brazilian peppertree) in the city of Recife might be potentiated when one species is indistinctly replaced [1]. Therefore, these species are interchangeably used, even with another. This phenomenon occurs because most of the though they belong to different genera, as the used parts do Brazilian medicinal plant species have not yet been subjected not allow for a clear differentiation. to appropriate studies that would establish their use in a It is possible that such homonym ethnospecies are being scientifically safe manner, so to speak, that is, describing overlapped in an unconscious and undocumented manner at 24 Evidence-Based Complementary and Alternative Medicine the points of sale, especially in the case of populations that [117]. With regard to the medicinal species whose safety arenolongereasilyfoundintheirnaturalreservoirsandthat and efficacy have been demonstrated, ethnobotanical studies precisely for that reason are subjected to substitution pro- that include their hidden diversity might contribute to the cesses. For example, the case of “espinheira santa” (Maytenus identification of more efficacious species as well as of those ilicifolia), which following its long-term indiscriminate har- more promptly available for consumption. vesting became a threatened species [109]andisassociated Therefore, the identification of homonym species with with several substitute species that currently occupy the same similarusesmightnotonlyreducethepressureofuseonthe semantic-therapeutic niche [110]. natural reservoirs but also allow for easier and more encom- Thistypeofapproachmustbetakenintoaccountupon passing access for a larger number of people. In this regard, it establishing conservation priorities and efficient manage- is worth stressing that to be efficient, public policies address- ment strategies, as accurate knowledge of the hidden diver- ing access to medicinal plants must take into consideration sity of medicinal plants and the possibilities for efficient the natural distribution of the species, when it is spontaneous, exchanges among homonym ethnospecies might favor a and the limits of its ecophysiological tolerance, in the case more balanced distribution of the extractivist pressure, thus of cultivated species. The identification of homonym species minimizing its impact, avoiding the collapse of populations might represent an alternative in both cases. and promoting greater resilience. Recently, the Brazilian government published a list of The applicability of hidden substitutions of species to 71 medicinal plant species recommended for use by the biological conservation is thus in keeping with explicative Unified Health System (Sistema Unico´ de Saude—SUS)´ [118]. modelsrelatedtotheutilitarianredundancyhypothesis[52], As a function of the continental size of Brazil and its according to which a larger number of species within one environmental diversity, the distributions of some of these utilitarian category leads to greater mutual support and medicinal species are not homogeneous across all regions. protection of the associated species as well as increased Species typical of the south and southeast regions are hardly resilience. found in the north and northeast regions, and vice versa. Thus, we might assert that the phenomenon of the hidden Therefore, in both cases, there are homonym ethnospecies diversity of medicinal plants gives support to utilitarian occupying the same semantic-therapeutic niche of many redundancy as an explanatory model for the pressure of use, species in the corresponding region. as the overlapping species are subsumed under one and the The case of Uncaria tomentosa is exemplary. This plant, same identity and consequently the same therapeutic indica- native to Amazonia (north region), is commonly known as tions, as their corresponding practical value is culturally well “unha de gato” (cat’s claw) and acknowledged for its remark- established. able anti-inflammatory activity. Although it was included in Because, based on the strength of tradition, the homonym the SUS list, access to this plant is extremely restricted in ethnospecies are functional analogs, the remaining task is to other Brazilian regions, which, however, will not prevent the distinguish each one of them and establish the level at which emergence of other types of “cat’s claw.” There are at least six the preference for and/or access to each particular species different species known as “cat’s claw” in the northeast region occurs and then to define the degree of utilitarian redun- alone, five of which are native and one subspontaneous, dancy, which is also hidden, so to speak. For that purpose, corresponding to four different families, thus denoting the once again the elaboration of a taxonomically reliable record generality of the common name and the particularity of the of this biodiversity is required. biological expression. Within that context, the assessment of hidden and redun- dant biodiversity becomes an important predictive ecolog- According to Albuquerque and Hanazaki [119]oneofthe ical tool, as a function of the perfect semantic-therapeutic basic rules in biological prospecting is to identify the criteria juxtaposition of the homonym ethnospecies at the regional used by people to select plants for medicinal use. According level. Public policies for the conservation, regulation, control, to those authors, the process underlying such selection might anduseofmedicinalplantsinBrazilshouldnotignorethe point to more efficacious strategies and shortcuts for the regional level and its implicit cultural and biological richness identification of key species relevant to bioprospecting. [111–113]. From this perspective, comparative ethnobotany A preliminary assessment of the distributions of the eth- willbecomeanindispensabletoolindecisionmakingand nospecies in the present study indicated that several species, actions aimed at the sustainable use of biodiversity. including exotic ones established centuries ago, have corresponding homonym ethnospecies from the local flora. This is 4.3. Implications for Bioprospecting. Several studies [114– the case for cinnamon, watercress, elder tree, artichoke, clove 116] have found similar biochemical compositions in related basil, plum, and rosemary, among others (see Table 1). Such species, which might point to similar uses within the range correspondences were also found when medicinal plant of applications already well established by tradition. The species were compared with the names of drugs (generic and biochemical constitutions of species in the same family trademarks names) with widely acknowledged therapeutic quitecommonlyincludethesamepatternofsecondary effects, such as Meracilina, penicillin, Novalgina, aspirin, components [114]. Terramycin, and ampicillin, among others [9, 11, 24, 93, 120, Nevertheless, the therapeutic efficacy and the risks associ- 121]. In such cases, the species is named after its correspond- ated with the use of the vast majority of species acknowledged ing drug name, thus representing a flagrant instance of as medicinal by the population have not yet been assessed classification based on analogical use. Evidence-Based Complementary and Alternative Medicine 25

Similarly, based on the wide variety and distribution of The fact that a significant percentage of the common homonym ethnospecies, we might infer that the development namesofplantsintheBraziliannortheastregionexhibits of knowledge at the local level seeks to subsume the available correspondences to multiple species is irrevocably estab- biodiversity under the already established and culturally lished. A more thorough understanding of the dynamics and consolidated semantic-therapeutic patterns. For that reason, dimensions of such semantic-biological variability and the when key species with high cultural relevance are absent, the corresponding implications requires the integration of sev- communities tend to opt for species substitutions [52]. eral areas of knowledge, including taxonomy, biochemistry, As a function of the existence of semantic-therapeutic population ecology, phytosociology, linguistics, and anthro- niches and the impossibility of filling them with tradition- pology. ally acknowledged species, an analogy-based local process The proportion of species found by ethnospecies (2.78) appearstobetriggered.Accordingtotheavailabledata,sev- was significant, although we recognize that a more compre- eral mechanisms of cultural selection are operative in this hensivecoverageofmarketsandfairsintheninenortheastern analogy-based local process, whereby the most fitting pieces statescouldleadtoanincreaseofthisproportionoreven of local knowledge become prevalent and amplified across the emergence of new ethnospecies not listed in this survey. the community, pointing to the locally accessible species, To what concerns the low number of respondents committed which thus come to be used as corresponding (homonym) to the study, in all the six markets visited, it should be ethnospecies. This hypothesis is corroborated by the high clarified that the purpose of the field survey was to catalogue frequency of homonym ethnospecies representing the native ethnospecies currently marketed in order to support the iden- flora (73%). tification of the corresponding species through the literature Comparative ethnobotanical studies of different regions search. might eventually elucidate the possible role of vernacular namesasmodelsforthemanifestationoftheexpressionof Acknowledgments local biodiversity or the measure and circumstances under which a peculiar regional classification system tends to pre- The authors thank all of the respondents who participated in vail at the expense of allochthonous and/or general sys- the study and the National Council of Scientific and Tech- tems. In addition, the identification of the level of semantic nological Development (Conselho Nacional de Desenvolvi- similarity of species at the local level might contribute to mento Cient´ıfico e Tecnologico—CNPq)´ for the scholarships a better understanding of the process of construction of granted to the authors and financial support to UPA (“Edital local/regional knowledge and make the planning of the Universal—2012”). use, prospection, and conservation of these resources more efficient [122]. References

[1] U. P. Albuquerque, J. M. Monteiro, M. A. Ramos, and E. L. C. 5. Conclusions de Amorim, “Medicinal and magic plants from a public market in northeastern Brazil,” Journal of Ethnopharmacology,vol.110, Regardless of being a frequent process, affecting either some no. 1, pp. 76–91, 2007. or the full set of species of a given region, the substitution [2]A.C.Pinto,D.H.Silva,V.S.Bolzani,N.P.Lopes,andR.A.Epi- of homonym ethnospecies denotes a novel consumption fanio, “Produtos naturais: atualidade, desafios e perspectivas,” option for a well-established cultural practice involving Qu´ımica Nova,vol.25,pp.45–61,2002. limited products within a commercial niche consolidated by [3] M. Veeman, “Conociendo los mercados locales y regionales tradition.Forthatreason,evenwherethelevel,frequency, para produtos forestales,” in Evaluando la Cosecha Oculta de los and circumstances under which such substitutions occur Bosques,B.M.CampbellandM.K.Luckert,Eds.,pp.81–116, mightnotbeidentifiedinthenearfuture,somerelevant Nordan-Comunidad, Montevideo, Uruguay, 2002. questions have already been raised. Such questions, which [4] A. B. Cunningham, Applied Ethnobotany—People, Wild Plant might contribute to optimizing the use of medicinal plants Use & Conservation, Earthscan Publications, London, UK, 2001. in a safe and more sustainable manner, include the following. [5] E. Hanlidou, R. Karousou, V. Kleftoyanni, and S. Kokkini, “The (1) How might the homonym species be alternately used for herbal market of Thessaloniki (N Greece) and its relation to the the same therapeutic action and how efficacious are they? ethnobotanical tradition,” Journal of Ethnopharmacology,vol. 91, no. 2-3, pp. 281–299, 2004. (2) For which homonym ethnospecies might divergent uses, [6] M. Krog, M. Falcao,˜ and C. S. Olsen, “Medicinal plants mar- absence, or differences in the level of therapeutic efficacy kets and trade in Maputo, Mozambique,” Forest e Landscape be currently listed? (3) What are the health risks to people Working Papers 16, Danish Centre for Forest, Landscape and who, either travelling or at their original place of residence, Planning, KVL, 2006. indiscriminately consume different species subsumed under [7] J. M. Monteiro, M. A. Ramos, E. L. Araujo,´ E. L. C. Amorim, and the same common name? (4) What tools might be created U. P. Albuquerque, “Collection and commerce of the Myracro- to support consumers and researchers in the understanding druon urundeuva Allemao˜ bark in the semi-arid region of Nor- and interpretation of the semantic plurality associated with theastern Brazil,” Bioremediation, Biodiversity & Bioavailability, medicinal plants? (5) Which bioprospecting actions and vol. 5, pp. 100–102, 2011. management plans have taken the hidden diversity of species [8]C.D.F.C.B.R.Almeida,E.L.C.deAmorim,U.P.deAlbu- at the regional level into consideration? querque, and M. B. S. Maia, “Medicinal plants popularly used in 26 Evidence-Based Complementary and Alternative Medicine

the Xingo´ region—a semi-arid location in Northeastern Brazil,” de classificaçao˜ folk,” Revista Brasileira de Plantas Medicinais, Journal of Ethnobiology and Ethnomedicine,vol.2,article15,pp. Botucatu,vol.7,pp.44–72,2005. 1–9, 2006. [25] P. Hiepko, “Eipo plant nomenclature and classification com- [9] S.L.Cartaxo,M.M.M.Souza,andU.P.deAlbuquerque,“Medi- pared with other folk taxonomic systems,” Willdenowia,vol.36, cinal plants with bioprospecting potential used in semi-arid pp. 447–453, 2006. northeastern Brazil,” Journal of Ethnopharmacology,vol.131,no. [26] M. Haverroth, Etnobotanica,ˆ uso e classificaçao˜ dos vegetais pelos 2, pp. 326–342, 2010. Kaingang,vol.3ofSeries´ de Estudos e Debates, NUPEEA, Recife, [10] I. G. C. Bieski, F. R. Santos, R. M. de Oliveira et al., “Ethnophar- Brazil, 2007. macology of medicinal plants of the pantanal region (Mato [27] K. Khasbagan and S. Soyolt, “Indigenous knowledge for plant Grosso, Brazil),” Evidence-Based Complementary and Alterna- species diversity: a case study of wild plants’ folk names used tive Medicine,vol.2012,ArticleID272749,36pages,2012. by the Mongolians in Ejina desert area, Inner Mongolia, P. [11] C. D. F. C. B. R. de Almeida, M. A. Ramos, R. R. V. Silva et al., R. China,” Journal of Ethnobiology and Ethnomedicine,vol.4, “Intracultural variation in the knowledge of medicinal plants in article 2, 2008. an urban-rural community in the Atlantic Forest from North- [28] M. A. Signorini, M. Piredda, and P. Bruschi, “Plants and tra- eastern Brazil,” Evidence-Based Complementary and Alternative ditional knowledge: an ethnobotanical investigation on Monte Medicine, vol. 2012, Article ID 679373, 15 pages, 2012. Ortobene (Nuoro, Sardinia),” Journal of Ethnobiology and Eth- [12] E. Lev and Z. Amar, “Ethnopharmacological survey of tradi- nomedicine,vol.5,article6,2009. tional drugs sold in the Kingdom of Jordan,” Journal of Ethno- [29] P. Burke, Hibridismo Cultural, Unisinos, Sao˜ Leopoldo, Brazil, pharmacology,vol.82,no.2-3,pp.131–145,2002. 2003. [13] M. A. Ramos, U. P.Albuquerque, and E. L. C. Amorim, “O com- [30] I. Nesheim, S. S. Dhillion, and K. A. Stølen, “What happens to ercio´ de plantas medicinais em mercados publicos´ e feiras livres: traditional knowledge and use of natural resources when people um estudo de caso,” in Topicos´ em conservaçao,˜ etnobotanicaˆ e migrate?” Human Ecology,vol.34,no.1,pp.99–131,2006. etnofarmacologia de plantas medicinais e magicas´ ,U.P.Albu- [31] International Institute for Enviromental and Development querque, C. F. C. B.R. Almeida, and J. F. A. Marins, Eds., (IIED), The Hidden Haverst: The Value of Wild Resources in pp. 127–163, NUPEEA/Sociedade Brasileira de Etnoecologia e Agricultural Systems, IIED, London, UK, 1995. Etnoecologia, Recife, Brazil, 2005. [32] International Institute for Enviromental and Development [14] S. Molares and A. Ladio, “Metodos´ micrograficos´ aplicados a` (IIED), “Valuing the hidden haverst: methodological approa- pesquisa etnobotanica,”inˆ Metodos´ e TecnicasnaPesquisaEtno-´ ches for local level economic analysis of wild resources,” biologica´ e Etnoecologica´ , U. P.Albuquerque, R. F. P.Lucena, and Research series, vol. 3, no. 4, Sustainable Agriculture and Envi- L. V. F. C. Cunha, Eds., pp. 381–399, NUPPEA, Recife, Brazil, ronmental Economics, IIED, London, UK, 1997. 2010. [33] E. Elisabetsky and L. Wannmacher, “The status of ethnophar- [15] M. Li, H. Cao, P. P.-H. But, and P.-C. Shaw, “Identification of macology in Brazil,” Journal of Ethnopharmacology,vol.38,no. herbal medicinal materials using DNA barcodes,” Journal of 2-3, pp. 137–143, 1993. Systematics and Evolution,vol.49,no.3,pp.271–283,2011. [34]“IBGEInstitutoBrasileirodeGeografiaeEstat´ıstica,” http:// [16] M. L. T. Nguyen, “Cultivated plant collections from markets www.ibge.gov.br/. places,” Ethnobotany Research e Applications,vol.3,pp.5–15, [35] I. R. Leal, M. Tabarelli, and J. M. C. Silva, Ecologia e conservaçao˜ 2005. da Caatinga, Editora Universitaria,´ Universidade Federal de [17] S. Lee, C. Xiao, and S. Pei, “Ethnobotanical survey of medicinal Pernambuco, Recife, Brazil, 2003. plants at periodic markets of Honghe Prefecture in Yunnan [36] D. Prado, “As caatingas da America´ do Sul,”in Ecologia e conser- Province, SW China,” Journal of Ethnopharmacology,vol.117,no. vaçao˜ da Caatinga,I.R.Leal,M.Tabarelli,andJ.M.C.Silva, 2, pp. 362–377, 2008. Eds., pp. 3–73, Editora Universitaria,´ Universidade Federal de [18] H. C. Conklin, “Lexicographical treatment of folk taxonomies,” Pernambuco, Recife, Brazil, 2003. Journal of American Linguistics, vol. 28, part 4, pp. 119–141, 1962. [37]A.M.Giulietti,A.L.BocageNeta,A.A.J.F.Castroetal.,“Dia- [19] D. G. Metzger and G. E. Williams, “Some procedurs and results gnostico´ da vegetaçao˜ nativa do bioma Caatinga,” in Biodiversi- in the study of native categories: tzeltal ‘firewood’,” American dade da Caatinga: areas´ e açoes˜ prioritarias´ para a conservaçao˜ , Anthropologist,vol.68,pp.389–407,1966. J. M. C. Silva, M. Tabarelli, M. T. Fonseca, and L. V. Lins, Eds., [20] B. Berlin, D. E. Breedlove, and P. H. Raven, “Covert categories pp.48–90,Ministerio´ do Meio Ambiente, Bras´ılia, Brazil, 2004. and folk taxonomies,” American Anthropologist,vol.70,pp. [38] N. Myers, R. A. Mittermeler, C. G. Mittermeler, G. A. B. da 290–299, 1968. Fonseca, and J. Kent, “Biodiversity hotspots for conservation [21] T. Hartmann, ANomenclaturaBotanicaˆ Bororoˆ ,Institutode priorities,” Nature,vol.403,no.6772,pp.853–858,2000. Estudos Brasileiros—USP, Sao˜ Paulo, Brazil, 1967. [39] C. Galindo-Leal and I. G. Camara,ˆ “Atlantic forest hotspots [22] B. Berlin, “Folk systematics in relationsystematics in relation to status: an overview,” in The Atlantic Forest of South America: biological classification and nomenclature,” Annual Review of Biodiversity Status, Threats, and Outlook, C. Galindo-Leal and Ecology and Systematics,vol.4,pp.259–271,1973. I. G. Camara,ˆ Eds., pp. 3–11, Center for Applied Biodiversity [23] B. Berlin, Ethnobiological Classification: Principles of Categoriza- Science e Island Press, Washington, DC, USA, 2003. tion of Plants and Animals in Traditional Societies, Princeton [40] C. T. Rizzini, Tratado de Fitogeografia de Brasil: Aspectos ecolo-´ University Press, Princeton, NJ, USA, 1992. gicos, sociologicos´ e flor´ısticos, Ambitoˆ Cultural Ediçoes˜ Ltda, [24] G. S. Vendruscolo, S. M. Eisinger, E. C. Soares, and R. A. Zachia, Rio de Janeiro, Brazil, 1997. “Estudo etnobotanicoˆ do uso dos recursos vegetais em Sao˜ Joao˜ [41] A.A.J.F.CastroandF.R.Martins,“CerradosdoBrasiledoNor- do Polesine,ˆ RS, Brasil, no per´ıodo de outubro de 1999 a junho deste: caracterizaçao,˜ area´ de ocupaçao˜ e consideraçoes˜ sobre a de 2073. II—Etnotaxonomia: criterios´ taxonomicosˆ e sistema sua fitodiversidade,” Pesquisa em Foco,vol.7,pp.147–178,1999. Evidence-Based Complementary and Alternative Medicine 27

[42] R. T. Pennington, M. Lavin, D. E. Prado, C. A. Pendry, S. K. Laginhas, munic´ıpio de Caico,RioGrandedoNorte(Nordeste´ Pell, and C. A. Butterworth, “Historical climate change and spe- do Brasil),” Revista Brasileira de Plantas Medicinais,vol.12,no. ciation: Neotropical seasonally dry forest plants show patterns 1,pp.31–42,2010. of both Tertiary and Quaternary diversification,” Philosophical [60] J. B. L. P. Medeiros, Zoneamento Fito-EcologicodaEstac´ ¸ao˜ Transactions of the Royal Society B, vol. 359, no. 1443, pp. 515– EcologicadeAiuaba:UmaContribuic´ ¸ao˜ aEducac` ¸ao˜ Ambiental 537, 2004. e aPesquisaCient` ´ıfica, [M.S. thesis], Universidade Federal do [43] M. Tabarelli and A. M. M. Santos, “Uma breve descriçao˜ sobre Ceara,´ Fortaleza, Brazil, 2004. ahistoria´ natural dos Brejos Nordestinos,” in Brejos de Alti- [61] J. M. Nascimento and G. M. Conceiçao, “Plantas Medicinais tude em Pernambuco e Para´ıba, Historia´ Natural, Ecologia e e indicaçoes˜ Terapeuticasˆ da Comunidade Quilombola Olho Conservaçao˜ ,K.C.Porto,J.J.P.Cabral,andM.Tabarelli,Eds., D’agua´ do Raposo, Caxias, Maranhao,˜ Brasil,” Revista de Biolo- vol.9ofserie´ Biodiversidade, pp. 17–24, Ministerio´ do Meio gia e Farmacia´ ,vol.6,no.2,pp.138–151,2011. Ambiente, Bras´ılia, Brazil, 2004. [62] M. S. Silva, A. R. Antoniolli, J. S. Batista, and C. N. Mota, “Plan- [44] M. Tabarelli, S. R. R. Pinto, and I. R. Leal, “Floresta Atlanticaˆ tas medicinais usadas nos disturbios´ do trato gastrintestinal nordestina: fragmentaçao,˜ degeneraçao˜ e conservaçao,”˜ Cienciaˆ no povoado Coloniaˆ Treze Lagarto, SE, Brasil,” Acta Botanica Hoje, vol. 44, pp. 36–41, 2009. Bras´ılica,vol.20,no.4,pp.815–829,2006. [45] I.R.Leal,J.M.Silva,M.Tabarelli,andT.E.LacherJr.,“Mudando o curso da conservaçao˜ da biodiversidade na Caatinga do [63] U. P. de Albuquerque, T. A. de Sousa Araujo,´ M. A. Ramos et Nordeste do Brasil,” in Megadiversidade,vol.1,pp.139–146, al., “How ethnobotany can aid biodiversity conservation: reflec- Conservaçao˜ Internacional do Brasil, Belo Horizonte, Brazil, tions on investigations in the semi-arid region of NE Brazil,” 2005. Biodiversity and Conservation,vol.18,no.1,pp.127–150,2009. [46] G. Freyre, Nordeste, Record, Rio de Janeiro, Brazil, 1989. [64] R. L. C. de Oliveira, E. M. F. Lins Neto, E. L. Araujo,´ and U. P. Albuquerque, “Conservation priorities and population struc- [47] D. Ribeiro, OPovoBrasileiro:AFormaçaoeoSentidodeBrasil˜ , ture of woody medicinal plants in an area of caatinga vegetation Companhia das Letras, Sao˜ Paulo, Brazil, 2nd edition, 1995. (Pernambuco State, NE Brazil),” Environmental Monitoring and [48] G. J. Martin, Ethnobotany, A Methods Manual,Chapman&Hall, Assessment,vol.132,no.1–3,pp.189–206,2007. London, UK, 1995. [65] U. P. Albuquerque, “Plantas Medicinais e Magicas´ Comercial- [49] Jardim Botanicoˆ do Rio de Janeiro, “Lista de Especies´ da Flora izadas Nos Mercados Publicos´ do Recife-PE,” Cienciaˆ e Tropico´ , do Brasil 2012,” http://floradobrasil.jbrj.gov.br/. vol. 25, no. 1, pp. 7–15, 1997. [50] “Missouri Botanical Garden (MOBOT)—W3 TROPICOS,” http://www.mobot.org/. [66] R. Monteles and C. U. B. Pinheiro, “Plantas medicinais em um quilombo maranhense: uma perspectiva etnobotanica,”ˆ Revista [51] R. R. Sokal and F. G. Rholf, Biometry,W.H.FreemanandCom- de Biologia e Cienciasˆ da Terra,vol.7,pp.38–48,2007. pany, New York, NY, USA, 1995. [67] F. D. S. Silva, M. A. Ramos, N. Hanazaki, and U. P.de Albuquer- [52] U. P. D. Albuquerque and R. F. D. Oliveira, “Is the use-impact que, “Dynamics of traditional knowledge of medicinal plants in on native caatinga species in Brazil reduced by the high species a rural community in the Brazilian semi-arid region,” Brazilian richness of medicinal plants?” Journal of Ethnopharmacology, Journal of Pharmacognosy, vol. 21, no. 3, pp. 382–391, 2011. vol. 113, no. 1, pp. 156–170, 2007. [53]J.M.Monteiro,M.A.Ramos,E.D.L.Araujo,´ E. L. C. Amorim, [68] I. M. Madaleno, “Plantas da medicina popular de Sao˜ Lu´ıs, and U. P. Albuquerque, “Dynamics of medicinal plants knowl- Brasil,” Boletim do Museu Paraense Emilio Goeldi. Ciencias edge and commerce in an urban ecosystem (Pernambuco, Humanas,vol.6,no.2,pp.273–286,2011. Northeast Brazil),” Environmental Monitoring and Assessment, [69]N.C.BarbozadaSilva,A.C.DelfinoRegis,M.A.Esquibel,J. vol.178,no.1–4,pp.179–202,2011. Esp´ırito Santo Santos, and M. Z. Almeida, “Uso de plantas [54] C. A. Agra, “Identificaçao˜ das plantas medicinais indicadas medicinais na comunidade quilombola da Barra II—Bahia, pelos raizeiros e utilizados pelas mulheres no combate a enfer- Brasil,” Bolet´ın Latinoamericano y del Caribe de Plantas Medici- midades do aparelho geniturinario´ da cidade de campina nales y Aromaticas´ , vol. 11, no. 5, pp. 435–453, 2012. grande-PB,” BIOFAR,vol.73,no.73,2007. [70] A. P.Leite, K. M. Pedrosa, C. M. Lucena, T. K. N. Carvalho, L. P. [55] S. M. Morais, J. D. P.Dantas, A. R. A. Silva, and E. F.Mangalhaes,˜ Felix, and R. F.P.Lucena, “Uso e conhecimento de especies´ vege- “Plantas medicinais usadas pelos ´ındios Tapebas do Ceara,”´ tais uteis´ em uma comunidade rural no vale do Pianco(Para´ ´ıba, Revista Brasileira de Farmacognosia,vol.15,no.2,pp.169–177, Nordeste, Brasil),” Biofar: Revista de Biologia e Farmacia´ ,pp. 2005. 133–157, 2012. [56] A. J. R. Silva and L. H. C. Andrade, “Etnobotanicaˆ Nordestina: [71] M. G. V. Marinho, C. C. Silva, and L. H. C. Andrade, “Levanta- estudo comparativo da relaçao˜ entre comunidades e vegetaçao˜ mento etnobotanicoˆ de plantas medicinais em area´ de caatinga na Zona do Litoral- Mata do Estado de Pernambuco, Brasil,” no munic´ıpio de Sao˜ Jose´ de Espinharas, Para´ıba, Brasil,” Revista Acta Botanica Bras´ılica,vol.19,no.1,pp.45–60,2005. Brasileira de Plantas Medicinais,vol.13,no.2,pp.170–182,2011. [57]F.C.S.Oliveira,R.F.M.Barros,andJ.M.MoitaNeto,“Plantas [72] M. M. Araujo, Estudo etnobotanicoˆ das plantas utilizadas como medicinais utilizadas em comunidades rurais de Oeiras, semia-´ medicinais no assentamento Santo Antonio,ˆ Cajazeiras, PB, rido piauiense,” Revista Brasileira de Plantas Medicinais,vol.12, Brasil [M.S. thesis], Universidade Federal de Campina Grande, no. 3, pp. 282–373, 2010. Para´ıba, Brazil, 2009. [58] W.J. Machado, A. P.N. Prata, and A. A. Mello, “Floristic compo- [73] R. F. P. Lucena, U. P. Albuquerque, J. M. Monteiro, C. D. F. B. R. sition in areas of caatinga and brejo de altitude in sergipe state, Almeida, A. T. N. Florentino, and J. S. F. Ferraz, “Useful plants Brazil,” Check List,vol.8,pp.1089–1173,2012. of the semi-arid northeastern region of Brazil—a look at their [59] A. A. Roque, R. M. Rocha, and M. I. B. Loiola, “Uso e diversi- conservation and sustainable use,” Environmental Monitoring dade de plantas medicinais da Caatinga na comunidade rural de and Assessment,vol.125,no.1–3,pp.281–290,2007. 28 Evidence-Based Complementary and Alternative Medicine

[74] J. R. Lemos, “Composiçao˜ flor´ıstica do Parque Nacional Serra Barra do Rio Mamanguape, Para´ıba, Brasil,” Revista de Biologia da Capivara, Piau´ı, Brasil,” Rodriguesia,vol.55,no.85,pp.55– eCienciasˆ da Terra,vol.6,pp.357–366,2006. 66, 2004. [90] M. I. B. Loiola, G. B. D. C. Paterno, J. A. Diniz, J. F. Calado, and [75] U. P. Albuquerque and L. H. C. Andrade, “Conhecimento bota-ˆ A. C. P. de OLiveira, “Leguminosae and its potencial of use in nico tradicional e conservaçao˜ em uma area´ de caatinga no the rural communities of Sao˜ miguel do gostoso—RN,” Revista estado de Pernambuco,” Acta Botanica Brasilica,vol.16,no.3, Caatinga,vol.23,no.3,pp.59–70,2010. pp. 273–285, 2002. [91] P. M. S. Silva, D. O. Brandao,˜ T. P. Chaves et al., “Study biopros- [76] U. P. de Albuquerque, V. A. da Silva, M. D. C. Cabral, N. pecting of medicinal plant extracts of the semi-arid northeast: Leal Alencar, and L. D. H. C. Andrade, “Comparisons between contribution to the control of oral microorganisms,” Evidence- the use of medicinal plants in indigenous and rural caatinga Based Complementary and Alternative Medicine,vol.2012, (dryland) communities in NE Brazil,” Boletin Latinoamericano Article ID 681207, 6 pages, 2012. ydelCaribedePlantasMedicinalesyAromaticas´ ,vol.7,no.3, [92] I. G. Oliveira, L. S. Cartaxo, and M. A. P. da Silva, “Plan- pp. 156–170, 2008. tas medicinais utilizadas na farmacopeia´ popular em Crato, [77]M.F.Agra,G.S.Baracho,K.Nurit,I.J.L.D.Bas´ılio, V. P. M. Juazeiro e Barbalha (Ceara,´ Brasil),” Revista Brasileira de Biocie-ˆ Coelho, and D. A. Barbosa, “Sinopse da flora medicinal do ncias,vol.5,no.1,pp.189–191,2007. Cariri Paraibano,” Oecologia Brasiliensis, pp. 323–330, 2007. [93] S. A. Texeira and J. I. M. Melo, “Plantas medicinais utilizadas no [78] A. L. Balcazar, Hipotese´ da aparencianadinˆ amicaˆ do uso munic´ıpio de Jupi, Pernambuco, Brasil,” Iheringiano,vol.61,no. de plantas medicinais na floresta nacional do Araripe (Ceara,´ 1-2, pp. 5–11, 2006. Noredeste do Brasil) [M.S. thesis], Universidade Federal Rural [94] G. L. Oliveira, Etnobotanicaˆ nordestina: plantas medicinais de Pernambuco, Recife, Brazil, 2012. utilizadas na comunidade Muribeca (Jaboatao˜ dos Guararapes, [79]J.A.Castro,B.P.Brasileiro,D.H.Lyra,D.deAlmeidaPereira, PE) [M.S. thesis], Universidade Federal de Pernambuco, Recife, J. L. Chaves, and C. L. F. Amaral, “Ethnobotanical study of Brazil, 2007. traditional uses of medicinal plants: the flora of caatinga in [95] A. C. C. Silva, MonumentoNaturalGrotadoAngico,Sergipe, the community of Cravolandia-BA,ˆ Brazil,” Journal of Medicinal Brasil: Flor´ıstica, estrutura da vegetaçao˜ e conservaçao˜ [M.S. Plant Research,vol.5,no.10,pp.1905–1917,2011. thesis], Universidade Federal de Sergipe, Sao˜ Cristov´ ao,˜ Brazil, 2010. [80] V. S. Almeida and F. P. S. F. Bandeira, “O significado cultural do uso de plantas da caatinga pelos quilombolas do Raso da [96] C. S. Santos, Diagnostico´ da flora ap´ıcola para sustentabildade Catarina, munic´ıpio de Jeremoabo, Bahia, Brasil,” Rodriguesia, da apicultura no Estado de Sergipe [M.S. thesis], Universidade vol. 61, pp. 195–209, 2010. Federal de Sergipe, Sao˜ Cristov´ ao,˜ Brazil, 2009. [81] A. M. N. M. Guerra, M. F.Pessoa, C. S. M. Souza, and P.B. Mara- [97] M. L. R. A. Omena, Estudo etnofarmacologico´ de plantas com caja,´ “Utilizaçao˜ de plantas medicinais pela comunidade rural açao˜ no sistema nervoso central: perspectiva de sustentabilidade Moacir Lucena, Apodi-RN,” Bioscience Journal,vol.26,pp.442– em Umbuzeiro do Matuto—Porto da Folha/SE [M.S. thesis], 450, 2010. Universidade Federal de Sergipe, Sao˜ Cristov´ ao,˜ Brazil, 2003. [98] I. R. Costa, F. S. Araujo,´ and L. W.Lima-Verde, “Flora e aspectos [82] R. C. Brito, Estudo Preliminar de Avaliaçao˜ Ambiental Estr- auto-ecologicos´ de um encrave de cerrado na chapada do ategica´ do Plano Diretor—Campus Ondina, Universidade Fed- Araripe, Nordeste do Brasil,” Acta Botanica Brasilica,vol.18,no. eral da Bahia, Salvador, Brazil, 2008. 4, pp. 759–770, 2004. [83] L. C. B. Costa, R. C. T. Moreira, R. C. S. Costa, and E. A. [99] V. M. Morais, Etnobotanicaˆ nos quintais da comunidade de R. M. Lucena, “Abordagem etnobotanicaˆ acerca do uso de Abderramant em Caraubas—RN´ [Ph.D. thesis], Universidade plantas medicinais na Vila Cachoeira, Ilheus,´ Bahia, Brasil,” Federal Rural do Semi-arido,` Rio Grande do Norte, Brazil. Acta Farmaceutica Bonaerense, vol. 21, pp. 205–211, 2002. [100]G.P.S.Sales,H.N.Albuquerque,andM.L.F.Cavalcanti, [84] M. Z. Almeida, Plantas Medicinais, EDUFBA, Salvador, Brazil, “Estudo do uso de plantas medicinais pela comunidade quilom- 2011. bola Senhor do Bonfim, Areia (PB),” Revista de Biologia e [85] D. B. O. Abreu, R. B. Oliveira Filho, C. F. A. Vasconcelos Netos, Cienciasˆ da Terra,vol.1,pp.55–66,2004. C. M. Lucena, L. P.Felix, and R. F.P.Lucena, “Classificaçao˜ etno- [101]T.S.SilvaandE.M.X.Freire,“Abordagemetnobotanicaˆ sobre botanicaˆ por uma comunidade rural em um brejo de altitude no plantas medicinais citadas por populaçoes˜ do entorno de uma Nordeste do Brasil,” Biofar: Revista de Biologia e Farmacia´ ,vol. unidade de conservaçao˜ da caatinga do Rio Grande do Norte, 6, pp. 55–74, 2011. Brasil,” Revista Brasileira de Plantas Medicinais,vol.12,no.4,pp. [86] F. X. Oliveira, L. A. Andrade, and L. P. Felix,´ “Comparaçoes˜ 427–435, 2010. flor´ısticas e estruturais entre comunidades de Floresta Ombro-´ [102]W.S.Judd,C.S.Campbell,E.A.Kellog,P.F.Stevens,and fila Aberta com diferentes idades, no Munic´ıpio de Areia, PB, M. J. Donoghue, Sistematica´ Vegetal: Um Enfoque Filogenetico´ , Brasil,” Acta Botanica Bras´ılica, vol. 20, pp. 861–873, 2006. Artmed, Porto Alegre, Brazil, 2009. [87] M. Q. Matos and J. M. Felili, “Flor´ıstica, fitossociologia e diversi- [103]J.Botha,E.T.F.Witkowski,andC.M.A.Shackleton,“Market dade da vegetaçao˜ arborea´ nas matas de galeria do Parque profiles and trade in medicinal plants in the Lowveld, South Nacional de Sete Cidades (PNSC), Piau´ı, Brasil,” Acta Botancia Africa,” Environmental Conservation,vol.31,no.1,pp.38–46, Brasilica,vol.24,no.2,pp.483–496,2010. 2004. [88]M.F.Agra,G.S.Baracho,K.Nurit,I.J.L.D.Bas´ılio, and V. P. [104] M.G.LusaandC.Bona,“Caracterizaçao˜ morfoanatomicaˆ e his- M. Coelho, “Medicinal and poisonous diversity of the flora of toqu´ımica de Cuphea carthagenensis (Jacq.) J.f. Macbr. (Lythra- “Cariri Paraibano”, Brazil,” Journal of Ethnopharmacology,vol. ceae),” Acta Botanica Brasilica,vol.25,no.2,pp.517–527,2011. 111, no. 2, pp. 383–395, 2007. [105] C.-L. Lee and S.-Y. Chen, “Classification of leaf images,” Inter- [89] M. S. Pereira and R. R. N. Alves, “Composiçao˜ flor´ıstica de um national Journal of Imaging Systems and Technology,vol.16,no. remanescente de Mata Atlanticaˆ na Area´ de Proteçao˜ Ambiental 1,pp.15–23,2006. Evidence-Based Complementary and Alternative Medicine 29

[106] Q. D. Wheeler, “Taxonomic triage and the poverty of phy- [122] A. C. S. Diegues, Etnoconservaçao—NovosRumosparaaCons-˜ logeny,” Philosophical Transactions of the Royal Society B,vol. ervaçao˜ da Natureza, Nupaub e HUCITEC, Sao˜ Paulo, Brazil, 359, no. 1444, pp. 571–583, 2004. 2000. [107] E. Mati and H. de Boer, “Ethnobotany and trade of medicinal plants in the Qaysari Market, Kurdish Autonomous Region, Iraq,” Journal of Ethnopharmacology,vol.133,no.2,pp.490–510, 2011. [108]P.D.N.Hebert,A.Cywinska,S.L.Ball,andJ.R.deWaard, “Biological identifications through DNA barcodes,” Proceedings of the Royal Society B,vol.270,no.1512,pp.313–321,2003. [109] M. P. Mariot and R. L. Barbieri, “Divergenciaˆ genetica´ entre acessos de espinheira-santa (Maytenus ilicifolia Mart. ex Reissek e M. aquifolium Mart.) com base em caracteres morfologicos´ e fisiologicos,”´ A Revista Brasileira de Plantas Medicinais,vol.12, no. 3, pp. 243–249, 2010. [110] S. Coulaud-Cunha, R. S. Oliveira, and W.Waissmmann, “Venda livre de Sorocea bonplandii Bailon como Espinheira-Santa no Munic´ıpio do Rio de Janeiro-RJ,” Revista Brasileira de Farma- cognosia,vol.4,supplement1,pp.51–53,2004. [111] A. C. S. Diegues and R. S. V. Arruda, Saberes tradicionais e biodiversidade no Brasil, Biodiversidade 4, Ministerio´ do Meio Ambiente, USP, Sao˜ Paulo, Brazil, 2001. [112] A. Begossi, N. Hanazaki, and J. Y. Tamashiro, “Medicinal plants in the Atlantic Forest (Brazil): knowledge, use, and conservation,” Human Ecology,vol.30,no.3,pp.281–299,2002. [113] M. Tabarelli and J. M. C. Silva, “Areas´ e açoes˜ prioritarias´ para aconservaçao,˜ utilizaçao˜ sustentavel´ e repartiçao˜ de benef´ıcios do bioma Caatinga,” in Biodiversidade, conservaçao˜ e uso sustentavel´ da flora do Brasil,E.L.Araujo,´ A. N. Moura, E. V. S. B. Sampaio,L.M.S.Gestinari,andJ.M.T.Carneiro,Eds.,vol.1,pp. 47–52, Imprensa Universitaria´ da UFRPE, Recife, Brazil, 2002. [114] D. E. Moerman and G. F. Estabrook, “Native Americans’ choice of species for medicinal use is dependent on plant family: confirmation with meta-significance analysis,” Journal of Ethnopha- rmacology,vol.87,no.1,pp.51–59,2003. [115] A. Smelcerovic and M. Spiteller, “Phytochemical analysis of nine Hypericum L. species from Serbia and the F.Y.R. Macedonia,” Pharmazie,vol.61,no.3,pp.251–252,2006. [116] M. L. Silva and V. Cechinel Filho, “Plantas do generoˆ Bauhinia: composiçao˜ qu´ımica e potencial farmacologico,”´ Qu´ımica Nova, vol. 25, pp. 449–454, 2002. [117] M. P. Guerra and R. O. Nodari, “Biodiversidade: aspectos biologicos,´ geograficos,´ legais e eticos,”´ in Farmacognisia: da planta ao medicamento,C.M.Simoes˜ et al., Ed., Editora da UFRGS; Editora da UFSC, Florianopolis,´ Brazil, 2004. [118] Brasil, Relaçao˜ Nacional de Plantas Medicinais de Interesse ao SUS, DAF/SCTIE/MS—RENISUS, Ministerio´ da Saude,´ Bras´ılia, Brazil, 2009, http://portal.saude.gov.br/portal/arquivos/pdf/RENISUS.pdf. [119] U. P. Albuquerque and N. Hanazaki, “As pesquisas etnodirigi- das na descoberta de novos farmacos´ de interesse medico´ e farmaceutico:ˆ fragilidades e pespectivas,” Revista Brasileira de Farmacognosia,vol.16,pp.678–689,2006. [120] G. S. Vendruscolo and L. A. Mentz, “Levantamento etnobota-ˆ nico das plantas utilizadas como medicinais por moradores do bairro Ponta Grossa, Porto Alegre, Rio Grande do Sul, Brasil,” Iheringia, Serie´ Botanicaˆ ,vol.61,no.1-2,pp.83–103,2006. [121]C.D.F.C.B.R.deAlmeida,M.A.Ramos,E.L.C.deAmorim, and U. P. de Albuquerque, “A comparison of knowledge about medicinal plants for three rural communities in the semi-arid region of northeast of Brazil,” Journal of Ethnopharmacology, vol. 127, no. 3, pp. 674–684, 2010. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 732760, 10 pages http://dx.doi.org/10.1155/2013/732760

Research Article The Key Role of Cultural Preservation in Maize Diversity Conservation in the Argentine Yungas

Norma I. Hilgert, Fernando Zamudio, Violeta Furlan, and Lucía Cariola

Instituto de Biolog´ıa Subtropical, CONICET, Facultad de Ciencias Forestales, Universidad Nacional de Misiones, Centro de Investigaciones del Bosque Atlantico,´ Bertoni 85, 3370 Puerto Iguazu,´ Argentina

Correspondence should be addressed to Norma I. Hilgert; [email protected]

Received 4 April 2013; Revised 14 June 2013; Accepted 22 July 2013

Academic Editor: Ana H. Ladio

Copyright © 2013 Norma I. Hilgert et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Farmers’ decisions on what to grow and why can contribute in understanding the conservation of agrobiodiversity. Culture and ethnicity are indicated as first-class factors leading preservation of heirloom cultivars but this has been little considered in studies examining factors that influence the loss or preservation of agrobiodiversity. We propose that corn’s ethnotaxa of less diverse uses, which are also key partners in local cultural reproduction, are usually cultivated by a few households. We analyse if there is a relationship between uses and richness of cultivated ethnotaxa at household level and describe corn’s medicinal and ritual uses. We found 25 cultivated ethnotaxa, heterogeneously distributed in the region, and we also found that ethnotaxa with less diverse uses are cultivated in fewer households. We identified that, at regional scale, richness is related with food use diversity. The most frequently cited medicinal uses were urinary and tract infections, diarrhoea, and liver disorders. Medicinal recipes involve combinations with other elements. Maize is an indispensable resource in the rituals that propitiate productive activity, to augur prosperity or misfortune according to signals. We have identified the vulnerability in preserving the richness of corn in the region and the factors that shape its cultivation at different scales.

1. Introduction At present, culture and ethnicity are recognized as first-class factors leading conservation of heirloom cultivars [8–12]. Conservation and loss of agrobiodiversity is a concern to Among the driving factors behind agrobiodiversity, persist- academics, development institutions (governmental or not), ence cultural salience has been identified in the first place and and to local populations [1–4], facing the modernization, utilitarian salience in the second. The first concept is under- socioeconomic, and environmental changes that occur both stood as the selection criteria that are specifically related globally and regionally. Migration, pests outbreaks, and cli- to culturally defined preferences and influences such as mate changes, among other variables, promote transformation in the composition and dynamics of agricultural parcels, foodways, cultural heritage, and memory; instead, the second changes that ultimately alter the functionality of contempo- one is understood as the selection criteria that are specifically rary rural landscapes. These changes occur in cascade related to market value, productivity, environment adapta- through complex mechanisms (top-down and bottom-up) tion, and resistance [8, 13–16]. and disseminate simultaneously in different directions and The assessment of criteria or the decisions that shape scales [5, 6]. local conservation of agrobiodiversity is central to guarantee Farmers’ decisions on what to grow and why can contrib- people’s food sovereignty and generation of in situ conserva- ute to understand the conservation of agrobiodiversity and tion plans which involve local peasants. Analytic categories can help scientists to realize farmers’ role in this process [2, 4, used to classify corpus of the local knowledge (religious, 7]. economic, alimentary, medicinal) are abstractions of a matrix Until not long ago it was held, from academia, that the of interconnected elements that, analyzed together, explain decisions of local people were guided by economic, agro- agricultural decision making and agrobiodiversity persis- nomic, and ecological variables (see review in Veteto [8]). tence [8, 17]. 2 Evidence-Based Complementary and Alternative Medicine

Indeed, the use of resources may differ depending on the family moves periodically along the year to the cerro, valle, context or the occasion: daily consumption of food within the and monte, where they cultivate and pasture their animals household, use in collective festivities, offering to supernatu- [26]. However, at present, transhumance in some areas has ral entities, and the act of sharing food with ancestors, among been reduced and abandoned in others [32]. This production others [18, 19]. In this context, a given resource: alimentary, system is usually complemented with paid work carried out medicinal, material can officiate as a symbol understood asan within or outside the community in nearby settlements or object represented by general consensus or something evoca- villages and allowances granted by the government have tive [20]. proven to be a useful contribution to household resources. The approach of this paper is supported by previous Local women also weave wool blankets, saddlebags, and crafts research in the study area, which found that the local health for their own households or for sale, as an other mean of get- concept that matches the Andean concept is understood as an ting some income. Nowadays, they are increasingly experi- holistic balance of a person environment [21–25]. According menting dyeing plant species in search for new colours and to our experience, to analyze cultural driving factors behind combining different species together, which is a new phenom- persistence of agrobiodiversity, it is necessary to develop a enon [33]. comprehensive analysis of the role of corn in the lives of the The most important crops are Zea mays (Poaceae) and Yungas residents. For this reason, we considered the general Solanum tuberosum (Solanaceae). In each altitudinal belt, uses of maize and the uses of herbal medicine which involves different varieties of the same species are produced, together maize varieties in particular, the ritual uses of maize (e.g., with Cucurbita spp. (Cucurbitaceae) and several varieties of meals prepared in agricultural celebrations or funerals), and Phaseolus vulgaris (Fabaceae). The currently cultivated crops those practices that reflect protective value assigned to maize include old maize landraces, recently incorporated foreign for being the legacy of La Pachamama, the principal chthonic elements, and mixed product of hybridization between some deity of the region. of the above mentioned [26, 34]. In the region of our study, a decrease in agrobiodiversity, Regarding local medicine, the current ethnomedical sys- the gradual abandonment of different production spaces, and tem involves, according to the classification proposed by the replacement of some species and varieties by others as an Molina [35], practices such as traditional, homemade, self- adaptive response to these changes in production have been treatment, religious, and biomedicine practices. As appropri- described [26]. In relation to this, and bearing in mind that ate, health practices are carried out by the rural doctor who the selection criteria affect the richness of grown ethnotaxa at has no formal education or degree of specialization, or by householdandregionallevels,weproposethatethnotaxaof people with graduate studies or degrees, as the local doctor less diverse uses, which are also key partners in local cultural andthenurseatthesanitarypost. reproduction for their medicinal value, symbolic and/or rit- Local people recognize three origins for ailments: natural, ual, are usually cultivated by a few households. This is consist- sociocultural (i.e., when a food taboo is not respected), and ent with those who assigned a leadership role to ritual values supernatural (i.e., witchcraft, air diseases, templar diseases) forconservationofagrobiodiversity[27, 28]. To evaluate this, [32, 36, 37].Thelasttwotypescanbediagnosedandtreated we analyze if there is a relationship between the uses assigned only by a rural doctor (called curandero locally). Diseases of to each ethnotaxon and the richness of cultivated ethnotaxa natural origin, like cough, dyspepsia, headache, and postpar- at household level and describe the medicinal and ritual uses tum pain, are usually solved within the family environment ofmaizeinthreeregionsoftheArgentineYungas. and it is not necessary to have any formal training or carry out any ritual process [36]. The diagnosis is made by the 2. Background rural doctor, based on an interview with the patient; other techniquesmaybealsoapplied.Thesetechniquesagreein The inhabitants of the communities under study are descen- general with the divinatory practices described by Amodio dants of Andean cultures, Chaco, and Hispanic who have [38]; they include the “reading” of Erythroxylum coca leaves, converged historically and culturally [29]. Due to its geopo- the“reading”oftheurine,thepulseo (or diagnosis by the litical history and geographical location in the Barituregion,´ unlike the other two, there are established social and com- pulse), the alumbriada,andrubbingofabodywithalum mercialtieswithBoliviancommunities[30]. At present they which is later burnt to interpret the ashes, among others. The identify themselves as criollos, although this name also iden- latter consists of simultaneous diagnosis and treatment, as the tifies people of different cultural roots. They maintain aset affection is considered to be transmitted to the mineral and of practices and beliefs, among which the cult of the Pacha- destroyed through burning [30, 36, 37]. mama, a characteristic element of Andean cultures, stands out, as mentioned above. They also profess Catholicism and 3. Material and Methods Evangelism (the latter more recent and in current expansion). The Spanish language is enriched with Quichua and Guaran´ı The study area covers three groups of settlements: settlements terms (to a lesser extent) [31]. located in the surroundings of Baritu´ National Park (Deptart- Regional economy is based on a system of shifting agri- ment Santa Victoria, Salta), settlements located in the former culture, transhumance, and gathering, to a lesser extent. Shift- large farm of San Andres´ (Deptartment Oran, Salta), and ing agriculture and transhumance which consists on the use those above the Calilegua National Park (hereinafter Los of different cultivation and cattle breeding areas along altitu- Toldos, San Andres, and Valle Colorado) (Figure 1). The sam- dinal gradients favour vertical use of the environment. Each pling unit was the nuclear family. Households were randomly Evidence-Based Complementary and Alternative Medicine 3

N Bolivia Bolivia Paraguay Los Toldos N

S 2 󳰀󳰀 0 󳰀

0 Salta ∘ 23 Jujuy San Andreś Valle Colorado 1

0 145 290 580 870 1160 0 10 20 40 60 80 (km) (km) 65∘0󳰀0󳰀󳰀W 64∘0󳰀0󳰀󳰀W International borderline Protected areas

Argentina 1 Calilegua Bolivia 2 Laguna Pintascayo Provincial borderline 3 PN Baritu´ Study area Towns

Figure 1: Studied area. selected within the group of people willing to participate in the remaining others were identified by the authors. The col- the study. lected samples were deposited in the Banco de Germoplasma Data were collected in three steps, covering a total of del INTA Pergamino (Argentina). 118 informants (in 150 surveys). Visits to the study area Regarding race and/or varieties of maize, in this paper were carried out from 1994 to 2000 on the first stage of we consider maize populations distinguished by farmers as the investigation, at that moment global ethnobotanical landraces according to the proposal by Perales et al. [11], practices of local medicinal knowledge were summarized populations that are often known locally as criollos, indicating in 59 surveys; the second stage visits took place from that it is local corn, while all landraces which have local name 2006 to 2008 accomplishing a total of 91 surveys; the third in the region are hereinafter referred to as ethnotaxa. We stage was made in 2012 and 10 surveys were carried out. considered only those ethnotaxa that have been cited by more In the first stage of the project, a semistructured interview than 5 people. In such ethnotaxa, besides vernacular name, was directed to the domestic uses of maize. In subsequent they recognize special characters (such as colour, cooking, stages, the interview deepened in different aspects of local and/or medicinal properties and plant size). Moreover, under maize varieties in each household (richness of cultivated the category of commercial maize, there is a pool of different ethnotaxa and assigned uses). In particular, the respondents ethnotaxa that were marked as purchased in markets (e.g., were asked about medicinal and ritual uses of maize, as hard corn, yellow corn, yellow corn Abajeno,˜ Cuban Corn, wellastheplantstheyusedtocombinewithit,theplant and Corn mule). Other ethnotaxa considered here, as the parts used, the methods of preparation and administration, overo or chesgua among others, although identified as hybrids dosage, treatment time, and illness duration. We took also by taxonomists, were considered a taxonomic unit in inter- into account observations made in festivals and religious and views because there was uniformity of criteria at selecting and propitiatory rituals developed in the context of agricultural namingthemandintheirusage. labor. Two variables were measured at household level: richness The plant specimens were collected in the presence of of maize grown at present (i.e., number of grown ethnotaxa in the study participants. Then, voucher specimens were identi- each household) and diversity of cited uses. Maize uses were fied by Norma Hilgert and were deposited in the herbarium grouped in three general categories, alimentary, fodder, and of the Museo de Ciencias Naturales of the Universidad Nacio- medicinal/ritual. The latter group includes citations of uses of nal de Salta, Argentina. The nomenclature used follows preparation of medicaments, preparation of ritual food, ritual Flora del Cono-Sur (http://www2.darwin.edu.ar/Proyectos/ practices, and/or propitiatory practices (such as a particular FloraArgentina/FA.asp)andTropicos(http://www.tropicos cultivation of an ethnotaxon as other protective element of .org/Home.aspx)database. crops or housing). Considering these categories, we estimated The majority of the collected maize varieties were iden- the diversity of use by ethnotaxa (i.e., number assigned to tified byamara C´ Hernandez,´ researcher of the Agronomical each ethnotaxon use as food, fodder, and medicinal/ritual, Faculty of the National University of Buenos Aires, and resp.). 4 Evidence-Based Complementary and Alternative Medicine

To analyze whether there is a relationship between the recipes involve its use in combination with other plants and/ ethnotaxa with less diverse uses and the number of house- or different resources. These mixtures include the use oftwo holds that cultivate them, we performed a diverse Spearman or more species. We registered the use of 25 plant species, correlation between the true diversity of uses of each ethno- belonging to 18 families. The affections most frequently taxon as ln 𝐷 = (Shannon-Wiener index) in Garc´ıa-Morales treated were urinary infections (6 different recipes), diar- et al. [39]andthenumberofhouseholdswhereeachethno- rhoea and liver disorders (3); for limpias and alumbriadas, taxon is grown. To identify the types of uses that model the forfever,andtoavoidairandcolddiseases(2);against richness of cultivated ethnotaxa, we performed a multiple cangrena (intensive urinary pain caused by hot imbalance), regression using the richness of crops by household as depen- against general pains, as dietary supplement, as a stimulant, dentvariableandthenumberofusesassignedtoeachethno- to heal pimples, and to remove the placenta (1). Toasted taxon by household for categories food, fodder, and medic- flour and/or stigma are used in different modes of prepara- inal/ritual as independent variable. In both cases, we used tion administrated orally (infusions, decoctions, masticatory, Excel spreadsheets and StatSoft, Inc. [40]. inhalation, and foods) and in topics (poultices, rubbing, and For more details on medicinal and therapeutic conditions ointment) (Table 1). Indistinct ethnotaxa is generally used in named in the current paper, see Hilgert [36] and Hilgert and these preparations, except for some ailments, or particular Gil [37] where a thorough analysis of ritual aspects can be cultural practices, described in the following section. found. Finally, more details of culinary preparations named inthepresentpapercanbefoundinHilgert[41]. 5. Maize Symbolism

4. Results Inthestudyregion,maizeisconsideredtherepresentation(or We have found 25 ethnotaxa of cultivated maize, heteroge- materialization) of the Pachamama, and it is an indispensable neously distributed in the region, 23 in Valle Colorado, 19 in resource in the rituals that propitiate or thank the productive Los Toldos, and 12 in San Andres. There is a low average of activity, for example, (1) in carnival dances, where local ethnotaxa cultivated by a household: 1.2, 2.3, and 3, respec- people brandish maize plants as a flag or handkerchief and tively.Alsotherangeofethnotaxagrownineachlocation (2) in San Isidro Labrador (patron of farmers) festivity in (1–10, 1–5, and 1–17, resp.) reflects an unequal distribution of mid-May, which celebrates the harvest and calls for work and prosperity in future harvests. On this saint day, a pilgrimage is varieties within each locality. When analyzing whether there is a relationship between performed from the temple to the household responsible for the ethnotaxa with less diverse uses and the number of house- the cornfield celebration. There, in the field, the image of the holds that grow them, at regional scale we found (considering saint is placed on the altar prepared for this particular pur- the three areas together) that ethnotaxa with less diverse uses pose. In the vicinity of the altar, the cobs are stacked cropped are grown in less households (coefficient: 0.888 (𝑃 < 0.001)). (in a mound called era) in its middle and one maize plant The same relationship was found locally (Los Toldos: Coef.: is left standing with two corn cobs. During the celebration, 0.908, 𝑃 < 0.001;SanAndres:Coef.:0.638,´ 𝑃 < 0.05;Valle around that era, people dance and sing, in gratitude for the Colorado: Coef.: 0.816, 𝑃 < 0.001). This is consistent with the production (Figure 2). proposed hypothesis. Culli landrace has a particular assigned property: the Moreover, we identified the types of uses that model ability to protect the cornfield and housing. In the first case, ethnotaxa richness of households cultivated in different situa- the culli is always grown in a plot—this sector can be in the tions.Wefoundthatrichnessofethnotaxacultivatedbya middle, a cross side, or somewhere else in the cornfield, in 2 household is related (adjusted 𝑟 :0.626,𝐹(3.104) = 60.715, order to prevent the winds and summer storms from pulling 𝑃 < 0.001,ES:2.219,𝛽: 0.788, 𝑃 < 0.001) to food uses downthecornfield(processcalledlocallyasel volteo de la diversity at regional scale. chacra). Culli is more often cultivated for its benefiting action For settlements in Los Toldos, richness of ethnotaxa cul- rather than for its culinary properties. The surface of the culli 2 tivatedbyahouseholdisrelated(adjusted𝑟 :0.739,𝐹(3.39) = cultivatedbyeachhouseholdissmallinrelationtoitstotal 40.722, 𝑃 < 0.001, ES: 1625) to food uses diversity (𝛽:0.602, production of corn. Moreover, when it is used for symbolic 𝑃 < 0.001) as fodder (𝛽:0.264,𝑃 < 0.01) and in medicinal/ protection of the housing, it is hanged on the outer frame of ritual (𝛽:0.240,𝑃 < 0.05). the kitchen door\as an eraquita or simbita that is to say as a For settlements in San Andres,´ richness of ethnotaxa pair of cobs tied through the braided husk. 2 cultivated by a household is related (adjusted 𝑟 :0.304, In addition, the listed benefactor properties are the 𝐹(3, 39) = 7.136, 𝑃 < 0.001, ES: 1.268) to the diversity of uses underlying use of this ethnotaxon in limpias and alumbriadas as fodder (𝛽:0.365,𝑃 < 0.01) and food (𝛽:0.375,𝑃 < 0.05). andinpreparingchicha (fermented beverage made from Finally, for settlements in Valle Colorado, richness of eth- corn) to be used in ritual contexts. A limpia is a curative pro- 2 notaxa cultivated by a household is related (adjusted 𝑟 :0.932, cedure that involves scrubbing the patient’s body with a mix- 𝐹(3.28) = 142.69, 𝑃 < 0.001,ES:1.477,𝛽:0.937,𝑃 < 0.001)to ture of coca, tabaco, culli cornmeal, and alum (see Table 1). In diversity of food uses. this case, this ethnotaxon is considered capable of providing In relation to its medicinal uses, maize is often used as protection to repel or remove bad air, witchcraft, and coldness the only component in preparations, but the most common (seen from templar medicine) of the patient’s body. Evidence-Based Complementary and Alternative Medicine 5

Table 1: Medicinal corn uses. Species, Family (herbarium) Local name Use Administration A handful of roots is boiled in 5 L of water with about 5cmofCortaderia selloana roots. Seven successive poultices are placed in the back with this mixture next Adesmia inflexa Griseb., To remove the to a hot corn cob tied with a black cloth. Each anagua˜ Fabaceae (1224; 1714) placenta application is left until it gets cold. This treatment is completed covering the back with two warm saddle cloth (one heated in the stove and the other in the back of a horse) Aninfusionispreparedinhalfaliterofwaterwith three or five fresh leaves, combined with a piece of Against urinary Equisetum bogotense (or E. giganteum), three fresh affections Artemisia absinthium L. leaves of Pluchea sagittalis,andatablespoonofcorn (2433); Tanacetum parthenium carqueja flour. It is drunk several times for several days (L.) Sch. Bip. (1479), A decoction in half a liter of water with three leaves Asteraceae mixed with three leaves of Erythroxylum coca var. coca a piece of Cinnamomum zeylanicum, and handful of Against diarrhea seeds of Pimpinella anisum is prepared. After being withdrawn from the fire, a little of cornflour is added. It is drunk lukewarm or cold several times for several days until symptoms disappear Against diarrhea See full recipe under Artemisia absinthium Cinnamomum zeylanicum A piece is boiled in 1 L of water; after being withdrawn canela Blume, Lauraceae (1575; 2330) Against hepatic from the fire, a little of flour is added. It is drunk affections lukewarm or cold several times for several days until symptoms disappear A few drops of juice are mixed in warm water with a Citrus x limon (L.) Osbeck, spoonofcornflourandahandfulofgroundflax limon´ Against fever Rutaceae (1590) (Linum usitatissimum) seeds. It is drunk several times until symptoms disappear A few drops of juice are mixed with a spoon of corn Against hepatic Citrus x sinensis (L.) Osbeck, naranja, n. flour in warm water. It is drunk several times for several affections Rutaceae (2053) dulce days until symptoms disappear Against diarrhea See full recipe under Artemisia absinthium Cortaderia selloana (Schult. To remove the and Schult. f.) Asch. and cortadera See full recipe under Adesmia inflexa placenta Graebn., Poaceae (1722) A piece of the plant with the stigmas of two spikes, one or two roots of Plantago australis—or P. major or P. Dolichandra unguis-cati (L.) myosurus—a piece of Equisetum bogotense (or E. L.G. Lohmann, Bignoniaceae una˜ de gato Against urinary giganteum), and handful of ground flax (Linum (2015, 2192) affections usitatissimum) seeds are boiled. When it reaches the boiling point, add half grated potato (Solanum tuberosum). It is drunk lukewarm or cold several times for several days until symptoms disappear See full recipe under Adesmia inflexa Equisetum bogotense Kunth, See full recipe under Dolichandra unguis-cati Against urinary (1394); E. giganteum L. (1617), cola de caballo affections A piece of the plants combined with the stigmas of two Equisetaceae spikes is boiled in 500 mL of water. It is drunk at own discretion, as a cold soft drink during the day An ointment is prepared with leaves of acullico—insalivated coca—and cornflour of culli roasted. This must be rubbed in the patient’s body and Erythroxylum coca Lam. var. For limpias and coca burn it. If treatment is alumbriada,thentothispaste coca, Erythroxylaceae (2108) alumbriadas alum is added. This metal, when burned, takes the form of the element that is causing the condition and orient to doctor about the treatment 6 Evidence-Based Complementary and Alternative Medicine

Table 1: Continued. Species, Family (herbarium) Local name Use Administration The grounded ashes of the burned branches are mixed Iresine diffusa Humb. & Bonpl. As dietary with mote, resulting in a paste known as lye. This ex Willd., Amaranthaceae sacha arborito supplement, as a product is used during the coca leaves insalivation, (1897) stimulant known as coqueo Against fever See full recipe under Citrus limon Linum usitatissimum L., linaza Against urinary Linaceae (1621; 2317) See full recipe under Dolichandra unguis-cati affections A cigar is prepared with dry crushed leaves of snuff Nicotiana tabacum L., For limpias and tabaco bundledincornhusksandsmokedwiththesmoke Solanaceae (1474; 1487) alumbriadas exhale on the body of the patient during the treatment Pimpinella anisum L., Apiaceae (manufactured an´ıs castilla Against diarrhea See full recipe under Artemisia absinthium product) The stigmas of two spikes are boiled in 500 mL of water Plantago australis Lam. (2534); Against urinary with two fresh leaves. It is drunk at own discretion, as a llanten´ P. major L. (2439); P. my osur u s affections cold soft drink during the day Lam. (2222), Plantaginaceae See full recipe under Dolichandra unguis-cati Pluchea sagittalis (Lam.) Against urinary Cabrera, Asteraceae (2392; cuatro cantos See full recipe under Artemisia absinthium affections 2571; 2530; 2179) An ointment with a few drops of almonds oil is mixed Against fever with a spoon of cornflour in warm water. It is put in the Prunus amygdalus Batsch, brow until fever disappear Rosaceae (manufactured almendra Mote—corn—and seeds of almonds are crushed. This product) To heal pimple ointment is placed on the pimple An infusion is prepared with two or three pieces of the Punica granatum L., skin of the dry fruit, with two spoonfuls of flour in Lythraceae (2469); Passiflora granda, Against diarrhea 250 mL of water. It is drunk lukewarm once or twice; if tenuifolia Killip, Passifloraceae granadilla symptoms persist, the treatment is repeated for two (1515) consecutive days Against cangrena Sambucus nigra L. subsp. Two or three flowers are boiled in 500 mL of water. A (intensive urinary peruviana (Kunth) R. Bolli, mololo spoon of corn flour and honey is added. It is drunk at pains caused by hot Adoxaceae (2142) own discretion, as a cold soft drink during the day imbalance) Solanum tuberosum L., Against urinary papa See full recipe under Dolichandra unguis-cati Solanaceae (1924; 1933; 1907) affections Duringthepuerperal,itisadvisableforthemotherto Origanum x appli (Domin) To avoid air and cold eat corn-based foods seasoned with oregano. This is oregano´ Boros, Lamiaceae (1448; 2242) diseases done to avoid coldness and the entrance of air which could cause a general weakness For all diseases cited See full recipes under all species above mentioned above The cornflour is mixed with egg yolk, salt, and porkfat Zea mays L., Poaceae (1386; Against general pains or chicken excrement. It is used as an ointment in ma´ız 1711; 2423; 2424) different painful parts of the body The stigmas of two spikes are boiled in 500 mL of water. Against hepatic and It is drunk at own discretion, as a cold soft drink during urinary affections the day For templar imbalances See recipe above mentioned manifested in urinary affections Evidence-Based Complementary and Alternative Medicine 7

Figure 3: In this picture an unusual cob corn is observed, called Pachamama or Sara. It is interpreted that the presence of these structures in a cornfield predicts good harvest in the coming years.

(i) Chicha. The use of 12 ethnotaxa was registered, with the highest number of citations for culli and morocho. In the case of culli landrace, since the preparation of thisdrinkrequiresalotofflour,whentheavailable Figure 2: In this picture the era of cobs is observed. The complete volume is scarce, medicinal preparation or any of the upright corn plant in the center of this mound represents the Pachamama. other ritual dishes is given priority rather than chicha. (ii) Tistinchas.Itisastewpreparedwithcobs,forwhich three alternate rows of grains are extracted (corn used is morocho, blanco boliviano, overo, tucumano, and It was also recorded that culli’s strength to scare the bad colorado). Lamb or beef are added, as well as broad air and misfortunes probably comes probably from its black beans (Vicia faba), beans (Phaseolus spp), peppers or dark colour. However, its dark colour is often mentioned as (Capsicum spp), green potatoes (Oxalis tuberosa), and an undesirable feature for everyday kitchen use (except for the potatoes (Solanum tuberosum), among other ingre- chichi; see below), as it is considered an unpleasant ingredient dients. The preparation is boiled overnight and pre- which turns everything black. paredespeciallyforthefirstofAugust,thedayofthe Anotherparticularfeatureassignedtomaizeisthecapac- Pachamama. ity to augur prosperity or misfortune, according to certain (iii) Pire (or Piri). It is prepared from roasted cornflour, signals. Among the positive signs is the appearance in the water, and onion; the last one is fried with abundant cornfield of unusual cobs with more than two cobs together oilorfat.Thedishisakindofsoup.Itcanbeseasoned (like a basket) (Figure 3). These cobs are called Pachamama or with salt, sugar, or both. Culli corn is employed, Sara and are interpreted as Mother Earth and her children. If together with sauceno˜ and overito. It is prepared espe- there are only two cobs together, one larger than the other, ciallyforthedayofthePachamama. That day, at it is interpreted as a representation of the semillera,which dawn, the pire is placed on the roof of the kitchen in is the woman in charge of seeding. The explanation of local order to feed the birds. On that day, approaching birds people is that the cob is the representation of the mother are considered mythical beings, able to intercede for with the baby carried on the back (as mothers carry usually the good fortune of households which have offered up. their young children in the region). It is interpreted that the presence of these corns in a cornfield predicts good harvest (iv) Tulpo. ItisaTypeofstewmadewithcornflourand in coming years. These rare cobs are stored in a special place locally produced vegetables (similar to tistinchas). in the house without removing the husk that is with la ropita Morochos and ma´ız blanco are preferred. This dish de la Pachamama (the clothes of Pachamama). is prepared especially for summer parties (Christmas Among the negative signals, the infection of corn plants and Carnival). with musura (Ustilago maydis) is frequently mentioned. It is believed that when this fungus appears in the cornfield it 6. Discussion is a sign that a family member will die. To avert the omen, infected plants are removed and pigs are fed with them, a In our study area, the maize is present in different contexts of practice that also prevents the spread of the fungus. family life, community ritual life, and the agricultural cycle. Finally, in all religious rituals or celebrations, corn is pre- In the literature, there are numerous examples with a similar pared in different meals and serves to entertain people, for the picture to the one found here, where the same resources are deceased (on the feast of Todos los Santos), and Pachamama, redefined as everyday food, medicine, and ritual resource in including the following. collective festivities and offerings3 [ , 18, 21, 42–44]. 8 Evidence-Based Complementary and Alternative Medicine

The observed relationship between the diversity of uses In relation to the positive (in the case of medicinal/ritual and the number of households that cultivate them can be uses) and negative (in everyday culinary uses assigned to interpreted as an indicator of regional vulnerability of con- culli due to its black colour) assessments, at present there is servation of less versatile ethnotaxa. This is consistent with increasing evidence of the importance of sensory perception the asymmetry in the number of cultivated ethnotaxa per when selecting resources, both cultivated and medicinal [3, family and the high number of very rare ethnotaxa found 51–54]. According to Leonti et al. [55]andBoster[56], these by Velasquez-Milla´ et al. [3]inAndeantownsinPeru. characteristics also officiate as mnemonic resources for the Our records also partially match with those observed in social transmission of knowledge and selection criteria. neighboring communities by Pinotti et al. [45]. These authors Undoubtedly, in the case of maize, visual appearance of noted a positive relationship between factors such as diet and ethnotaxa is an essential tool when selecting seeds for the conservation of local resources, like us, but identified the next planting, although further studies would be necessary informal trade and exchange networks as the main means of to define what other aspects are involved. obtaining different corns, to the detriment of local agriculture. We found that at the regional level alimentary uses are the Acknowledgments ones which influence mostly the cultivation of ethnotaxa, so their conservation is strongly associated with the traditional The authors wish to dedicate this paper to the inhabitants of cuisine conservation. This aspect has been pointed out previ- the settlements under study in recognition of their valuable ously in numerous occasions. D´ıaz et al. [46] define maize and cooperation, patience, and goodwill. The authors thank potato as diacritic elements of present regional food (both CONICET (PIP 191) and INTA (Project: Filogeograf´ıa e traditional and tourism-related innovations). In line with the implementacion´ de un sistema de monitoreo molecular de la diversity of food uses found in the present, Camara´ Hernan- identidad genetica´ de cultivos andinos) for their financial dez et al. [34], Velasquez-Milla´ et al. [3], and D´ıaz et al. [46] supporttoNormaHilgert.SpecialthanksgotoMonika explain the preferential and alternative uses of ethnotaxa in Kujawska and Haydee´ Gonzalez´ for their valuable idiomatic different preparations, consistent with the particular charac- contributions, to Guillermo Gil for advise in statistical anal- teristics of each ethnotaxon (oily, mealy, hard, or soft, etc.). yses, and to anonymous reviewers for improving the paper. Instead, we found that local crop modellers of ethnotaxa varybyregion.WehavenotedthatLosToldosistheonly References region in which the medicinal and ritual uses have significant importance in the conservation of ethnotaxa. This could be [1] F. Wolff, “Legal factors driving agrobiodiversity loss,” ELNI interpreted as an indicator of greater conservation of medici- Review,vol.1,11pages,2004,http://www.agrobiodiversitaet nalandritualpracticesassociatedwithcorn,althoughitcould .net/site/page/downloads/dateien/ABD.Elni.pdf. also be a result of the long association between villagers and [2] S. B. Brush, M. B. Corrales, and E. Schmidt, “Agricultural devel- researchers, which facilitates the obtention of information opment and maize diversity in Mexico,” Human Ecology,vol.16, not typically shared with outsiders, in accordance with the no. 3, pp. 307–328, 1988. experienced by Pinotti et al. [45] in neighbouring regions. [3] D. Velasquez-Milla,´ A. Casas, J. Torres-Guevara, and A. Cruz- In San Andres for a change, the use of maize as a fodder Soriano, “Ecological and socio-cultural factors influencing in has the principal application among the cultivated ethnotaxa. situ conservation of crop diversity by traditional Andean house- This is probably due to the changes in land use experienced holds in Peru,” Journal of Ethnobiology and Ethnomedicine,vol. duringthelast20years.Itisspeciallyevidentintheaban- 7, article no. 40, 2011. donment of the vertical use of the environment and the slow [4] D.Louette,A.Charrier,andJ.Berthaud,“Insituconservationof abandonment of transhumance [26, 43]. maize in Mexico: genetic diversity and maize seed management Regarding the medicinal applications, it has been in a traditional community,” Economic Botany,vol.51,no.1,pp. observed that the corn is used in many recipes that include 20–38, 1997. other plant species (Table 1). In these recipes, we observed [5] A. E. Izquierdo and H. R. Grau, “Agriculture adjustment, land- indiscriminate frequent employment of marketed cornstarch use transition and protected areas in Northwestern Argentina,” JournalofEnvironmentalManagement,vol.90,no.2,pp.858– or flour of any ethnotaxon. This suggests that the corn is 865, 2009. used as a binder or thickener, rather than for its medicinal [6]J.W.JanusekandA.L.Kolata,“Top-downorbottom-up:rural properties. In other regions, this flexibility and replacement settlement and raised field agriculture in the Lake Titicaca of products in medicinal preparations have been recorded Basin, Bolivia,” Journal of Anthropological Archaeology,vol.23, as common in domestic medical practice and is generally no. 4, pp. 404–430, 2004. associated with the availability of products [47–49]. [7] F. Berkes, J. Colding, and C. Folke, “Rediscovery of traditional Therefore, it is important to conduct in the future more ecological knowledge as adaptive management,” Ecological investigations on the role assigned to different resources to Applications,vol.10,no.5,pp.1251–1262,2000. prepare medicines and identify the ethnotaxa with preferen- [8]J.R.Veteto,Seeds of persistence: agrobiodiversity, culture, and tial medicinal use and also those which function as replace- conservation in the American Mountanin South [Ph.D. disserta- ment alternatives for the most important ones. In respect to tion],UniversityofGeorgia,Athens,2010. the protective or propitiatory role assigned to corn culli, [9] V. D. Nazarea, “Local knowledge and memory in biodiversity similar uses have been cited for other ethnotaxa in Mexico conservation,” Annual Review of Anthropology,vol.35,pp.317– [50]. 335, 2006. Evidence-Based Complementary and Alternative Medicine 9

[10] V. D. Nazarea, Cultural Memory and Biodiversity, University of [26] N. I. Hilgert and G. E. Gil, “Traditional andean agriculture and Arizona Press, Tucson, Ariz, USA, 1998. changing processes in the Zenta river basin, Salta, Northwestern [11] H. R. Perales, B. F. Benz, and S. B. Brush, “Maize diversity and Argentina,” Darwiniana,vol.43,no.1–4,pp.30–43,2005. ethnolinguistic diversity in Chiapas, Mexico,” Proceedings of the [27] J. Tuxill, Agrarian change and crop diversity in Mayan milpas of National Academy of Sciences of the United States of America, Yucatan, Mexico: implications for in situ conservation [Ph.D. dis- vol. 102, no. 3, pp. 949–954, 2005. sertation], Yale School of Forestry and Environmental Studies [12] G. P. Nabhan, “Agrobiodiversity change in a Saharan desert and The New York Botanical Garden, 2005. oasis, 1919–2006: historic shifts in Tasiwit (Berber) and Bedouin [28] B. C. Campbell, Developing dependence, encountering resistance: crop inventories of Siwa, Egypt,” Economic Botany,vol.61,no.1, the historical ethnoecology of farming in the Missouri Ozarks pp.31–43,2007. [Ph.D. dissertation], Department of Anthropology, University [13] V. D. Nazarea, Local Knowledge and Agricultural Decision Mak- of Georgia, 2005. ing in the Philippines: Class, Gender, and Resistance,Cornell [29] V.Nu´nez˜ Regueiro and M. Tartusi, “Aproximacion´ al estudio del University Press, Ithaca, NY, USA, 1995. area´ pedemontana de Sudamerica,”´ Cuadernos del INA,vol.12, [14] S. B. Brush, “Cultural research on the origin and maintenance pp.125–160,1990. of agricultural diversity,” in Nature Knowledge: Ethnoscience, [30] N. I. Hilgert and G. E. Gil, “Medicinal plants of the Argentine Cognition, and Utility,G.SangaandG.Ortalli,Eds.,pp.379– Yungas plants of the Las Yungas biosphere reserve, Northwest of 385, Berghahn Books, New York, NY, USA, 2005. Argentina, used in health care,” Biodiversity and Conservation, [15]S.B.Brush,H.J.Carney,andZ.Human,´ “Dynamics of Andean vol. 15, no. 8, pp. 2565–2594, 2006. potato agriculture,” Economic Botany,vol.35,no.1,pp.70–88, [31] N. I. Hilgert, “La salud en las Yungas. ¿Cuales´ son los principales 1981. problemas segun´ la medicina tradicional y la formal?” in Avan- [16] R. E, Rhoades, Breaking New Ground: Agricultural Anthropology, ces Sobre Plantas Medicinales Andinas,V.N.DoraandP.M. International Potato Center, Lima, Peru, 1984. Lelia, Eds., vol. 1, pp. 1–43, RISAPRET/CYTED, S. S. de Jujuy, Argentina, 2009. [17] T. Ingold, The Perception of the Environment: Essays in Liveli- hood, Dwelling, and Skill,Routledge,London,UK,2000. [32] N. I. Hilgert and G. E. Gil, “Los cambios de uso del ambiente y la medicina herbolaria. Estudio de caso en Yungas Argentina,” [18] G. S. Torino, “El ritual del d´ıa de las almas como una forma de BLACPMA,vol.7,no.3,pp.130–140,2008. comunicacion´ social: Ya vienen las almitas, esperemos la senal,˜ preparemos comidita con hambre han de llegar,” Cuadernos de [33] D. A. Lambare,´ N. I. Hilgert, and R. S. Ramos, “Dyeing plants la Facultad de Humanidades y Ciencias Sociales,vol.36,pp.109– and knowledge transfer in the yungas communities of North- 120, 2009. west Argentina,” Economic Botany,vol.65,no.3,pp.315–328, 2011. [19] M. P. Babot, M. Marschodd, and F. Pazzarelli, Las manos en la masa: arqueolog´ıas, antropolog´ıas e historias de la alimentacion´ [34] J. Camara´ Hernandez, A. M. Miante Alzogaray, R. Bellon,´ and en Suramerica´ , Facultad de Filosof´ıa y Humanidades, Univer- J. A. Galmarini, Razas de ma´ız nativas de la Argentina,Facultad sidad Nacional de Cordoba,´ Museo de Antropolog´ıa UNC, de Agronom´ıa,UBA,BuenosAires,Argentina,2012. Instituto Superior de Estudios Sociales UNT, Cordoba,´ Spain, [35] I. Molina, “Reflexiones sobre la clasificacion´ de medicinas. 1st edition, 2012. Analisis´ de una propuesta conceptual,” Scripta Ethnologica,vol. [ 2 0 ] V. W. Tu r n e r, La Selva de los S´ımbolos. Aspectos del ritual 27, pp. 111–147, 2005. ndembu, Siglo Veintiuno Ediciones, Madrid, Spain, 1980. [36] N. I. Hilgert, “Plants used in home medicine in the Zenta River basin, Northwest Argentina,” Journal of Ethnopharmacology, [21] O. Sturzenegger, “Area´ de la Selva Tucumano-Oranense. San vol.76,no.1,pp.11–34,2001. Andres:´ actividades de subsistencia tradicionales y ritual prop- iciatorio,” in Programa de Investigaciones sobre Epidemiolog´ıa [37] N. I. Hilgert and G. E. Gil, “Reproductive medicine in northwest Psiquiatrica´ , Documenta Laboris 27, Consejo Nacional de Argentina: traditional and institutional systems,” Journal of Investigaciones Cient´ıficas y Tecnicas,´ Buenos Aires, Argentina, Ethnobiology and Ethnomedicine,vol.3,article19,2007. 1982. [38] E. Amodio, “Cocacha mamacha. Practicas´ adivinatorias y mito- [22] G. Torres, “Curanderismo y Brujer´ıa en el area´ de la selva Tucu- log´ıa de la coca entre los quechuas del Peru,”´ Societesuissedes´ mano-Oranense,”in Programa de Investigaciones sobre Epidemi- Americanistes´ ,vol.57-58,pp.123–137,1993. olog´ıa Psiquiatrica´ (PEPSI-CONICET),vol.2,pp.1–38,Docu- [39] R. Garc´ıa-Morales,C.E.Moreno,andJ.Bello-Gutierrez,´ “Ren- menta Laboris, Buenos Aires, Argentina, 1982. ovando las medidas para evaluar la diversidad en comunidades [23] M. C. Bianchetti, Cosmovision´ sobrenatural de la locura: pautas ecologicas:´ el numero´ de especies efectivas de murcielagos´ en el populares de salud mental en la puna argentina,V´ıctor Manuel sureste de Tabasco, Mexico,”´ Therya,vol.2,no.3,pp.205–215, Hanne, Salta, Argentina, 1999. 2011. [24] N. I. Hilgert, “Las plantas en las festividades religiosas de la selva [40] StatSoft, STATISTICA (data analysis software system), version andina argentina,” Societ´ esuissedesAm´ ericanistes´ ,vol.68,pp. 8.0., 2007, http://www.statsoft.com/ . 37–49, 2004. [41] N. I. Hilgert, “Las plantas comestibles en una zona de las Yun- [25] I. Lantos, M. Maier, and N. Ratto, “Recreando recetas: primeros gas meridionales (Argentina),” Anales del Jard´ın Botanico´ de resultados de una experimentacion´ con variedades nativas de Madrid,vol.55,no.1,pp.117–138,1999. ma´ız del noroeste argentino,”in Las manos en la masa: arqueolo- [42] J. C. Hernandez´ and D. A. de Cabezas, Ma´ıces andinos y sus usos g´ıas, antropolog´ıas e historias de la alimentacion´ en Suramerica´ , en la Quebrada de Humahuaca, Facultad de Agronom´ıa, UBA, M. P.Babot, M. Marschodd, and F. Pazzarelli, Eds., pp. 527–552, Buenos Aires, Argentina, 2007. Facultad de Filosof´ıa y Humanidades, Universidad Nacional de [43] M. G. Moritan´ and A. D. Brown, “Organizacion´ social: conflic- Cordoba,´ Museo de Antropolog´ıa UNC, Instituto Superior de tos sociales y diversidad de actores,” in Finca San Andres.´ Un Estudios Sociales UNT, Cordoba,´ Spain, 1st edition, 2012. espacio de cambios ambientales y sociales en el Alto Bermejo, 10 Evidence-Based Complementary and Alternative Medicine

A. D. Brown, M. Garc´ıa Moritan,B.N.Ventura,N.I.Hilgert,´ andL.R.Malizia,Eds.,vol.5,pp.129–158,2007. [44] V. M. Toledo and N. Barrera-Bassols, Agroecolog´ıa y Sabidur´ıas Tradicionales: Un Panorama Mundial en la Memoria Biocul- tural, Icaria editorial, Barcelona, Spain, 2008. [45] L. V. Pinotti, R. L. Pinto, M. A.´ Ferrari et al., “Modalidades ali- mentarias en la quebrada de humahuaca patrimonializada,” in Las manos en la masa: arqueolog´ıas, antropolog´ıas e historias de la alimentacion´ en Suramerica´ ,M.P.Babot,M.Marschodd, and F. Pazzarelli, Eds., pp. 185–205, Facultad de Filosof´ıa y Humanidades, Universidad Nacional de Cordoba,´ Museo de Antropolog´ıa UNC, Instituto Superior de Estudios Sociales UNT, Cordoba,´ Spain, 1st edition, 2012. [46] D. D´ıaz, S. Guerrero, S. Naumann, and G. Sammartino, “Ali- mentacion´ en la quebrada de humahuaca continuidad y discon- tinuidades desde el poblamiento hasta nuestros d´ıas. Un aporte desdelaantropolog´ıa alimentaria,” in Las manos en la masa: arqueolog´ıas, antropolog´ıas e historias de la alimentacion´ en Suramerica´ , M. P. Babot, M. Marschodd, and F. Pazzarelli, Eds., pp. 163–184, Facultad de Filosof´ıa y Humanidades, Universidad Nacional de Cordoba,´ Museo de Antropolog´ıa UNC, Instituto Superior de Estudios Sociales UNT, Cordoba,´ Spain, 1st edition, 2012. [47] K. C. Ford, LasYerbasdelaGente:AStudyofHispano-American Medicinal Plants,vol.60ofAnthropological Papers,Museumof Anthropology University of Michigan, Ann Arbor, Mich, USA, 1975. [48] F. Zamudio and N. I. Hilgert, “Mieles y plantas en la medicina criolla del Norte de Misiones, Argentina,” Bonplandia,vol.20, no. 2, pp. 165–184, 2011. [49] M. Kujawska, F. Zamudio, and N. I. Hilgert, “Honey-based mixtures used in home medicine by nonindigenous population of Misiones, Argentina,” Evidence-based Complementary and Alternative Medicine,vol.2012,ArticleID579350,15pages, 2012. [50] E. Hernandez´ X, “Maize and man in the greater southwest,” Economic Botany,vol.39,no.4,pp.416–430,1985. [51] B. Frei, O. Sticher, T.Carlos Viesca, and M. Heinrich, “Medicinal and food plants: isthmus Sierra Zapotec criteria for selection,” Journal of Applied Botany,vol.72,no.3-4,pp.82–86,1998. [52] B. Frei, O. Sticher, and M. Heinrich, “Zapotec and mixe use of tropical habitats for securing medicinal plants in Mexico,” Eco- nomic Botany,vol.54,no.1,pp.73–81,2000. [53] A. Ankli, O. Sticher, and M. Heinrich, “Yucatec Maya medicinal plants versus nonmedicinal plants: indigenous characterization and selection,” Human Ecology,vol.27,no.4,pp.557–580,1999. [54] S. Molares, Flora medicinal aromaticadelaPatagonia:carac-´ ter´ısticas anatomicas´ y propiedades organolepticas´ utilizadas en el reconocimiento por parte de la terapeutica´ popular [Ph.D. thesis], Universidad Nacional del Comahue, Bariloche, Argentina, 2010. [55] M. Leonti, O. Sticher, and M. Heinrich, “Medicinal plants of the Popoluca, Mexico:´ organoleptic properties as indigenous selection criteria,” Journal of Ethnopharmacology,vol.81,no.3,pp. 307–315, 2002. [56] J. S. Boster, “Selection for perceptual distinctiveness: evidence from aguaruna cultivars of manihot esculenta,” Economic Bot- any,vol.39,no.3,pp.310–325,1985. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 670352, 12 pages http://dx.doi.org/10.1155/2013/670352

Research Article Wild Animals Used as Food Medicine in Brazil

Rômulo Romeu Nóbrega Alves,1 Tacyana Pereira Ribeiro Oliveira,2,3 and Ierecê Lucena Rosa3 1 Departamento de Biologia, Universidade Estadual da Para´ıba, Avenida das Baraunas,´ 351/Campus Universitario,´ Bodocongo´ 58109-753, Campina Grande, PB, Brazil 2 Centro de Cienciasˆ Biologicas´ e Sociais Aplicadas, Universidade Estadual da Para´ıba, Rua Horacio´ Trajano de Oliveira, s/n, Campus V, 58020-540 Joao˜ Pessoa, PB, Brazil 3 Departamento de Sistematica´ e Ecologia, Universidade Federal da Para´ıba, 58059-900 Joao˜ Pessoa, PB, Brazil

Correspondence should be addressed to Romuloˆ Romeu Nobrega´ Alves; romulo [email protected]

Received 5 April 2013; Accepted 4 July 2013

Academic Editor: Maria Franco Trindade Medeiros

Copyright © 2013 Romuloˆ Romeu Nobrega´ Alves et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The connection between eating and healing is common in traditional folk medical systems, and the multiple possibilities resulting from the combination of biodiversity and culture confer a wealth and complexity in terms of knowledge of the flora and fauna as to their potential as food medicine. The growing awareness of the links between traditional therapeutic-alimentary uses of wildlife and conservation has drawn attention to the gaps in knowledge on the social, economic, and biological contexts in which different forms of traditional wildlife uses take place, particularly with regard to zootherapeutic resources. In this study we interviewed 124 merchants and 203 traditional users of animal-derived remedies in Brazil, aiming at documenting the animal species used as foods and medicines in urban and rural areas of the country. At least 354 wild animal species are used in Brazilian traditional medicine, of which 157 are also used as food. The high degree of overlap between medicinal and alimentary uses of wild animals highlights the importance of understanding the socioeconomic, cultural, and ecological contexts in which those traditional uses take place for elucidating their potential impact on public health and biodiversity conservation.

1. Introduction oldastheuseofbiodiversitybyhumans[6]. For ancient Assyrians, Greeks, and Chinese, cultivated grains, roots, Nature-based traditional food and medicine are generally fruits, and spices were frequently employed in the curing viewed as interchangeable, diet being highly regarded as the arts, while, in pharaonic Egypt, plants such as lettuce, sesame, primary basis for sustaining and/or restoring health and well- onions, leeks, cucumbers, plums, watermelon, and many being. Consequently, foods are considered and often times other edibles were included in the healer’s arsenal [11]. Histor- chosen for their distinctive medicinal or healing values [1–4]. ical sources of ancient Egypt also mention the medicinal uses For many traditional societies, now and in the past, food is— of animal-derived substances, such as cattle milk, bee honey, at least partially—medicine, and medicine is food [5]. Medic- lizard blood, ox organs, swallow’s liver, bat limbs, ambergris inal cuisines and consumption of health edibles have held a fromthespermwhale,andtheglandsofthemuskdeer[12– central position in traditional folk medical systems [6–10]. 15]. Much of the plant material that is consumed by animals Atthedawnofrecordedhistoryitisknownthatman inthewildcontainsanarrayofsecondarycompounds.Johns often ate or wore on his person some portion of an animal [6] argues that the herbal medicines and modern pharmaceu- that was thought to have a healing or protecting influence ticals used by humans today have replaced the nonnutritive [16], and this aspect highlights that the origin of the medicinal chemicals commonly present in our primate ancestors’ diets, useoffaunalelementsisintertwinedwiththeiruseasfood.In the connection between eating and healing likely being as this same direction, Chemas [17] remarked that the treatment 2 Evidence-Based Complementary and Alternative Medicine of illnesses using animal-based remedies is an extremely old geographical isolation contributes to strengthen traditional practice, whose most remote ancestor is a carnivore diet, and local medical practices and, also, to prompt selection closelyfollowedbytheritualingestionofdeceasedpersons of natural resources for the treatment of new diseases [25, (e.g., close relatives, warriors) as a means to absorb their 33]. Interest in animal-derived remedies, however, extends virtues (e.g., courage, virility) and subsequently by a true beyond people lacking access to medical services in Brazil. medicinal use indissociable from magic-religious elements. AsshownbyAlvesandRosa[26, 27], even in cities where Since ancient times, the thinking of “food as medicine” such services are more accessible, many people still resort to has existed in Chinese medical theories and Chinese food traditional healers, showing the cultural acceptability of such therapy [18, 19]. Books on Chinese cooking often empha- practices. size the medicinal value of foods and the importance of In this study we explored the medicinal and alimentary “nutritional therapies” dating from earliest times, and many uses of wildlife in Brazil, aiming to (1) document the animal of the vegetable and animal products decocted in Chinese species used and the illnesses to which they were prescribed medicines are used routinely in cooking. During the 1980s, and (2) to discuss resource use in a conservationist context. talk at banquets frequently revolved around the healthful properties of foods being consumed, and nutritional and food preparation advice was commonly tendered in clinics along 2. Methods with herbal prescriptions [20]. Research in several regions of the world has illustrated Data were collected from January 2002 to June 2012. Data that many wild plants retained in local food cultures are consisted in (a) interviews in markets/shops located in the inseparable from traditional therapeutic systems [7, 9, 21, cities of Belem´ (Para´ State), Sao˜ Lu´ıs (Maranhao˜ State), 22]. For example, in a rural Hausa community in northern Teresina (Piau´ı State), Goianiaˆ (Goias´ State), Natal (Rio Nigeria,ofthe119plantsidentifiedasfood,allbutfiveare Grande do Norte State), Joao˜ Pessoa, Campina Grande includedamongthetotal374medicinals.This,however,does (Para´ıba State), Recife (Pernambuco State), Maceio(Alagoas´ notmeanthatHausaintermixesthedomainsoffoodand State), Aracaju´ (Sergipe State), Salvador (Bahia State), Vitoria´ medicine [7]. Examples of a number of food animals also (Esp´ırito Santo State), Niteroi´ (Rio de Janeiro State), Flo- used as remedies can be found in the literature [23–28]. Yet rianopolis´ (Santa Catarina State), and Porto Alegre (Rio our knowledge about the practice of food medicine is limited, Grande do Sul State), where we documented the animal particularly with regard to the traditional consumption of medicinal foods traded, (b) interviews in outdoor markets animal food medicines [29]. to 124 merchants about the use and commercialization of Although often regarded as supplementary to local peo- medicinal animals (23 interviewees in Belem,´ 21 in Sao˜ Lu´ıs, ples’ diet, wild food and medicine are essential in times of cri- 21inTeresina,16inSantaCruz,11inCaruaru,10inJoao˜ sis and play an important nutritional role. Hence, the neglect Pessoa, and 22 in Campina Grande), (c), and interviews with of traditional food and medicines may seriously deteriorate 203 traditional users of animal-derived remedies (67 men and the health and well-being of traditional peoples [30, 31]. 70 women) in the following rural communities: Municipality Further, nature-based traditional food and medicine are gen- of Cajueiro da Praia (Piau´ıState)(𝑛=36), Pesqueiro Beach, erally viewed as interchangeable, diet being highly regarded Municipality of Soure (ParaState)(´ 𝑛=41), Environmental as the primary basis for sustaining and/or restoring health Protected Area Barra do Rio Mamanguape, Municipality and well-being. Consequently, foods are considered and often of Rio Tinto (Para´ıbaState)(𝑛=30), Municipality of times chosen for their distinctive medicinal or healing values. Queimadas (Para´ıba State) (𝑛=66), and Municipality of Animal medicinal foods have been broadly used since Raposa (Maranhao˜ State) (𝑛=30), as described in Alves and ancient times and have played a significant role in healing Rosa [1, 25, 26]. practices in Brazil [3, 32], where elements of indigenous, In cities, the sampling method was nonrandom, and European, and African cultures met and produced a singular the interviewees were predefined [34]. Despite attempts to repertoire of species that are used as food and often also as interview all animal merchants in the markets visited, some medicine. interviews were cancelled. Others proved to be fruitless, Brazil provides an interesting setting for several reasons: because interviewees were reluctant to answer questions. (a) the country possesses between 15 and 20% of all the world’s At the surveyed fishing communities, we identified local biological diversity, as well as a significant cultural diversity, people with a specialized knowledge of medicinal animal use. represented by more than 200 indigenous groups and by a Additional interviewees were chosen by using the snowball large number of local communities which detain a consid- technique [35], based on information initially provided by the erableknowledgeofthefloraandfaunaandoftraditional specialists. systems of renewable natural resources management [25]; Torespect intellectual property rights, we adopted the fol- (b) the multiple possibilities resulting from this combination lowing protocol in the field: before the survey, we introduced of biodiversity and culture confer a wealth and complexity ourselves, explained the nature and objectives of our research, in terms of knowledge of the Brazilian flora and fauna as and asked the respondents for permission to record the to its therapeutic potential; (c) in addition, Brazil is vast, information. The ethical approval for the study was obtained with parts of the territory of difficult access; this precludes from the Ethics Committee of Paraiba University State. some local populations from accessing services provided by The information obtained through semistructured inter- the government’s health care network. In many cases this views was complemented by free interviews [36], and, Evidence-Based Complementary and Alternative Medicine 3 for each animal cited, respondents were requested to fur- Fishes 77 nish vernacular name, folk use, parts used, preparation and administration of remedy, and which animal species are Mammals 35 also used as food. Zoological material was identified with the aid of specialists, through (1) examination of voucher Reptiles 20 specimens (donated by the interviewees) (2) photographs of the animals or their parts, taken during interviews; and (3) Birds 11 vernacular names, with the aid of taxonomists familiar with the study areas’ fauna. Only wild animals and taxa that could Crustaceans 9 be identified to species level were included in the database. Records of animal-based folk remedies were gathered Molluscs 5 from scientific articles, books, and book chapters, the- ses, and dissertations, as well as from reports available 0 20 40 60 80 100 in international online databases such as Science Direct Number of animals (http://www.sciencedirect.com/), Scirus (http://www.scirus .com/), Google Scholar, Scopus (http://www.scopus.com/), Figure 1: Number of animal species, per taxonomic category, used Web of Science (http://www.isiknowledge.com/), and Bio- as food and medicines in Brazil. logical Abstracts (http://science.thomsonreuters.com/)using the following search terms: medicinal animals + use + Brazil, zootherapy + commercialization + Brazil, hunting + of the medicinal uses of animals in human history are clear: medicinal animals + Brazil, and, and zootherapy + Brazil. animals and their products were part of the primary resources Whenever applicable, scientific names provided in pub- that ancient peoples could use as food or for treating their lications were updated using ITIS Catalogue of Life: 2012 illnesses. Annual Checklist (http://www.catalogueoflife.org/). Most of the time, the hunted or fished animal whose Information on the conservation status of animal species meat is consumed as food also provides byproducts that are was obtained from the International Union for Conser- used for medicinal purposes, such as skin or fats. In fact, vation of Nature’s Red List (http://www.iucnredlist.org/), the utilization of remainings or by-products seems to be the Convention on the International Trade in Endangered widespread and one of the most striking characteristics of the SpeciesofWildFaunaandFlora(http://www.cites.org/eng/ Brazilian folk medicine, in terms of medicinal animals [41]. In resources/species.html), Brazil’s Official List of Endangered this sense, it is remarkable that in general the animal-based Species [37] and National List of Species of Aquatic Inverte- medicinal products constitute by-products from animals brates and Fishes Endangered, Overexploited, or Threatened hunted for other purposes. Such multiple uses (including of Exploitation [38]. medicinal) of fauna and their impact on animal populations must be properly assessed and taken into consideration when implementing recovery plans for these target species, 3. Results and Discussion especially those that are highly exploited [3, 27, 42]. The high number of fish species recorded as medicinal Animals used as medicine food recorded in our study were foods in this study was expected, given their high consump- distributed in six zoological groups. As shown in Figure 1, tion as food, mainly in coastal areas. As pointed by Burger the taxon with the largest number of species was fishes andGochfeld[43], in many parts of the world more than (77 species; 49.0%), followed by mammals (35; 22.3%) and halfofthepeopleliveincoastalcommunitieswherefishis reptiles (20; 12.7%). These results are in line with previ- prominent in their diets. Although people who live near the ous studies carried out elsewhere (eg., 28, 40, and 41– sea eat more seafood than those who live in the hinterland, 46), further highlighting the widespread use of wild-caught seafood, both fresh and frozen, has become increasingly vertebrates in the diets and medical systems of different available and is gaining in popularity throughout the world. societies. In addition to being an important and available source of Generally, species are harvested through fishing or hunt- protein, the popularity of fish as food is also due to the ing, mainly for alimentary purposes. In this sense, their fact that they are considered healthy. Fishes are considered utilization as remedies potentializes resource use. Meat, the an excellent and low-fat source of protein, provide many principal product consumed as food, in some cases was health benefits, such as omega-3 fatty acids that reduce also consumed due to a perceived medicinal value. This cholesterol levels and the incidence of stroke, heart disease, distinction, however, was not always clear-cut, as the same and preterm delivery, and enhance cognitive development animal can be one, the other, or both categories at the [44–52]. same time, depending on the parts used, the method of Among terrestrial vertebrates, mammals are the most preparation, and the state of health or pathology of the hunted taxon for alimentary purposes. The frequent use of individual being treated. This result is in line with Huffman those animals as medicinal foods was expected, given their [39], who remarked that in traditional human societies, the comparatively larger body size (when compared with other difference between food and medicine may not always be terrestrial vertebrates) and the possibility they offer of a clear. In fact, according to O’Hara-May [40], the beginnings higher energetic intake. In the neotropical region, mammals 4 Evidence-Based Complementary and Alternative Medicine

Table 1: Animal species used as food and medicine in Brazil and conditions to which remedies are prescribed.

Taxonomic category/species Conditions to which remedies are prescribed Molluscs Phacoides pectinata (Gmelin, 1791) Sexual impotence Mytella charruana (Orbigny, 1842) Ophthalmological problems Mytella guyanensis (Lamarck, 1819) Weakness Osteoporosis, pneumonia, stomach ache, cancer, flu, Crassostrea rhizophorae (Guilding, 1828) weakness, pain relief in injuries caused by the dorsal fin spine of a species of catfish, anemia, and tuberculosis Anomalocardia brasiliana (Gmelin, 1791) Asthma, flu, stomach and ache Crustaceans Cardisoma guanhumi (Latreille, 1825)NE/IN Asthma, bronchitis, wounds, and boils Goniopsis cruentata (Latreille, 1802) Epilepsy, venereal disease Hemorrhage in women, incontinence, osteoporosis, Ucides cordatus (Linnaeus, 1763)DD/IN cough, asthma, tuberculosis, womb disorders, arthrosis, and bronchitis Macrobrachium carcinus (Linnaeus, 1758) Amnesia Macrobrachium acanthurus (Wiegmann, 1836) Irritation when milk teeth are erupting Macrobrachium borellii (Nobili, 1896) Irritation when milk teeth are erupting Xiphopenaeus schmitti (Burkenroad, 1936) Irritation when milk teeth are erupting, skin spots Xiphopenaeus kroyeri (Heller, 1862) Irritation when milk teeth are erupting, skin spots Epilepsy, to alleviate the symptoms of intoxication with Aratus pisonii (H. Milne Edwards, 1837) poison of Colomesus psittacus (a species of pufferfish) Fishes Trachelyopterus galeatus (Linnaeus, 1766) Umbilical hernia, asthma, and sexual impotence Leporinus friderici (Bloch, 1794) Earache Schizodon knerii (Steindachner, 1875) Leucoma, edema Bagre bagre (Linnaeus, 1766) Injuries caused by itself Genidens barbus (Lacepede,` 1803) Pain relief caused in injuries by the species’ sting Genidens genidens (Cuvier, 1829) Injuries caused by itself Sciadeichthys luniscutis (Valenciennes, 1837) Pain relief caused in injuries by the species’ sting Aspredo aspredo (Linnaeus, 1758) Asthma Aspredinichthys tibicen (Valenciennes, 1840) Asthma Balistes capriscus (Gronow, 1854)DD/IN Bronchitis Stroke, asthma, thrombosis, earache, pain relief caused in injuries by the species’ sting, hemorrhage, ascites, Balistes vetula (Linnaeus, 1758)VU schistosomiasis, appendicitis, menstrual cramps, and gastritis Thalassophryne nattereri (Steindachner, 1876) Pain relief caused in injuries by the species’ sting Callichthys callichthys (Linnaeus, 1758) Asthma, umbilical hernia Carcharhinus limbatus (Muller¨ and Henle, 1839)LC Osteoporosis Asthma, rheumatism, wounds, inflammations, Carcharhinus porosus (Ranzani, 1840) osteoporosis, and anemia Galeocerdo cuvier (Peron´ and Lesueur, 1822)LC Osteoporosis Rhizoprionodon lalandii (Muller¨ and Henle, 1839) Rheumatism Rhizoprionodon porosus (Poey, 1861) Rheumatism Sphyrna lewini (Griffith and Smith, 1834)LC/IN Asthma, wounds, rheumatism, and inflammation Centropomus parallelus (Poey, 1860) Nephritis Centropomus undecimalis (Bloch, 1792) Edema in the legs Alcoholism, leishmaniasis, skin burns, wounds, and Astyanax bimaculatus (Linnaeus, 1758) rheumatism Brycon nattereri (Gunther,¨ 1864) Flu Colossoma macropomum (Cuvier, 1818)DD/IN Paralysi of arms and legs Evidence-Based Complementary and Alternative Medicine 5

Table 1: Continued. Taxonomic category/species Conditions to which remedies are prescribed Hydrolycus scomberoides (Cuvier, 1816) Earache Opisthonema oglinum (Lesueur, 1818) Alcoholism Asthma, pain relief caused in injuries by the species’ Dasyatis guttata (Bloch and Schneider, 1801) sting, and burns Asthma, pain relief caused in injuries by the species’ Dasyatis marianae (Gomes, Rosa, and Gadig, 2000) sting, and burns Franciscodoras marmoratus (Reinhardt, 1874) Injuries caused by itself Lithodoras dorsalis (Valenciennes, 1840) Swelling Megalodoras uranoscopus (Eigenmann and Eigenmann, 1888) Rheumatism Platydoras costatus (Linnaeus, 1758) Rheumatism Pterodoras granulosus (Valenciennes, 1821) Rheumatism Oxydoras niger (Valenciennes, 1821) Rheumatism Echeneis naucrates Linnaeus, 1758 Asthma, bronchitis Erythrinus erythrinus (Bloch and Schneider, 1801) Asthma Ophthalmological problems, rheumatism, cataracts, wounds, snake bite, conjunctivitis, stroke, thrombosis, Hoplias malabaricus (Bloch, 1794) asthma, toothache, fever, earache, diarrhea, deafness, boils, bleedings, alcoholism, tetanus, sore throat, itching, sprains, and leucoma Gadus morhua (Linnaeus, 1758)VU Boils Ginglymostoma cirratum (Bonnaterre, 1788)DD Rheumatism Pimelodella brasiliensis (Steindachner, 1876) Injuries caused by that fish species Holocentrus adscensionis (Osbeck, 1765) Wounds Stroke, headache, asthma, shortness of breath, Megalops atlanticus (Valenciennes, 1847) thrombosis, chest pain, and injuries caused by bang Gymnothorax funebris (Ranzani, 1840) Bleeding (wounds) Gymnothorax moringa (Cuvier, 1829) Bleeding (wounds) Gymnothorax vicinus (Castelnau, 1855) Bleeding (wounds) Asthma, pain relief caused in injuries by the species’ Aetobatus narinari (Euphrasen, 1790)LC sting, burns, and hemorrhage Narcine brasiliensis (Olfers, 1831) Toothache Arapaima gigas (Schinz, 1822)DD/II/IN Asthma, pneumonia Asthma, wounds, hernia, burns in the skin, Phractocephalus hemioliopterus (Bloch and Schneider, 1801) rheumatism, flu, and cough Pseudoplatystoma corruscans (Spix and Agassiz, 1829) Flu Pseudoplatystoma fasciatum (Lunnaeus, 1776) Cold Sorubimichthys planiceps (Spix and Agassiz, 1829) Leishmaniasis, tuberculosis Asthma, toothache, earache, wounds, athlete’s foot, Zungaro zungaro (Humboldt, 1821)DD/IN burns in the skin, rheumatism, and flu Asthma,hernia,flu,pneumonia,cough,earache,and Paratrygon aiereba (Muller¨ and Henle, 1841) burns Asthma,hernia,flu,pneumonia,cough,earache,and Potamotrygon hystrix (Muller¨ and Henle, 1834) burns Asthma,hernia,flu,pneumonia,cough,earache,and Potamotrygon motoro (Muller¨ and Henle, 1841) burns Potamotrygon orbignyi (Castelnau, 1855) Pain relief caused in injuries by that species’ sting Pain relief caused in injuries by the species’ sting, Plesiotrygon iwamae (Rosa, Castello, and Thorson, 1987) wounds, and cracks in the sole of the feet Pristis pectinata (Latham, 1794)CR Asthma, rheumatism, and arthritis Pristis perotteti (Muller¨ and Henle, 1841)CR Asthma, rheumatism, and arthritis Prochilodus argenteus (Spix and Agassiz, 1829) To avoid swelling of the breast feeding, mycosis Chilblain, skin burns, wounds, rheumatism, and eye Prochilodus nigricans (Spix and Agassiz, 1829) pains 6 Evidence-Based Complementary and Alternative Medicine

Table 1: Continued. Taxonomic category/species Conditions to which remedies are prescribed Atlantoraja cyclophora (Regan, 1903)VU Hemorrhage after delivery Mylossoma duriventre (Cuvier, 1818) Venereal disease Serrasalmus brandtii (Lutken,¨ 1875) Inflammations, sexual impotence Cynoscion acoupa (Lacepede,` 1801) Renal failure Cynoscion leiarchus (Cuvier, 1830) Renal failure Pain relief caused in injuries by the species’ sting, Micropogonias furnieri (Desmarest, 1823) cough, asthma, and bronchitis Pachyurus francisci (Cuvier, 1830) Asthma, urinary incontinence, and backache Plagioscion surinamensis (Bleeker, 1873) Urinary disorders, hemorrhage, and snake bites Plagioscion squamosissimus (Heckel, 1840) Urinary disorders, hemorrhage, and snake bites Calamus penna (Valenciennes, 1830) Asthma Synbranchus marmoratus (Bloch, 1795) Bronchitis Colomesus psittacus (Bloch and Schneider, 1801) Breast cancer, backache, and warts Sphoeroides testudineus (Linnaeus, 1758) Rheumatism Trichiurus lepturus (Linnaeus, 1758) Asthma Asthma, pain relief caused in injuries by the species’ Urotrygon microphthalmum (Delsman, 1941) sting, and burns Reptiles Earache, erysipelas, asthma, rheumatism, edema, abscesses, joint pain, wounds, acne, athlete’s foot, sore Iguana iguana (Linnaeus, 1758)DD/II throat,swelling,burn,tumor,tosuckasplinteroutof skin or flesh, boil, injuries caused by the spines of the “arraia” and others fishes, inflammation, and hernia Earache, deafness, rheumatism, erysipelas, skin thorns and wounds, respiratory diseases, sore throat, snake Tupinambis merianae (Dumeril´ and Bibron, 1839)DD/II bite, asthma, tumor, swelling, infection, and bronchitis

Sexual impotence, rheumatism, erysipelas, dermatitis, snake bites, asthma, tetanus, earache, thrombosis, wounds, paronychia, swelling, herpes zoster, irritation when milk teeth are erupting, jaundice, inflammation, Tupinambis teguixin (Linnaeus 1758)DD/II tumor, sore throat, infection, bronchitis, injuries caused by the spines of the “arraia,” pain relief in injuries caused by snake bites, toothache, suck a splinter out of skin or fresh, headache, cough, stroke, and coarse throat Rheumatism, lung disease, thrombosis, boils, tuberculosis, stomach ache, edema, snake bite, cancer, ache, swelling, to prevent abort, pain in the body, Boa constrictor (Linnaeus, 1758)DD/II inflammation, athlete’s foot, calluses, tumors, cracks in the sole of the feet, goiter, sore throat, arthrosis, insect sting, dog bite, erysipelas, thrombosis, asthma, neck strain, and strain muscle Wounds, skin problems, bruises, sprains, arthrosis, rheumatism, boils, sexual impotence, headache, sore Eunectes murinus (Linnaeus, 1758)DD/II throat, thrombosis, swelling, tumour, asthma, muscle strain, numbness, syphilis, to reduce pain, and luxation Injuries caused by bang, toothache, diabetes, headache, backache, wounds, cough, bronchitis, asthma, Caretta caretta (Linnaeus, 1758)VU/I thrombosis, rheumatism, stroke, hoarseness, flu, backache, earache, sore throat, and swelling Injuries caused by bang, toothache, diabetes, headache, backache, wounds, cough, bronchitis, asthma, flu, thrombosis, rheumatism, toothache, stroke, hoarseness, Chelonia mydas (Linnaeus, 1758)VU/I earache, sore throat, swelling, whooping cough, arthritis,erysipelas,boil,wounds,arthrosis,and inflammation Evidence-Based Complementary and Alternative Medicine 7

Table 1: Continued. Taxonomic category/species Conditions to which remedies are prescribed Injuries caused by bang, toothache, diabetes, headache, backache, wounds, cough, bronchitis, asthma, Eretmochelys imbricata (Linnaeus, 1766)EN/I thrombosis, stroke, hoarseness, flu, rheumatism, earache, sore throat, and swelling

Injuries caused by bang, toothache, diabetes, headache, Lepidochelys olivacea (Eschscholtz, 1829)EM/I backache, wounds, cough, flu, bronchitis, asthma, thrombosis, rheumatism, stroke, and hoarseness Dermochelys coriacea (Vandelli, 1761)CR/I Rheumatism, earache, sore throat, and swelling Rhinoclemmys punctularia (Daudin, 1802) Wounds, tumor, erysipelas, earache, and rheumatism Inflammation, acne, tumor, boil, rheumatism, Podocnemis expansa (Schweiger, 1812)LC/II pterygium, skin spots, backache, earache, arthrosis, arthritis, swelling, and wrinkle Podocnemis unifilis (Troschel, 1848)VU/II Wounds, tumor, erysipelas, earache, and rheumatism Podocnemis sextuberculata (Cornalia, 1849) Blackhead; acne Peltocephalus dumerilianus (Schweigger, 1812) Blackhead; acne Asthma, stroke, bronchitis, backache, earache, rheumatism, thrombosis, sexual impotence, snake bites (antidote), evil eye, irritation when milk teeth are Caiman crocodilus (Linnaeus, 1758)LC/II erupting,discharge,swelling,scratch,athlete’sfoot, ophthalmological problems, asthma, sore throat, amulet used as a protection against snake bite, rheumatism, hernia, prostate problems, infection, and thrombosis Asthma, sore throat, amulet used as a protection against Caiman latirostris (Daudin, 1801)LC/II snake bite, rheumatism, irritation when milk teeth are erupting, hernia, and prostate problems Thrombosis, infection, swelling, asthma, amulet used as a protection against snake bite, injuries caused by spines Melanosuchus niger (Spix, 1825)LC/II of the “arraia,” and pain relief in injuries caused by snake bites Snake bite, asthma, stroke, rheumatism, thrombosis, backache, sexual impotence, edema, mycosis, evil eye, irritation when milk teeth are erupting, snake bite Paleosuchus palpebrosus (Cuvier, 1807)LC/II (antidote), discharge, sore throat, amulet used as a protection against snake bite, hernia, and prostate problems Paleosuchus trigonatus (Schneider, 1801)DD/II Rheumatism Birds Anser anser (Linnaeus, 1758) Laryngitis, pharyngitis, and tonsillitis Swelling, inflammation, injuries caused by the spines of Ardea cocoi (Linnaeus, 1766) the “arraia” and others fishes, asthma, boil, tumor, inflammation, rheumatism, and earache Penelope jacucaca (Spix, 1825)VU Insomnia Injuries caused by the spines of the “arraia” and others Ciconia maguari (Gmelin, 1789) fishes, and thrombosis Leptotila rufaxilla (Richard and Bernard, 1792) Thrombosis Columba livia (Gmelin, 1789) Asthma, laryngitis, pharyngitis, and tonsillitis Meleagris gallopavo (Linnaeus, 1758) Asthma Rhea americana (Linnaeus, 1758)LC/II General aches, rheumatism, thrombosis, and strokes Crypturellus noctivagus (Wied, 1820)VU Thrombosis, stroke Nothura boraquira (Spix, 1825) Thrombosis, stroke Rhynchotus rufescens (Temminck, 1815) Snake bite, thrombosis, and snake bites (antidote) 8 Evidence-Based Complementary and Alternative Medicine

Table 1: Continued. Taxonomic category/species Conditions to which remedies are prescribed Mammals Wound in the breast caused by suckling, ophthalmological problems, stomach disorders, Agouti paca (Linnaeus, 1766)LC/III pterygium, to suck a splinter out of skin or flesh, injuries caused by the spines of “arraia,” and control cholesterol level Bubalus bubalis (Linnaeus, 1758) Rheumatism, osteoporosis, and thrombosis Capra hircus (Linnaeus, 1758) Evil eye, snake bite, and muscle strain Bradypus variegatus (Shinz, 1825) Thrombosis Bradypus tridactylus (Linnaeus, 1758) Thrombosis, insects bite, and scorpions bite Cavia aperea (Erxleben, 1777) Inflammation Kerodon rupestris (Wied-Neuwied, 1820) Constipation Alouatta belzebul (Linnaeus, 1766)CR Whooping cough, sore throat, and asthma Alouatta nigerrima (Lonnberg,¨ 1941) Whooping cough, inflammation Whooping cough, inflammation, and to accelerate Alouatta seniculus (Linnaeus, 1766)LC/II parturition Cebus apella (Linnaeus, 1758)LC/II Insect sting Blastocerus dichotomus (Illiger, 1815)VU/I Diarrhea, vomit Mazama americana (Erxleben, 1777)DD/III Stroke Diarrhea, verminosis, and evil eye Mazama simplicicornis (Illinger, 1811) Asthma, edema, rheumatism, snake bite, thrombosis, to Mazama cf. gouazoubira (G. Fischer, 1814) assist children who take longer than usual to start walking, toothache, wounds, and sprains Ozotocerus bezoarticus (Linnaeus, 1758) Diarrhea, verminosis, and evil eye Thrombosis, insects bite, scorpions bite, edema, Dasypus novemcinctus (Linnaeus, 1758) asthma, deafness, earache, and evil eye Wounds, earache, evil eye, asthma, sore throat, Euphractus sexcinctus (Linnaeus, 1758) pneumonia, sinusitis, deafness, and coarse throat Tolypeutes tricinctus (Linnaeus, 1758)VU Thrombosis, rheumatism Dasyprocta prymnolopha (Wagler, 1831) Asthma, thrombosis Asthma, headache, rheumatism, hernia, womb disorders, sore throat, injuries caused by the spines of Sotalia fluviatilis (Gervais and Deville, 1853)DD/I the “arraia,” swelling, hemorrhoids inflammation, wounds, earache, erysipelas, athlete’s foot, tumor, and cancer Asthma, headache, rheumatism, hernia, womb disorders, sore throat, injuries caused by the spines of Sotalia guianensis (P.J.VanBen´ eden,´ 1864) the “arraia,” swelling, hemorrhoids inflammation, wounds, earache, erysipelas, athlete’s foot, tumor, and cancer Bronchitis, thrombosis, epilepsy, stroke, abscesses, Coendou prehensilis (Linnaeus, 1758) conjunctivitis, and asthma Thrombosis, conjunctivitis, venereal disease, Hydrochaeris hydrochaeris (Linnaeus, 1766) rheumatism, earache, strengthen bones, liver pain, bronchitis, asthma, wounds, erysipelas, and cough Asthma, headache, rheumatism, hernia, womb disorders, sore throat, injuries caused by the spines of Inia geoffrensis (Blainville, 1817)VU/II the “arraia,” swelling, hemorrhoids inflammation, wounds, earache, erysipelas, athlete’s foot, tumor, and cancer Sylvilagus brasiliensis (Linnaeus, 1758) Thrombosis, conjunctivitis, boils, and burns Conepatus semistriatus (Boddaert, 1785) Rheumatism Evidence-Based Complementary and Alternative Medicine 9

Table 1: Continued. Taxonomic category/species Conditions to which remedies are prescribed Lontra longicaudis (Olfers, 1818)DD/I Thrombosis Myrmecophaga tridactyla (Linnaeus, 1758)VU/II Thrombosis, stroke Myrmecophaga tetradactyla (Linnaeus, 1758) Edema, thrombosis Rheumatism, arthrosis, osteoporosis, bursitis, muscular Tapirus terrestris (Linnaeus, 1758)VU/II pain, asthma, and tonsillitis Pecari tajacu (Linnaeus 1758)LC/II/III Thrombosis, bronchitis, and stroke Tayassu pecari (Link, 1795)LC/II Thrombosis, stroke Sprains, vaginal discharge, injuries caused by bang, burns, asthma, menstrual cramps, rheumatism, sore Trichechus inunguis (Natterer, 1883)VU/I throat, wounds, muscle strain, suck a splinter out of skin or fresh, tumor, backache, hernia, arthrosis, luxation, menstrual cramps, and insects bite Sprains, vaginal discharge, injuries caused by bang, burns, asthma, menstrual cramps, rheumatism, sore Trichechus manatus (Linnaeus, 1758)CR/I throat, wounds, muscle strain, suck a splinter out of skin or fresh, tumor, backache hernia, arthrosis, luxation, menstrual cramps, and insects bite Categories of IUCN Red List: CR: critically endangered, EN: endangered, VU: vulnerable, LC: least concern, DD: data deficient, and NE: not evaluated. Cites appendices (I, II, and III); IN: Anexo 2–Instruc¸ao˜ Normativa n. 5/2004/MMA. clearly constitute the most important taxonomic group in least 354 wild animal species are used in Brazilian traditional terms of the number of species used by rural communities medicine [71], of which 157 (44.3%) are also used as food [53–60]. (Table 1),aresultthatmirrorsthecentralroleplayedby Reptiles ranked third in the number of species recorded wildlife as a source of protein in different parts of the world. used as food and medicine in this study and are among As shown by previous studies, in at least 62 countries world- the animals most frequently used in folk medicine; the con- wide, wildlife (including fish) provides significant proteins, sumption of reptile meat is often intertwined with cultural or calories, and essential fats to rural communities [58–60, 72– medicinal beliefs [61–67]. In this study, chelonians stood out 76]. It should be noted, however, that the number of animal as the reptiles most used as medicinal food (𝑛=13species), species used as medicinal food in Brazil was higher than the a result in line with their extensive use as food in Brazil. As number of species recorded for those purposes elsewhere (see shown by Alves et al. [66], of the 36 species of chelonians in [23]), possibly as a result of the country’s significant biological Brazil,20(55.5%)areeatenbyhumans.Thoseanimalsare and cultural diversity [77]. commonly sought after as food in the northern region of the The overlap between alimentary and medicinal use can country where they achieve the highest species richness and be exemplified by the use of caymansCaiman ( latirostris, abundance. In a smaller proportion, lizards and caymans are C. crocodilus, Paleosuchus palpebrosus, and Melanosuchus also important as food medicine; on the other hand, only a niger). While their meat was primarily consumed as food, fewsnakespecieshavebeenusedasfood,despitethereported their teeth, skin, fat, and penis were used for treating dis- use of several species in Brazilian traditional medicine [64, eases such as asthma, stroke, bronchitis, backache, earache, 66].Thesmallnumberofsnakespeciescurrentlyusedasfood rheumatism, thrombosis, sexual impotence, swelling, oph- in Brazil is not surprising given the negative images attributed thalmological problems, sore throat, infection, thrombosis, to these animals in myths, legends, and popular beliefs [66– swelling, injuries caused by spines of stingray, and pain 68]. Rea [69] noted that not only snakes are rejected because relief in injuries caused by snake bites. Interestingly, caymans oftheirdisagreeablenature,butalsoanyothercreaturewith were also used as amulets to protect against snake bite or asimilarshapeorbehaviorwillreceivesimilartreatment. against evil eye [64, 65]. Likewise, the meat of armadillos A study undertaken among human populations living along (Euphractus sexcintus and Dasypus novemcinctus)wasused the banks of the Rio Negro River (Amazonas State, Brazil) as food, while their tail and skin were used for treating showed that the electric eel (Electrophorus electricus)was earacheandasthmaandasanamulettoprotectagainstevil one of the least favored meats because of its strong smell eye. andtheshapeofitsbody—“itlooksjustlikeasnake” Another interface between the use of animals as food and [70]. medicine was expressed through the need, by those taking Some of the animals quoted by interviewees were mainly animal-based medicines, to control their diet—otherwise the hunted for medicinal purposes, an example being the boa medication would not work. Similar findings were described snake (Boa constrictor), which is eventually also used as food by Begossi [4]andSeixasandBegossi[78] who recorded [66]. Conversely, other species are hunted for consumption, the use of the word “carregado” to encompasses a set of and their byproducts are utilized for medicinal purposes. At supposed attributes of an animal (such as teeth, blood, 10 Evidence-Based Complementary and Alternative Medicine aggressive behavior, “strong flesh,” and fattiness) and factors 2007. that could provoke an inflammation if the animal was eaten [2] A. Begossi and F.Braga, “Food taboos and folk medicine among by a wounded or unhealthy person. fishermen from the Tocantins River,” Amazoniana,vol.12,pp. Although the main part used for alimentary purposes 341–352, 1992. was the flesh, the eggs and viscera of some species were also [3]R.R.N.Alves,I.L.Rosa,andG.G.Santana,“Therole used. Examples include the Amazon River turtle Podocne- of animal-derived remedies as complementary medicine in mis expansa (Schweigger, 1812), the Black Vulture Coragyps Brazil,” BioScience,vol.57,no.11,pp.949–955,2007. atratus (Bechstein, 1793), the smooth-billed ani Crotophaga [4] A. Begossi, “Food taboos at Buzios´ Island (SE Brazil): their sig- ani (Linnaeus, 1758), the red-footed tortoise Chelonoidis car- nificance and relation to folk medicine,” Journal of Ethnobiology, bonaria (Spix, 1824), the yellow-footed tortoise Chelonoidis vol.12,no.1,pp.117–139,1992. denticulata (Linnaeus, 1766), and the domestic chicken Gallus [5] T. Johns, The Origins of Human Diet and Medicine, University gallus (Linnaeus, 1758). of Arizona Press, Tucson, Arizona, 1996. The consumption of the meat of reptiles, mammals, birds, [6] T. Johns, With Bitter Herbs They Shall Eat It,UniversityArizona and fishes is often related to the purported medicinal or Press, Tucson, Arizona, 1990. cultural benefits derived from the animal parts [62, 63, 74, 79– [7] N. L. Etkin and P. J. Ross, “Food as medicine and medicine 82], and this enduring relationship between food animals and as food. An adaptive framework for the interpretation of plant utilization among the Hausa of northern Nigeria,” Social Science medicinal therapy goes well beyond the understanding that and Medicine,vol.16,no.17,pp.1559–1573,1982. adequate nutrition sustains a person’s health. For instance, [8] N. L. Etkin, Eating on the Wild Side: The Pharmacologic, Eco- Werner [83] noted that much of the variation in the use logic and Social Implications of Using Noncultigens, University or nonuse of lizards as food apparently stems from cultural Arizona Press, Tucson, Arizona, 2000. beliefs concerning the medicinal or other benefits of their [9] A. Pieroni and L. L. Price, Eating and Healing: Traditional Food flesh. As an example of such cultural beliefs, in our study we as Medicine, CRC Press, New York, NY, USA, 2006. foundthatsomespeciesusedasfood(e.g.,Crassostrea rhi- [10] N. L. Etkin, “Medicinal cuisines: diet and ethopharmacology,” zophorae, Anomalocardia brasiliana,andEunectes murinus) Pharmaceutical Biology,vol.34,no.5,pp.313–326,1996. were also considered to be aphrodisiacs. [11] L. Manniche, An Ancient Egyptian Herbal,UniversityofTexas Of the animals used as remedies and food, 52 (33.1%) are Press, Austin, Tex, USA, 1989. under some form of legal protection, a result that clearly indi- [12] C. P.Bryan, Ancient Egyptian Medicine: The Papyrus Ebers,Ares, cates the need for bringing all relevant stakeholders together Chicago, Ill, USA, 1930. to develop strategies that can more effectively deal with the [13]J.W.Estes,The Medical Skills of Ancient Egypt, Science History issues related to the harvesting of wildlife for alimentary Publication, Canton, Mass, USA, 1989. and/or medicinal purposes in Brazil. As discussed by Alves [14] C. Stetter, The Secret Medicine of the Pharaohs-Ancient Egyptian and Rosa [1], sustainability of harvesting of medicinal animals Healing, Edition Q, Chicago, Ill, USA, 1st edition, 1993. is challenged by many factors, from both social and ecological [15] J. F. Nunn, Ancient Egyptian Medicine, University of Oklahoma perspectives, and it is important to respect differing views Press, Norman, Okla, USA, 2002. of the value of wildlife, while, at the same time, conserving [16] L. C. Mackinney, “Animalsubstances in materia medica: a study biodiversity. in the persistence of the primitive,” Journal of the History of Connections between traditional medicine, biodiversity, Medicine and Allied Sciences,vol.1,no.1,pp.149–170,1946. and human health have recently been addressed by different [17] R. C. Chemas, “A zooterapia no ambitoˆ da medicina civilizada. authors [84–88] and have drawn attention to the fact that I. Organoterapia humana e animal stricto sensu,”in Zooterapia: biodiversity loss can have indirect and direct effects on Os Animais na Medicina Popular Brasileira,E.M.Costa-Neto humanwell-beingaswell.Therelianceontraditionaluses andR.R.N.Alves,Eds.,pp.75–102,NUPEEA,Recife,Brazil, of animals as food and as medicine by communities around 2010. the world highlights the need for further interdisciplinary [18] J. Chen, “Regulatory control of functional food in China,” research in ethnozoology which can be used in strategies to Scandinavian Journal of Nutrition/Naringsforskning,vol.44,no. conserve biodiversity. 3, pp. 130–131, 2000. [19] W. Weng and J. Chen, “The Eastern perspective on functional foods based on traditional Chinese medicine,” Nutrition Acknowledgments Reviews, vol. 54, no. 11, pp. S11–S16, 1996. [20] J. Farquhar, “Eating chinese medicine,” Cultural Anthropology, ThefirstandlastauthorswouldliketoacknowledgeCon- vol. 9, no. 4, pp. 471–497, 1994. selho Nacional de Desenvolvimento Cient´ıfico e Tecnologico´ [21] A. Fleuret, “Dietary and therapeutic uses of fruit in three Taita (CNPq) for providing a research fellowship. The authors communities,” in Forests, Trees and Food,FAO,Ed.,Foodand also thanked the interviewees who contributed with the Agriculture Organization, Rome, Italy, 1993. information to this research. [22] G. Moreno-Black, P. Somnasang, and S. Thamathawan, “Culti- vating continuity and creating change: women’s home garden References practices in Northeastern Thailand,” Agriculture and Human Values,vol.13,no.3,pp.3–11,1996. [1] R. R. N. Alves and I. L. Rosa, “Zootherapeutic practices among [23] A. Pieroni and A. Grazzini, “Alimenti-medicina di origine fishing communities in North and Northeast Brazil: a compar- animale,” in Herbs, Humans and Animals/Erbe, uomini e bestie, ison,” Journal of Ethnopharmacology, vol. 111, no. 1, pp. 82–103, A.Pieroni,Ed.,pp.155–171,VDM,London,UK,1999. Evidence-Based Complementary and Alternative Medicine 11

[24] A. Pieroni, M. E. Giusti, and A. Grazzini, “Animal remedies in [43] J. Burger and M. Gochfeld, “Ocean ecosystems and human the folk medicinal practices of the Lucca and Pistoia Provinces, health,” in Oceans and Human Health: Risks and Remedies from Central Italy,” in Des sources du savoir aux medicaments´ du the Seas,P.J.Walsh,S.L.Smith,L.E.Fleming,H.Solo-Gabriele, futur/From the Sources of Knowledge to the Medicines of the andW.H.Gerwick,Eds.,pp.145–160,AcademicPress,New Future,J.Fleurentin,J.M.Pelt,andG.Mazars,Eds.,pp.371– York, NY, USA, 20 08. 375, IRD Editions, Paris, France, 2002. [44]C.Bouzan,J.T.Cohen,W.E.Connoretal.,“Aquantitative [25] R. R. N. Alves and I. L. Rosa, “From cnidarians to mammals: the analysis of fish consumption and stroke risk,” American Journal use of animals as remedies in fishing communities in NE Brazil,” of Preventive Medicine,vol.29,no.4,pp.347–352,2005. JournalofEthnopharmacology,vol.107,no.2,pp.259–276,2006. [45] J. T. Cohen, D. C. Bellinger, and B. A. Shaywitz, “A quantitative [26] R. R. N. Alves and I. L. Rosa, “Zootherapy goes to town: the use analysis of prenatal methyl mercury exposure and cognitive of animal-based remedies in urban areas of NE and N Brazil,” development,” American Journal of Preventive Medicine,vol.29, Journal of Ethnopharmacology,vol.113,no.3,pp.541–555,2007. no.4,p.353,2005. [27] R. R. N. Alves and I. L. Rosa, “Trade of animals used in Brazilian traditional medicine: trends and implications for conservation,” [46] J. T.Cohen, D. C. Bellinger, W.E. Connor, and B. A. Shaywitz, “A Human Ecology,vol.38,no.5,pp.691–704,2010. quantitative analysis of prenatal intake of n-3 polyunsaturated fatty acids and cognitive development,” American Journal of [28]F.S.Ferreira,N.L.G.Silva,E.F.F.Matiasetal.,“Potentiation Preventive Medicine,vol.29,no.4,p.366,2005. of aminoglycoside antibiotic activity using the body fat from the snake Boa constrictor,” Brazilian Journal of Pharmacognosy,vol. [47] M. Daviglus, J. Sheeshka, and E. Murkin, “Health benefits from 21,no.3,pp.503–509,2011. eating fish,” Comments on Toxicology,vol.8,no.4–6,pp.345– [29] A. Begossi, “Food taboos-a scientific reason?” in Plants for Food 374, 2002. and Medicine, H. D. V.Pendergast, N. Etkin, D. R. Harris, and P. [48] A. Konig,¨ C. Bouzan, J. T. Cohen et al., “A quantitative analysis J.Houghton,Eds.,pp.41–46,RoyalBotanicGardens,Kew,UK, of fish consumption and coronary heart disease mortality,” 1998. American Journal of Preventive Medicine,vol.29,no.4,pp.335– [30] FAO, Forests, Trees and Food, Food and Agriculture Organiza- 346, 2005. tion, Rome, Italy, 1992. [49] A. J. McMichael and C. D. Butler, “Fish, health, and sustainabil- [31] IIED, Whose Eden? An Overview of Community Approaches to ity,” American Journal of Preventive Medicine,vol.29,no.4,pp. Wildlife Management, Overseas Development Administration, 322–323, 2005. London, UK, 1994. [50] J. Patterson, “Introduction—comparative dietary risk: balance [32] R. R. N. Alves, “Fauna used in popular medicine in Northeast the risk and benefits of fish consumption,” Comments on Brazil,” Journal of Ethnobiology and Ethnomedicine,vol.5,article Toxicology,vol.8,no.4–6,pp.337–344,2002. 1, 30 pages, 2009. [51] W. C. Willett, “Fish: balancing health risks and benefits,” [33] E. Rodrigues, “Plants and animals utilized as medicines in AmericanJournalofPreventiveMedicine,vol.29,no.4,pp.320– the Jau´ National Park (JNP), Brazilian Amazon,” Phytotherapy 321, 2005. Research,vol.20,no.5,pp.378–391,2006. [34] U. P. Albuquerque and R. F. P. Lucena, “Metodos´ e tecnicas´ [52] J. Burger and M. Gochfeld, “Perceptions of the risks and benefits para coleta de dados,” in Metodos´ e Tecnicas´ na Pesquisa of fish consumption: individual choices to reduce risk and Etnobotanicaˆ , U. P. Albuquerque and R. F. P. Lucena, Eds., pp. increase health benefits,” Environmental Research,vol.109,no. 37–62, NUPEEA/Livro Rapido,´ Recife, Brazil, 2004. 3, pp. 343–349, 2009. [35] K. Bailey, Methods of Social Research, Free Press, New York, NY, [53] J. Ojasti, Wildlife Utilization in Latin America: Current Situation USA, 1994. and Prospects for Sustainable Management, Food and Agricul- [36] H. P. Huntington, “Using traditional ecological knowledge in ture Organization of the United Nations, Rome, Italy, 1997. science: methods and applications,” Ecological Applications,vol. [54] K. H. Redford and J. G. Robinson, “The game of choice: patterns 10,no.5,pp.1270–1274,2000. of Indian and colonist hunting in the neotropics,” American [37] A. B. M. Machado, G. M. Drummond, and A. P. Paglia, Livro Anthropologist,vol.89,no.3,pp.650–667,1987. Vermelho da fauna brasileira ameaçada de extinçao˜ , MMA, [55] J. G. Robinson and K. H. Redford, Neotropical Wildlife Use and Fundaçao˜ Biodiversitas, Bras´ılia, Brasil, 2008. Conservation, University of Chicago Press, Chicago, Ill, USA, [38] MMA, “Lista Nacional das especies´ de invertebrados aquaticos´ 1991. e peixes sobreexplotadas ou ameaçadas de sobreexplotaçao.˜ [56] K. H. Redford and J. G. Robinson, “Subsistence and commercial Instruçaonormativa5,de21demaiode2004,”˜ Diario´ Oficial uses of wildlife in Latin America,” in Neotropical Wildlife Use da Uniao˜ ,vol.102,pp.136–142,2004. and Conservation, J. G. Robinson and K. H. Redford, Eds., pp. [39] M. A. Huffman, “Self-medicative behavior in the African great 6–23, 1991. apes: an evolutionary perspective into the origins of human traditional medicine,” BioScience,vol.51,no.8,pp.651–661, [57] M. S. Alvard, J. G. Robinson, K. H. Redford, and H. Kaplan, 2001. “The sustainability of subsistence hunting in the neotropics,” [40] J. O’Hara-May, “Food or medicine,” Transactions of the British Conservation Biology, vol. 11, no. 4, pp. 977–982, 1997. Society for the History of Pharmacy,vol.1,pp.61–70,1971. [58] N.Hanazaki,R.R.N.Alves,andA.Begossi,“Huntinganduseof [41] F. D. B. P. Moura and J. G. W. Marques, “Folk medicine using terrestrial fauna used by Caiçaras from the Atlantic Forest coast animals in the Chapada Diamantina: incidental medicine?” (Brazil),” Journal of Ethnobiology and Ethnomedicine,vol.5,no. Ciencia e Saude Coletiva,vol.13,no.2,pp.2179–2188,2008. 1, article 36, 2009. [42] R. R. N. Alves and I. L. Rosa, Animals in Traditional Folk [59] J. A. A. Barbosa, V. A. Nobrega, and R. R. N. Alves, “Hunting Medicine: Implications for Conservation, Springer, Berlin, Ger- practices in the semiarid region of Brazil,” Indian Journal of many, 2013. Traditional Knowledge, vol. 10, no. 3, pp. 486–490, 2011. 12 Evidence-Based Complementary and Alternative Medicine

[60] R. R. N. Alves, L. E. T. Mendonça,M.V.A.Confessor,W.L.S. [77] “MMA Status of the National Biodiversity Strategy Advances,” Vieira, and L. C. S. Lopez, “Hunting strategies used in the semi- in Proceedings of the 2003 Meeting for Identification of Issues arid region of northeastern Brazil,” Journal of Ethnobiology and on Biodiversity for Cooperation and Interchanging among South Ethnomedicine,vol.5,no.12,article56,2009. American countries, Rio de Janeiro, Brazil, December 2003. [61] L. R. Angeletti, U. Agrimi, C. Curia, D. French, and R. Mariani- [78] C. Seixas and A. Begossi, “Ethnozoology of caiçaras from Costantini, “Healing rituals and sacred serpents,” The Lancet, Aventureiro, Ilha Grande,” Journal of Ethnobiology,vol.21,no. vol. 340, no. 8813, pp. 223–225, 1992. 1,pp.107–135,2001. [62] J. B. Thorbjarnarson, C. J. Lagueux, D. Bolze, M. W. Klemens, [79] H. S. Fitch, R. W. Henderson, and D. M. Hillis, “Exploitation and A. B. Meylan, “Human use of turtle: a worldwide perspec- of iguanas in Central America,” in Iguanas of the World Their tive,” in Turtle Conservation,M.W.Klemens,Ed.,pp.33–84, Behavior, Ecology and Conservation, pp. 397–415, Noyes, Saddle Smithsonian Institution Press, London, UK, 2000. River, NJ, USA, 1982. [63] M. W. Klemens and J. B. Thorbjarnarson, “Reptiles as a food [80] R.R.N.Alves,W.M.S.Souto,andR.R.D.Barboza,“Primatesin resource,” Biodiversity and Conservation,vol.4,no.3,pp.281– traditional folk medicine: a world overview,” Mammal Review, 298, 1995. vol.40,no.2,pp.155–180,2010. [64]R.R.N.Alves,N.A.Neto,G.G.Santana,W.L.S.Vieira,andW. [81] R. R. N. Alves, R. R. D. Barboza, and W.M. S. Souto, “AGlobal O. Almeida, “Reptiles used for medicinal and magic religious overview of canids used in traditional medicines,” Biodiversity purposes in Brazil,” Applied Herpetology,vol.6,no.3,pp.257– and Conservation,vol.19,no.6,pp.1513–1522,2010. 274, 2009. [82] H. Hilaluddin, R. Kaul, and D. Ghose, “Conservation implica- [65] R. R. D. Alves, W. L. D. Vieira, and G. G. Santana, “Reptiles tions of wild animal biomass extractions in Northeast India,” used in traditional folk medicine: conservation implications,” Animal Biodiversity and Conservation,vol.28,no.2,pp.169– Biodiversity and Conservation,vol.17,no.8,pp.2037–2049, 179, 2005. 2008. [83] D. I. Werner, “The rational use of green iguanas,” in Neotropical [66] R. R. N. Alves, K. S. Vieira, G. G. Santana et al., “A review Wildlife Use and Conservation,J.G.RobinsonandK.H. on human attitudes towards reptiles in Brazil,” Environmental Redford, Eds., pp. 181–201, The University of Chicago Press, Monitoring and Assessment,vol.184,no.11,pp.6877–6901,2012. Chicago, Ill, USA, 1991. [67] R. R. N. Alves, G. A. Pereira Filho, K. Silva Vieira et al., “A [84] R. R. N. Alves and I. L. Rosa, “Biodiversity, traditional medicine zoological catalogue of hunted reptiles in the semiarid region of andpublichealth:wheredotheymeet?”Journal of Ethnobiology Brazil,” Journal of Ethnobiology and Ethnomedicine,vol.8,no.1, and Ethnomedicine,vol.3,article14,9pages,2007. pp. 1–27, 2012. [85] C. Anyinam, “Ecology and ethnomedicine: exploring links [68] H. Fernandes-Ferreira, R. L. Cruz, D. M. Borges-Nojosa, and between current environmental crisis and indigenous medical R. R. N. Alves, “CrençasAssociadasaSerpentesnoEstadodo practices,” Social Science and Medicine,vol.40,no.3,pp.321– Ceara,´ Nordeste do Brasil,” Sitientibus,vol.11,no.2,pp.153–163, 329, 1995. 2011. [86] A. J. McMichael and R. Beaglehole, “The changing global [69] A. M. Rea, “Resource utilization and food taboos of Sonoran context of public health,” The Lancet,vol.356,no.9228,pp.495– desert peoples,” Journal of Ethnobiology,vol.1,pp.69–83,1981. 499, 2000. [70] A. L. Silva, “Comida de gente: preferenciasˆ e tabus alimentares entre os ribeirinhos do Medio´ Rio Negro (Amazonas, Brasil),” [87] E. Chivian, Biodiversity: Its Importance to Human Health,Cen- Revista de Antropologia,vol.50,no.1,pp.125–179,2007. ter for Health and the Global Environment. Harvard Medical School Cambridge, Cambridge, Mass, USA, 2002. [71] R. R. N. Alves, G. G. Santana, and I. L. Rosa, “The role of animal-derived remedies as complementary medicine in [88] R. R. N. Alves and I. L. Rosa, “Why study the use of animal Brazil,”in Animals in Traditional Folk Medicine: Implications for products in traditional medicines?” Journal of Ethnobiology and Conservation,R.R.N.AlvesandI.L.Rosa,Eds.,pp.289–301, Ethnomedicine,vol.1,article5,5pages,2005. Springer, Berlin, Germany, 2013. [72] R. R. N. Alves, “Relationships between fauna and people and the role of ethnozoology in animal conservation,” Ethnobiology and Conservation,vol.1,pp.1–69,2012. [73] E. L. Bennett and J. G. Robinson, “Hunting for the Snark,” in Hunting for Sustainability in Tropical Forests,J.G.Robinsonand E. L. Bennett, Eds., pp. 1–9, Columbia University Press, New York, NY, USA, 2000. [74] E. L. Bennett and J. G. Robinson, “Hunting of wildlife in tropical forests,” The World Bank Environment Department Papers,vol. 76, pp. 1–42, 2000. [75]R.Nasi,D.Brown,D.Wilkieetal.,Conservation and Use of Wildlife-Based Resources: The Bushmeat Crisis,Secretariat of the Convention on Biological Diversity and Center for International Forestry Research (CIFOR), Bogor, Indonesia, 1st edition, 2008. [76] W. R. Townsend, “The sustainability of subsistence hunting by the SirionoIndiansofBolivia,”in´ Hunting for Sustainability in Tropical Forests, J. G. Robinson and E. L. Bennett, Eds., pp. 267– 281, Columbia University Press, New York, NY, USA, 2000. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 841580, 9 pages http://dx.doi.org/10.1155/2013/841580

Research Article Acute and Chronic Toxicity of an Aqueous Fraction of the Stem Bark of Stryphnodendron adstringens (Barbatimão) in Rodents

Marco Antonio Costa,1 João Carlos Palazzo de Mello,1,2 Edílson Nobuyoshi Kaneshima,3 Tânia Ueda-Nakamura,1,3 Benedito Prado Dias Filho,1,3 Elisabeth Aparecida Audi,1,4 and Celso Vataru Nakamura1,3,5

1 Programa de Pos-Graduac´ ¸ao˜ em Cienciasˆ Farmaceuticas,ˆ Universidade Estadual de Maringa,´ Avenida Colombo 5790, 87020-900 Maringa,´ PR, Brazil 2 Departamento de Farmacia,´ Universidade Estadual de Maringa,´ Avenida Colombo 5790, 87020-900 Maringa,´ PR, Brazil 3 Departamento de Cienciasˆ Basicas´ da Saude,´ Universidade Estadual de Maringa,´ Avenida Colombo 5790, 87020-900 Maringa,´ PR, Brazil 4 Departamento de Farmacologia e Terapeutica,ˆ Universidade Estadual de Maringa,´ Avenida Colombo 5790, 87020-900 Maringa,´ PR, Brazil 5 Laboratorio´ de Inovac¸ao˜ Tecnologica´ no Desenvolvimento de Farmacos´ e Cosmeticos,´ Avenida Colombo 5790, Bloco B08, 87020-900 Maringa,´ PR, Brazil

Correspondence should be addressed to Celso Vataru Nakamura; [email protected]

Received 4 April 2013; Revised 25 June 2013; Accepted 25 June 2013

Academic Editor: Ulysses Paulino Albuquerque

Copyright © 2013 Marco Antonio Costa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Stryphnodendron adstringens has a high tannin content and is used as an antiseptic and antimicrobial and in the treatment of leucorrhea, gonorrhea, wound healing, and gastritis. The present study evaluated the toxic effects of the heptamer prodelphinidin (F2) from the stem bark of S. adstringens in rodents. In the acute toxicity test, the mice that received oral doses exhibited reversible −1 effects, with an LD50 of 3.015 mg ⋅ kg . In the chronic toxicity test at 90 days, Wistar rats were treated with different doses of F2 (10, −1 100, and 200 mg ⋅ kg ). In the biochemical, hematological, and histopathological examinations and open-field test, the different dose groups did not exhibit significant differences compared with controls. The present results indicate that F2 from the stem bark of S. adstringens caused no toxicity with acute and chronic oral treatment in rodents at the doses administered.

1. Introduction has shown that it has antiulcerogenic potential, antiprotozoan activity, anti-inflammatory effects, antimicrobial activity, and The bioactivity of various compounds in medicinal plants has wound healing effects6 [ , 10–18]. been assessed. These compounds are isolated and analyzed The bark of S. adstringens is rich in proanthocyanidin to determine biological activity, mechanisms of action, and polymers, including several flavan-3-ols, such as prodelphini- toxicity [1, 2]. The genus Stryphnodendron sp belongs to the dins and prorobinetinidins [19–22]. The chemical composi- family Fabaceae (native savanna), approximately 48 species tion of prodelphinidin (F2) has been partially defined as a of which have been identified, including Stryphnodendron heptamer compound [16]. adstringens (Mart.) Coville (known as “barbatimao”).˜ This The toxicity of S. adstringens was the subject of a study speciesisfoundinthecentralsavannahregionofBrazil[3–5]. by Rebecca et al. [23, 24]. The crude extract of stem bark This plant is popularly used as an antiseptic and antimicrobial was administered at high doses in mice and tested in liver and in the treatment of leucorrhea, gonorrhea, gastritis, diar- mitochondria, showing signs of liver toxicity. Other studies rhea, bleeding, and wound healing [6–9]. Scientific research have been conducted with other parts of the plant [25, 26]. 2 Evidence-Based Complementary and Alternative Medicine

Studies by De Sousa et al. [27]andCostaetal.[28]showed removed, weighed, and evaluated for macroscopic abnormal- that S. adstringens had no genotoxic effects in Drosophila ities. When changes were observed in the autopsies, further melanogaster or micronuclei (bone marrow) and Artemia histological examination of the organs was performed. salina tests in mice, respectively. Thus, considering the wide use of this plant and that few 2.4.2. Repeated-Dose Oral Toxicity Study in Rats. Wistar rats studies have been conducted to determine the toxicological were divided into four groups (11 males and 11 females). profile of S. adstringens, the present study sought to obtain Onegroupservedasthecontrolandreceivedonlywater. more information about toxicity at therapeutic doses. The other groups received F2 of S. adstringens (10, 100, or −1 200 mg⋅kg ) suspended in water and administered orally by 2. Materials and Methods gavage daily for 90 days. The volume administered by gavage in the rats was approximately 0.5 mL per animal. The choice 2.1. Plant Material. Stem bark from Stryphnodendron ad- of the doses was based on the estimated oral dose in popular stringens was collected in Sao˜ Jeronimoˆ da Serra, Parana,´ ⋅ −1 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 use (10 mg kg )[34] and 10- and 20-times the effective dose Brazil (S23 43 7. 8 ,W5045 23.5 ; altitude 926 m), in March [29, 30]. 2008. A voucher herbarium specimen was deposited at the All of the animals received food and water ad libitum Universidade Estadual de Maringa(no.HUM14321).´ during the treatment. They were observed daily with regard to behavior and weighed weekly. At the end of the 90-day 2.2. Aqueous Fraction. The bark was dried at room temper- period, the animals were deprived of food for 15 h and then ature and then pulverized. The crude extract was obtained sacrificed. Their blood was collected for biochemical and by turboextraction of the bark at 1,000 ×g with 70% acetone hematological examination. The organs were carefully dis- in water for 15 min. The organic solvent was eliminated by sected and removed for weighing, macroscopic examination, rotavapor and lyophilized to yield a crude extract (F1; 300 g). and histopathological analysis. F1 (50 g) was suspended in water (500 mL) and partitioned with ethyl acetate (500 mL; 1 : 1) to obtain a proanthocyanidin Blood Analysis. Biochemical analyses were performed to polymer-rich fraction (aqueous fraction; F2; 35 g). determine glucose, aspartate aminotransferase (AST), ala- nine aminotransferase (ALT), alkaline phosphatase (ALP), 2.3. Animals. Adult Wistar rats (90 days old), weighing 230– total protein, creatinine, uric acid, blood urea nitrogen (BUN), triglycerides, total cholesterol, 𝛾-glutamyltransferase 240 g (female) and 355–365 g (male), and Swiss mice (60 days 𝛾 old), weighing 35–45 g (female) and 45–60 g (male), were ( -GT), bilirubin, sodium, and potassium. These were evalu- used and housed in groups of five per cage, with food and ated using the Dimension RXL Max system (Siemens). water freely available. The animals were maintained on a Hematological analyses were performed using the auto- 12 h/12 h light/dark cycle under controlled temperature (22 ± matic counter Pentra 60 ABX (ABX Diagnostics) to evaluate ∘ 1 C). The protocol was approved by the Ethical Committee of the following parameters: erythrocyte count (red blood cells the State University of Maringa(Approvalno.026/2009).´ (RBCs)), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, 2.4. Toxicity Studies. The toxicity studies were performed and leucocyte count (white blood cells (WBCs)). Differ- according to the Brazilian National Health Surveillance ential WBC counts (nonsegmented neutrophils, segmented Agency (ANVISA) [29–32]. neutrophils, basophils, eosinophils, lymphocytes, and mono- cytes) were made using a glass-slide method. Immediately 2.4.1. Acute Toxicity Study in Mice. Swissmiceweredivided after collection, blood smears were air-dried and stained with into seven groups, with 10 animals per group (five males and Leishman’s stain. One hundred cells were randomly counted five females). Six groups were orally treated by gavage with in each smear, and the percentage of each type was calculated. different doses of F2 (500, 1,000, 2,000, 3,000, 4,000, and −1 Any morphological change in the blood cells was noted. 5,000 mg⋅kg ). One group that received distilled water was includedasanegativecontrol.Thevolumeadministeredby Organ Weights and Histopathological Analyses.Theorgans gavage in the mice was approximately 0.3 mL per animal. (thymus, esophagus, stomach, duodenum, lung, heart, kid- Water and food were freely available to the animals. The neys,liver,spleen,adrenals,andsexorgans)ofalloftheani- general behavior and number of survivors were observed mals were examined macroscopically. The positions, shapes, at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h and daily sizes, and colors of the internal organs were visually observed thereafter until day 14. Toxicological effects were assessed, for signs of macroscopic abnormalities. The organs were including changes in locomotion, respiration, piloerection, weighed and fixed in Bouin’s fixative and preserved in 70% −1 diarrhea, drooling, altered muscle tone, hypnosis, convul- ethanol. For the 200 mg⋅kg dose of F2 and controls, tissue sions, hyperexcitability, writhing (abdominal constrictions), slides were prepared and stained with hematoxylin and eosin and mortality (which is expressed as the median lethal dose for microscopic examination. [LD50]) [33]. From the 24th hour until day 14, the weights of the animals were recorded. Open-Field Test (OFT). Locomotor behavior was assessed At the end of this period, all of the animals were on day 86 of treatment in the open-field test. Each animal sacrificed. The kidneys, heart, lungs, spleen, and liver were was placed in a round wooden arena (70 cm diameter) Evidence-Based Complementary and Alternative Medicine 3

Table 1: Acute toxicity of F2 of Stryphnodendron adstringens administered orally in mice.

F2 S. Observed changes adstringens dose No. of Motor Hypnosis/ − Diarrhea Piloerection Hypoactivity Hyperventilation Ptosis Hypothermia Catatonia (mg⋅kg 1) deaths impairment sedation Control 0 No No No No No No No No No 500 0 No No No No No No No No No 1,000 0 No No No No No No No No No Reversible Reversible 2,000 2 after 8 h after 72 h No No No No No No No (2) (5) Reversible Reversible Reversible Reversible after Reversible Reversible Reversible 3,000 7 after 8 h after 7 d after 48 h 48 h after 48 h after 48 h after 48 h No No (1) (6) (4) (2) (1) (1) (1) Reversible Reversible Reversible Reversible after Reversible Reversible 4,000 9 after 8 h after 7 d after 48 h 4h No after 48 h No after 1 h No (6) (12) (9) (2) (4) (1) Reversible Reversible Reversible Reversible after Reversible Reversible Reversible Reversible 5,000 11 after 48 h after at 14 d after 48 h 12 h after 48 h after 48 h after 1 h No after 12 h (6) (10) (5) (8) (1) (4) (1) (1) No: no toxic symptoms observed. The numbers in parentheses indicate the number of animals that showed the changes. The mice were observed daily forsigns of toxicity (behavioral changes and mortality) for 14 days. with 30 cm high walls. Luminosity at the center of the open lower doses. Only liver tissue damage was observed at the −1 field was 60 lux during a 5-min period. Rearing, self-cleaning, 5,000 mg⋅kg dose. urination, the number of fecal pellets, and locomotion were recorded [35]. For the evaluation of locomotion, the total 3.2. Repeated-Dose Oral Toxicity Study in Rats distance traveled was analyzed using a video tracking system (Ethovision). 3.2.1. Behavior and Body Weight Gain. During treatment, no signs of toxicity were observed in the animals. Body weight 2.5. Statistical Analyses. The results are expressed as mean gain as a function of time is shown in Figures 2 and 3.All ± of the groups showed weight gain compared with their initial standard deviation (SD). The data were analyzed using −1 weight, with the exception of the 100 mg⋅kg dose in females. Statistica8.0software.Thestatisticalanalyseswereperformed −1 using one-way analysis of variance (ANOVA) followed by Furthermore, the doses of 100 and 200 mg⋅kg in males and −1 the Dunnett post hoc test. When the variance was not con- 100 mg⋅kg in females caused less weight gain compared stant, the nonparametric Kruskal-Wallis test was performed with controls (Figures 2 and 3; Table 3). to determine significant differences. The histopathological results were analyzed using Fisher’s exact test. Differences 3.2.2. Biochemical Analyses. The effects of F2 of S. adstringens were considered significant at 𝑃 ≤ 0.05. on biochemical profiles showed specific changesTable ( 4). In male rats, some parameters were significantly increased compared with the control group, including potassium 3. Results −1 −1 (10.7 ± 2.4 mEq⋅L at 100 mg⋅kg ), uric acid (2.7 ± 1.1 −1 −1 3.1. Acute Toxicity Study in Mice. The effects are summarized and 2.0 ± 0.8 mg⋅dL at 100 and 200 mg⋅kg , resp.), and −1 −1 −1 in Table 1. The doses of 500 and 1,000⋅ mg kg showed no AST (186.0 ± 47.0 U⋅L at 100 mg⋅kg ). Other param- ⋅ −1 eters were significantly decreased, including triglyceride signs of toxicity. Only the 2,000 mg kg dose caused signs of −1 −1 (85.5 ± 28.3 mg⋅dL at 200 mg⋅kg ) and creatinine (0.4 ± toxicity, beginning with diarrhea and piloerection. At doses of −1 −1 ⋅ −1 0.1 mg⋅dL at 100 mg⋅kg ). In female rats, only triglycerides 3,000–5,000 mg kg , hypoactivity, hyperventilation, ptosis, −1 −1 hypothermia, motor impairment, sedation, and catatonia (58.9 ± 12.5 and 72.7 ± 21.3 mg⋅dL at 100 and 200 mg⋅kg , were observed, all of which were reversible after 48 h. Death resp.) were significantly decreased compared with the control −1 occurred in the groups that received 2,000–5,000 mg⋅kg , group. −1 resultinginanLD50 of 3,015 mg⋅kg .Allofthegroupshad lower body weights in the first 24 h. After 7 days, however, 3.2.3. Haematological Analyses. The effects of F2 of S. adstrin- body weights recovered (Figure 1). With regard to the weights gens on hematological parameters showed specific changes of the organs, only the liver showed a decrease at doses but only in male rats (Table 5). Significant differences from −1 of 4,000 and 5,000 mg⋅kg (Table 2). In the macroscopic the control groups were found, including decreased MCH and histopathological analyses, no changes were observed at (20.0 ± 0.4, 19.9 ± 0.5,and20.0 ± 0.4 pg at 10, 100, 4 Evidence-Based Complementary and Alternative Medicine

50 70 45 60 40 35 50 30 40 25 20 30 Weight (g) Weight Weight (g) Weight 15 20 10 10 5 0 0 Female 500 1.000 2.000 3.000 4.000 Male 500 1.000 2.000 3.000 4.000 control control −1 −1 mg·kg of F2 mg·kg of F2

Initial weight 7 days Initial weight 7 days 24 h 14 days 24 h 14 days (a) (b)

−1 Figure 1: Body weight gain (g) in female mice (a) and male mice (b) treated orally with 500, 1,000, 2,000, 3,000, 4,000, and 5,000 mg⋅kg F2 in the acute toxicity study.

Table 2: Effect of F2 of Stryphnodendron adstringens on the weights of organs of mice.

− F2 S. adstringens (mg⋅kg 1) Organs Control 500 1,000 2,000 3,000 4,000 5,000 Male ∗ ∗ Liver 3.27 ± 0.4 2.57 ± 0.3 2.87 ± 0.2 2.75 ± 0.3 2.58 ± 0.2 2.32 ± 0.3 2.45 ± 0.6 Spleen 0.31 ± 0.04 0.24 ± 0.02 0.27 ± 0.06 0.30 ± 0.07 0.20 ± 0.05 0.20 ± 0.03 0.20 ± 0.06 Lungs 0.48 ± 0.01 0.35 ± 0.03 0.40 ± 0.04 0.35 ± 0.04 0.46 ± 0.11 0.39 ± 0.06 0.46 ± 0.16 Heart 0.31 ± 0.03 0.25 ± 0.04 0.27 ± 0.04 0.28 ± 0.04 0.34 ± 0.05 0.29 ± 0.07 0.32 ± 0.04 Kidneys 0.95 ± 0.09 0.67 ± 0.05 0.82 ± 0.05 0.70 ± 0.09 0.81 ± 0.06 0.64 ± 0.10 0.72 ± 0.12 Female ∗ ∗ Liver 2.22 ± 0.3 1.94 ± 0.2 2.04 ± 0.3 1.93 ± 0.3 1.98 ± 0.5 1.92 ± 0.4 1.68 ± 0.2 Spleen 0.25 ± 0.04 0.20 ± 0.06 0.28 ± 0.06 0.29 ± 0.09 0.24 ± 0.07 0.26 ± 0.08 0.19 ± 0.03 Lungs 0.38 ± 0.07 0.32 ± 0.06 0.33 ± 0.04 0.36 ± 0.07 0.43 ± 0.15 0.50 ± 0.15 0.47 ± 0.14 Heart 0.20 ± 0.04 0.17 ± 0.04 0.21 ± 0.06 0.24 ± 0.03 0.25 ± 0.07 0.25 ± 0.06 0.19 ± 0.03 Kidneys 0.51 ± 0.06 0.44 ± 0.08 0.49 ± 0.03 0.55 ± 0.07 0.52 ± 0.07 0.51 ± 0.09 0.49 ± 0.03 ∗ Values expressed as mean ± DP (standard deviation). ANOVA or Kruskal-Wallis tests. 𝑃 < 0.05 compared to control.

Table 3: Percentage of weight gain in the animals after 90 days of significant differences were observed in female rats compared treatment with the aqueous fraction (F2) of S. adstringens. with the control group. Weight gain (%) Groups 3.2.4. Organ Weights and Histopathological Analyses. No Male Female macroscopic alterations, differences in relative body weight, Control 26.2 ± 3.3 20.8 ± 4.3 − or histopathological parameters were observed compared 10 mg⋅kg 1 22.7 ± 6.2 15.5 ± 4.2 − ∗ ∗ with controls. 100 mg⋅kg 1 18.4 ± 6.9 12.8 ± 5.5 ⋅ −1 15.2 ± 5.6∗ 14.8 ± 3.9 200 mg kg 3.2.5. Open-Field Test. Locomotion (Figure 4), rearing, uri- ∗ ∗ Values are mean ± S.E.M. ANOVA test. 𝑃 < 0.05 compared to the control nation, the number of fecal pellets, and self-cleaning behavior group. were not significantly different between the treatment groups and controls.

−1 4. Discussion and 200 mg⋅kg , resp.), decreased MCHC (35.1 ± 0.3%at −1 100 mg⋅kg ), and increased platelet counts (929 ± 108 and The indiscriminate use of S. adstringens without con- 3 −3 −1 961 ± 105 × 10 mm at 10 and 100 mg⋅kg ,resp.).No sidering possible toxicity or efficacy can lead to serious Evidence-Based Complementary and Alternative Medicine 5

600 350

300 a aa 500 a a b a, b a, b 250 400 200 300 150 Weight (g) Weight (g) 200 100

100 50

0 0 Male control 10 100 200 Female control 10 100 200 −1 −1 mg·kg of F2 mg·kg of F2

Initial weight Initial weight Final weight (90 days) Final weight (90 days) (a) (b)

−1 Figure 2: Body weight gain (g) in male rats (a) and female rats (b) treated orally for 90 days with F2 of S. adstringens (10, 100, and 200 mg⋅kg ) and controls. a𝑃 < 0.05, compared with initial values; b𝑃 < 0.05, compared with control values (ANOVA).

480 300 460 a a 280 a 440 a 420 a, b b 400 260 380 240 360 Body (g) weight 340 Body (g) weight 220 320 300 200 0 7 14 21 28 35 42 49 56 63 70 77 84 90 0 7 14 21 28 35 42 49 56 63 70 77 84 90 Days Days Male control 100 Female control 100 10 200 10 200 (a) (b)

−1 −1 Figure 3: Body weight (g) in male rats (a) and female rats (b) treated orally for 90 days with F2 of S. adstringens (10 mg⋅kg ∙,100mg⋅kg 󳵳, −1 and 200 mg⋅kg ◼)andcontrols(Q). a𝑃 < 0.05, compared with initial values; b𝑃 < 0.05, compared with controls (ANOVA).

2500 health risks. The proanthocyanidin polymer-rich fraction 2000 (heptamer compound) of the stem bark of S. adstringens (F2) offers possible advantages, including antifungal activity 1500 against Candida spp., especially C. albicans [16]. Vulvovaginal 1000 candidiasisisoneofthemostfrequentpathologiesobserved in daily gynecology practice. Alternative herbal treatments 500 Locomotion (cm) Locomotion may be less costly and lead to greater adherence to treatment, 0 thus improving the patients quality of life [36–38]. −1 −1 −1 Control 10 mg·kg 100 mg·kg 200 mg·kg The results of the acute toxicity study indicated that Treatment thetoxicityofF2fromthestembarkofS. adstringens is low (Tables 1 and 2). Some signs of toxicity were observed, Figure 4: Open-field test results. The figure shows locomotion in which increased progressively with increasing dose but were rats that orally received F2 of Stryphnodendron adstringens at day 86 quickly reversible. No significant changes in organ weight ∗ of treatment. The data are expressed as mean ± SEM. 𝑃 < 0.05, or macroscopic and histological parameters were observed compared with control group (ANOVA). compared with the control group. 6 Evidence-Based Complementary and Alternative Medicine

Table 4: Biochemical parameters in male and female rats orally treated with F2 of S. adstringens for 90 days.

− F2 (mg⋅kg 1) Parameters Controls 10 100 200 Male − Sodium (mEq⋅L 1) 142.2 ± 5.0 144.5 ± 1.6 142.1 ± 3.3 141.7 ± 2.7 − ∗ Potassium (mEq⋅L 1) 6.3 ± 0.4 7.3 ± 0.4 10.7 ± 2.4 7.5 ± 1.2 − Glucose (mg⋅dL 1) 122.6 ± 11.6 127.9 ± 12.5 129.4 ± 15.5 123.4 ± 29.0 − ∗ Triglyceride (mg⋅dL 1) 119.9 ± 23.4 134.5 ± 23.4 95.7 ± 23.5 85.5 ± 28.3 − Cholesterol (mg⋅dL 1) 99.3 ± 13.9 104.5 ± 19.1 97.1 ± 10.2 92.7 ± 21.1 − ∗ ∗ Uric acid (mg⋅dL 1) 1.2 ± 0.2 1.5 ± 0.2 2.7 ± 1.1 2.0 ± 0.8 − ALP (U⋅L 1) 76.0 ± 18.3 68.4 ± 9.6 84.0 ± 15.8 89.2 ± 13.0 − ∗ AST (U⋅L 1) 106.7 ± 19.5 148.6 ± 26.3 186.0 ± 47.0 151.4 ± 67.0 − ALT (U⋅L 1) 56.6 ± 7.7 60.9 ± 12.7 90.0 ± 26.1 62.3 ± 13.0 Total protein (g/dL) 6.5 ± 0.6 6.4 ± 0.2 6.8 ± 0.3 6.9 ± 0.5 − Total bilirubin (mg⋅dL 1) 0.11 ± 0.02 0.07 ± 0.04 0.11 ± 0.08 0.05 ± 0.04 − BUN (mg⋅dL 1) 44.1 ± 3.2 43.9 ± 4.1 50.9 ± 8.9 48.6 ± 8.6 − ∗ Creatinine (mg⋅dL 1) 0.6 ± 0.2 0.5 ± 0.06 0.4 ± 0.1 0.5 ± 0.1 − 𝛿-GT (U⋅L 1) 1.6 ± 0.5 1.7 ± 0.7 2.8 ± 1.6 1.4 ± 0.7 Female − Sodium (mEq⋅L 1) 132.7 ± 6.0 132.7 ± 5.8 132.4 ± 2.6 136.2 ± 3.2 − Potassium (mEq⋅L 1) 6.8 ± 1.1 8.4 ± 1.9 8.6 ± 1.4 6.0 ± 0.3 − Glucose (mg⋅dL 1) 116.6 ± 22.9 123.5 ± 14.9 119.5 ± 17.1 131.9 ± 16.4 − ∗ ∗ Triglyceride (mg⋅dL 1) 99.9 ± 20.6 78.5 ± 22.7 58.9 ± 12.5 72.7 ± 21.3 − Cholesterol (mg⋅dL 1) 98.4 ± 13.3 88.2 ± 11.9 92.0 ± 19.5 96.9 ± 22.2 − Uric acid (mg⋅dL 1) 1.4 ± 0.4 1.8 ± 0.8 1.6 ± 0.4 1.1 ± 0.5 − ALP (U⋅L 1) 59.5 ± 9.3 57.8 ± 13.6 67.4 ± 16.8 54.5 ± 19.1 − AST (U⋅L 1) 173.4 ± 71.9 233.9 ± 90.9 193.5 ± 58.6 136.4 ± 65.9 − ALT (U⋅L 1) 59.0 ± 12.9 73.7 ± 20.0 68.5 ± 14.9 51.9 ± 6.1 Total protein (g/dL) 7.4 ± 0.8 7.4 ± 0.2 7.9 ± 0.5 7.3 ± 0.5 − Total bilirubin (mg⋅dL 1) 0.13 ± 0.05 0.15 ± 0.08 0.12 ± 0.09 0.13 ± 0.05 − (mg⋅dL 1) 49.5 ± 5.9 59.3 ± 12.7 55.2 ± 6.6 45.3 ± 6.8 − Creatinine (mg⋅dL 1) 0.6 ± 0.2 0.5 ± 0.1 0.5 ± 0.1 0.6 ± 0.1 − 𝛿-GT (U⋅L 1) 1.9 ± 0.9 3.3 ± 1.4 3.4 ± 1.4 2.5 ± 0.9 ∗ The data are expressed as mean ± SEM. 𝑃 < 0.05,comparedwithcontrolgroup(ANOVAorKruskal-Wallistest).

−1 Thedecreaseinliverweightatdosesof4,000and and 200 mg⋅kg , significant differences in weight gain were −1 5,000 mg⋅kg andtissueliverdamageatadoseof observed. −1 5,000 mg⋅kg indicate a direct action of the plant in the According to Lewis et al. [41], “some factors can be useful liver at high doses, which is consistent with Rebecca et al. in differentiating a significant change from control values, −1 [23, 24], who studied doses >800 mg⋅kg in rats. from a treatment-related effect. This difference is less likely to −1 be an effect of treatment if: there is no obvious dose response; The high LD50 values of F2 (3,015 mg⋅kg )confirm its low acute toxicity that has been indicated by other it is due to finding(s) in one or more animals that could be considered outlier(s) and it is within normal biological parameters [39]. Moreover, Rebecca et al. [23]foundanLD50 −1 of 2,700 mg⋅kg of the crude extract with acute treatment, variation (within the range of reference values).” demonstrating that the aqueous fraction has less toxicity. The In the present study, the biochemical results varied widely high LD50 values indicate that the extract can be administered between doses and between sexes, with no linear profile. The with a high degree of safety. alteredvalueswerewithinnormallimits,andtheresultsare In the repeated-dose oral toxicity experiment, no signifi- considered normal for this animal species. Other values that cant changes in behavior or mortality were observed in rats. changed did not show a dose response [42, 43]. In male rats, potassium, AST, and creatinine at AccordingtoRazaetal.[40], changes in body weight can −1 −1 100 mg⋅kg , triglycerides at 200 mg⋅kg , and uric acid indicate adverse effects when the animal shows a loss greater −1 than 10% of initial weight. In the present study, however, at 100 and 200 mg⋅kg significantly changed. In female −1 neither group lost weight during treatment. At doses of 100 rats, only triglycerides at 100 and 200 mg⋅kg significantly Evidence-Based Complementary and Alternative Medicine 7

Table 5: Hematological results in male and female rats orally treated confirmed by the absence of histopathological changes in with F2 of S. adstringens for 90 days. this organ. − F2 (mg⋅kg 1) To evaluate liver function, AST, ALT, ALP, total bilirubin, Parameters Controls and 𝛾-GT were assessed because these are considered markers 10 100 200 of liver function, and liver changes have been reported after Male phytotherapeutic product use [45–48]. In the present study, WBC 10.6 ± 3.7 7.1 ± 0.6 8.7 ± 2.8 7.2 ± 1.6 a significant increase in AST levels was only observed in ×103 −3 ( mm ) male rats at the intermediate dose, with no dose response. Segmented (%) 20.9 ± 8.9 16.0 ± 6.5 15.9 ± 4.9 16.1 ± 4.6 As noted earlier, the high doses used in the acute study Eosinophil (%) 2.3 ± 0.8 1.9 ± 0.8 1.5 ± 0.5 2.3 ± 0.9 altered liver tissue, which could justify the increase in AST Lymphocyte 66.6 ± 11.8 75.2 ± 9.7 79.3 ± 5.2 78.6 ± 5.6 through the release of enzymes from the cells of the damaged (%) organ or changes in cellular membrane permeability [23]. Monocyte (%) 3.4 ± 0.7 3.1 ± 0.8 3.1 ± 0.5 3.0 ± 0.9 However, this did not appear to be the case at present. Our RBC histopathological examinations did not indicate any cellular 6 −3 8.16 ± 0.3 8.16 ± 0.4 8.22 ± 0.5 8.50 ± 0.3 (×10 mm ) lesions. Haemoglobin 17.1 ± 0.6 16.0 ± 0.9 16.4 ± 1.1 17.0 ± 0.6 The levels of total protein, glucose, cholesterol, and (g/dL) triglycerides were measured to assess the general biochem- Haematocrit 46.7 ± 1.8 46.3 ± 2.5 46.5 ± 3.2 47.7 ± 2.1 icalprofileoftheanimalsanddeterminethepresenceof (%) metabolic changes. The values were not significantly different MCV (fL) 57.3 ± 1.2 56.7 ± 1.2 56.5 ± 1.2 55.9 ± 1.4 between the control and treatment groups in both female and ∗ ∗ ∗ MCH (pg) 20.9 ± 0.5 20.0 ± 0.4 19.9 ± 0.5 20.0 ± 0.4 male rats, with the exception of triglycerides, which appeared ∗ to be reduced in both females and males but remained within MCHC (%) 36.5 ± 0.7 35.6 ± 0.8 35.1 ± 0.3 35.8 ± 0.8 the normal range [43]. High doses of the crude extract of Platelet 679 ± 171 929 ± 108∗ 961 ± 105∗ 895 ± 106 ×103 −3 stem bark can cause liver mitochondrion toxicity [23, 24]. ( mm ) Mitochondrial lesions can alter lipid metabolism. Female In the present study, hematological changes did not WBC 3 −3 7.2 ± 1.1 10.5 ± 4.3 8.3 ± 2.6 7.9 ± 1.9 appear to be related to treatment with F2. No changes were (×10 mm ) observed in females, but changes in MCH (10, 100, and Segmented (%) 18.2 ± 8.0 20.2 ± 6.9 14.3 ± 4.3 19.0 ± 9.0 −1 −1 200 mg⋅kg ), MCHC (100 mg⋅kg ), and platelet counts (10 2.5 ± 0.9 2.1 ± 0.9 2.2 ± 0.9 2.0 ± 1.0 −1 Eosinophil (%) and 100 mg⋅kg ) were observed in males. However, the Lymphocyte 76.2 ± 8.3 74.3 ± 8.0 76.5 ± 15.2 75.6 ± 10.3 changesinMCHCandplateletsdidnotshowadose-response (%) 3.2 ± 0.8 3.1 ± 0.7 2.9 ± 0.8 3.2 ± 1.0 relationship, and the changes in MCH, despite being evident Monocyte (%) at the three doses tested, remained within the normal range RBC 6 −3 7.35 ± 0.3 7.27 ± 0.6 7.26 ± 0.7 7.5 ± 0.4 (19.0 ± 1.09)[43]. These results suggest that F2 did not (×10 mm ) exert effects on blood cells or bone marrow, which are both Haemoglobin 15.6 ± 0.5 15.3 ± 1.2 15.3 ± 1.6 15.8 ± 0.9 sensitive to toxicity in animals [49].Thisresultissupported (g/dL) by the absence of F2 genotoxicity in mice in another study Haematocrit 43.7 ± 1.8 42.9 ± 3.9 45.6 ± 5.3 44.6 ± 2.4 [28]. (%) The relative weights of the organs did not show significant MCV (fL) 59.5 ± 1.1 58.9 ± 1.8 59.9 ± 1.9 59.4 ± 1.1 21.2 ± 0.4 21.0 ± 0.5 21.2 ± 1.1 21.1 ± 0.3 changes in the macroscopic and histopathological exami- MCH (pg) nations in the treatment groups compared with controls in 35.7 ± 0.8 35.6 ± 1.3 35.3 ± 1.9 35.7 ± 0.5 MCHC (%) either sex. In contrast to the study by Rebecca et al. [23], Platelet 3 −3 878 ± 124 884 ± 187 856 ± 168 730 ± 202 which found thymus involution at high doses, lower doses did (×10 mm ) ∗ not affect this organ. The data are expressed as mean ± SEM. 𝑃 < 0.05,comparedwithcontrol Another interesting result was found in the open-field group (ANOVA or Kruskal-Wallis test). test,inwhichnoneoftheparametersshowedsignificant changes, indicating that F2 did not exert neurobehavioral alterations in the animals. changedcomparedwiththecontrolgroup.Toevaluatekidney The low toxicity of F2 from the stem bark of S. adstringens, function, creatinine, urea, sodium, potassium, and uric acid reflected by high LD50 values, suggests a wide safety mar- were assessed [44]. Urea and sodium were not different from gin at therapeutic doses. In the repeated-dose oral toxicity controls in both female and male rats. In males, significant study, no serious signs or significant changes in hemato- differences in creatinine and potassium levels in the groups logical, biochemical, and histopathological parameters or treated with F2 did not show a dose response, and uric acid other remarkable effects were observed in rats. These toxicity −1 remained within the normal range (1.2–7.5mg⋅dL )[42]. studies suggest that the fraction is safe at the doses adminis- The same parameters also did not change in females. This tered. However, further studies are needed to evaluate other suggests that F2 does not adversely affect kidney function, parameters, including carcinogenicity, teratogenicity, and 8 Evidence-Based Complementary and Alternative Medicine neurotoxicity. Future clinical pharmacology studies should the region Northeast of Brazil,” Brazilian Journal of Pharmacog- also be conducted to determine tolerance and substantiate its nosy,vol.18,no.3,pp.472–508,2008. pharmacological use. [8] M. G. Brandao,˜ N. N. Zanetti, P. Oliveira, C. F. Grael, A. C. Santos, and R. L. M. Monte-Mor,´ “Brazilian medicinal plants described by 19th century European naturalists and in the 5. Conclusions Official Pharmacopoeia,” JournalofEthnopharmacology,vol. 120, no. 2, pp. 141–148, 2008. The low toxicity of F2 obtained from stem bark of S. adstringens in the acute and repeated-dose oral (chronic) [9] Z. V. Pereira, R. M. Mussury, A. B. de Almeida, and A. Sangalli, “Medicinal plants used by Ponta Pora˜ community, Mato Grosso toxicity studies suggests that F2 obtained from the stem bark do Sul State,” Acta Scientiarum—Biological Sciences,vol.31,no. of S. adstringens is safe at the concentrations tested. 3,pp.293–299,2009. [10]E.A.Audi,D.P.Toledo,P.G.Peresetal.,“Gastricantiulcero- Glossary genic effects of Stryphnodendron adstringens in rats,” Phytother- apy Research, vol. 3, pp. 264–266, 1999. F1: Crude extract obtained from the stem bark [11] E. A. Audi, C. E. Mendes De Toledo, F. Solera Dos Santos et of Stryphnodendron adstringens al.,“Biologicalactivityandqualitycontrolofextractandstem F2: Aqueous fraction obtained from the stem bark from Stryphnodendron adstringens,” Acta Farmaceutica bark of Stryphnodendron adstringens Bonaerense,vol.23,no.3,pp.328–333,2004. LD50: Dose that inactivates 50% of individuals. [12] J. D. Herzog-Soares, R. K. Alves, E. Isac et al., “Atividade tri- panocida in vivo de Stryphnodendron adstringens (barbatimao˜ verdadeiro) e Caryocar brasiliensis (pequi),” Revista Brasileira Conflict of Interests de Farmacognosia,vol.12,pp.1–2,2002. The authors declare that there is no conflict of interests. [13] P. S. Luize, T. S. Tiuman, L. G. Morello et al., “Effects of medicinal plant extracts on growth of Leishmania (L.) amazonensis and Trypanosoma cruzi,” Brazilian Journal of Pharmaceutical Acknowledgments Sciences,vol.41,no.1,pp.85–94,2005. [14]F.B.Holetz,T.Ueda-Nakamura,B.P.DiasFilhoetal.,“Bio- The authors thank Maristela Gabriel for technical assistance. logical effects of extracts obtained from Stryphnodendron ThisstudywassupportedbyCAPES,CNPQ,FINEP,and adstringens on Herpetomonas samuelpessoai,” Memorias do PRONEX/Fundac¸ao˜ Araucaria.´ Instituto Oswaldo Cruz,vol.100,no.4,pp.397–401,2005. [15]A.M.M.Felipe,V.P.Rincao,F.J.Benatietal.,“Antiviral˜ References effect of Guazuma ulmifolia and Stryphnodendron adstringens on poliovirus and bovine herpesvirus,” Biological and Pharma- [1] S. C. Udem, O. Obidoa, and I. U. Asuzu, “Acute and chronic ceutical Bulletin,vol.29,no.6,pp.1092–1095,2006. toxicity studies of Erythrina senegalensis DC stem bark extract [16] K. Ishida, J. C. Palazzo de Mello, D. A. Garcia Cortez, B. P. in mice,” Comparative Clinical Pathology,vol.19,no.3,pp.275– Dias Filho, T. Ueda-Nakamura, and C. V.Nakamura, “Influence 282, 2010. of tannins from Stryphnodendron adstringens on growth and [2] G. K. Singh and V.Kumar, “Acute and sub-chronic toxicity study virulence factors of Candida albicans,” Journal of Antimicrobial of standardized extract of Fumaria indica in rodents,” Journal of Chemotherapy,vol.58,no.5,pp.942–949,2006. Ethnopharmacology,vol.134,no.3,pp.992–995,2011. [17] K. Ishida, S. Rozental, J. C. P. de Mello, and C. V. Nakamura, [3]J.B.Calixto,“Efficacy,safety,qualitycontrol,marketingand “Activity of tannins from Stryphnodendron adstringens on Cryp- regulatory guidelines for herbal medicines (phytotherapeutic tococcus neoformans: effects on growth, capsule size and pig- agents),” Brazilian Journal of Medical and Biological Research, mentation,” Annals of Clinical Microbiology and Antimicrobials, vol.33,no.2,pp.179–189,2000. vol. 8, p. 29, 2009. [4] U. P.de Albuquerque, J. M. Monteiro, M. A. Ramos, and E. L. C. [18]T.M.DeSouza,J.A.Severi,V.Y.A.Silva,E.Santos,andR.C. de Amorim, “Medicinal and magic plants from a public market L. R. Pietro, “Bioprospection of antioxidant and antimicrobial in northeastern Brazil,” Journal of Ethnopharmacology,vol.110, activities in the bark of Stryphnodendron adstringens (Mart.) no. 1, pp. 76–91, 2007. Coville (Leguminosae-Mimosoidae),” Revista de Ciencias Far- [5]A.C.C.Sanches,G.C.Lopes,C.E.M.Toledo,L.V.S. maceuticas Basica e Aplicada, vol. 28, no. 2, pp. 221–226, 2007. Sacramento, C. M. Sakuragui, and J. C. P. Mello, “Estudo Mor- [19] J. Palazzo De Mello, F. Petereit, and A. Nahrstedt, “Flavan- fologico´ Comparativo das Cascas e Folhas de Stryphnodendron 3-ols and prodelphinidins from Stryphnodendron adstringens,” adstringens, S. polyphyllum e S. obovatum- Leguminosae,” Latin Phytochemistry,vol.41,no.3,pp.807–813,1996. American Journal of Pharmacy, vol. 26, no. 3, pp. 362–368, 2007. [20] J. Palazzo De Mello, F. Petereit, and A. Nahrstedt, “Prorobi- [6]G.C.Lopes,A.C.C.Sanches,C.V.Nakamura,B.P.Dias netinidins from Stryphnodendron adstringens,” Phytochemistry, Filho, L. Hernandes, and J. C. P.De Mello, “Influence of extracts vol.42,no.3,pp.857–862,1996. of Stryphnodendron polyphyllum Mart. and Stryphnodendron [21] J. C. Palazzo De Mello, F. Petereit, and A. Nahrstedt, “A obovatum Benth. on the cicatrisation of cutaneous wounds in dimeric proanthocyanidin from Stryphnodendron adstringens,” rats,” Journal of Ethnopharmacology, vol. 99, no. 2, pp. 265–272, Phytochemistry,vol.51,no.8,pp.1105–1107,1999. 2005. [22] G. C. Lopes, F. A. Vieira Machado, C. E. Mendes de Toledo, C. [7]M.D.F.Agra,K.N.Silva,I.J.L.D.Bas´ılio, P. F. De Freitas, M. Sakuragui, and J. C. Palazzo de Mello, “Chemotaxonomic and J. M. Barbosa-Filho, “Survey of medicinal plants used in significance of 5-deoxyproanthocyanidins in Stryphnodendron Evidence-Based Complementary and Alternative Medicine 9

species,” Biochemical Systematics and Ecology,vol.36,no.12,pp. [39] T. A. Loomis and A. W. Hayes, Loomis Essentials of Toxicology, 925–931, 2008. Academic Press, New York, NY, USA, 1996. [23] M. A. Rebecca, E. L. Ishii-Iwamoto, R. Grespan et al., “Tox- [40] M. Raza, O. A. Al-Shabanah, T. M. El-Hadiyah, and A. A. Al- icological studies on Stryphnodendron adstringens,” Journal of Majed, “Effect of prolonged vigabatrin treatment on hemato- Ethnopharmacology,vol.83,no.1-2,pp.101–104,2002. logical and biochemical parameters in plasma, liver and kidney [24] M. A. Rebecca, E. L. Ishii-Iwamoto, A. M. Kelmer-Bracht et al., of Swiss albino mice,” Scientia Pharmaceutica,vol.70,no.2,pp. “Effect of Stryphnodendron adstringens (barbatimao)˜ on energy 135–145, 2002. metabolism in the rat liver,” Toxicology Letters,vol.143,no.1,pp. [41] R. W.Lewis, R. Billington, E. Debryune, A. Gamer, B. Lang, and 55–63, 2003. F. Carpanini, “Recognition of adverse and nonadverse effects in [25] M. E. Burger,¨ N. Ahlert, B. Baldisserotto, A. Langeloh, B. toxicity studies,” Toxicologic Pathology,vol.30,no.1,pp.66–74, Schirmer,andR.Foletto,“Analysisoftheabortiveand/or 2002. infertilizing activity of Stryphnodendron adstringens (Mart.) [42] B. M. Mitruka and H. M. Rawnsley, “Clinical biochemical Coville,” Brazilian Journal of Veterinary Research and Animal and hematological reference values,” in Normal Experimental Science, vol. 36, pp. 296–299, 1999. Animals, Masson Publishing, New York, NY, USA, 1977. [26] P. Cintra, O. Malaspina, and O. C. Bueno, “Toxicity of bar- [43] S. T. Wolford, R. A. Schroer, F. X. Gohs et al., “Reference batimao˜ to Apis mellifera and Scaptotrigona postica,under range data base for serum chemistry and hematology values in laboratory conditions,” Journal of Apicultural Research,vol.42, laboratory animals,” Journal of Toxicology and Environmental no.1-2,pp.9–12,2003. Health,vol.18,no.2,pp.161–188,1986. [27] N. C. De Sousa, S. De Carvalho, M. A. Spano,´ and U. Graf, [44] G. Chandramohan, K. S. Al-Numair, and K. V. Pugalendi, “Absence of genotoxicity of a phytotherapeutic extract from “Effect of 3-hydroxymethyl xylitol on hepatic and renal func- Stryphnodendron adstringens (Mart.) Coville in somatic and tional markers and protein levels in streptozotocindiabetic rats,” germ cells of Drosophila melanogaster,” Environmental and African Journal of Biochemistry Research, vol. 3, pp. 198–204, Molecular Mutagenesis, vol. 41, no. 4, pp. 293–299, 2003. 2009. [28] M. A. Costa, K. Ishida, V. Kaplum et al., “Safety evaluation of [45] C. M. Corns, “Herbal remedies and clinical biochemistry,” proanthocyanidin polymer-rich fraction obtained from stem Annals of Clinical Biochemistry,vol.40,no.5,pp.489–507,2003. bark of Stryphnodendron adstringens (Barbatimao)˜ for use as a [46] J. El Hilaly, Z. H. Israili, and B. Lyoussi, “Acute and chronic pharmacological agent,” Regulatory Toxicology and Pharmacol- toxicological studies of Ajuga iva in experimental animals,” ogy,vol.58,no.2,pp.330–335,2010. Journal of Ethnopharmacology,vol.91,no.1,pp.43–50,2004. [29] Brasil. Ministerio´ da Saude.´ Anvisa—Agenciaˆ Nacional de [47] S. O. Aniagu, F. C. Nwinyi, B. Olanubi et al., “Is Berlina gran- Vigilanciaˆ Sanitaria,´ “Resoluçao˜ RDC nº 48. Dispoe˜ sobre o diflora (Leguminosae) toxic in rats?” Phytomedicine,vol.11,no. registro de medicamentos fitoterapicos,”´ 2004. 4, pp. 352–360, 2004. [30] Brasil. Ministerio´ da Saude.´ Anvisa—Agenciaˆ Nacional de [48] M.-F. Yuen, Y. Tanaka, D. Y.-T. Fong et al., “Independent risk Vigilanciaˆ Sanitaria,´ “Resoluçao-RE˜ nº 90. Guia para a factors and predictive score for the development of hepatocel- realizaçao˜ de estudos de Toxicidade Pre-Cl´ ´ınica de Fit- lular carcinoma in chronic hepatitis B,” Journal of Hepatology, oterapicos,”´ 2004. vol. 50, no. 1, pp. 80–88, 2009. [31] T. M. Antonelli-Ushirobira, E. N. Kaneshima, M. Gabriel, E. A. [49] H. A. Harper, Review of Physiological Chemistry, Lange Medical Audi, L. C. Marques, and J. C. P. Mello, “Acute and subchronic Publications, Los Altos, Calif, USA, 14th edition, 1973. toxicological evaluation of the semipurified extract of seeds of guarana´ (Paullinia cupana) in rodents,” Food and Chemical Toxicology,vol.48,no.7,pp.1817–1820,2010. [32]C.-Y.Li,R.K.Devappa,J.-X.Liu,J.-M.Lv,H.P.S.Makkar,and K. Becker, “Toxicity of Jatropha curcas phorbol esters in mice,” Food and Chemical Toxicology,vol.48,no.2,pp.620–625,2010. [33]L.C.MillerandM.L.Tainter,“EstimationoftheED50andits error by means of logarithmic probit graph paper,” Proceedings of the society for Experimental Biology and Medicine,vol.57,pp. 261–264, 1994. [34] Plantas que Curam, 2013, http://www.plantasquecuram.com .br/ervas/barbatimao.html. [35] J. R. Royce, “On the construct validity of open-field measures,” Psychological Bulletin, vol. 84, no. 6, pp. 1098–1106, 1977. [36] F.C.Odds,A.J.P.Brown,andN.A.R.Gow,“Antifungalagents: mechanisms of action,” Trends in Microbiology,vol.11,no.6,pp. 272–279, 2003. [37] P. L. Fidel Jr., “History and update on host defense against vaginal candidiasis,” American Journal of Reproductive Immunology, vol. 57, no. 1, pp. 2–12, 2007. [38] L. Ostrosky-Zeichner, “Combination antifungal therapy: a critical review of the evidence,” Clinical Microbiology and Infection, vol.14,no.4,pp.65–70,2008. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 851621, 8 pages http://dx.doi.org/10.1155/2013/851621

Research Article Comparison of Brazilian Plants Used to Treat Gastritis on the Oxidative Burst of Helicobacter pylori-Stimulated Neutrophil

Cibele Bonacorsi,1 Luiz Marcos da Fonseca,2 Maria Stella Gonçalves Raddi,2 Rodrigo Rezende Kitagawa,3 and Wagner Vilegas4

1 Instituto de Cienciasˆ da Saude,´ Universidade Federal de Mato Grosso, 78557-267 Sinop, MT, Brazil 2 Faculdade de Cienciasˆ Farmaceuticas,ˆ Universidade Estadual Paulista (UNESP), 14801-902 Araraquara, SP, Brazil 3 Departamento de Cienciasˆ Farmaceuticas,ˆ Universidade Federal do Esp´ırito Santo, 29040-090 Vitoria,´ ES, Brazil 4 Campus Experimental do Litoral Paulista, Universidade Estadual Paulista (UNESP), 11330-900 Sao˜ Vicente, SP, Brazil

Correspondence should be addressed to Maria Stella Gonc¸alves Raddi; [email protected]

Received 12 March 2013; Revised 25 June 2013; Accepted 26 June 2013

Academic Editor: Ulysses Paulino Albuquerque

Copyright © 2013 Cibele Bonacorsi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ten Brazilian medicinal plants used to treat gastritis and ulcers were carefully selected on the basis of ethnopharmacological importance and antiulcerogenic activity previously described. The antioxidant activity of the methanolic extracts was determined in analysis conditions that simulate a real biological activity on inhibition of the oxidative burst induced in neutrophils using Helicobacter pylori as activator, by a luminol-amplified chemiluminescence assay. The extracts, at low concentration (5 𝜇g/mL), exhibited a large variation in inhibitory effects of H. pylori-induced oxidative burst ranging from 48% inhibition to inactive, but all extracts, excluding Byrsonima intermedia, had inhibitory activity over 80% at the concentration of 100 𝜇g/mL. The total suppressive antioxidant capacity measured as the effective concentration, which represents the extract concentration producing 50% inhibition of the chemiluminescence induced by H. pylori, varies from 27.2 to 56.8 𝜇g/mL and was in the following order: Qualea parviflora > Qualea multiflora > Alchornea triplinervia > Qualea grandiflora > Anacardium humile > Davilla elliptica > Mouriri pusa > Byrsonima basiloba > Alchornea glandulosa > Byrsonima intermedia. The main groups of compounds in tested extracts are presented. Differences in the phytochemical profile, quantitatively and qualitatively, of these plants can explain and justify their protective effect on the gastric mucosa caused by the neutrophil-generated ROS that occurs when H. pylori displays its evasion mechanisms.

1. Introduction Infiltration of the gastric mucosa with neutrophils, macrophages, and lymphocytes is a hallmark of H. pylori Helicobacter pylori infection is recognized as an important infection [6]. To generate the microbicidal oxidants, poly- causative agent of gastroduodenal diseases, causing chronic morphonuclear neutrophils demonstrate a burst of oxidative gastritis, peptic ulcer disease, and increased risk of cancer activity, which is known as the respiratory burst, by releasing − [1]. H. pylori infects about one-half of the world’s population large quantities of superoxide anion (O2 )asaresultofthe and usually persists lifelong unless eradicated by antibiotic activation of NADPH oxidase [7]. The enzyme superoxide treatment [2]. In Brazil, the overall prevalence of H. pylori dismutase reduces the superoxide anion radical to form is high when compared to that of developed countries [3]. hydrogen peroxide (H2O2)andoxygen(O2)[8]. Myeloper- The rate of H. pylori prevalence in adults in the south of oxidase, an enzyme released from the azurophilic granules − Brazil is about 63% [4],whileinapoorurbancommunity in neutrophils, uses H2O2 andchlorideions(Cl)assub- in northeastern it is 80% [5]. strates to produce hypochlorous acid (HOCl), an important 2 Evidence-Based Complementary and Alternative Medicine antibacterial compound, but an extremely strong oxidant 2. Material and Methods that can also attack host biomolecules [9]. Although the neutrophils recruited to the gastric mucosa during infec- 2.1. Plant Material and Preparation of the Extracts. The tion represent one obvious source of oxidative stress, H. MeOH extracts used in this study from Alchornea glandulosa pylori itself also generates reactive oxygen species (ROS) (Euphorbiaceae) [11], Alchornea triplinervia (Euphorbiaceae) to resist oxidative damage from chronic inflammation that [12], Anacardium humile (Anacardiaceae) [13], Byrsonima accumulatesingastricepithelialcells[10]. Paradoxically, basiloba, Byrsonima intermedia (Malpighiaceae) [14], Davilla this robust immune/inflammatory response cannot clear elliptica (Dilleniaceae) [15], Qualea grandiflora (Vochysi- the infection, thus leaves the host prone to complications aceae) [16], Qualea parviflora (Vochysiaceae) [17], Qualea resulting from chronic inflammation. In addition, extensive multiflora (Vochysiaceae), and Mouriri pusa (Melastomat- studies have revealed that H. pylori-induced ROS production aceae) [18, 19]werethesameastheusedinourprevious in gastric epithelial cells might affect gastric epithelial cell studies. Briefly, the dried powdered plant material (leaves signal transduction, resulting in gastric carcinogenesis [10]. or bark) was extracted exhaustively with successions of methanol at room temperature. The extract was filtered and In Brazil, a large number of herbal extracts are used in ∘ folk medicine to treat various types of digestive disorders, concentrated under reduced pressure at 60 Cwitharotary and several studies have documented the beneficial effects evaporator to yield the MeOH extract. The reference material, of Brazilian plant in the prevention of gastric injury [11–19]. extract, and phytochemical screening of these plants are The mechanisms by which H. pylori infection leads to gastric described in Bonacorsi et al. [22]. mucosal damage include the direct effects of virulent factors produced by bacterium, the propagation and perpetuation of 2.2. Animals. Male rats (Rattus norvegicus albinus)weigh- inflammation, oxidative stress, and the induction of apoptosis ing 290 ± 20 g were obtained from the Animal House of in infected gastric epithelial cells [20]. Herbs that can pro- the Sao˜ Paulo State University “Julio´ de Mesquita Filho” ∘ tect cells from oxidative stress and antioxidant therapeutic (UNESP). The animals were maintained at 23 ± 2 Canda approaches may play an important role by protecting the relative humidity of 50 ± 5% under a 12 h light/12 h dark gastric mucosa from oxidative damage or by accelerating cycle. The Ethical Committee of the Pharmaceutical Sciences healing of gastric ulcers [21]. at Araraquara—UNESP—approved the experimental proce- In our previous study, the antioxidant activity of some dure of this study (resol 05/2008). Brazilian plants was investigated in a cell-free system using the DPPH radical scavenging activity [22]. The DPPH assay is classified as a single-electron transfer reaction that may 2.3. Experimental Protocols be neutralized by the antioxidant either by direct reduction 2.3.1. Collection of Polymorphonuclear Neutrophils. Suspen- via electron transfers or by radical quenching via H atom sions of polymorphonuclear neutrophils were obtained from transfer [23]. One of the limitations of this assay is the rats by intraperitoneal (i.p.) injection of 10 mL of a solution of poor correlation between DPPH chemical structure with free sterile oyster glycogen 0.5% (w/v) in saline followed, 12 h later, radicals produced in biological systems [24]. Therefore, to by lavage with 20 mL Dulbecco’s phosphate-buffered saline study the potential antioxidant effects of natural products, it (D-PBS) without calcium containing 10 IU heparin/mL. The is very important to add a cellular-based assay considering cells were washed twice in D-PBS and were carefully layered the complexity involved in their in vivo mechanisms of action onto 5 mL of Ficoll-Paque (𝑑 = 1077) and centrifuged at [25]. 800 ×g for 30 min. Subsequently, the neutrophils were washed Chemiluminescence assays that measure the production again with D-PBS and adjusted to a concentration of 2.0 × of ROS have been widely used as a sensitive assay for 6 10 cells/mL. The proportion of neutrophils (over 95%) and monitoring free radicals and reactive metabolites produced cell viability in the peritoneal exudate were determined by cell by enzymes, cells, or organ systems [26]. The use of chemilu- staining with May-Grunwald-Giemsa.¨ minescent probes amplifies chemiluminescence by allowing the detection of low levels of light emission, thereby increases the sensitivity of the reaction [27]. Antioxidants affect the 2.3.2. Helicobacter Pylori Strain, Culture Conditions, and intensity of luminol-dependent chemiluminescence [28]. Luminol Chemiluminescence Assay. H. pylori type strain Since the release of ROS by polymorphonuclear, neutrophils ATCC 43504, which is metronidazole resistant and amox- arebelievedtobeanimportantpartofthepathogenesis icillin susceptible, was obtained from the American Type of H. pylori-associatedgastritisandduodenalulcerare;in Culture Collection (Manassas, VA, USA). The bacterium ∘ the present study, the methanolic extracts (MeOH) obtained was grown in a microaerophilic atmosphere at 37 Con from Brazilian medicinal plants used to treat gastritis and Columbia agar containing 5% sheep blood for 3 days. H. ulcer were investigated for their antioxidants effects on pylori organisms were collected and suspended in 0.01 M the neutrophil oxidative burst generated by H. pylori as a phosphate buffered 0.15 M saline, pH 7.4 (PBS) at different stimulant, using luminol-amplified chemiluminescence. This absorbance (0.15, 0.2, and 0.3) with a 620 nm filter in study is a part of a larger survey in which other functional order to follow the respiratory burst of polymorphonuclear properties of these extracts such as their antimicrobial, neutrophils exposed to bacteria using luminol-enhanced antiinflammatory, and antiulcerogenic activities were also chemiluminescence assay performed following the protocol 6 evaluated [11–19]. described by Bonacorsi et al. [29]. Briefly, 5.0 × 10 cells/mL Evidence-Based Complementary and Alternative Medicine 3

× −5 and 2.0 10 M luminol (Sigma Chemical Co., St. Louis, 80 MO) were added to a tube containing PBS-D. This vial was placed in a lightproof chamber of a Bio-Orbit model 70 1251 luminometer (Bio-Orbit, Finland), and the carousel was 60 rotated to bring the sample in line with the photomultiplier 50 tube to record background activity. The control stimulus (1 mg/mL zymozan A (Sigma Chemical Co., St. Louis, MO)) 40 or bacteria were added to the suspension at a final volume 30 of 1.0 mL. The chemiluminescence emission of each vial, 20 expressed in mV, was recorded for 90 min to obtain kinetic Chemiluminescence (mV) Chemiluminescence curves. All experiments were carried out in triplicate and 10 repeated at least three times. 0

0 20406080100 2.3.3. Luminol-Dependent Chemiluminescence for Determi- Time (min) nation of Total Antioxidant Reactivity of Extracts. The inhibitory effect of the MEOH extracts on the chemilumi- Figure 1: Luminol-enhanced chemiluminescence kinetic profile of nescence emission by H. pylori-stimulated neutrophils was stimulated neutrophils with Helicobacter pylori and nonopsonized determined using the chemiluminescence assay described zymosan. Background (solid circles), zymosan 700 𝜇g/mL (open above. For the inhibition experiments, the stimulus (H. pylori circles), H. pylori suspension 0.15 ODU (solid triangles), H. pylori suspension at an optical density of 0.2) was added to the tubes, suspension 0.20 ODU (solid rectangles (◼)), and H. pylori suspen- and light release (in mV) was measured for 15 min. After sion 0.30 ODU (open rectangles). Each curve was constructed from this, D-PBS containing the MEOH extract at noncytotoxic 90 points, each point representing the mean of three replicates. concentrations(5,50,and100𝜇g/mL) was added, and the oxidative burst was continuously monitored for another 75 min. The chemiluminescence response was quantified as The results revealed that the tested extracts, at low the integrated area below the resulting chemiluminescence concentration, have a large variation in antioxidant activity curve, over a period from 0 to 90 min. The background ranging from 48% to inactive at 5 𝜇g/mL. The inhibition of chemiluminescence from neutrophils in the absence of stim- the oxidative burst of these plant extracts was in the following ulus was also measured. The effective concentration50 (EC ), order: Qualea parviflora > Qualea multiflora > Alchornea that is, the extract concentration producing 50% inhibition of triplinervia > Alchornea glandulosa > Byrsonima intermedia the chemiluminescence induced by H. pylori,wascalculated > Qualea grandiflora > Anacardium humile > Davilla elliptica; using a log-plot transformation of the data. Quercetin was Mouriri pusa and Byrsonima basiloba were inactive. All used as a standard antioxidant. All tests were performed in examined extracts, excluding B. intermedia, had antioxidant triplicateandrepeatedatleastthreetimes.Noneofthese activity over 80% at the concentration of 100 𝜇g/mL. In terms extracts affected neutrophils viability (over 95%) upon 90 min of efficient concentration, the EC50 values ranged from 27.2 𝜇 incubation with 100 g/mL of plant extracts. to 56.8 𝜇g/mL. The suppressive activity was in the following order: Qualea parviflora > Qualea multiflora > Alchornea 2.4. Statistical Analysis. The parameters were expressed as triplinervia > Qualea grandiflora > Anacardium humile > the mean (SD). Data were analyzed by analysis of variance Davilla elliptica > Mouriri pusa > Byrsonima basiloba > (ANOVA). The differences were considered statistically sig- Alchornea glandulosa > Byrsonima intermedia.Although nificant when the test yielded a value of 𝑃 ≤ 0.05 compared the EC50 for A. glandulosa and A. humile is close (43.4 to the standard antioxidant. and 40.2 𝜇g/mL, resp.), A. humile was less active than A. glandulosa at 5 𝜇g/mL (9.1% and 21.6%, resp.). B. basiloba and M. pusa were inactive at the 5 𝜇g/mL but they showed high 3. Results activity at the concentration of 100 𝜇g/mL (93.4% and 93.0% Challenge of neuthrophils with H. pylori resulted in a inhibition). strong luminol chemiluminescence response (exemplified in Figure 1). It was found that a significant kinetic profile occurs 4. Discussion following exposures of neutrophils to different concentrations of H. pylori. H. pylori suspension 0.20 ODU was used Despite years of experience with Helicobacter pylori treat- in the tests. ment, the ideal regimen for treating this infection remains to The capacity of the MEOH extracts to inhibit the oxida- be found [1]. Antibiotics are not the only factor of the success tive burst induced by H. pylori is shown in Table 1.The of H. pylori eradication therapy; another factor influencing extracts inhibited the oxidative burst in a concentration- H. pylori eradication rate is microenvironment created by the dependent manner, that is, as the concentration of the sample bacteria. The more severe clinical manifestation associated increased, the percentage inhibition of oxidative burst also with some H. pylori strains may be attributed to the higher increased to a certain extent and then leveled off with further grade of inflammation that they induce10 [ ]. It has been increase in extracts concentration. suggested that antioxidants increase the effectiveness of 4 Evidence-Based Complementary and Alternative Medicine

Table 1: Effects of methanolic extract of Brazilian plants used to treat gastritis on luminol-dependent chemiluminescence response of neutrophil stimulated by H. pylori.

Botanical name a b c Concentration (𝜇g/mL) IA % reduction in IA EC50 (𝜇g/mL) (popular name) 5 0(control) 2.96 × 10 ± 12542 — 5 ∗ Alchornea glandulosa 5 2.32 × 10 ± 9838 21.6 5 ∗ 43.4 (tapia)´ 50 0.54 × 10 ± 2279 81.6 5 ∗ 100 0.52 × 10 ± 2197 82.4 5 0(control) 2.34 × 10 ± 11565 — 5 ∗ Alchornea triplinervia 5 1.67 × 10 ± 8245 28.6 5 ∗ 36.6 (tanheiro) 50 0.26 × 10 ± 1301 88.9 5 ∗ 100 0.23 × 10 ± 1144 90.2 5 0(control) 2.20 × 10 ± 13718 — 5 Anacardium humile 5 2.00 × 10 ± 8077 9.1 5 ∗ 40.2 (cajuzinho-do-cerrado) 50 0.21 × 10 ± 2047 90.5 5 ∗ 100 0.11 × 10 ± 1113 95.0 5 0(control) 2.90 × 10 ± 3502 — 5 Byrsonima basiloba 5 2.92 × 10 ± 8077 0 5 ∗ 42.9 (murici-de-ema) 50 0.30 × 10 ± 2376 89.7 5 ∗ 100 0.19 × 10 ± 464 93.4 5 0(control) 1.43 × 10 ± 9123 — 5 Byrsonima intermedia 5 1.22 × 10 ± 7767 14.6 5 ∗ 56.8 (murici-do-campo) 50 0.57 × 10 ± 3648 60.1 5 ∗ 100 0.37 × 10 ± 2373 74.1 5 0(control) 2.98 × 10 ± 16869 — 5 Davilla elliptica 5 2.80 × 10 ± 15811 6.0 5 ∗ 41.1 (cipo-de-carij´ o)´ 50 0.29 × 10 ± 1651 90.3 5 ∗ 100 0.16 × 10 ± 891 94.6 5 0(control) 2.18 × 10 ± 7691 — 5 Qualea grandiflora 5 1.95 × 10 ± 6878 10.5 5 ∗ 39.3 (pau-da-terra) 50 0.15 × 10 ± 867 93.1 5 ∗ 100 0.14 × 10 ± 765 93.6 5 0(control) 2.20 × 10 ± 18681 — 5 ∗ Qualea parviflora 5 1.14 × 10 ± 9655 48.0 5 ∗ 27.2 (ipe-cascudo)ˆ 50 0.14 × 10 ± 1194 93.6 5 ∗ 100 0.11 × 10 ± 931 95.0 5 0(control) 2.60 × 10 ± 22417 — 5 ∗ Qualea multiflora 5 1.37 × 10 ± 11586 47.3 5 ∗ 27.7 (cerrado-campo) 50 0.17 × 10 ± 1432 93.5 5 ∗ 100 0.13 × 10 ± 1117 95.0 5 0(control) 2.30 × 10 ± 4038 — 5 Mouriri pusa 5 2.30 × 10 ± 4127 0 5 ∗ 42.4 (puc¸a)´ 50 0.18 × 10 ± 520 92.2 5 ∗ 100 0.16 × 10 ± 459 93.0 5 0(control) 2.48 × 10 ± 5987 — 5 ∗ 1 1.11 × 10 ± 3211 55.2 5 ∗ Quercetin (standard) 5 0.18 × 10 ± 1101 92.7 <1.0 5 ∗ 50 0.15 × 10 ± 578 94.0 5 ∗ 100 0.13 × 10 ± 499 94.8 ∗ aIntegrated area of chemiluminescence curve: mean of triplicate readings ± SD (𝑛=3); bcompared to the control; cefficient concentration; statistically significant difference𝑃 ( < 0.05). Evidence-Based Complementary and Alternative Medicine 5 the antibiotics by reducing inflammation and oxidative stress support an unpaired electron [39]. The position of hydroxyl in the gastric mucosa [30]. Determination of the antioxidant groups seems more important than their number for the activity of plant extracts and compounds often gives different antioxidantcapacityofphenolics;forexample,hydroxyl results since the methods used are based on different reaction groups in the ortho position of the B ring can greatly enhance mechanisms [31]. the antioxidant capacity, such as in catechins [40]. Themainaimofthispaperwastostudyinanalysis The leaves of Alchornea triplinervia are commonly conditions that simulate, as much as possible, a real antiox- used in Brazilian folk medicine in tea form to treat idantactivityofsomeBrazilianmedicinalspeciesonROS gastric disturbances. In order to better comprehend the induced in neutrophils exposure to H. pylori in order to give effect of MEOH extract on gastric injuries, Lima et al. a contribution to the pharmacological validation for their [12] separated the MEOH extract into ethyl acetate and usetotreatulcersandgastritis.Theantioxidantcapacity water, thus obtaining two fractions. Oral pretreatment rats of MeOH extracts on the neutrophil oxidative burst was with ethyl acetate fraction decreased the gastric injuries evaluated through chemiluminescence assay using luminol induced by ethanol resulting in more efficient gastro- as probe. The chemiluminescence method is a direct method protective effect than with MEOH extract. The authors of radical investigation, though the advantage of the method observed that the ethyl acetate fraction contains primarily five consists in the fact that chemiluminescence intensity is phenolic compounds: ellagic acid, quercetin-3-O-beta-D- directly proportional to a steady-state concentration of the galactopyranoside, quercetin-7-O-beta-D-glucopyranoside, radicals responsible for luminescence irrespective of the quercetin-3-O-beta-D-glucopyranoside, and quercetin-3-O- activity of these radicals [32]. It is enable to measure the alpha-L-arabinopyranoside. level of free radicals and estimate antioxidant protection Higher level of gallic acid derivatives than catechins and parameters and antioxidant action. Luminol tracks the pro- flavonoids was detected in Anacardium humile, and methyl duction of reactive oxygen species formed in the intra- and gallate has been found to be the major component [13]. − extracellular environment, such as HOCl, H2O2,andO2 Gallic acid and its derivatives are commonly used as food [33]. From the results showed in Table 1, the extracts inhibited additives as antioxidants [41]. Plants containing substances the respiratory burst of neutrophils induced by H. pylori in like quercetin and gallic acid are effective in preventing ulcers, concentration-dependent manner. mainly because of their antioxidant properties [42]. In nature, there are a wide variety of natural antioxi- According to ethnopharmacological studies, infusion dants which are different in their composition, physical and from leaves of Davilla elliptica is employed in folk medicine chemical properties, mechanism, and site of action [34]. as tea form to treat gastric pain, diarrhea, inflamma- Among the extracts, Qualea parviflora and Qualea multiflora tion, and ulcer. The chromatographic profile obtained by were found to be the most potent as they showed inhibition HPLC-PAD analyses led to the recognition of three main ranges between 48% and 95% for the tested concentrations classes of secondary metabolites in the methanolic extracts (5 and 100 𝜇g/mL, resp.). The action mechanisms involved from leaves of Davilla elliptica: phenolic acid derivatives, in the gastroprotective effects from Qualea parviflora consist flavonoids, and condensed tannins, myricetin-3-O-alpha- in reducing the gastric lesion by increasing the antioxidant rhamnopyranoside has been reported to be the main capacity of the gastric mucosa, though, in turn, main- flavonoid with percentage of 36.9% of the total flavonoid taining the GSH levels, increasing sulfhydryl compounds, content;however,themostabundantclassofsecondary and stimulating the gastric PGE2 synthesis [17]. Qualea metabolites found was the condensed tannins (41.2%) [15]. parviflora has been found to contain ellagic acid as its Tannins are potent scavengers of peroxyl radicals, and they major constituent. Phytochemical studies have shown that can also interact with mucus proteins, improving their 󸀠 methanolic extract contains 3,3 -di-O-methylellagic acid- cytoprotective effect by forming a protein lining over the 󸀠 4-O-beta-D-glucopyranoside, 3-O-methylellagic acid-4 -O- gastrointestinal mucosa [43]. alpha-L-rhamnopyranoside, 3,3-4-tri-O-methylellagic acid- Mouriri pusa was effective in experimentally healing rat 󸀠 󸀠 4 -O-beta-D-glucopyranoside, 3,3 -di-O-methylellagic acid, ulcers after 14 or 30 days of treatment [19]. Phytochemical triterpenes, and saponins [35]. Gastroprotective properties of investigation of the MeOH extract of Mouriri pusa yielded ellagic acids were evaluated by Beserra et al. [36] for gastric tannins, flavonoids, and (−)-epicatechin. The effect of tannins ulceration caused by ethanol, indomethacin, and acetic acid and flavonoids fractions from Mouriri pusa leaves methanolic treatments. extract on the prevention and cicatrisation process of gastric Qualea grandiflora showed a strong antiulcer effect on the ulcers was also demonstrated [18]. surface of the gastric mucosa and the phytochemical inves- Byrsonima basiloba is a native arboreal type from tigation proved the presence of tannins, catechins, steroids, the Brazilian “cerrado” (tropical American savanna), and terpenoids, and saponins [16]. Tannins and flavanoids con- the local population uses it to treat diseases, such as tain a variety of phenolic hydroxyl groups and show the diarrhea and gastric ulcer. Phytochemical analysis of the strongest antioxidant capacity and free radical-scavenging extracts revealed the presence of n-alkanes, lupeol, urso- activity among around a hundred phenolic compounds [37, lic and oleanolic acid, (+)-catechin, quercetin-3-O-alpha-L- 38]. The basic structural orientation of the compounds arabinopyranoside, gallic acid, methyl gallate, amentoflavone, 󸀠󸀠 determines the antioxidant activity of phenolics, such as quercetin, quercetin-3-O-(2 -O-galloyl)-beta-D-galactopy- 󸀠󸀠 how easily a hydrogen atom from a hydroxyl group can be ranoside, and quercetin-3-O-(2 -O-galloyl)-alpha-L-arabi- donated to a free radical, and the ability of the compounds to nopyranoside [44]. 6 Evidence-Based Complementary and Alternative Medicine

Previous investigations regarding the chemical composi- become useful as phytodrugs for the treatment of gastric tion of Byrsonima intermedia leaves indicated the presence of ulcers induced by H. pylori. quercetin-3-O-beta-D-galactopyranoside, (+)-catechin, (−)- epicatechin, gallic acid, methyl gallate, quercetin-3-O-alpha- Conflict of Interests L-arabinopyranoside, and amentoflavone [45]. Since most medicinal herbs are prepared for consumption of herbal tea, The authors declare that they have no conflict of interests. Rinaldo et al. [46]haveevaluatedthedifferencebetweenthe methanolic extract and the infusible form from Byrsonima intermedia and showed that the extract presents higher Acknowledgment amounts of flavan-3-ols than the infusible form per gram of This study was supported by Fundac¸ao˜ de Amparo a leaves. Gupta and Sharma [34] provided evidence that the hot ∘ Pesquisa do Estado de Sao˜ Paulo (BIOTA-FAPESP) and Con- water (80 C) extraction is a useful method with extracting selho Nacional de Desenvolvimento Cient´ıfico e Tecnologico´ efficiency of 83.7% for antioxidant activity and of 77.4% for (CAPES). total phenolic content, as compared with 80% methanolic extraction. Although, the phytochemical investigation of A. glandu- References losa led to the isolation of phenolic compounds like quercetin, gallic acid, amentoflavone, methyl gallate, myricetin- [1] J. G. Kusters, A. H. M. van Vliet, and E. J. Kuipers, “Pathogenesis 3-O-alpha-L-rhamnopyranoside, quercetin-3-O-alpha-L-ar- of Helicobacter pylori infection,” Clinical Microbiology Reviews, abinopyranoside, quercetin-3-O-beta-D-galactopyranoside, vol.19,no.3,pp.449–490,2006. and pterogynidine [11],thisextractreachedanEC50 of [2] M. J. Blaser and J. C. Atherton, “Helicobacter pylori persistence: 43.4 𝜇g/mL higher than that observed for A. triplinervia. biology and disease,” Journal of Clinical Investigation, vol. 113, The differences in the chemical constituents as well asin no.3,pp.321–333,2004. the quantity of several components might justify differences [3] T. Moujaber, C. R. MacIntyre, J. Backhouse, H. Gidding, H. in the antioxidant activity. Bonacorsi et al. [29], under Quinn, and G. L. Gilbert, “The seroepidemiology of Heli- the same working conditions used in this study, reported cobacter pylori infection in Australia,” International Journal of the inhibition of luminol oxidation on neutrophil oxidative Infectious Diseases,vol.12,no.5,pp.500–504,2008. burst generated by H. pylori by some phenolic compounds. [4] I. S. Santos, J. Boccio, A. S. Santos et al., “Prevalence of At 5 𝜇g/mL concentration, the most potent inhibitor was Helicobacter pylori infectionandassociatedfactorsamong methyl gallate (73.1%) compared to (+)-catechin (28.3%), adults in Southern Brazil: a population-based cross-sectional study,” BMC Public Health,vol.5,article118,2005. amentoflavone (16.7%), quercetin 3-O-alpha-L-arabinopyranoside (8.9%), and quercetin 3-O-beta-D-galactopyranoside [5]M.N.Rodrigues,D.M.M.Queiroz,R.T.Rodrigues,A.M. (7.2%). Although the antioxidant activity of constituents of C. Rocha, M. B. B. Neto, and L. L. B. C. Braga, “Helicobacter pylori infection in adults from a poor urban community in plant extracts can be demonstrated, the possible synergistic Northeastern Brazil: demographic, lifestyle and environmental effects of these compounds should be considered. factors,” Brazilian Journal of Infectious Diseases,vol.9,no.5,pp. Even though intensive studies on the chemical contents in 405–410, 2005. numerous Brazilian plants commonly used in folk medicine [6]H.M.ScottAlgood,J.Gallo-Romero,K.T.Wilson,R.M. to the treatment of gastritis and ulcers have been conducted, PeekJr.,andT.L.Cover,“HostresponsetoHelicobacter pylori the complete composition data are yet insufficient to predict infection before initiation of the adaptive immune response,” the antioxidant activity on the oxidative burst induced by H. FEMS Immunology and Medical Microbiology,vol.51,no.3,pp. pylori in neutrophils. There is enough evidence to conclude 577–586, 2007. that these Brazilian plants might exert a beneficial effect [7] D. B. Graham, C. M. Robertson, J. Bautista et al., “Neutrophil- in gastric diseases related to generation of reactive oxygen mediated oxidative burst and host defense are controlled by a species. The use of medicinal plants as phytoceuticals orin Vav-PLC𝛾2 signaling axis in mice,” Journal of Clinical Investiga- combination with antibiotics for the treatment of H. pylori is tion,vol.117,no.11,pp.3445–3452,2007. an active research field. [8]W.L.Lee,R.E.Harrison,andS.Grinstein,“Phagocytosisby neutrophils,” Microbes and Infection,vol.5,no.14,pp.1299– 5. Conclusions 1306, 2003. [9] J. M. Robinson, “Reactive oxygen species in phagocytic leuko- As part of a continuous study on the benefits of medicinal cytes,” Histochemistry and Cell Biology,vol.130,no.2,pp.281– plants regarding the gastrointestinal tract, in this work, we 297, 2008. demonstrated the protective effect of some Brazilian plants by [10] O. Handa, Y. Naito, and T. Yoshikawa, “Helicobacter pylori:a which H. pylori and neutrophils collaborate to cause gastric ROS-inducing bacterial species in the stomach,” Inflammation mucosal damage. Although all studied plants showed antiox- Research,vol.59,no.12,pp.997–1003,2010. idant activity, there are enough evidences to conclude that the [11] T. R. Calvo, Z. P. Lima, J. S. Silva et al., “Constituents and most effective species confirmed for their lowest antioxidant antiulcer effect of Alchornea glandulosa: activation of cell efficient concentration were Qualea parviflora and Qualea proliferation in gastric mucosa during the healing process,” multiflora. The present results justify the ethnomedical use Biological and Pharmaceutical Bulletin,vol.30,no.3,pp.451– of these plants, which appears to have a great potential to 459, 2007. Evidence-Based Complementary and Alternative Medicine 7

[12]Z.P.Lima,T.R.Calvo,E.F.Silvaetal.,“Brazilianmedicinal [28]O.Hirayama,M.Takagi,K.Hukumoto,andS.Katoh,“Evalu- plant acts on prostaglandin level and Helicobacter pylori,” ation of antioxidant activity by chemiluminescence,” Analytical Journal of Medicinal Food, vol. 11, no. 4, pp. 701–708, 2008. Biochemistry,vol.247,no.2,pp.237–241,1997. [13] A. Luiz-Ferreira, A. C. A. De Almeida, M. Cola et al., “Mech- [29]C.Bonacorsi,M.S.G.Raddi,L.M.daFonseca,M.Sannomiya, anisms of the gastric antiulcerogenic activity of Anacardium and W. Vilegas, “Effect of Byrsonima crassa and phenolic con- humile St. Hil on ethanol-induced acute gastric mucosal injury stituents on Helicobacter pylori-induced neutrophils oxidative in rats,” Molecules,vol.15,no.10,pp.7153–7166,2010. burst,” International Journal of Molecular Sciences,vol.13,no.1, [14] R. C. Santos, H. Kushima, C. M. Rodrigues et al., “Byrsonima pp. 133–141, 2012. intermedia A. Juss.: gastric and duodenal anti-ulcer, antimicro- [30] J. M. B. V´ıtor and F. F. Vale, “Alternative therapies for Helicobac- bial and antidiarrheal effects in experimental rodent models,” ter pylori: probiotics and phytomedicine,” FEMS Immunology Journal of Ethnopharmacology,vol.140,no.2,pp.203–212,2012. and Medical Microbiology,vol.63,no.2,pp.153–164,2011. [15] H. Kushima, C. M. Nishijima, C. M. Rodrigues et al., “Davilla [31]S.P.Wong,L.P.Leong,andJ.H.WilliamKoh,“Antioxidant elliptica and Davilla nitida: gastroprotective, anti-inflammatory activities of aqueous extracts of selected plants,” Food Chem- immunomodulatory and anti-Helicobacter pylori action,” Jour- istry,vol.99,no.4,pp.775–783,2006. nal of Ethnopharmacology,vol.123,no.3,pp.430–438,2009. [32] Y. A. Vladimirov and E. V. Proskurnina, “Free radicals and [16] C. A. Hiruma-Lima, L. C. Santos, H. Kushima et al., “Qualea cell chemiluminescence,” Biochemistry,vol.74,no.13,pp.1545– grandiflora, a Brazilian “Cerrado” medicinal plant presents an 1566, 2009. important antiulcer activity,” Journal of Ethnopharmacology,vol. [33] N. Parij, A. Nagy, P. Fondu, and J. Neve,` “Effects of non- 104, no. 1-2, pp. 207–214, 2006. steroidal anti-inflammatory drugs on the luminol and lucigenin [17] L. P. Mazzolin, A. L. M. Nasser, T. M. Moraes et al., “Qualea amplified chemiluminescence of human neutrophils,” European parviflora Mart.: an integrative study to validate the gastropro- Journal of Pharmacology,vol.352,no.2-3,pp.299–305,1998. tective, antidiarrheal, antihemorragic and mutagenic action,” [34] V. K. Gupta and S. K. Sharma, “Plants as natural antioxidants,” Journal of Ethnopharmacology,vol.127,no.2,pp.508–514,2010. Natural Products Radiance,vol.5,no.4,pp.326–334,2006. [18] P. C. P. Vasconcelos, M. A. Andreo, W. Vilegas, C. A. Hiruma- [35]A.L.M.Nasser,C.B.A.Carli,C.M.Rodriguesetal., Lima, and C. H. Pellizzon, “Effect of Mouriri pusa tannins and “Identification of ellagic acid derivatives in methanolic extracts flavonoids on prevention and treatment against experimental from Qualea species,” Zeitschrift fur Naturforschung C,vol.63, gastric ulcer,” Journal of Ethnopharmacology,vol.131,no.1,pp. no. 11-12, pp. 794–800, 2008. 146–153, 2010. [36] A. M. S. E. S. Beserra, P. I. Calegari, M. D. C. Souza et [19] P. C. P. Vasconcelos, H. Kushima, M. Andreo et al., “Studies of al., “Gastroprotective and ulcer-healing mechanisms of ellagic gastric mucosa regeneration and safety promoted by Mouriri acid in experimental rats,” JournalofAgriculturalandFood pusa treatment in acetic acid ulcer model,” Journal of Ethnophar- Chemistry,vol.59,no.13,pp.6957–6965,2011. macology,vol.115,no.2,pp.293–301,2008. [37] V. Koleckar, K. Kubikova, Z. Rehakova et al., “Condensed and [20]P.C.Konturek,S.J.Konturek,andT.Brzozowski,“Helicobacter hydrolysable tannins as antioxidants influencing the health,” pylori infection in gastric cancerogenesis,” Journal of Physiology Mini-Reviews in Medicinal Chemistry,vol.8,no.5,pp.436–447, and Pharmacology,vol.60,no.3,pp.3–21,2009. 2008. [21] M. G. Repetto and S. F. Llesuy, “Antioxidant properties of [38] Y. Cai, Q. Luo, M. Sun, and H. Corke, “Antioxidant activity natural compounds used in popular medicine for gastric ulcers,” and phenolic compounds of 112 traditional Chinese medicinal Brazilian Journal of Medical and Biological Research,vol.35,no. plants associated with anticancer,” Life Sciences,vol.74,no.17, 5, pp. 523–534, 2002. pp.2157–2184,2004. [22] C. Bonacorsi, L. M. da Fonseca, M. S. G. Raddi, R. R. Kitagawa, [39] V. Roginsky, “Chain-breaking antioxidant activity of natural M. Sannomiya, and W. Vilegas, “Relative antioxidant activity of polyphenols as determined during the chain oxidation of brazilian medicinal plants for gastrointestinal diseases,” Journal methyl linoleate in Triton X-100 micelles,” Archives of Biochem- of Medicinal Plant Research, vol. 5, no. 18, pp. 4511–4518, 2011. istry and Biophysics,vol.414,no.2,pp.261–270,2003. [23] A. Jimenez,A.Selga,J.L.Torres,andL.Juli´ a,` “Reducing activity [40] C. A. Rice-Evans, N. J. Miller, and G. Paganga, “Structure- of polyphenols with stable radicals of the TTM series. Electron antioxidant activity relationships of flavonoids and phenolic transfer versus H-abstraction reactions in flavan-3-ols,” Organic acids,” Free Radical Biology and Medicine,vol.20,no.7,pp.933– Letters,vol.6,no.24,pp.4583–4586,2004. 956, 1996. [24]V.RoginskyandE.A.Lissi,“Reviewofmethodstodetermine [41] Y. Ow and I. Stupans, “Gallic acid and gallic acid deriva- chain-breaking antioxidant activity in food,” Food Chemistry, tives: effects on drug metabolizing enzymes,” Current Drug vol. 92, no. 2, pp. 235–254, 2005. Metabolism,vol.4,no.3,pp.241–248,2003. [25] C. Lopez-Alarc´ ona´ and A. Denicolab, “Evaluating the antiox- [42]S.Sumbul,M.A.Ahmad,M.Asif,andM.Akhtar,“Roleof idant capacity of natural products: a review on chemical and phenolic compounds in peptic ulcer: an overview,” Journal of cellular-based assays,” Analytica Chimica Acta,vol.763,pp.1– Pharmacy and Bioallied Sciences,vol.3,no.3,pp.361–367,2011. 10, 2013. [43] T. Okuda, “Systematics and health effects of chemically distinct [26] M. Nakano, “Detection of active oxygen species in biological tannins in medicinal plants,” Phytochemistry,vol.66,no.17,pp. systems,” Cellular and Molecular Neurobiology,vol.18,no.6,pp. 2012–2031, 2005. 565–579, 1998. [44] W.D.M.Lira,F.V.DosSantos,M.Sannomiya,C.M.Rodrigues, [27] Y. A. Vladimirov, E. V. Proskurnina, and D. Y. Izmailov, W. Vilegas, and E. A. Varanda, “Modulatory effect of Byrsonima “Chemiluminescence as a method for detection and study of basiloba extracts on the mutagenicity of certain direct and free radicals in biological systems,” Bulletin of Experimental indirect-acting mutagens in Salmonella typhimurium assays,” Biology and Medicine,vol.144,no.3,pp.390–396,2007. Journal of Medicinal Food, vol. 11, no. 1, pp. 111–119, 2008. 8 Evidence-Based Complementary and Alternative Medicine

[45] M. Sannomiya, C. R. P. Cardoso, M. E. Figueiredo et al., “Mutagenic evaluation and chemical investigation of Byrsonima intermedia A. Juss. leaf extracts,” Journal of Ethnopharmacology, vol. 112, no. 2, pp. 319–326, 2007. [46] D. Rinaldo, J. M. Batista Jr., J. Rodrigues et al., “Determination of catechin diastereomers from the leaves of Byrsonima species using chiral HPLC-PAD-CD,” Chirality,vol.22,no.8,pp.726– 733, 2010. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 727042, 7 pages http://dx.doi.org/10.1155/2013/727042

Research Article In Vitro Antileishmanial Activity of Essential Oil of Vanillosmopsis arborea (Asteraceae) Baker

Aracélio Viana Colares,1,2,3,4 Fernando Almeida-Souza,4 Noemi Nosomi Taniwaki,5 Celeste da Silva Freitas Souza,4 José Galberto Martins da Costa,6 KátiadaSilvaCalabrese,4 and Ana Lúcia Abreu-Silva7

1 Universidade Federal do Maranhao˜ (UFMA), 65080-805 Sao˜ Lu´ıs, MA, Brazil 2 Rede Nordeste de Biotecnologia (RENORBIO), 65080-805 Sao˜ Lu´ıs, MA, Brazil 3 Faculdade Leao˜ Sampaio (FALS), 63180-000 Juazeiro do Norte, CE, Brazil 4 Laboratorio´ de Imunomodulac¸ao˜ e Protozoologia, Instituto Oswaldo Cruz (FIOCRUZ), 21040-900 Rio de Janeiro, RJ, Brazil 5 Unidade de Microscopia Eletronica,ˆ Instituto Adolf Lutz (IAL), 01246-000 Sao˜ Paulo, SP, Brazil 6 Laboratorio´ de Pesquisa de Produtos Naturais, Universidade Regional do Cariri (URCA), 63105-000 Crato, CE, Brazil 7 Departamento de Patologia, Universidade Estadual do Maranhao˜ (UEMA), 65055-310 Sao˜ Lu´ıs, MA, Brazil

Correspondence should be addressed to Katia´ da Silva Calabrese; [email protected]

Received 5 April 2013; Revised 13 June 2013; Accepted 15 June 2013

Academic Editor: Ulysses Paulino Albuquerque

Copyright © 2013 Aracelio´ Viana Colares et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The search for new immunopharmacological chemical agents to treat various diseases caused by bacteria, fungi, and protozoa, such as leishmaniasis, for example, has led to the exploration of potential products from plant species and their main active ingredients. Antimonial drugs are the current treatment for leishmaniasis. These drugs cause major side effects and frequent discontinuation of treatment. In this study, we evaluated the in vitro leishmanicidal activity of essential oil of Vanillosmopsis arborea (VAEO) and its major compound 𝛼-bisabolol against Leishmania amazonensis.Theessentialoiland𝛼-bisabolol showed activity against promastigotes (IC50 7.35 and 4.95 𝜇g/mL resp.) and intracellular amastigotes (IC50 12.58 and 10.70 𝜇g/mL, resp.). Neither product showed any cytotoxicity on treated macrophages. The ultrastructural analysis of promastigotes incubated with VAEO or 𝛼-bisabolol at 30 𝜇g/mL, showed morphological changes with the accumulation of vesicles electrodense lipid inclusions. The results give evidence that both VAEO and 𝛼-bisabolol have potential as new therapeutic agents against leishmaniasis.

1. Introduction threatening about 350 million people in more than 90 countries [5]. Leishmania species have a wide distribution Natural products obtained from plants have been used in among tropical and subtropical countries, including several the treatment of infectious diseases, especially in developing countries in Latin America, Africa, India, part of Western countries. It is estimated that about 25% of medicines are Asia, and some Central European countries bordering the derived directly or indirectly from herbal products [1]. Mediterranean. The disease presents two clinical forms: Several studies have shown that various plant species have cutaneous and visceral and the former can be subdivided inhibitory action against certain types of parasites such as into localized cutaneous leishmaniasis, diffuse cutaneous Leishmania amazonensis [2], Leishmania chagasi [3], and and mucocutaneous [6]. The cutaneous form is the most Leishmania infantum [4]. common, affecting about 1 to 1.5 million people per year Leishmaniasisisachronicdiseasecausedbyprotozoan [7, 8]. In Brazil, cutaneous leishmaniasis is present in almost parasites belonging to the genus Leishmania. It is an endemic all states of the federation, and L. amazonensis is the main disease that constitutes a serious public health problem, etiologic agent [7]. 2 Evidence-Based Complementary and Alternative Medicine

Drugs such as sodium stibogluconate (Pentostam), am- 2.3. Essential Oil Analysis. Oil analysis was performed photericin B, pentamidine, and antimony N-methylglucam- using a Shimadzu GC MS-QP2010 series (GC/MS system) ine (Glucantime) are used for leishmaniasis treatment. The under the following conditions: Rtx-5MS capillary column pentavalent antimonials have been the mainstay of treatment (30 m × 0.25 mm, 0.25 𝜇m film thickness); carrier gas— ∘ for human leishmaniasis in recent decades, with cure rates helium at 1.5 mL/min; injector temperature—250 C; detector ∘ ∘ ∘ ranging from 60 to 90% [9, 10]. However, treatment with temperature—290 C; column temperature—60 C–180 Cat ∘ ∘ ∘ ∘ pentavalent antimonial is generally long expensive and toxic 5 C/min, then 180 –280 Cat10C/min (10 min); scanning and this prolonged course of treatment has caused the speed—0.5 scan/sec from 𝑚/𝑧 40 to 350; split ratio—1 : 200; development of resistance to these drugs [11, 12]. injected volume—1 𝜇Lof[25𝜇L (essential oil)/5 mL CHCl3] Thus the development of antileishmanial agents from (1 : 200); solvent cut time—2.5 min; and mass spectrometer— natural products has become important to find an alternative operated at 70 eV of ionization energy. The identification drug with low toxicity and high efficacy [13]. of individual components was based on their mass spectral Essential oils (EOs) are natural compounds and complex fragmentation according to the mass spectral library NIST 08, mixtures obtained from different parts of plants such as the retention indices, and comparison with published data. flowers, leaves, bark, stems, wood, roots, fruits, or seeds and are commonly used in folk medicine to treat various 2.4. Parasites. Promastigote forms of Leishmania amazonen- typesofdiseases[14, 15]. Most of the EOs are comprised ∘ of terpenes, especially monoterpenes and sesquiterpenes. sis (MHOM/BR/76/MA-76) were cultured at 26 CinLIBHIT Several studies by different research groups have shown media supplemented with 10% fetal bovine sera (Gibco, USA) biological activity of EOs and their major compounds against and 100 U/mL of penicillin (Gibco, USA). Cultures were used protozoal diseases such as leishmaniasis. For example, the only up to a maximum of ten in vitro passages [23]. essential oil of Chenopodium ambrosioides [16], Cymbopogon citratus [17] that both contain citral, have been evaluated for their antileishmanial properties. Also the studies of Escobar 2.5. Animals. Female BALB/c mice 4–6 weeks old were pur- et al. [18]whotested19essentialoils,fiveofwhichwerefrom chased from Centro de Criac¸ao˜ de Animais de Laboratorio,´ different species of Lippia andallofthemwereevaluatedfor InstitutoOswaldoCruz,RiodeJaneiro,andmaintained their antileishmanial properties. under pathogen-free conditions. The animals were handled in accordance with Guidelines for Animal Experimentation of Vanillosmopsis arborea Baker is native to the Araripe the Colegio´ Brasileiro de Experimentac¸ao˜ Animal. The local National Forest, in the Northeast of Brazil in the state of Ethics Committee on Animal Care and Utilization approved Ceara[´ 19]. There are few studies concerning the traditional all procedures involving the animals (CEUA FIOCRUZ- use of this plant. However, biological and pharmacological LW72/12). studies have shown that the essential oil of V. arborea (VAEO) presents antimicrobial [14], anti-inflammatory [20], and gastroprotective activity [21]. The present study characterized 2.6. Cell Cultures. The macrophage J774.G8 line was cultured the essential oil of Vanillosmopsis arborea chemically and in RPMI 1640 medium (Sigma, USA) supplemented with 10% evaluated its leishmanicidal and cytotoxic activity. Its major fetal bovine sera, penicillin (100 U/mL), and streptomycin compound 𝛼-bisabolol was also evaluated. ∘ (100 𝜇g/mL) at 37 Cand5%CO2. Female BALB/c mice were inoculatedwith1mLofsodiumthioglycolate3%,andafter 2. Materials and Methods 72 hours the peritoneal macrophages were harvested with PBS solution. The harvest was centrifuged at 4000 rpm, and the cells were suspended in RPMI medium supplemented as 2.1. Plant Material. Vanillosmopsis arborea Baker (Aster- ∘ aceae) was collected in Crato County, CearaState,Brazil.´ described before and cultured at 37 Cand5%CO2. The plant material was identified by Dr. Arlene Pessoa, and a voucher specimen was deposited under number 9493 at the 2.7. Activity against Promastigote Forms. Promastigote forms Herbarium “Dardano´ de Andrade Lima” of the Universidade 6 Regional do Cariri (URCA). of L. amazonensis (10 parasites/mL) from a 2–4-day-old culturewereplacedin96-wellplatesinthepresenceof different concentrations of VAEO and 𝛼-bisabolol (Sigma- 2.2. Essential Oil Extraction. The oil was extracted using a Aldrich, ≥95% (GC)) (1.9–60 𝜇g/mL, for both products) to a Clevenger-type apparatus. The analysis of the volatile con- final volume of 200 𝜇L per well for 24 hours. Wells without stituentswascarriedoutinaHewlett-PackardGC/MS,model parasiteswereusedasblank,andwellswithonlyparasites 5971. The stems were dried (850 g), according to (traditional) were used as control. After the treatment, the viability of folk medicine, and reduced in size, and EO extraction was parasites was evaluated by the tetrazolium-dye (MTT) colori- carried out by hydrodistillation for 2 h using a Clevenger- metric method modified by Mosmann [24]. MTT (5 mg/mL), type apparatus, resulting in an essential oil yield of 0.4% a volume equal to 10% of the total, was added to each well. (3.40 mL). After the oil was collected, it was dried using After 2 hours, the plate was centrifuged at 4000 rpm; then anhydrous sodium sulfate and subsequently stored under low 150 𝜇L supernatant was removed from each well and 100 𝜇Lof ∘ light conditions at 10 Cuntilanalysis[22]. DMSOwereaddedtodissolvetheformazan.Theabsorbance Evidence-Based Complementary and Alternative Medicine 3 was read on a spectrophotometer at a wavelength of 540 nm. 0.1 M sodium-cacodylate buffer and postfixed in a solution The data were normalized according to the formula: containing 1% osmium tetroxide, 0.8% ferrocyanide, and 5mMcalciumchloride,washedin0.1Msodium-cacodylate DO sample − DO blank %survival= × 100. (1) buffer, dehydrated in graded acetone, and embedded in epoxy DO control − DO blank resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a transmission electron TheresultswereusedtocalculatetheIC50 (50% inhibition of microscope JEM-1011 (JEOL, Japan). parasite growth). Amphotericin B was used as the reference drug. 2.11. Statistical Analysis. The values were expressed as ± 2.8. Activity against Intracellular Amastigotes. Peritoneal average S.D. The results were analyzed by Analysis of 5 macrophageswereculturedin24-wellplates(10 cells/well), Variance (ANOVA) followed by the Tukey test. The analyses ∘ were performed with the software GraphPad Prism 5.0.4. with coverslips, at 37 Cand5%CO2.Thecellswereinfected 𝑃 < 0.05 with promastigotes forms of L. amazonensis using a ratio of Differences were considered significant at . 10 : 1 parasite/cell, and after 2 hours the cells were washed three times with PBS to remove free parasites. The infected 3. Results and Discussion cells were treated with different concentrations of VAEO and 𝛼-bisabolol (60–1.9 𝜇g/mL, for both products) in triplicate for The chemical analysis revealed that 𝛼-bisabolol constituted 24 hours. The coverslips with the infected and treated cells 97.9% (25.5 min.) of VAEO (Figures 1 and 2). Other com- were fixed with Bouin, stained with Giemsa, and examined pounds identified were o-methyl eugenol (1.6%, 18.5 min.) by light microscopy. The inhibition percentage was calculated and bisabolol oxide (0.5%, 24.9 min.). In another study by using the formula described by Guru et al. [25]: PI = 100 Santos et al. [26]theessentialoilofV. arborea showed − (AN × 100)/(INA × TI), where NA is actual number of 𝛼-bisabolol as a major compound, however, at a different amastigotes/100 spleen cell nuclei after treatment; INA is concentration (80.43%). The chemical characterization of initial number of amastigotes/100 spleen cell nuclei; TI is fold the essential oil of Vanillosmopsis pohlii,too,identified𝛼- increase in the number of amastigotes in control animals on bisabolol as a major compound at a concentration of 79.0% the corresponding day of the biopsy in treated animals; and [27]. These differences may be due to seasonality and the time PI is percentage inhibition. The IC50 was calculated with the of collection of the plant sample [28]. GraphPad Prism. Amphotericin B was used as the reference The incubation of VAEO and its major compound, 𝛼- drug. bisabolol, efficiently inhibited the growth of Leishmania amazonensis promastigotes (Figure 3). The IC50/24 h values were 2.9. Cytotoxicity Assay. Macrophages J774.G8 were cultured 7. 3 5 a n d 4 . 9 5 𝜇g/mL, respectively (Table 1). The 𝛼-bisabolol 5 in 96-well plates (5 × 10 cells/mL) with different con- wasmoreeffectivethanthymol,themajorcompound centrations of VAEO and 𝛼-bisabolol (60–1.9 𝜇g/mL, both of Lippia sidoides,whichshowedanIC50 of 22.63 𝜇g/mL ∘ products) to a final volume of 200 𝜇Lperwell,at37Cand against promastigotes of L. amazonensis [15]. In another 𝛼 𝜇 5% CO2. Wells without cells were used as blank, and wells study, -bisabolol showed an IC50 of 10.99 g/mL against withonlycellswereusedascontrol.After24hours,the promastigotes of Leishmania infantum, which demonstrated viability of the cells was evaluated by the tetrazolium-dye its antiparasitic potential [29]. Despite having IC50 greater (MTT) colorimetric method modified by Mosmann [24]. than 𝛼-bisabolol, VAEOshowed significant inhibition against MTT(5mg/mL),avolumeequalto10%ofthetotal,was promastigotes of Leishmania, especially when compared to added to each well. After 2 hours, the plate was centrifuged other plant species such as Lippia sidoides (IC50 44.38 𝜇g/mL), at 4000 rpm; then the supernatant was removed from each Cordia verbenacea, Cajanus cajan, Lantana camara (IC50 120, well and 100 𝜇LofDMSOwasaddedtodissolvetheformazan. 62, and 14 𝜇g/mL, resp.) [30], and Plectranthus amboinicus, The absorbance was read on a spectrophotometer at 540 nm Aristolochia cymbifera,andLippia alba (IC50 > 500 𝜇g/mL) wavelength.Thedatawerenormalizedfollowingtheformula [30]. [24]: Our results also show that, when the two products were compared, VAEO was less effective against promastigotes and DO sample − DO blank intracellular amastigotes than 𝛼-bisabolol (Table 1 and Fig- %survival= × 100. (2) DO control − DO blank ure 4). The selectivity index (SI), especially for intracellular amastigotes, showed that 𝛼-bisabolol (9.383) was less toxic Theresultswereusedtocalculatethecellcytotoxicityby50% than VAEO (11.526). These values (9.383 and 11.526) were (CC50)withGraphPadPrism5. not statistically different, which confirms the role of the 𝛼- bisabolol in the leishmanicidal action of VAEO. 2.10. Transmission Electron Microscopy. Promastigote forms Both products tested showed no cytotoxicity against of L. amazonensis were treated with VAEO and 𝛼-bisabolol macrophages with CC50 145 and 100.4 𝜇g/mL, respectively at concentrations of 30, 15, 7.5, and 3.75 𝜇g/mL for 24 hours (Table 1), indicating no cytotoxicity, although the essential for both products. The parasites were fixed with 2.5% glu- oilsandtheircompoundshavebeenreportedtopresent taraldehyde (Sigma, USA) in 0.1 M sodium cacodylate buffer, cytotoxic effect when incorporated into the cell membrane pH 7.2 overnight. Parasites were washed three times with [31]. The 𝛼-bisabolol is a sesquiterpene with low toxicity 4 Evidence-Based Complementary and Alternative Medicine

Table 1: Leishmanicidal and cytotoxic activity of VAEO and its major constituent 𝛼-bisabolol.

IC 𝜇g/mL CC 𝜇g/mL Compounds 50 50 SI a Promastigote Amastigote intracellular J774.G8 ama VAEO 7.35 ± 0.050 12.58 ± 0.068 145 ± 0.023 11.526 𝛼-Bisabolol 4.95 ± 0.054 10.70 ± 0.085 100.4 ± 0.025 9.383 Amphotericin B 3.1 ± 0.048 7.8 ± 0.059 1.688 ± 0.4993 0.216 a SIama (selectivity index) = CC50 macrophage/IC50 amastigote.

×105 2,258,460 100 20 25.525

10 Intensity

5 50 TIC ∗ 1.00 18.525 0 24.848 Parasite survival (%) Parasite 3.0 10.0 20.0 30.0 40.0 44.0 (min)

Figure 1: Chromatogram of the essential oil of Vanillosmopsis arborea Baker. 0 1.9 3.8 7.5 15.0 30.0 60.0 (𝜇g/mL) OH VAEO 𝛼-Bisabolol

Figure 3: Effects of VAEO and 𝛼-bisabolol on Leishmania amazonensis promastigote forms. Each bar represents the mean ± standard deviation of three independent experiments in triplicate.

300

Figure 2: Chemical structure of 𝛼-bisabolol.

200 andisusedinthecosmeticindustry[32, 33]whereit presents a pro-apoptotic activity [34]. Its leishmanicidal action against promastigotes and intracellular amastigotes Survival index 100 may occur directly or indirectly through the production of cellular mechanisms, such as the production of nitric oxide (NO), which is the major effector molecule that participates in the intracellular killing of Leishmania [35]. The 0 hydrophobic nature and the toxic effects of essential oils and 1.9 3.8 7.5 15.0 30.0 60.0 𝜇 their major constituents against various microorganisms are ( g/mL) a common feature of plants which have volatile oils. These VAEO compounds can preferably incorporate in the cell membranes 𝛼-Bisabolol by inducing a loss of permeability to protons and ions and can induce changes considered vital for the cell; for example, the Figure 4: Effects of VAEO and 𝛼-bisabolol on Leishmania ama- ergosterol biosynthetic pathway causes extensive lesions on zonensis intracellular amastigotes. Each bar represents the mean ± the membranes of the Candida [31]. According to the results standard deviation of three independent experiments in triplicate. Evidence-Based Complementary and Alternative Medicine 5

(a) (b) (c)

(d) (e) (f)

Figure 5: Transmission electron microscopy of Leishmania amazonensis promastigote with no treatment (a) or incubated with 𝛼-bisabolol (b and c) and VAEO (d, e, and f) at concentrations of 30 𝜇g/mL for both compounds. Also both treatments show the interaction of lipid droplets with swelling of the plasma membrane. N: nucleus; k: kinetoplast; m: mitochondria; pf:pocketflagellar.Bars1.0𝜇m (a); 0.5 𝜇m (b); 0.2 𝜇m (c); 1.0 𝜇m (d); 0.5 𝜇m (e); 200 nm (f). shown here, it is possible that 𝛼-bisabolol and VAEO have the cellular membranes and can thus produce changes in the ability to increase cell permeability to exogenous compounds, integrity of cell structures and mitochondrial membranes, for since some sesquiterpenes (particularly 𝛼-bisabolol) can example [40]. induce changes in membranes allowing microorganisms to The promastigotes treated for 24 h with VAEO at a enter the cells and thus augmenting the microbial permeabil- concentration of 30 𝜇g/mL (Figures 5(d), 5(e) and 5(f)) ity to antimicrobial agents [36–38]. showed increased volumes of flagellar pockets with conse- The ultrastructural analysis of the promastigote control quent breakage, increased volumes and changes in mito- shows the parasite with an elongated cell body and the chondrial kinetoplasts, abnormal condensation of chromatin presence of a well-defined kinetoplast and nucleus (Figure 5). in the nucleus (small arrow—Figure 5(f)), discontinuity The promastigotes treated with 𝛼-bisabolol for 24 hours, of the nuclear membrane, lipid inclusions in the presence at a concentration of 30 𝜇g/mL (Figures 5(b) and 5(c)) of electrondense vesicles (white asterisk—Figure 5(f)), and presented severe cell damage with the loss of parasite mor- visualization of the inclusion of a lipid envelope within phology, discontinuity of the nuclear membrane, increased the plasma membrane (thin arrow—Figure 5(f)), with the mitochondrial volume and kinetoplast, and presence of vesi- consequent loss of parasite morphology. Similar structural cles with an electrondense display (white asterisk—Figures changes have also been described in Trypanosoma cruzi 5(b) and 5(c)) with lipid inclusion in the plasma membrane [38] and other parasites treated with volatile compounds. (arrow—Figures 5(b) and 5(c)). Volatile compounds from plants have hydrophobic char- Many terpenes derived from essential oils, such as 𝛼- acteristics with excellent affinity for cell membranes of bisabolol, are bioactive, especially against different types of different microorganisms, including Leishmania parasites, pathogens [39, 40]. Terpenes, hydrocarbons formed from which contribute significantly to the toxicity attributed to units of isoprene, can easily penetrate the lipid bilayer of these compounds [29]. The lipid inclusions observed and the 6 Evidence-Based Complementary and Alternative Medicine consequent accumulation of lipid precursors can result from [5] J. Alvar, I. D. Velez, C. Bern et al., “Leishmaniasis worldwide theinterferenceoftheoilcomponents,including𝛼-bisabolol, and global estimates of its incidence,” PloS ONE,vol.7,no.5, on the lipid biosynthesis pathways, such as ergosterol [37], pp. 1–12, 2012. thus leading to morphological and structural changes affect- [6] B. L. Herwaldt, “Leishmaniasis,” The Lancet,vol.354,no.9185, ing the survival of the parasite. pp. 1191–1199, 1999. Our results suggest that the essential oil of Vanillosmopsis [7]H.W.Murray,J.D.Berman,C.R.Davies,andN.G.Saravia, arborea and its major compound, 𝛼-bisabolol, show great “Advances in leishmaniasis,” The Lancet,vol.366,no.9496,pp. antileishmanial potential, with the future possibility of a new 1561–1577, 2005. alternative in the treatment for leishmaniasis or acting as an [8] M. Ameen, “Cutaneous leishmaniasis: advances in disease adjuvant antiprotozoan. pathogenesis, diagnostics and therapeutics,” Clinical and Exper- imental Dermatology,vol.35,no.7,pp.699–705,2010. [9]J.Alvar,S.Yactayo,andC.Bern,“Leishmaniasisandpoverty,” 4. Conclusion Trends in Parasitology, vol. 22, no. 12, pp. 552–557, 2006. TheresultsshowthattheessentialoilVanillosmopsis arborea [10] P.Minodier and P.Parola, “Cutaneous leishmaniasis treatment,” Travel Medicine and Infectious Disease,vol.5,no.3,pp.150–158, exhibits leishmanicidal activity in vitro against L. amazo- 2007. nensis andthatthisactivityisrelatedtothepresenceof its major compound 𝛼-bisabolol. This major compound, 𝛼- [11] W. Mayrink, A. C. De Carvalho Botelho, P. A. Magalhaes˜ et al., “Immunotherapy, immunochemotherapy and chemotherapy bisabolol, represents over 97% of the composition of VAEO, for American cutaneous leishmaniasis treatment,” Revista da and, when tested separately, it also showed similar leishmani- SociedadeBrasileiradeMedicinaTropical,vol.39,no.1,pp.14– cidal activity. The ultrastructural analysis showed that both 21, 2006. products induced morphological changes with the presence [12] R. Reithinger, J.-C. Dujardin, H. Louzir, C. Pirmez, B. Alexan- of cytoplasmic lipid inclusions suggesting an action on the der,andS.Brooker,“Cutaneousleishmaniasis,”The Lancet lipid metabolism of the parasite due to increased exocytic Infectious Diseases,vol.7,no.9,pp.581–596,2007. activity in the region of the flagellar pocket. Moreover, no [13] A. Dube, N. Singh, A. Saxena, and V. Lakshmi, “Antileish- cytotoxic effects were observed on the macrophages treated manial potential of a marine sponge, Haliclona exigua (Kirk- by either product. Further studies with the essential oil of V. patrick) against experimental visceral leishmaniasis,” Parasitol- arborea against cutaneous leishmaniasis will be carried out to ogy Research,vol.101,no.2,pp.317–324,2007. demonstrate the potential of natural products derived from [14]J.G.DaCosta,A.R.Campos,S.A.Brito,C.K.B.Pereira, this medicinal plant species, thus contributing to advances in E. O. Souza, and F. F. G. Rodrigues, “Biological screening of allopathic medicine as well as the development of techniques araripe basin medicinal plants using Artemia salina Leach and for the conservation of species with potential therapeutic pathogenic bacteria,” Pharmacognosy Magazine,vol.6,no.24, use. pp. 331–334, 2010. [15] M. D. G. F. de Medeiros, A. C. da Silva, A. M. D. G. L. Citoetal.,´ “In vitro antileishmanial activity and cytotoxicity of essential oil Acknowledgments from Lippia sidoides Cham,” Parasitology International,vol.60, no. 3, pp. 237–241, 2011. This work was supported by BIONORTE (CNPq, pro- [16] L. Monzote, A. M. Montalvo, R. Scull, M. Miranda, and J. cess 555077/2010-5) and FAPEMA (process 01593/12). Katia´ Abreu, “Combined effect of the essential oil from Chenopodium da Silva Calabrese (CNPq no. 308630/2008-4) and Ana ambrosioides and antileishmanial drugs on promastigotes of Lucia Abreu-Silva (CNPq no. 306218/2010-0) are senior Leishmania amazonensis,” RevistadoInstitutodeMedicina researchers. Tropical de Sao Paulo,vol.49,no.4,pp.257–260,2007. [17] M. R. Santin, A. O. dos Santos, C. V.Nakamura, B. P.Dias Filho, References I. C. P.Ferreira, and T. Ueda-Nakamura, “In vitro activity of the essential oil of Cymbopogon citratus and its major component [1] O. F. Kunle, H. O. Egharevba, and P. O. Ahmadu, “Standard- (citral) on Leishmania amazonensis,” Parasitology Research,vol. ization of herbal medicines—a review,” International Journal of 105, no. 6, pp. 1489–1496, 2009. Biodiversity and Conservation,vol.4,no.3,pp.101–112,2012. [18]P.Escobar,S.M.Leal,L.V.Herrera,J.R.Martinez,andE. [2]T.S.Tiumana,M.A.Brenzana,T.Ueda-Nakamuraa,B.P.D. Stashenko, “Chemical composition and antiprotozoal activities Filho, D. A. G. Corteza, and C. V. Nakamura, “Intramuscular of Colombian Lippia spp essential oils and their major compo- and topical treatment of cutaneous leishmaniasis lesions in nents,” Memorias do Instituto Oswaldo Cruz,vol.105,no.2,pp. mice infected with Leishmania amazonensis using coumarin (−) 184–190, 2010. mammea A/BB,” Phytomedicine,vol.19,no.13,pp.1196–1199, [19] M. E. O. Matos, M. P. De Sousa, F. J. A. Matos, and A. A. 2012. Craveiro, “Sesquiterpenes from Vanillosmopsis arborea,” Journal [3] D. S. Correa,ˆ A. G. Tempone, J. Q. Reimao˜ et al., “Anti- of Natural Products, vol. 51, no. 4, pp. 780–782, 1988. leishmanial and anti-trypanosomal potential of polygodial [20] G. D. O. Leite, L. H. I. Leite, R. D. S. Sampaio et al., “Modulation isolated from stem barks of Drimys brasiliensis Miers (Winter- of topical inflammation and visceral nociception by Vanillos- aceae),” Parasitology Research, vol. 109, no. 1, pp. 231–236, 2011. mopsis arborea essential oil in mice,” Biomedicine and Preventive [4] M. E. Ferreira, A. Rojas de Arias, G. Yaluff et al., “Antileish- Nutrition,vol.1,no.3,pp.216–222,2011. manial activity of furoquinolines and coumarins from Helietta [21] G. O. Leite, A. R. Penha, C. N. Fernandes, H. H. F. Souza, J. G. apiculata,” Phytomedicine,vol.17,no.5,pp.375–378,2010. M. da Costa, and A. R. Campos, “Gastroprotective mechanism Evidence-Based Complementary and Alternative Medicine 7

of Vanillosmopsis arborea bark essential oil,” Fitoterapia,vol.80, Memorias´ Do Instituto OswalDo Cruz,vol.108,no.2,pp.172– no. 1, pp. 77–80, 2009. 177, 2013. [22] F. F. G. Rodrigues, J. G. M. da Costa, and H. D. M. Coutinho, [36] Z. Mart´ın-Quintal, M. D. R. Garc´ıa-Miss, M. Mut-Mart´ın, A. “Synergy effects of the antibiotics gentamicin and the essential Matus-Moo, L. W. Torres-Tapia, and S. R. Peraza-Sanchez,´ oil of Croton zehntneri,” Phytomedicine,vol.16,no.11,pp.1052– “The leishmanicidal effect of (3S)-16,17-didehydrofalcarinol, an 1055, 2009. oxylipin isolated from Tridax procumbens, is independent of [23] S. C. Gonçalves da Costa and P. H. Lagrande, “Development of NO production,” Phytotherapy Research,vol.24,no.7,pp.1004– cell mediated immunity to flagellar antigens and acquired resis- 1008, 2010. tance to infection by Trypanosoma cruzi in mice,” Memorias do [37] E. Nibret and M. Wink, “Trypanocidal and antileukaemic Instituto Oswaldo Cruz,vol.76,no.4,pp.367–381,1981. effects of the essential oils of Hagenia abyssinica, Leonotis [24] T. Mosmann, “Rapid colorimetric assay for cellular growth and ocymifolia, Moringa stenopetala, and their main individual survival: application to proliferation and cytotoxicity assays,” constituents,” Phytomedicine,vol.17,no.12,pp.911–920,2010. Journal of Immunological Methods,vol.65,no.1-2,pp.55–63, [38] B. F. Brehm-Stecher and E. A. Johnson, “Sensitization of 1983. Staphylococcus aureus and Escherichia coli to antibiotics by the [25]P.Y.Guru,A.K.Agrawal,U.K.Singha,A.Singhal,andC.M. sesquiterpenoids nerolidol, farnesol, bisabolol, and apritone,” Gupta, “Drug targeting in Leishmania donovani infections using Antimicrobial Agents and Chemotherapy,vol.47,no.10,pp. tuftsin-bearing liposomes as drug vehicles,” FEBS Letters,vol. 3357–3360, 2003. 245, no. 1-2, pp. 204–208, 1989. [39] M. D. S. S. Rosa, R. R. Mendonça-Filho, H. R. Bizzo et al., [26] N. K. A. Santos, H. D. M. Coutinho, G. S. B. Viana, F. F. G. “Antileishmanial activity of a linalool-rich essential oil from Rodrigues, and J. G. M. da Costa, “Chemical characterization Croton cajucara,” Antimicrobial Agents and Chemotherapy,vol. and synergistic antibiotic activity of volatile compounds from 47, no. 6, pp. 1895–1901, 2003. the essential oil of Vanillosmopsis arborea,” Medicinal Chemistry [40] G. F. Santoro, M. G. Cardoso, L. G. L. Guimaraes,J.M.Freire,˜ Research, vol. 20, no. 5, pp. 637–641, 2011. and M. J. Soares, “Anti-proliferative effect of the essential oil of [27]I.L.Andrade,J.N.S.Bezerra,M.A.A.Limaetal.,“Chemical Cymbopogon citratus (DC) Stapf (lemongrass) on intracellular composition and insecticidal activity of essential oils from amastigotes, bloodstream trypomastigotes and culture epi- Vanillosmopsis pohlii baker against Bemisia argentifolii,” Journal mastigotes of Trypanosoma cruzi (Protozoa: Kinetoplastida),” of Agricultural and Food Chemistry,vol.52,no.19,pp.5879– Parasitology,vol.134,no.11,pp.1649–1656,2007. 5881, 2004. [28] E. O. Sousa, A. V. Colares, F. F. G. Rodrigues, A. R. Campos, S. G. Lima, and J. G. M. da Costa, “Effect of collection time on essential oil composition of Lantana camara Linn (Verbe- naceae) growing in Brazil Northeastern,” Records of Natural Products,vol.4,no.1,pp.31–37,2010. [29] M. Morales-Yuste, F. Morillas-Marquez,J.Mart´ ´ın-Sanchez,´ A. Valero-Lopez,´ and M. C. Navarro-Moll, “Activity of (-)𝛼- bisabolol against Leishmania infantum promastigotes,” Phy- tomedicine,vol.17,no.3-4,pp.279–281,2010. [30] F.G. Braga, M. L. M. Bouzada, R. L. Fabri et al., “Antileishmanial and antifungal activity of plants used in traditional medicine in Brazil,” Journal of Ethnopharmacology, vol. 111, no. 2, pp. 396– 402, 2007. [31] A. Ahmad, A. Khan, F. Akhtar et al., “Fungicidal activity of thymol and carvacrol by disrupting ergosterol biosynthesis and membrane integrity against Candida,” European Journal of Clinical Microbiology and Infectious Diseases,vol.30,no.1,pp. 41–50, 2011. [32] M. Wink, “Evolutionary advantage and molecular modes of action of multi-component mixtures used in phytomedicine,” Current Drug Metabolism,vol.9,no.10,pp.996–1009,2008. [33]L.F.Villegas,A.Marçalo, J. Martin et al., “(+)-epi-𝛼-bisbolol is thewound-healingprincipleofPeperomia galioides:investigation of the in vivo wound-healing activity of related terpenoids,” Journal of Natural Products, vol. 64, no. 10, pp. 1357–1359, 2001. [34] A. Hernandez-Ceruelos,´ E. Madrigal-Bujaidar, and C. de la Cruz, “Inhibitory effect of chamomile essential oil on the sister chromatid exchanges induced by daunorubicin and methyl methanesulfonate in mouse bone marrow,” Toxicology Letters, vol. 135, no. 1-2, pp. 103–110, 2002. [35] E. N. Lor´ıa-Cervera, E. I. Sosa-Bibiano, L. E. Villanueva-Lizama et al., “Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana,” Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 308980, 9 pages http://dx.doi.org/10.1155/2013/308980

Research Article Which Approach Is More Effective in the Selection of Plants with Antimicrobial Activity?

Ana Carolina Oliveira Silva,1,2 Elidiane Fonseca Santana,3 Antonio Marcos Saraiva,4 Felipe Neves Coutinho,4 Ricardo Henrique Acre Castro,4 Maria Nelly Caetano Pisciottano,4 Elba Lúcia Cavalcanti Amorim,5 and Ulysses Paulino Albuquerque2

1 Rede Nordeste de Biotecnologia (RENORBIO), Programa de Pos-Graduac´ ¸ao,UniversidadeFederalRuraldePernambuco,˜ Rua Dom Manoel de Medeiros, s/n, Dois Irmaos,˜ 52171-900 Recife, PE, Brazil 2 Departamento de Biologia, Laboratorio´ de Etnobotanicaˆ Aplicada, Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmaos,˜ 52171-900 Recife, PE, Brazil 3 Departamento de Biologia, Universidade de Pernambuco, Avenida Agamenon Magalhaes,˜ s/n, Santo Amaro, 50100-010 Recife, PE, Brazil 4 Departamento de Farmacia,´ Laboratorio´ de Analises´ Microbiologicas,´ Universidade Federal de Pernambuco, Avenida Professor Moraes Rego, 1235 Cidade Universitaria,´ 50670-901 Recife, PE, Brazil 5 Departamento de Farmacia,´ Laboratorio´ de Produtos Naturais, Universidade Federal de Pernambuco, Avenida Professor Moraes Rego, 1235 Cidade Universitaria,´ 50670-901 Recife, PE, Brazil

Correspondence should be addressed to Ana Carolina Oliveira Silva; [email protected] and Ulysses Paulino Albuquerque; [email protected]

Received 2 February 2013; Revised 3 June 2013; Accepted 8 June 2013

Academic Editor: Romuloˆ Romeu da Nobrega´ Alves

Copyright © 2013 Ana Carolina Oliveira Silva et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The development of the present study was based on selections using random, direct ethnopharmacological, and indirect ethnopharmacological approaches, aiming to evaluate which method is the best for bioprospecting new antimicrobial plant drugs. A crude extract of 53 species of herbaceous plants collected in the semiarid region of Northeast Brazil was tested against 11 microorganisms. Well-agar diffusion and minimum inhibitory concentration (MIC) techniques were used. Ten extracts from direct, six from random, and three from indirect ethnopharmacological selections exhibited activities that ranged from weak to very active against the organisms tested. The strain most susceptible to the evaluated extracts was Staphylococcus aureus. The MIC analysis revealed the best result for the direct ethnopharmacological approach, considering that some species yielded extracts classified as active or moderately active (MICs between 250 and 1000 𝜇g/mL). Furthermore, one species from this approach inhibited the growth of the three Candida strains. Thus, it was concluded that the direct ethnopharmacological approach is the most effective when selecting species for bioprospecting new plant drugs with antimicrobial activities.

1. Introduction increased significantly, mainly due to the extensive use of drugs and the rapid genetic transfer of resistance. It is esti- The search for new, natural compounds is growing, mainly mated that microbial resistance develops within seven to due to the acquired resistance of microorganisms to com- eight years of regular antibiotic use [2, 4]. Therefore, the monly used drugs [1–3] and because nosocomial infections development of new drugs that are capable of overcoming causedbythesemicroorganismshaveincreasinglyresulted microbial resistance is critical. in public health problems. Although several antibiotics are Given these circumstances, bioprospection studies have available on the market, microbial resistance to them has been developed, aiming to identify plants from which new 2 Evidence-Based Complementary and Alternative Medicine drugs may be produced, either using crude plant extracts or involving compound isolation and identification for the pro- byisolatingandcharacterizingtheisolatedactivecompo- duction of antimicrobial phytopharmaceuticals. nents; moreover, the goal of these investigations is to understand the role of these components as the basis for the devel- 1.1. Selection and Collection of Species. For species selection, a opment of new drugs (both natural and synthetic) from database was created from 10 floristic and/or phytosociologi- plants [5].Itisestimatedthat30to40%ofthemostrecent cal studies conducted in the “Caatinga” (savanna-like vegeta- antimicrobial drugs available on the market are derived from tion) in the semiarid region of Northeast Brazil. Six hundred natural products. However, these resources have been poorly forty-five species belonging to 81 families and 319 genera explored to produce additional antimicrobial drugs from were included. Subsequently, the folk use for each species plants [6]. was assessed in the literature, totaling 147 species with indi- It is known that plants provide an unlimited range of cations for use. For the random selection, all species without compounds because of their high chemical diversity and medicinal uses were considered. For the direct ethnopharma- becausetheyhavebeenusedforcenturiesbyseveraldifferent cological selection, plants with indications of use for infec- peoples. For example, Arctostaphylos uva-ursi (L.) Spreng and tious and parasitic diseases, such as pleuritis, helminthiasis, Vaccinium macrocarpon Aiton are used to treat urinary tract general inflammation, urinary disorders, prostate infection, infections, and Melissa officinalis L., Allium sativum L., and and uterine inflammation, were considered. For the indirect Melaleuca alternifolia Cheel are well-known broad-spectrum ethnopharmacological selection, all of the species with any antimicrobial agents [7]. indications of medicinal use were selected, excluding those Currently, one of the major challenges is the selection species that were related to the direct ethnopharmacological of plants for a bioprospecting study because this process approach. Thereafter, the Biostat 5.0 software was used for is the first research step. The various methods include the species selection. Initially, 20 species were randomly selected selection approach, which is used to choose plants based on for each type of approach. However, due to the unavailability certain criteria: random selection, which involves the arbi- of certain species during the collection period, 19 species trary collection of the species without consideration of, for of herbs were selected for the random approach, 20 for the instance, taxonomic affinities and ethnobotanical informa- direct ethnopharmacological selection, and 14 for the indirect tion; ethnodirected selection, which includes ethnobotanical ethnopharmacological selection. and ethnopharmacological approaches and applies informa- In total, 53 species of plants were collected from April tion about the traditional use of plants to treat specific dis- to July 2011 at the Experimental Station of the Agricultural eases; and the chemotaxonomic approach, which is based on Research Company of Pernambuco, Agronomic Institute of Pernambuco (Instituto Agronomicoˆ de Pernambuco (IPA)) thestudyofplantsfromthesamefamilyorgenusofaspecies ∘ 󸀠 ∘ 󸀠 from which active compounds have been already isolated [8]. (8 14 Sand3555 W, 537 m altitude) in Caruaru, Agreste Several studies have been focused on selecting plants region of Pernambuco, Northeast Brazil. Voucher specimens through the ethnodirected approach [1, 9, 10], corroborating are deposited in the Herbarium of Professor Vasconcelos the folk use of many species that are traditionally used. Sev- Sobrinho of the Federal Rural University of Pernambuco eral authors also suggest that random selection should be (PEUFR). The area was selected because it is well studied in avoided in favor of ethnodirected selection because the latter terms of floristics and provides easy access for obtaining the appears to be a more efficient method of selecting species for various species. bioprospecting [7]. Despite the large body of published data (namely, the 1.2. Extract Preparation. Becausethespecieswereherba- studies cited previously) suggesting that ethnodirected selec- ceous, all of the extracts were prepared from the leaves. tion is more efficient, few studies have compared the different Twenty grams of dried and powdered leaves was macerated in selection approaches (e.g., see Melo [11] and Svetaz et al. [12]) absolute methanol for 24 hours. The process was extensively repeated. The extract obtained was then evaporated under to provide a more realistic scenario for bioprospecting stud- ∘ reduced pressure at 45 C until dried. The samples were placed ies. in a desiccator for a one-week period. The extracts were The main aim of the present study was to assess wheth- diluted in 20% dimethyl sulfoxide (DMSO) and used at er the probability of discovering plants with antimicro- concentrations of 100 mg/mL and 50 mg/mL. As there is no bial potentials is greater when they already have indications standard regarding the maximum concentration of crude of ethnopharmacological use for infectious and parasitic extract of plants for screening, the protocol of the Laboratory diseases (direct ethnopharmacological selection) than when of Microbiological Analyses, from Federal University of Per- they are randomly selected (random selection) or do not have nambucowasadopted,wheretheassayswereperformed. direct indications of use for infectious and parasitic diseases (indirect ethnopharmacological selection). Furthermore, we 1.3. Antimicrobial Assay. The antimicrobial activity was eval- aimed to determine the selection group to which the plants uatedintwotrials.First,theantimicrobialactivitywas belonged with the lowest minimum inhibitory concentration determined by the agar diffusion method [13]. Next, for the (MIC), that is, to assess whether plants with higher antimicro- species that displayed inhibition halo greater than 13 mm, the bialactivitiesareamongthosewithahistoryoffolkuseinthe MIC [13] was determined. treatment of infectious diseases. Finally, we aimed to define The crude extracts were tested against standard strains of which species should be selected for future investigations Staphylococcus aureus (ATCC 6538), S. epidermidis (sperm), Evidence-Based Complementary and Alternative Medicine 3

S. saprophyticus (LACEN 07), Bacillus subtilis (ATCC 6633), 2. Results and Discussion Enterococcus faecalis (ATCC 51299), Klebsiella pneumoniae (secretion), Pseudomonas aeruginosa (ATCC 14502), Ofthe20plantextractsobtainedbydirectethnopharma- Escherichia coli (ATCC 35218), Candida albicans (urine), C. cological selection, 10 (50%) exhibited activity against the krusei (blood), and C. tropicalis (rectal swab). These strains microorganisms tested. Of the 19 plant extracts obtained were selected because of their clinical relevance. by random selection, seven (36.84%) showed antimicrobial Inoculumswereprepared24hoursinadvanceandkeptin activity against at least one strain. Of the 14 plant extracts Mueller-Hinton agar (bacteria) and Sabouraud agar (yeast). obtained by indirect ethnopharmacological selection, two The inoculums were suspended in sterile saline solution using (14.28%) exhibited antimicrobial activity (Table 1). The active 8 extracts that were tested exhibited antimicrobial activity only a 0.5 McFarland standard (10 UFC/mL) [14]. against the Gram-positive bacteria and the Candida strains. For the agar diffusion method, sterile swabs were used to × Of the 10 extracts obtained by direct ethnopharmacolog- inoculate sterile petri plates (20 100 mm) containing 20 mL ical selection, 40% exhibited moderate antimicrobial activity, of Mueller-Hinton agar for bacteria and 20 mL Sabouraud 30% were active, and 30% were highly active. Of the extracts agar for yeast. On each plate, four wells (6 mm diameter) were obtained by random selection, 33.33% exhibited moderate 𝜇 created, to which 100 L of the extracts at concentrations of activity, and 66.66% were highly active. Of the extracts 100 mg/mL and 50 mg/mL was added, as well as the positive obtained by indirect ethnopharmacological selection, 70% controls tetracycline (30 mg/mL) for bacteria and ketocona- were moderately active, and 30% were active. Based on the zole (50 mg/mL) for yeast. Twenty percent DMSO was used as G-test (𝐺 = 127.1860), significant differences𝑃 ( < 0.005) thenegativecontrol.Theplateswereincubatedaerobicallyat ∘ existed between the proportions of active species among the 37 ± 1 C for 24 hours. The antimicrobial activity was assessed three approaches, indicating that direct ethnopharmacolog- by measuring the inhibition halo of microbial growth around ical selection is the most effective for the selection of plants thewell,andtheresultswereclassifiedaccordingtothe with greater antimicrobial activities. following scale: inhibition zones down to 9 mm, inactive; 9– The plant species obtained by direct ethnopharmacolog- 12 mm, moderately active; 13–18 mm, active; above 18 mm, ical selection were more effective in terms of the number of very active [15]. The G-test was performed (𝑃 < 0.05) strains with inhibited growth. Four of the plants, Acanthos- to determine significant differences between the selection permum hispidum, Euphorbia hyssopifolia, Hyptis suaveolens, approaches and the antimicrobial activity. All of the assays and Indigofera suffruticosa,displayedantimicrobialactivity were performed in triplicate. against four microorganisms. Two of the plants, Ludwigia The MIC of the extracts and the reference antibiotic octovalvis and Momordica charantia,wereactiveagainst (tetracycline) were determined using the Mueller-Hinton five microorganisms. Centratherum punctatum,whichwas brothmicrodilutiontechniquefollowingtheprotocolestab- obtained by random selection, inhibited the growth of lished by the Clinical and Laboratory Standards Institute [16] five microorganisms, whereas Blainvillea acmella inhibited for bacteria. Inoculums were prepared in the same medium four microorganisms. Only Tillandsia recurvata,whichwas 8 and were adjusted to a 0.5 McFarland standard (10 UFC/mL) obtained by indirect ethnopharmacological selection, dis- and diluted 1 : 10 for the broth dilution method. The micro- played antimicrobial activity against five microorganisms. ∘ plates were incubated at 37 C, and the MIC was read after Although the plant species that were selected by the three a 24-hour incubation period. The MIC was defined asthe approaches exhibited the same versatility in the inhibition of lowest compound concentration at which the microorganism various strains (i.e., five microorganisms from the 11 that were tested showed no visible growth. tested), the selection approaches were distinguished by the antimicrobial activities presented by the species; this activity The MIC for yeasts was performed by the broth microdi- was greater in the species obtained by direct ethnopharma- lution technique according to the CLSI [17], and ketoconazole cological selection, resulting in the largest inhibition halo was used as the positive control. The inoculum was used ata 6 (ranging from 22 to 30 mm). Furthermore, of the 53 plant concentration of 1.0 × 10 UFC/mL. The MIC was determined species studied, only four of the extracts could inhibit five in Roswell Park Memorial Institute (RPMI) 1640 medium microorganisms; two of these plant species were obtained (Gibco,InvitrogenCo.,NewYork,USA),withMOPSbuffer, ∘ by direct ethnopharmacological selection. The findings of pH 7.0. The plates were incubated at 37 C, and the readings the present study indicate greater success when plant species were obtained after a 24-hour incubation period. All of the are selected based on their direct indications of use for assays were performed in duplicate. infectious and parasitic diseases, which is similar to the < 𝜇 Plant extracts with MICs 100 g/mL were considered findings of Phongpaichit et al. [18], who investigated the highly active antimicrobial agents; MICs ranging from 100 to use of plants to treat fungal infections in AIDS patients. 500 𝜇g/mL were classified as active; MICs ranging between By selecting species based on folk use, the authors achieved 500 and 1000 𝜇g/mL were considered moderately active; a success rate of 40%. Cruz et al. [9], analyzing Brazilian MICs ranging from 1000 to 2000 𝜇g/mL were considered to plants traditionally used to treat mycoses, achieved a success have low activity; and MICs >2000 𝜇g/mL were classified as rate of 50%, confirming the results of the present study. van inactive [14]. The G-test was conducted (𝑃 < 0.005)toeval- Vuuren and Naidoo [10] analyzed plants (selected from the uate significant differences between the selection approaches ethnobotanical literature) used in the treatment of sexually and the MIC. transmitteddiseasesandfoundthat90%oftheextracts 4 Evidence-Based Complementary and Alternative Medicine

Table 1: Antimicrobial activity of herbaceous plants from the semiarid region, Northeast of Brazil, based on random, direct, and indirect ethnopharmacological approaches (Inhibition halo in mm).

𝐶. Species Sa Se Ss Bs Ef Ec Kp Pa Ca Ck Ct mg/mL Random approach 100 Astraea lobata (L.) Klotzsch ——————————— 50 100 23 20 20 22 Blainvillea acmella (L.) Philipson ——————— 50 18 13 16 20 100 30 20 22 21 22 Centratherum punctatum Cass. —————— 50 25 17 14 16 14 100 Croton hirtus L’ H er.´ ——————————— 50 100 Cyperus uncinulatus Schrad. ex. Nees ——————————— 50 100 Delilia biflora (L.) Kuntze ——————————— 50 100 7 Drymaria cordata (L.) Willd. ex Roem. & Schult. —————————— 50 100 Euphorbia heterophylla L. ——————————— 50 100 Lepidium ruderale L. ——————————— 50 100 Melanthera latifolia (Gardner) Cabr. ——————————— 50 100 Mollugo verticillata L. ——————————— 50 100 9107 9 Parthenium hysterophorus L. ——————— 50 7 100 Ruellia asperula (Mart.&Nees)Benth.&Hook. 10—————————— 50 100 8 Ruellia geminiflora Kunth ——— ——————— 50 100 20 20 18 Sida urens L. ———————— 50 20 20 15 100 10 8 10 6 Spermacoce verticillata L. — —————— 50 10 6 7 100 8 Stylosanthes scabra Vogel —————————— 50 8 100 Talinum triangulare (Jacq.) Willd. ——————————— 50 Species 1 (Malvaceae) ————————303030 Indirect ethnopharmacological approach 100 Alternanthera brasiliana (L.) Kuntze ——————————— 50 100 Alternanthera tenella Colla ——————————— 50 100 Commelina erecta L. ——————————— 50 100 Commelina obliqua Vahl ——————————— 50 100 Hypenia brachystachys (Pohl ex Benth.) Harley ——————————— 50 Evidence-Based Complementary and Alternative Medicine 5

Table 1: Continued. 𝐶. Species Sa Se Ss Bs Ef Ec Kp Pa Ca Ck Ct mg/mL 100 14 12 11 Hypenia salzmannii (Benth.) Harley — ——————— 50 12 12 9 100 Polygala paniculata L. ——————————— 50 100 Polygala violacea Aubl. ——————————— 50 100 Portulaca elatior Mart. ex Rohrb. ——————————— 50 100 Portulaca oleracea L. ——————————— 50 100 Solanum agrarium Sendtn. ——————————— 50 100 Solanum americanum Mill. ——————————— 50 100 17 18 18 13 15 Tillandsia recurvata (L.) L. —————— 50 15 17 16 13 11 100 Tillandsia usneoides (L.) L. ——————————— 50 Direct ethnopharmacological approach 100 12 10 10 Acalypha multicaulis Mull. Arg ———————— 50 10 10 8 100 22 19 19 17 Acanthospermum hispidum DC. ——————— 50 20 15 15 15 100 Ageratum conyzoides L. ——————————— 50 100 Aosa rupestris Gardner ——————————— 50 100 8 7 7 15 Argemone mexicana L. —————— — 50 7 7 7 15 100 Boerhavia diffusa L. ——————————— 50 100 20 16 18 19 6 Euphorbia hyssopifolia L. —————— 50 16 13 15 16 0 100 Conocliniopsis prasiifolia (DC.) R.M. King & H. Rob. ——————————— 50 100 Heliotropium indicum L. ——————————— 50 100 18 15 18 15 Hyptis suaveolens (L.) Poit. ——————— 50 16 13 16 12 100 30 22 22 30 Indigofera suffruticosa Mill. — —————— 50 30 22 20 30 100 14 Leonotis nepetifolia (L.) R. Br.6 —————————— 50 13 100 13 13 15 13 13 Ludwigia octovalvis (Jacq.) P.H. Raven —————— 50 11 12 15 12 12 100 10 14 8 8 11 Melochia tomentosa L. —————— 50 10 11 7 7 10 100 20 15 15 18 18 Momordica charantia L. —————— 50 17 15 14 15 15 6 Evidence-Based Complementary and Alternative Medicine

Table 1: Continued. 𝐶. Species Sa Se Ss Bs Ef Ec Kp Pa Ca Ck Ct mg/mL 100 Physalis angulata L. ——————————— 50 100 Rhaphiodon echinus Schauer ——————————— 50 100 Richardia grandiflora (Cham. & Schltdl.) Steud. ——————————— 50 100 12 12 Scoparia dulcis L. —— ——————— 50 12 12 100 Waltheria indica L. ——————————— 50 𝐶: concentration; Sa: Staphylococcus aureus;Se:S. epidermidis;Ss:S. saprophyticus;Bs:Bacillus subtilis;Ef:Enterococcus faecalis;Ec:Escherichia coli;Pa: Pseudomonas aeruginosa;Kp:Klebsiella pneumonia;Ca:Candida albicans;Ck:C. krusei e; Ct: C. tropicalis;—:noinhibition. exhibited significant antimicrobial activity against the tested For species obtained by direct ethnopharmacological selec- microorganisms, thereby validating the ethnomedicinal use tion, four extracts were classified as moderately active: A. of species. Comparing the results obtained from the different hispidum, E. hyssopifolia, I. suffruticosa,andMomordica selection approaches (Table 1), direct ethnopharmacological charantia. Ludwigia octovalvis from the direct ethnopharma- selection yielded a greater number of species that were active cological selection and species 1 from the random selection against the tested microorganisms—plants such as Acanthos- were the only species with MICs that were classified as active permum hispidum, Euphorbia hyssopifolia,andI. suffruticosa, andhighlyactiveforC. albicans (125 𝜇g/mL and 31.25 𝜇g/mL, which caused inhibition halo ranging from 17 to 30 mm for resp.). L. octovalvis also exhibited a moderately active extract the S. aureus, S. epidermidis, S. saprophyticus,andB. subtilis against C. krusei and C. tropicalis (1000 𝜇g/mL), while the strains. Another example of the efficiency of plant species extract from species 1 was highly active against C. krusei obtained by direct ethnopharmacological selection was their (62.5 𝜇g/mL) and C. tropicalis (62.5 𝜇g/mL). According to action against E. faecalis. Whereas three of the plant species Fabry et al. [19], plants with MICs below 8 mg/mL are con- were able to inhibit the growth of E. faecalis,onlyonespecies sidered to display some antimicrobial activity. Moreover, our obtained by random selection and one species obtained findings indicate that these species are potential candidates by indirect ethnopharmacological selection inhibited this for further investigations (of the isolation and identification microorganism. of compounds with antimicrobial activities) because the The MIC was determined for four species obtained by MICs were below 1 mg/mL, confirming other studies indi- random selection, nine species obtained by direct ethnophar- cating that plant extracts and natural products with MICs macological selection, and two species obtained by indirect below 1 mg/mL deserve special attention and must therefore ethnopharmacological selection: Blainvillea acmella, Cen- be carefully analyzed [7, 20]. tratherum punctatum, Sida urens, and species 1 that is in The results obtained in the present study indicate that process of patent registration, and so its name cannot be direct ethnopharmacological selection is an effective bio- disclosed, (random selection); Acanthospermum hispidum, prospecting tool for antimicrobial activity. Svetaz et al. [12] Argemone mexicana, E. hyssopifolia, Hyptis suaveolens, I. suf- evaluated ethnomedical information on the discovery of fruticosa, Leonotis nepetifolia, Ludwigia octovalvis, Melochia plants with antifungal activity and determined that the proba- tomentosa,andMomordica charantia (direct ethnopharma- bility of finding plants with this activity is significantly higher cological selection); and Tillandsia recurvata and Hypenia when reports exist of their use as antifungal agents compared salzmannii (indirect ethnopharmacological selection). The with the absence of such reports. The authors categorically MICsforalloftheanalyzedspeciesandstrainsarepresented affirmed that the ethnopharmacological approach is useful in Table 2.BasedontheG-test (𝐺 = 76.5443), a significant in detecting plants with antifungal activity. Furthermore, difference (𝑃 < 0.005) existed among the MICs of the in studies on the potential antimalarial effect of Nigerian three selection approaches, with direct ethnopharmacolog- plants, Adebayo and Krettli [21] discussed the difficulty, high ical selection again being distinct because extracts of plant cost, and low efficacy of the random selection approach, species belonging to this group exhibited superior MICs. The which was the selection method that had been used in that extracts were tested only against the strain to which they country decades prior. The current method is ethnobotanical showed activity up to 13 mm. selection, based on information regarding indigenous uses of All of the species obtained by indirect ethnopharma- the species, which has reduced costs and time compared with cological selection were considered to have low activities random selection. becausetheydisplayedMICsabove1000𝜇g/mL. In the ran- The low probability of finding promising plants for dom selection, only Sida urens was considered moderately bioprospection through random selection suggests that this active, presenting an MIC of 500 𝜇g/mL against S. aureus. approach is not recommended for the discovery of new Evidence-Based Complementary and Alternative Medicine 7

Table 2: Minimal inhibitory concentration (𝜇g/mL) of herbaceous species from the semiarid region, Northeast of Brazil, based on random, direct, and indirect ethnopharmacological approaches.

Species Sa Se Ss Bs Ef Ca Ck Ct Random approach Blainvillea acmella (L.) Philipson >1000 >1000 >1000 >1000 NT NT NT NT Centratherum punctatum Cass. >1000 >1000 >1000 >1000 >1000 NT NT NT Sida urens L. 500 1000 1000 NT NT NT NT NT Species 1 (Malvaceae) NT NT NT NT NT 31.25 62.5 62.5 Indirect ethnopharmacological approach Tillandsia recurvata (L.) L. >1000 >1000 >1000 >1000 >1000 NT NT NT Hypenia salzmannii (Benth.) Harley >1000 NT NT NT NT NT NT NT Direct ethnopharmacological approach Acanthospermum hispidum DC. 1000 >1000 >1000 >1000 NT NT NT NT Argemone mexicana L. NT NT NT NT NT NT >1000 NT Euphorbia hyssopifolia L. 1000 >1000 >1000 1000 NT NT NT NT Hyptis suaveolens (L.) Poit. >1000 >1000 >1000 NT NT NT NT NT Indigofera suffruticosa Mill. 500 >1000 >1000 1000 >1000 NT NT NT Leonotis nepetifolia (L.) R. Br. >1000 NT NT NT NT NT NT NT Ludwigia octovalvis (Jacq.) P.H. Raven 250 500 NT NT NT 125 1000 1000 Melochia tomentosa L. >1000 >1000 NT >1000 NT NT NT NT Momordica charantia L. 1000 >1000 >1000 1000 >1000 NT NT NT Sa: Staphylococcus aureus;Se:S. epidermidis;Ss:S. saprophyticus;Bs:Bacillus subtilis;Ef:Enterococcus faecalis;Ca:Candida albicans;Ck:C. krusei e; Ct: C. tropicalis; NT: extract not tested for the strain. antimicrobial agents. Although this approach was responsible included C. krusei and C. tropicalis, which are species that are for the discovery of taxol [22, 23], it is currently known more resistant to commonly used drugs. Among the species that only one in 10,000 plants will be a promising source of that we investigated, L. octovalvis and species 1 inhibited the new drugs, while the ethnodirected selection is responsible growth of the three Candida species, with the most active for74%ofallthedrugsofplantorigin[24]. Nevertheless, MICs ranging from 62.5 𝜇g/mL to 31.25 𝜇g/mL. This finding controversy exists regarding the efficacy of this approach of the present study is of great importance, considering the because the selected plants appear not to be effective in the high incidence of C. albicans (70%) and C. tropicalis (20%) treatment of cancer [11] and mycoses caused by yeasts and in Latin America [28], the emergence of other Candida Aspergillus spp.; indeed, for these types of fungi, Svetaz et al. species resistant to antifungal agents, and, mainly, the limited [12] found no significant differences between the antimicro- number of drugs available to treat fungal infections [29]. bial activities of plants obtained by random selection versus Due to the cosmopolitan nature of the species analyzed ethnopharmacological selection. Moreover, the fact that in herein, studies on the antimicrobial activity involving some of the present study, only one species obtained by random these species have been conducted in various regions world- selection was highly active against the three Candida strains wide, such as Ageratum conyzoides in Malaysia [30], which is indicates that the random approach should not be completely used against cough and has displayed an MIC of 1600 𝜇g/mL abandoned but should instead be adapted to other selection for the Mycobacterium strains. However, in the present study, approaches, such as chemosystematic or ecological methods. all of the tested strains were resistant to the extract from A. The species that were obtained by direct ethnopharma- conyzoides. cological, random, and indirect ethnopharmacological selec- Gachet et al. [31], who tested plants traditionally used tions were active only against Gram-positive bacteria. The against leishmaniasis in Ecuador, found that the extract of S. inactivity against Gram-negative bacteria might be observed dulcis is effective against axenic amastigotes of L. donovani. becauseofthelipophilicouterlayer,amongotherreasons, In the present study, this species displayed moderate activity as this layer most likely prevents the access of the extract to against S. aureus and B. subtilis.Wiartetal.[26]testedthe the interior of the bacteria, as observed by Nantitanon et al. methanol extract in an antimicrobial screening of plants from [25] in their assessment of the antimicrobial activity of Hyptis Malaysia including, among others, A. conyzoides, C. hirtus, E. suaveolens. Regarding antifungal activity, it is noteworthy prostrate, and H. suaveolens; these species are effective against that most of the studies were conducted using C. albicans various microorganisms such as B. cereus, P. ae r ug ino s a , [26]. The exceptions were the studies by Cruz et al. [9], B. subtilis, S. aureus,andC. albicans. In the present study, who tested the use of plants from the Caatinga against C. however, except for H. suaveolens, which inhibited the growth guilliermondii, andDeToledoetal.[27], who tested the use of S. aureus, S. saprophyticus, S. epidermidis, and B. subtilis of plants against C. parapsilosis strains. The present study also (resulting in inhibition halo between 15 and 18 mm), the 8 Evidence-Based Complementary and Alternative Medicine other species displayed no activity against any of the analyzed and ethnopharmacological selection in the search for new microorganisms. pharmaceutical products. Matsuse et al. [32] studied plants from Panama with potential antiviral activity and found that both the crude 3. Conclusions extractandisolatedcompoundsfromE. hyssopifolia were effective against HIV. In the present study, this species isone It may be concluded that direct ethnopharmacological selec- ofthemosteffectivewithanMICof1000𝜇g/mL for S. aureus, tion is an important bioprospecting tool and that the Caatinga which qualifies it as a potential candidate for studies aiming is a type of vegetation that should be included in future to develop drugs obtained from plants of the Caatinga that studies on the bioprospection of new antimicrobial plant are more effective against resistant strains. In addition, this drugs. Additionally, the above-mentioned species should be finding supports the folk use of this species against microbial included in the studies investigating the production of new infections in the semiarid region of Northeast Brazil [33]. phytomedicines. In Brazil, especially the Caatinga, few studies exist on antimicrobial activity based on ethnobotanical data, and Conflict of Interests investigations are almost nonexistent regarding species from The authors declare that there is no conflict of interests. the Caatinga, whether native or spontaneous. Cruz et al. [9] studied the extract of Ziziphus joazeiro Mart., Caesalpinia Acknowledgments pyramidalis Tul., Bumelia sartorum Mart., and Hymenaea courbaril L.,whicharetraditionallyusedtotreatmycosesand The authors are grateful to Lucilene Lima dos Santos for found that Z. joazeiro and C. pyramidalis display significant species identification and Edna Santos and Ingrid Suely Lima antifungal activities, making them potential candidates for for technical assistance, to FACEPE for scholarship to ACOS, the development of new strategies to treat fungal infections. and to CNPq for financial support (Edital Universal 2012) and Almeida et al. [1], in a study comparing the antimicrobial productivity grant given to Ulysses Paulino Albuquerque. efficiency of species selected in the Caatinga and Atlantic Forest, tested the crude extract of B. diffusa,whichdisplayed moderate activity against S. aureus, Streptococcus faecalis,and References Mycobacterium smegmatis when collected in the Caatinga. [1]C.D.F.CasteloBrancoRangelDeAlmeida,D.L.DeVascon- However, the extract of the species from the Atlantic Forest celosCabral,C.C.B.RangelDeAlmeida,E.L.CavalcantiDe exhibited no activity against any microorganism. In the Amorim, J. M. De Arajo, and U. P. De Albuquerque, “Com- present study, all of the tested strains were resistant to the parative study of the antimicrobial activity of native and exotic extract of this plant. The authors concluded that the Caatinga plants from the Caatinga and Atlantic Forest selected through region appears to be a promising source in the search for an ethnobotanical survey,” Pharmaceutical Biology,vol.50,no. new compounds of plant origin, due to the larger size of 2,pp.201–207,2012. the inhibition halo generated when using extracts from these [2] D. O. Guimaraes,L.S.Momesso,andM.T.Pupo,“Antibi˜ oticos:´ species and due to their ability to inhibit a greater number of importanciaˆ terapeuticaˆ e perspectivas para a descoberta e microorganisms. desenvolvimento de novos agentes,” Qu´ımica Nova,vol.33,no. Despite the fact that species 1 from the random selec- 3,pp.667–679,2010. tion displays an MIC that classifies it as highly active [3]G.G.F.Nascimento,J.Locatelli,P.C.Freitas,andG.L.Silva, (≤62.5 𝜇g/mL) for the three Candida species tested, the “Antibacterial activity of plant extracts and phytochemicals on findings in the present studies regarding species obtained by antibiotic-resistant bacteria,” Brazilian Journal of Microbiology, direct ethnopharmacological selection (i.e., Acanthospermum vol. 31, no. 4, pp. 247–256, 2000. hispidum, Euphorbia hyssopifolia, I. suffruticosa, Ludwigia [4]G.P.Silveira,F.Nome,J.C.Gesser,andM.M.Sa,´ “Estrategias´ octovalvis,andMomordica charantia, which exhibited MICs Utilizadas no Combate a Resistenciaˆ Bacteriana,” Qu´ımica ranging from 250 to 1000 𝜇g/mL) indicate that this selection Nova,vol.29,no.4,pp.844–855,2006. approach is an effective strategy for bioprospecting new [5] H.-F. Ji, X.-J. Li, and H.-Y. Zhang, “Natural products and drug drugs with antimicrobial activity. Furthermore, additional in- discovery: can thousands of years of ancient medical knowledge lead us to new and powerful drug combinations in the fight depth studies should be conducted using compounds isolated against cancer and dementia?” EMBO Reports,vol.10,no.3,pp. from the cited species. The present study included only 194–200, 2009. spontaneous herbaceous species of the Caatinga, which is a [6]D.Chattopadhyay,M.Chawla-Sarkar,T.Chatterjeeetal.,“Re- practice that is still undervalued in bioprospecting but that, cent advancements for the evaluation of anti-viral activities of based on our findings, appears to be potentially useful in natural products,” New Biotechnology,vol.25,no.5,pp.348– thesearchforcompoundswithantimicrobialactivity;indeed, 365, 2009. the plant extracts used were able to inhibit yeasts that occur [7]J.L.R´ıos and M. C. Recio, “Medicinal plants and antimicrobial with high incidence in Latin America and that exhibit high activity,” Journal of Ethnopharmacology,vol.100,no.1-2,pp.80– resistance to regular antibiotics. Additionally, these species 84, 2005. are characterized by wide distribution, high population num- [8] U. P. Albuquerque and N. Hanazaki, “As pesquisas etnodirigi- bers, and rapid growth, which would facilitate their study. das na descoberta de novos farmacos´ de interesse medico´ e Our findings are supported by the investigations of Cruz farmaceutico:ˆ fragilidades e perspectivas,” Revista Brasileira De et al. [9] and Almeida et al. [1] in considering the Caatinga Farmacognosia,vol.16,pp.678–689,2006. Evidence-Based Complementary and Alternative Medicine 9

[9]M.C.S.Cruz,P.O.Santos,A.M.BarbosaJr.etal.,“Antifungal [26] C. Wiart, S. Mogana, S. Khalifah et al., “Antimicrobial screening activity of Brazilian medicinal plants involved in popular of plants used for traditional medicine in the state of Perak, treatment of mycoses,” Journal of Ethnopharmacology, vol. 111, Peninsular Malaysia,” Fitoterapia,vol.75,no.1,pp.68–73,2004. no. 2, pp. 409–412, 2007. [27] C. E. M. De Toledo, E. A. Britta, L. F. Ceole et al., “Antimicrobial [10] S. F. van Vuuren and D. Naidoo, “An antimicrobial investigation and cytotoxic activities of medicinal plants of the Brazilian of plants used traditionally in southern Africa to treat sexually cerrado, using Brazilian cachac¸a as extractor liquid,” Journal of transmitted infections,” Journal of Ethnopharmacology,vol.130, Ethnopharmacology,vol.133,no.2,pp.420–425,2011. no. 3, pp. 552–558, 2010. [28] M. A. Pfaller and D. J. Diekema, “Epidemiology of invasive [11] J. G. Melo, Estrategias´ de bioprospecc¸ao˜ e agentes anticancer´ıg- candidiasis: a persistent public health problem,” Clinical Micro- enosapartirdafloranordestina[Ph.D.thesis], Rede Nordeste biology Reviews,vol.20,no.1,pp.133–163,2007. de Biotecnologia, Recife, Brazil, 2010. [29] B. P. Mathew and M. Nath, “Recent approaches to antifungal [12] L. Svetaz, F. Zuljan, M. Derita et al., “Value of the ethnomedical therapy for invasive mycoses,” ChemMedChem,vol.4,no.3,pp. information for the discovery of plants with antifungal proper- 310–323, 2009. ties. A survey among seven Latin American countries,” Journal [30] S. Mohamad, N. M. Zin, H. A. Wahab et al., “Antituberculosis of Ethnopharmacology,vol.127,no.1,pp.137–158,2010. potential of some ethnobotanically selected Malaysian plants,” [13] Clinical and Laboratory Standards Institute (CLSI), Perfor- Journal of Ethnopharmacology,vol.133,no.3,pp.1021–1026, mance Standards for Antimicrobial Disk Susceptibility Tests,M2- 2011. A8, Wayne, Pa, USA, 8th edition, 2003. [31] M. S. Gachet, J. S. Lecaro, M. Kaiser et al., “Assessment of anti- [14] A. M. Saraiva, R. H. A. Castro, R. P. Cordeiro et al., “In vitro protozoal activity of plants traditionally used in Ecuador in the evaluation of antioxidant, antimicrobial and toxicity properties treatment of leishmaniasis,” Journal of Ethnopharmacology,vol. of extracts of Schinopsis brasiliensi engl. (Anacardiaceae),” 128,no.1,pp.184–197,2010. African Journal of Pharmacy and Pharmacology,vol.5,no.14, [32] I. T. Matsuse, Y.A. Lim, M. Hattori, M. Correa, and M. P.Gupta, pp.1724–1731,2011. “A search for anti-viral properties in Panamanian medicinal [15]T.M.A.Alves,A.F.Silva,M.Brandao˜ et al., “Biological plants. The effects on HIV and its essential enzymes,” Journal screening of brazilian medicinal plants,” Memorias´ do Instituto of Ethnopharmacology, vol. 64, no. 1, pp. 15–22, 1998. Oswaldo Cruz,vol.95,pp.367–373,2000. [33] U. P. de Albuquerque, P. M. de Medeiros, A. L. S. de Almeida et [16] Clinical and Laboratory Standards Institute (CLSI), Reference al., “Medicinal plants of the caatinga (semi-arid) vegetation of Method for Broth Dilution Antifungal Susceptibility Testing of NE Brazil: a quantitative approach,” Journal of Ethnopharmacol- Yeasts:ApprovedStandard, M27-A3, NCCLS, Wayne, Pa, USA, ogy,vol.114,no.3,pp.325–354,2007. 2008. [17] Clinical and Laboratory Standards Institute (CLSI), Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard, M07-A8, CLSI, Wayne, Pa, USA, 8th edition, 2009. [18] S. Phongpaichit, S. Subhadhirasakul, and C. Wattanapirom- sakul, “Antifungal activities of extracts from Thai medicinal plants against opportunistic fungal pathogens associated with AIDS patients,” Mycoses,vol.48,no.5,pp.333–338,2005. [19] W.Fabry, P.O. Okemo, and R. Ansorg, “Antibacterial activity of East African medicinal plants,” Journal of Ethnopharmacology, vol.60,no.1,pp.79–84,1998. [20] S. Gibbons, “Anti-staphylococcal plant natural products,” Natu- ral Product Reports,vol.21,no.2,pp.263–277,2004. [21] J. O. Adebayo and A. U. Krettli, “Potential antimalarials from Nigerian plants: a review,” Journal of Ethnopharmacology,vol. 133, no. 2, pp. 289–302, 2011. [22] G. M. Cragg, M. R. Boyd, J. H. Cardellina et al., “The search for new pharmaceutical crops: drug discovery and development at the National Cancer Institute,” in New Crops,J.JanickandJ. E. Simon, Eds., pp. 161–167, John Wiley & Sons, New York, NY, USA, 1993. [23] P. A. Cox and M. J. Balick, “The ethnobotanical approach to drug discovery,” Scientific American,vol.270,no.6,pp.82–87, 1994. [24]E.Douwes,N.R.Crouch,T.J.Edwards,andD.A.Mulhol- land, “Regression analyses of southern African ethnomedicinal plants: informing the targeted selection of bioprospecting and pharmacological screening subjects,” Journal of Ethnopharma- cology,vol.119,no.3,pp.356–364,2008. [25] W.Nantitanon,S.Chowwanapoonpohn,andS.Okonogi,“Anti- oxidant and antimicrobial activities of Hyptis suaveolens essential oil,” Scientia Pharmaceutica,vol.75,no.1,pp.35–46,2007. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 579346, 10 pages http://dx.doi.org/10.1155/2013/579346

Research Article Antiulcerogenic Activity of the Hydroalcoholic Extract of Leaves of Croton campestris A. St.-Hill in Rodents

Francisco E. B. Júnior, Dayanne R. de Oliveira, Elizângela B. Bento, Laura H. I. Leite, Daniele O. Souza, Ana Luiza A. Siebra, Renata S. Sampaio, Anita O. P. B. Martins, Andreza G. B. Ramos, Saulo R. Tintino, Luiz J. Lacerda-Neto, Patricia R. L. Figueiredo, Larissa R. Oliveira, Cristina K. S. Rodrigues, Valterlúcio S. Sales, Francisco R. S. D. N. Figueiredo, Emmily P. Nascimento, Alefe B. Monteiro, Érika N. Amaro, José G. M. Costa, Henrique Douglas Melo Coutinho, Irwin R. A. de Menezes, and Marta R. Kerntopf Department of Biological Chemistry, Regional University of Cariri, Crato, CE, Brazil

Correspondence should be addressed to Henrique Douglas Melo Coutinho; [email protected]

Received 20 February 2013; Revised 24 May 2013; Accepted 27 May 2013

Academic Editor: Ulysses Paulino de Albuquerque

Copyright © 2013 Francisco E. B. Junior´ et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Croton campestris A. St.-Hill., popularly known as “velame do campo,” is a species native to the savannah area of Northeast Brazil, which is used by traditional communities in folk medicine for variety of health problems, especially detoxification, inflammation, and gastritis. The hydroalcoholic extract of C. campestris leaves (HELCC) was assessed for its antiulcerogenic effect in gastric lesion models and effect on intestinal motility in mice, and possibleechanisms m of action were examined. HELCC showed significant gastroprotective action in all models of gastric ulcer evaluated; the results suggest that this action probably involves the nitric oxide pathway. HELCC did not show alteration of intestinal motility in mice. It was also found that C. campestris represents a promising natural source with important biological potential, justifying some of its uses in folk medicine.

1. Introduction 100% remission of ulcers, with reduced side effects and without compromising the patient’s wellbeing, which usually Peptic ulcer is a term used to describe a group of ulcerative results in chronic use of these drugs. Published studies have disorders that occur in areas of the upper gastrointestinal reported the widespread identification of new drugs derived tract, which are exposed to acidic secretions and pepsin. It from natural antiulcerogenic sources [2]. represents a chronic health problem. Approximately 10% of Studies have shown that lipid peroxidation and oxidative the population have or will develop peptic ulcer. Its incidence reactions are implicated in the pathogenesis of lesions is slightly higher in men than women (1.3 : 1), and although induced by ethanol by attacking biological molecules and it occurs at any age, duodenal ulcer occurs most often in the range of 30–55 years, whereas gastric ulcer occurs in the range prostaglandins. Thus, research in animal models has attracted of 50–70 years [1]. the interest of many investigators as a promising source for In Brazil, despite the absence of epidemiological records the discovery of new drugs [3–5]. of ulcer cases, it is known that there are numerous cases in- The genus Croton, whose name means “tick,”is the second volving this disease, which means a significant public health largest in the family Euphorbiaceae and belongs to the sub- problem prompting the search for new substances with anti- family Crotonoidae Crotoneae and tribe [6]. It is subdivided ulcerogenic activity. Although there is a large arsenal of drugs into 40 sections and has more than 1,300 species of mainly with antiulcerogenic activity on the market, none produces pantropical distribution, of which 350 species in 29 sections 2 Evidence-Based Complementary and Alternative Medicine occur in Brazil [7]. Of these, a total of 52 species in 18 sections 2.3. Phytochemical Screening. The material was submitted to are referred to Northeast Brazil [8]. phytochemical screening for the presence of tannins, Several species of Croton have long played an important flavonoids, saponins, steroids, triterpenes, coumarins, quin- role in traditional uses of medicinal plants in Africa, Asia, ones, organic acids, and alkaloids. Accordingly, the hydro- and South America, including the treatment of cancer, con- alcoholic extract (0.3 g) was diluted in distilled water (9 mL) stipation, diarrhea, and other digestive problems, diabetes, and 70% ethanol (21 mL) and after dilution, distributed into external wounds, fever, hypercholesterolemia, hypertension, six flasks, in which the tests for different compounds were inflammation, intestinal worms, malaria, pain, ulcers, and performed. Phenols and tannins: 3 drops of ferric chloride obesity [9]. were added to the flask, and development of a blue color Studies involving several species of the genus Croton have denotedthepresenceofpyrogallictannins,whichcouldbe shown different pharmacological activities such as anti- further confirmed with the addition of gelatin, resulting in a inflammatory and antioxidant activities of C. celtidifolius precipitate with the sample. Anthocyanins, anthocyanidins, [10], antileishmanial effect of C. cajucara [11], antinociceptive and flavonoids: the sample was acidified (pH 3) by the addi- effect of C. cajucara [12], antiulcerogenic and cytotoxic activi- tion of 18 drops of HCl, and no change in color of the flask ties of C. cajucara [13], hypoglycemic and hypolipidemic pro- demonstrated the presence of flavones, flavonols, xanthones, perties of C. cajucara [14], antimicrobial and the antimalarial and flavanonols. effects C. kongensis [15], antinociceptive and anti-inflam- The test for leucoanthocyanidins, catechins, and flavon- matory properties of C. malambo [16], antimicrobial effect of ones was performed by alkalinization to pH 8.5 with 1% C. sonderianus [17], purgative effect of C. campestris [18], and sodium hydroxide in the first flask, acidification (pH 3)by hypotensive and narcotic activities of C. eluteria [19]. means of 18 drops of HCl and heating for 2 min in the second AstudyofC. campestris in the traditional community of flask, and alkalinization (pH 11) with 18 drops of 1% NaOH the bioregion of Chapada do Araripe revealed the popular use andalsoheatingfor2mininthethirdflask.Thus,flask2 of the leaves and roots of this plant for gastric, hematological, didnotshowachangeincolor,demonstratingthepresence and inflammatory disturbances, wounds, and respiratory of flavonones, and still the orange red color of flask 3 also problems. For their medicinal uses, the local population revealed the presence of flavonones. The test for chalcones utilizes infusion in alcoholic beverages, teas, and macerates, and aurones, by alkalinization (pH 11) with 18 drops of 1% which justifies the use of the hydroalcoholic extract for the NaOH,gaveapurpleredcolorintheflask,indicatingthe verification of the antiulcer activity. presence of these compounds. The hydroalcoholic extract of leaves of C. campestris A. The test for alkaloids was done by diluting the extract St.-Hill (HELCC) was assessed for its antiulcerogenic effect (0.3 g) in 30 mL of 5% acetic acid and 15 mL of ammonia for in models of gastric lesions using absolute ethanol, acidified alkalinization. The preparation was heated until boiling, and ethanol, and indomethacin, and mechanisms of action were 10 mL of 10% ammonium hydroxide was then added, along examined. The effect of HELCC on gastric motility was also with 15 mL of chloroform. After homogenization and allow- ruled out. ing to stand in a separatory funnel, the chloroform phase was collectedinabeaker,andthesolventwasevaporated.The residuewasresuspendedin1%HClandhomogenized,and 2. Materials and Methods solution was placed on a slide along with a drop of Dragen- dorff reagent, where the formation of a precipitate was indi- 2.1. Plant Material. The leaves of the plant species Croton cativeofthepresenceofalkaloids. campestris A. St.-Hill were collected in the municipality of The test for steroids and triterpenoids was carried out by ∘ 󸀠 󸀠󸀠 ∘ 󸀠 Crato, Ceara,´ Brazil, using a GPS device (7 22 2.8 S; 39 28 dilution of the extract (0.3 g) in 6 mL of chloroform and filtra- 󸀠󸀠 42.4 W; altitude: 892 meters). The procedures for specimen tion using cotton covered with sodium sulfate, collecting the preparation followed the recommendations described by filtrate in a test tube. After the addition of 0.1 mL of anhydrous Ming 1996 apud Di Stasi, 1996. The material was deposited acetic acid and three drops of sulfuric acid, the development in the Herbarium of the Federal University of Rio Grande do of a green color indicated the presence of steroids [20]. Norte (UFRN) and registered under no. 7095. The research was reviewed by the Ethics Committee for Animal Research, CEPA, Faculty of Medicine of Juazeiro do Norte (FMJ), and 2.4. Assays was approved (case no. 2009 0432 FR 271610). 2.4.1. Assessment of Oral Acute Toxicity. Swiss mice were used throughout to evaluate HELCC for toxicity and effect on 2.2. Preparation of Hydroalcoholic Extract of the Leaves of Cro- gastric lesions. The animals were fasted for 3 h prior to the tonCampestrisA.St.-Hill(HELCC). The plant material (fresh experiment and were given a single dose of extract dissolved leaves: 852 g C. campestris) was washed under running water, in 2% w/v Tween 80 at 17.5, 55, 175, 550, 2000, and 5000 mg/kg crushed and macerated, and then submitted to cold extrac- and observed for mortality for up to 48 h (short-term toxi- tion with 99.9% ethanol and water in a 1 : 1 proportion (8.7 L of city). Based on the short-term toxicity, the dose of the next solvent). The solvent was removed using a rotary evaporator animal was determined as per OECD guideline 425. All ∘ under reduced pressure and temperature of 40–50 C, and a animals were also observed for long-term toxicity (14 days). yield of 70.89 g crude extract was obtained using a lyophilizer. The LD50 of the test extracts was calculated using “AOT 425” Evidence-Based Complementary and Alternative Medicine 3 software provided by the Environmental Protection Agency, (saline, 0.1 mL/10 g, p.o.), or atropine (0.01 g/kg, p.o.) and 10% USA [21]. activated charcoal (0.1 mL/10 g, p.o.). HELCC, atropine, or vehicle was administered first, followed by activated char- 2.5. Gastric Lesions Induced by Ethanol [22]. The mice were coal 1 h later. Thirty minutes after charcoal administration, divided into groups (𝑛 = 6),fastedforaperiodof15hand the animals were sacrificed, and their small intestine was treated with HELCC (50, 75, 125, 250, 500, and 750 mg/kg, removed. The total length of the intestine (the pyloric region p.o.), omeprazole (30 mg/kg, p.o.), or vehicle (saline, 0.1 mL/ to the ileocecal junction) was then measured; the distance 10 g, p.o.) 1 h before administration of absolute ethanol traveled by the charcoal was determined based on the dis- (0.2 mL/animal, p.o.). After 30 min, the animals were sacri- tance from the pylorus to the last portion of the intestine that ficed by cervical dislocation. Their stomach was removed, containedatleast1cmofcontinuouscharcoal.Thirtyminutes opened along the greater curvature, and rinsed with saline after administration of the charcoal, the animals were sacri- and digitized; the ulcerated area was expressed as a percent- ficed and analyzed as described above. age relative to the total area of the gastric body using ImageJ software. 2.10. Statistical Analysis. Data are presented as the mean ± standard deviation. The level of significance was 0.05. Dif- 2.6. Gastric Lesions Induced by Acidified Ethanol [23]. The ferences between the means were determined by analysis of mice were treated with HELCC (50, 75, 125, 250, 500, and variance (ANOVA), and the Newman-Keuls test was used to 750 mg/kg, p.o.), omeprazole (30 mg/kg, p.o.), or vehicle determine statistical significance. Analyses were performed (saline, 0.1 mL/10 g, p.o. for the control lesion group). One using GraphPad Prism software version 5.0. hour after treatment, the animals received 0.2 mL of 0.3 M hydrochloric acid (HCl) in 60% ethanol and were sacrificed 1 h later. The percentage of stomach ulceration was deter- 3. Results mined as described above. Phytochemical prospecting [20] of HELCC identified the pre- 2.7. Gastric Lesions Induced by Indomethacin [24]. The mice sence of tannins flavonols, flavones, flobabenics, flavanone, were pretreated with HELCC (250, 500, and 750 mg/kg, p.o.), flavonals, xanthones, terpenes, and alkaloidsTable ( 1), possi- omeprazole (30 mg/kg, p.o.), or vehicle (saline, 0.1 mL/10 g, blyinvolvedinthebioactivepropertiesevaluated. p.o. for the control lesion group). Six hours after administration of the ulcerogenic agent (indomethacin, 10 mg/kg, s.c.), the animals were sacrificed. The percentage of stomach 3.1. Toxicological Data. HELCC did not show evidence of ulceration was determined as described above. acutetoxicity.Thedailymonitoringofweightofanimalsdid not indicate a significant variation compared to the control 2.8. Evaluation of the Mechanism of Action of Gastroprotective group (saline). There were no signs of morbidity or death just HELCC. To determine the mechanism of action, experi- after extract administration, or during the period of obser- ments were performed to separately examine the involvement vation and weighing (14 days after treatment) in the group of 𝛼-2 receptors, prostaglandins [23], nitric oxide [25], and treated orally. The median lethal dose (LD50) for the oral + ATP-dependent K channel activation [26]inthegastropro- administration of HELCC was greater than or equal to tective effect of HELCC (75 mg/kg, p.o.). These experi- 5000 mg/kg, with a 95% confidence interval. ments used the appropriate antagonists including yohim- The animals showed decreased activity and mild dyspnea, bine (2 mg/kg, i.p.), indomethacin (10 mg/kg, p.o.), L-NAME showing recovery within four hours. (10 mg/kg, i.p.), and glibenclamidee (5 mg/kg, p.o.) or ago- nists including L-arginine (600 mg/kg, p.o.) as a positive con- 3.2. Gastroprotection Tests. The effects of HELCC on gastric trol for L-NAME and misoprostol (0.016 mg/kg, p.o.) as a lesions induced by absolute ethanol (0.2 mL/animal, p.o.) are control for indomethacin before the oral administration of shown in Figure 1.Theanimalsthatreceivedonlyvehicle 0.2 mL of 96% ethanol. combined with oral administration of absolute ethanol In each case, the animals were pretreated with the specific showed an extensive area of gastric lesion (18.57 ± 1.87%). antagonist or agonist for 30 min before the administration HELCC produced a marked reduction in the area damaged by of HELCC. Finally, 0.2 mL of 96% ethanol was orally admin- absolute ethanol at all doses tested: 1.34 ± 0.66% at 50 mg/kg isteredonehourafterHELCC.Theanimalswerethensacri- (92.78% reduction), 1.11±0.67% at 75 mg/kg (94.02%), 0.93± ficed, and their stomach was removed, opened along the 0.43%at125mg/kg(94.99%),0.87 ± 0.54%at250mg/kg greatercurvature,washedinsaline,andcompressedbetween (95.31%), 0.82 ± 0.31% at 500 mg/kg (95.58%), and 0.53 ± glass slides for better viewing. The slides were scanned at 0.16% at 750 mg/kg (97.14%), when compared to the control 1200 dpi. The percentage area of gastric lesions (glandular (𝑃 < 0.001 in all cases). The animals that received omeprazole portion) was determined with the aid of ImageJ software. The (30 mg/kg, p.o.) also showed a significant decrease in gastric injured area is expressed as a relative percentage of the total lesion area and a substantial reduction percentage in ulcer area of the gastric body [23, 26]. area, that is, 0.78 ± 0.34% and 95.79%, respectively, compared to control (𝑃 < 0.001). 2.9. Effect of HELCC on Intestinal Motility27 [ ]. The ani- The effects of HELCC on gastric lesions induced by acidi- mals were treated with HELCC (75 mg/kg, p.o.), vehicle fied ethanol (0.2 mL/animal, p.o.) are shown in Figure 2. 4 Evidence-Based Complementary and Alternative Medicine

Table 1: Prospecting photochemistry of hydroalcoholic extract of 30 lyophilized of leaves of Croton campestris A. St.-Hill.

(+) presence Metabolites (−) absence Phenols − 20 Pyrogallic tannins − Flobabenic tannins + Anthocyanins − Anthocyanidins − 10

Flavones + (%) area Gastric ulcerated + Flavonols ∗∗∗ ∗∗∗ ∗∗∗ + ∗∗∗ ∗∗∗ Xanthones ∗∗∗ ∗∗∗ Chalcones − 0 Aurones − CL 30 50 75 125 250 500 750 (mg/kg) Flavanonols + Leucoanthocyanidin − Omeprazole HEFCC − Catechin Ethanol-HCL (0.1 mL/10 g, v.o.) Flavonone + + CL 125 Alkaloids 30 250 Terpenes + 50 500 75 750 Steroids − Figure 2: Effect of oral administration of the HEFCC in gastric ∗∗∗𝑃 < 0.001 25 lesions induced by acid ethanol in mice. Significance when compared with control group.

20 (97.45%), respectively, when compared to the control (𝑃< 15 0.001 in all cases). The animals that received omeprazole (30 mg/kg, p.o.) also showed a significant reduction in the 10 injured areas and a high percentage reduction in ulcer area, that is, 3.58 ± 0.56% and 84.56%, respectively, compared to control (𝑃 < 0.001).

Gastric ulcerated area (%) area Gastric ulcerated 5 The effects of HELCC on gastric lesions induced by indo- ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ methacin (10 mg/kg, s.c.) are shown in Figure 3.Theanimals 0 that received only vehicle combined with s.c. administration CL 30 50 75 125 250 500 750 of indomethacin showed a large area of gastric lesion (12.61± (mg/kg) 3.27%). HELCC produced a significant reduction in the areas Omeprazole HEFCC damaged by indomethacin, at all doses tested: 0.63 ± 0.11%at Ethanol absolute (0.1 mL/10 g, v.o.) 250 mg/kg (reduction of 95.00%), 0.33 ± 0.08%at500mg/kg CL 125 (97.38%), and 0.47 ± 0.17% at 750 mg/kg (97.38%), respec- 30 250 tively,whencomparedtothecontrol(𝑃 < 0.001 in all cases). 50 500 The animals that received omeprazole (30 mg/kg p.o.) also 75 750 showed significant reduction in gastric lesion area and a substantial percentage reduction in ulcer area, that is, 0.40 ± Figure 1: Effect of oral administration of the HEFCC in gastric 0.24 (𝑃 < ∗∗∗ < 0.001 % and 96.82%, respectively, compared to control lesions induced by ethanol abs in mice. Significance 𝑃 0.001) when compared with control group. . In the evaluation of the role of nitric oxide (NO) in the protective effect of HELCC against gastric lesions induced by absolute ethanol in mice, it was observed that the animals that The animals that received only vehicle combined with oral received only vehicle combined with oral administration of administration of acidified ethanol showed an extensive area absolute ethanol showed gastric lesion (20.52 ± 2.29%).The of gastric lesion (23.19 ± 3.09%).HELCCshowedsubstantial animals that received L-NAME (10 mg/kg, i.p.), an inhibitor reduction in the areas damaged by acidified ethanol, at all of nitric oxide synthase (NOS), along with absolute ethanol doses tested: 3.04 ± 0.80% at 50 mg/kg (reduction of 86.89%), (0.2 mL/animal, p.o.), also showed a large percentage of 1.94 ± 0.70% at 75 mg/kg (91.63%), 1.67 ± 0.75%at125mg/kg ulcerated area (28.73 ± 3.83%).However,theanimalsthat (92.79%), 2.40 ± 0.99%at250mg/kg(89.65%),0.52 ± 0.30% received L-arginine (600 mg/kg, p.o.) in combination with at 500 mg/kg (97.75%), and 0.59 ± 0.26%at750mg/kg absolute ethanol exhibited reductions in injured area (1.47 ± Evidence-Based Complementary and Alternative Medicine 5

20 40

30 15

20 10

10 Gastric ulcerated area (%) area Gastric ulcerated 5 a/b/c Gastric ulcerated area (%) area Gastric ulcerated a/b/c 0 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ CL 10 600 10 75 (mg/kg) 0 CL 30 250 500 750 (mg/kg) L-NAMEL-arginine L-NAME HEFCC

Omeprazole HEFCC 75 (mg/kg)

Indomethacin (10 mg/kg, s.c.) HEFCC

CL 500 Ethanol absolute (0.1 mL/10 g, v.o.) 30 750 CL 250 10 10 75 600 Figure 3: Effect of oral administration of the HEFCC in gastric ∗∗∗𝑃 < 0.001 lesions induced by indomethacin in mice. Significance Figure 4: The role of nitric oxide (NO) in the gastroprotector effect when compared with control group. of the HEFCC in gastric lesions model induced by ethanol absolute in mice. Significance a𝑃 < 0.001 versus CL, b𝑃 < 0.001 versus L- NAME (10 mg/Kg, v.o.), and c𝑃 < 0.001 versus L-NAME (10 mg/kg, 0.38%),withpercentageofulcerreductionof92.83%.HELCC v.o.) + EHFCC (75 mg/kg, v.o.). (75 mg/kg, p.o.) produced a significant (𝑃 < 0.001) reduction in ulcerated area to 1.11 ± 0.67% or a percentage reduction of 25 94.59%, compared to control. However, HELCC (75 mg/kg, p.o.), when given with L-NAME (10 mg/kg, i.p.), had its effect 20 blocked, resulting in a percentage of ulcerated area of 30.65 ± 6.55 %(Figure 4). 15 Intheinvestigationoftheroleofprostaglandinsinthe protective effect of HELCC against gastric lesions induced by 10 absolute ethanol in mice, it was seen that the animals that a/c received only vehicle in combination with oral administra- 5

18.57± (%) area Gastric ulcerated tion of absolute ethanol showed a gastric lesion area of a/c 1.87%. The animals that received indomethacin (10 mg/kg, a/c 0 s.c.) together with absolute ethanol (0.2 mL/animal, p.o.) CL 10 0.016 10 75 (mg/kg) displayed a percentage of ulcerated area of 13.09 ± 1.92%. However, the animals that received misoprostol (0.016 mg/kg, p.o.) combined with absolute ethanol showed reductions in Indometacine MisoprostolIndomethacinHEFCC injured area (0.57 ± 0.25%), resulting in a percentage reduction of 96.95%. HELCC (75 mg/kg, p.o.) produced a signi- 75 (mg/kg) (𝑃 < 0.001) 1.11 ± 0.67 ficant reduction in ulcerated area to % HEFCC or a percentage reduction of 94.02%, compared to the control. HELCC(75mg/kg,p.o.)whencombinedwithindomethacin Ethanol absolute (0.1 mL/10 g, v.o.) (10 mg/kg, s.c.) resulted in a percentage of ulcerated area of CL 10 3.95 ± 1.15% and a percentage reduction in ulcer area of 10 75 78.72% (Figure 5). 0.016 In the investigation of the role of the alpha-2 noradrenergic receptor in the protective effect of HELCC against gastric Figure 5: The role of prostaglandins in gastroprotector effect of the lesions induced by absolute ethanol in mice, it was seen that HEFCC in gastric lesion model induced by ethanol absolute in mice. a b c the animals that received only vehicle combined with oral Significance 𝑃 < 0.001 versus CL, 𝑃 < 0.01 versus CL, 𝑃 < 0.001 administration of absolute ethanol showed a gastric lesion versus indomethacin (10 mg/Kg, s.c.). area of 23.22 ± 2.08%. The animals that received yohimbine 6 Evidence-Based Complementary and Alternative Medicine

(2 mg/kg, i.p.) together with absolute ethanol (0.2 mL/animal, 40 p.o.) exhibited an extensive ulcerated area of 30.34 ± 4.96%. HELCC (75 mg/kg, p.o.) produced a significant (𝑃 < 0.001) reduction in ulcerated area to 0.91 ± 0.63%orapercentage 30 reduction of 96.34%, compared to the control. Also, HELCC (75 mg/kg, p.o.) given together with yohimbine (2 mg/kg, i.p.) 20 produced a significant (𝑃 < 0.01) gastroprotective effect with a percentage of ulcerated area of 9.30 ± 0.59%orapercentage a/b reduction in ulcer area of 59.94% (Figure 6). 10

In the evaluation of the involvement of ATP-dependent (%) area Gastric ulcerated + K channels in the protective effect of HELCC against gastric b/c lesions induced by absolute ethanol in mice, it was observed 0 that the animals that received only vehicle in combination CL 2275(mg/kg) with oral administration of absolute ethanol displayed a gastric lesion area of (17.17 ± 2.44%). The animals that received glibenclamidee (5 mg/kg, i.p.) along with absolute ethanol Yohimbine Yohimbine HEFCC (0.2 mL/animal, p.o.) showed an ulcerated area of 13.30 ± 75 (mg/kg) 2.25%). HELCC (75 mg/kg, p.o.) produced a significant (𝑃 < HEFCC 0.001) reduction in ulcerated area to 1.11 ± 0.67%orper- centage reduction of 91.65%, compared to the control. Also, Ethanol absolute (0.1 mL/10 g, v.o.) HELCC (75 mg/kg, p.o.) when given with glibenclamidee (5 mg/kg, i.p.) still exerted a significant gastroprotective effect CL 2 (𝑃 < 0.001) with a percentage of ulcerated area of 1.23±0.53% 2 75 or percentage reduction in ulcer area of 90.75% (Figure 7). Figure 6: The role of noradrenergic receptor alpha2 in the gastroprotector effect of HEFCC in gastric lesion model induced by ethanol abs in mice. Significance a𝑃 < 0.001 versus CL, b𝑃 < 0.001 3.3. Evaluation of Intestinal Motility. The percentage of the versus glibenclamide (5 mg/kg, i.p.). distance traveled by the marker (activated charcoal) in the small intestine of mice was (82.77 ± 4.29%).Theanimalsthat received HELCC (75 mg/kg) orally had a distance traveled usedacutemodelsfortheevaluationofantiulcerogenicsub- by the marker (84.20 ± 3.94%) that was similar to that stancesinanimalsarethemodelsofgastriclesioninducedby obtained by vehicle group, demonstrating that HELCC did absolute ethanol or indomethacin [30]. In this work, besides not significantly affect gastrointestinal motility. However, in the use of these methodologies, the effects of HELCC were the group treated with atropine (0.01 g/kg, p.o.), a muscarinic also evaluated with the gastric ulceration model using acidi- antagonist, there was a decrease in intestinal motility with a fied ethanol. distance traveled of 63.21 ± 2.05%, compared to the vehicle Ethanol-induced gastric ulcer occurs predominantly in control group (saline, p.o.), showing a significant effect (𝑃 < the glandular portion of the stomach, as a result of a direct 0.01), as seen in the data presented in Figure 8. necrotizing action and, moreover, the lack of defense factors such as secretion of bicarbonate and mucus and increased oxidative stress [31]. HELCC at all doses tested by oral admin- 4. Discussion istration prevented, in a dose-dependent manner, gastric lesion induced by the administration of absolute ethanol. The Several species of the genus Croton have played an important doses decreased the percentage of gastric ulcer area in the role in traditional uses of medicinal plants in Africa, Asia, same manner as with omeprazole, a well-known proton and South America. Such uses include the treatment of pump inhibitor, when compared to the vehicle-treated con- cancer, constipation, diarrhea and other digestive problems, trol lesion group. It was also demonstrated that the gastro- external wounds, diabetes, dyslipidemia, hypertension, fever, protective action of HELCC at all orally administered doses, inflammation, intestinal worms, malaria, pain, ulcers, and against gastric ulcer induced by acidified ethanol, resulted obesity [9]. In the toxicological evaluation, HELCC did not in a percentage reduction in ulcer area similar to that with show signs of significant acute toxicity in the animals tested omeprazole, in comparison with the vehicle-treated control and at all doses administered, corroborating a previous study lesion group. that demonstrated the low acute toxicity of Croton campestris Indomethacin like the majority of nonsteroidal anti- A. St.-Hill. in two guinea pigs [28]. inflammatory drugs (NSAIDs) acts as an inhibitor of pros- As seen in the evaluation of the gastroprotective activity taglandin synthesis and consequently diminishes the defense of HELCC, experimental animals play an important role in mechanisms of gastric mucus [32–34]. It is the primary the search for new drugs with protective properties. Consid- mechanism by which this class of drugs cause, damage to the ering that the etiology of ulcer is multifactorial, lesions in gastrointestinal tract [35]. the gastric mucosa can be induced in different experimental This research demonstrates the ability of the HELCC to models through various mechanisms [29]. Some of the most inhibitthegastricdamagecausedbyNSAIDs,wherewe Evidence-Based Complementary and Alternative Medicine 7

25 100

20 80

15 ∗∗ 60 10 40 5 Gastric ulcerated area (%) area Gastric ulcerated a/b a/b 20 0

CL 5575(mg/kg) (%) charcoal pelo by travelled Distance 0 C 0.01 75 Glibenclamida Glibenclamida HEFCC

75 (mg/kg) Atropine (0.01 g/Kg, v.o.) HEFCC (75 mg/kg, v.o.) HEFCC Activate charcoal 10% (0.1 mL/10 g, v.o.) Ethanol absolute (0.1 mL/10 g, v.o.) C 0.01 CL 5 75 5 75 Figure 8: The effect of HEFCC on intestinal motility in mice. Figure 7: The role of ATP-dependent K+ channels in the gastropro- ∗∗ 𝑃 < 0.01 tector effect of HEFCC in gastric lesion model induced by ethanol Significance versus C (vehicle control). absolute in mice. Significance a𝑃 and a𝑃 < 0.001 versus CL, b𝑃 < 0.001 versus glibenclamide (5 mg/kg, i.p.). synthase), showed a large ulcerated gastric area, after admin- used a classic model of induction of gastric lesion by s.c. istration of absolute ethanol, as in the control lesion group. indomethacin. Orally administered HELCC, at the doses HELCC managed to inhibit the appearance of gastric lesions tested, was able to prevent the appearance of gastric lesions in in the gastroprotection model using L-NAME. The extract in animals subjected to the treatment with this NSAID, showing this situation showed a non-significant percentage reduction significant results when compared to the gastric lesion con- in ulcer area. Similarly, it was seen that the L-arginine (a sub- trol. strate of NO synthase) significantly reduced the percentage Among the various factors involved in maintaining the of ulcerated gastric area when compared with the vehicle- integrity of the gastric mucus and protection against injury treated control lesion group. This suggests that the com- caused by ulcerogenic agents, we examined the contribution pounds present in the extract that exert a gastroprotective of endogenous nitric oxide, prostaglandins, ATP-sensitive effect involving the cytoprotective effect of NO. potassium channels (KATP), and alpha-2 noradrenergic Considering the traditional use of the leaves of the species receptor [36, 37] to the action of HELCC on the gastric Croton campestris A. St.-Hill. for gastric disturbances, in the mucosa. formofteasandinfusion,andinviewofthepresenceofsuch The discovery of nitric oxide (NO) as an agent of cell secondary metabolites as tannins, flavonoids, and alkaloids in signaling was one of the most important events in human the extract, it is possible to correlate the antiulcerogenic effect physiology of the last 80–90 years. NO is a free radical gas, demonstrated by the plant with these compounds. Also, it is which has drawn attention because of its role in signal trans- possible to correlate studies that demonstrate an association duction related to various physiological processes such as between plant species that contain tannins, flavonoids, and smooth muscle relaxation and vasodilation, neurotransmis- alkaloids and gastroprotective effects44 [ , 45], as probably sion, platelet aggregation mechanisms, regulation of pro- and occur with Croton campestris A. St.-Hill. antiapoptotic mechanisms, and control of blood pressure and The protective action of prostaglandins (PGs) on the blood flow38 [ , 39]. gastric mucosa is mediated by an increase in mucus produc- Related to the modulation of the physiological compo- tion and secretion of bicarbonate, modulation of gastric acid nents of gastrointestinal tract, NO plays an important role in secretion, inhibition of the release of inflammatory mediators the control of gastrointestinal motility [40], gastric blood flow by mast cells, and the maintenance of blood flow during expo- [41], recruitment of neutrophils [42], and secretion of mucus sure to irritants. It is known that prostaglandin E2 (PGE2) [43]. has a gastroprotective action against lesions caused by ethanol In the present research, the assessment of the role of NO and that this protection stems from an increase in intracellu- 󸀠 󸀠 in the gastroprotective effect of HELCC in models of gastric lar guanosine-3 ,5-cyclic monophosphate (cGMP), which is lesions, induced by ethanol in mice, showed that the ani- mediated by an increase in intracellular free calcium concen- mals that received L-NAME (a nonspecific inhibitor of NO tration and nitric oxide production [46]. 8 Evidence-Based Complementary and Alternative Medicine

In the evaluation of a possible involvement of prostaglan- a protective effect against acute gastric lesion induced by dins in the gastroprotective effect of HELCC, we used miso- absolute ethanol, acidified ethanol, or indomethacin in mice, prostol, an analogue of prostaglandin E1 (PGE1), as well as thus indicating a cytoprotective action of HELCC, which indomethacin, and an inhibitor of PG synthesis. Misoprostol occurs through the potentiation of the NO/cGMP pathway. significantly inhibited the development of gastric lesions The leaves of C. campestris contain compounds (metabolites) when compared with the vehicle-treated control lesion group. that possibly act in synergy in the activation of defense factors Pretreatment with indomethacin did not reverse the cytopro- and in reducing aggressor factors of gastric mucosa, which tective effect of HELCC, demonstrating just a trend in this makes this extract promising for the development of new direction. On the basis of this result, we cannot state that therapies to combat gastropathy associated with NSAID- prostaglandins play a role in the relaxant effect of HELCC. induced peptic ulcer disease. The modulation of 𝛼2 receptors located in the intramural peripheral parasympathetic ganglia decreases the discharge References of vagal acetylcholine, which reduces gastric secretion and motility and increases blood flow. The 𝛼2-noradrenergic [1] E. Rubin and J. P. Palazzo, “Trato gastrointestinal,” in Patologia: receptors and the 𝛼2presynapticareinvolvedintheregula- Bases Clinicopatologicas´ ,E.Rubin,Ed.,chapter13,pp.673–750, tion of gastric acid secretion and are effective in protection Guanabara Koogan, Rio de Janeiro, Brazil, 2006. against chemical agents such as NSAIDs and ethanol, where [2]A.Jamal,K.Javed,M.Aslam,andM.A.Jafri,“Gastroprotective their effects may be mediated central and peripheral receptors effect of cardamom, Elettaria cardamomum Maton. fruits in [47]. rats,” Journal of Ethnopharmacology,vol.103,no.2,pp.149–153, In our study, the administration of yohimbine (10 mg/kg, 2006. i.p.), an indole alkaloid that promotes the release of neuro- [3] J. E. Carvalho, Atividade antiulcerogenicaˆ e anticancerˆ de produ- transmitters by blocking the presynaptic 𝛼2 receptors [48], tos naturais e de s´ıntese,Divisao˜ de Farmacologia e Toxicologia, did not reverse the HELCC effect. This indicates that the Universidade de Campinas, 2006. gastroprotective effect of HELCC does not act by modulation [4] A. H. Gilani and Atta-ur-Rahman, “Trends in ethnopharmacol- of 𝛼2receptoractivity. ogy,” Journal of Ethnopharmacology,vol.100,no.1-2,pp.43–49, KATP channels are regulated by ligands and their mecha- 2005. nism is defined based on their sensitivity to intracellular ATP, [5] D. J. Newman, G. M. Cragg, and K. M. Snader, “Natural prod- which inhibits its activity. It has been postulated that KATP ucts as sources of new drugs over the period 1981–2002,” Jour- channels are involved in a variety of pathophysiological func- nal of Natural Products,vol.66,no.7,pp.1022–1037,2003. tions in the stomach such as regulation of blood flow, gastric [6] R. Braga, PlantasdoNordeste,especialmentedoCeara´,Escola acid secretion, and gastric muscle contractility [49]. In the Superior de Agricultura de Mossoro,´ Fortaleza, Brazil, 3rd edi- vascular system, these channels are related to the relaxation tion, 1976. of vascular smooth muscle, having an important role in blood [7] P. Berry, Croton Research Network, University of Wisconsin pressure control. This vasodilation can be blocked by gly- Board of Regents, Madison, Wis, USA, 2006. buride, a sulfonylurea that blocks KATP channels [50]. Our [8] D. S. Carneiro-Torres, I. Cordeiro, and F. A. Franc¸a, “A fam´ılia studies showed that HELCC, when combined with gliben- Euphorbiaceae na flora de inselbergs da regiao˜ de Milagres, clamidee, significantly retained its gastroprotective effect, Bahia, Brasil,” Boletim de Botanicaˆ da Universidade de Sao˜ indicating that the mechanism of action of the active ingredi- Paulo,vol.20,pp.31–47,2002. ents of the extract may not involve the stimulation of KATP [9] A. Salatino, M. L. F. Salatino, and G. Negri, “Traditional uses, channels. chemistry and pharmacology of croton species (Euphor- Another way in which HELCC could promote protection biaceae),” Journal of the Brazilian Chemical Society,vol.18,no. 1,pp.11–33,2007. ofthegastricmucosawouldbethroughincreasedgastro- intestinal motility, so increased intestinal transit would acce- [10] G. M. Nardi, R. Felippi, S. DalBo´ et al., “Anti-inflammatory and antioxidant effects of Croton celtidifolius Bark,” Phytomedicine, lerate gastric emptying, thereby decreasing the effect of vol. 10, no. 2-3, pp. 176–184, 2003. aggressor acid in the stomach and duodenum. Cholinergic innervation of the circular muscle layer of TGI, acting on [11] M. D. S. S. Rosa, R. R. Mendonc¸a-Filho, H. R. Bizzo et al., “Anti- leishmanial activity of a linalool-rich essential oil from Croton muscarinic receptors M1 and M3, is primarily responsible for cajucara,” Antimicrobial Agents and Chemotherapy,vol.47,no. gastrointestinal motility. 6, pp. 1895–1901, 2003. In this sense, HELCC did not significantly alter the intes- [12]A.R.Campos,F.A.A.Albuquerque,V.S.N.Rao,M.A.M. tinal transit of mice when compared to the vehicle group Maciel, and A. C. Pinto, “Investigations on the antinociceptive (10%activatedcharcoal)andthegroupthatreceivedatropine, activity of crude extracts from Croton cajucara leaves in mice,” which is a drug blocking the muscarinic action of acetyl- Fitoterapia,vol.73,no.2,pp.116–120,2002. choline. [13]A.A.B.Almeida,P.S.Melo,C.A.Hiruma-Limaetal.,“Anti- ulcerogenic effect and cytotoxic activity of semi-synthetic cro- tonin obtained from Croton cajucara Benth,” European Journal 5. Conclusion of Pharmacology,vol.472,no.3,pp.205–212,2003. [14]R.M.Silva,F.A.Santos,V.S.N.Rao,M.A.Maciel,andA.C. In short, we demonstrate here for the first time that HELCC, Pinto, “Blood glucose- and triglyceride-lowering effect of trans- an extract of leaves of Croton campestris A. St.-Hill., has dehydrocrotonin, a diterpene from Croton cajucara Benth., in Evidence-Based Complementary and Alternative Medicine 9

rats,” Diabetes,ObesityandMetabolism,vol.3,no.6,pp.452– [31] C. Rujjanawate, D. Amornlerdpison, S. Pojanagaroon, and D. 456, 2001. Kanjanapothi, “Anti-gastric ulcer effect of Kaempferia parvi- [15]J.Thongtan,P.Kittakoop,N.Ruangrungsi,J.Saenboonrueng, flora,” Journal of Ethnopharmacology,vol.102,no.1,pp.120–122, and Y. Thebtaranonth, “New antimycobacterial and antimalar- 2005. ial 8,9-secokaurane diterpenes from Croton kongensis,” Journal [32] D. A. Lewis and P. J. Hanson, “Anti-ulcer drugs of plant origin,” of Natural Products, vol. 66, no. 6, pp. 868–870, 2003. in Progress Medicinal Chemistry,G.P.EllisandG.B.West,Eds., [16] A. I. Suarez,´ R. S. Compagnone, M. M. Salazar-Bookaman et vol. 28, pp. 201–231, Elsevier Science Publishers, Amsterdam, al., “Antinociceptive and anti-inflammatory effects of Croton The Netherlands, 1991. malambo Bark aqueous extract,” Journal of Ethnopharmacology, [33] Y. Morimoto, K. Shimohara, S. Oshima, and T. Sukamoto, vol. 88, no. 1, pp. 11–14, 2003. “Effects of the new anti-ulcer agent KB-5492 on experimental [17] J. D. Mcchesney, A. M. Clark, and E. R. Silveira, “Antimicrobial gastric mucosal lesions and gastric mucosal defensive factors, as diterpenes of Croton sonderianus. II. ent-Beyer-15-en-18-oic compared to those of teprenone and cimetidine,” The Japanese acid,” Pharmaceutical Research,vol.8,no.10,pp.1243–1247,1991. Journal of Pharmacology,vol.57,no.4,pp.495–505,1991. [18] F. E. Babili, C. Moulis, M. Bon, M.-J. Respaud, and I. Fouraste,´ [34] F. Evans, “The gastro-intestinal Tract,” in Selection, Preparation “Three furano-diterpenes from the Bark of Croton campestris,” and Pharmacological Evaluation of Plant Material,pp.25–45, Phytochemistry, vol. 48, no. 1, pp. 165–169, 1998. 1996. [19] C. Vigor, N. Fabre, I. Fouraste,´ and C. Moulis, “Three clerodane [35] J. L. Wallace, “Nonsteroidal anti-inflammatory drugs and gas- diterpenoids from Croton eluteria Bennett,” Phytochemistry, troenteropathy: the second hundred years,” Gastroenterology, vol.57,no.8,pp.1209–1212,2001. vol. 112, no. 3, pp. 1000–1016, 1997. [20] F. J. A. Matos, Introduçao˜ afitoqu` ´ımica experimental, Ediçoes˜ [36] O. S. Zayachkivska, S. J. Konturek, D. Drozdowicz, T. Brzo- UFC, Fortaleza, Brazil, 1997. zowski, and M. R. Gzhegotsky, “Influence of plant-originated [21] OECD—Guidlines for the testing of chemicals. Acute Oral gastroproteciive and antiulcer substances on gastric mucosal Toxicity-Up-and-Down-Procedure (UDP),2008. repair,” Fiziologicheski˘ıZhurnal,vol.50,no.6,pp.118–127,2004. [22] A.Robert,J.E.Nezamis,C.Lancaster,andA.J.Hanchar,“Cyto- [37] T. Brzozowski, P. C. Konturek, D. Drozdowicz et al., “Grape- protection by prostaglandins in rats. Prevention of gastric fruit-seed extract attenuates ethanol-and stress-induced gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, lesions via activation of prostaglandin, nitric oxide and sensory and thermal injury,” Gastroenterology,vol.77,no.3,pp.433–443, nerve pathways,” World Journal of Gastroenterology, vol. 11, no. 1979. 41, pp. 6450–6458, 2005. [23] T. Mizui, N. Shimono, and M. Doteuchi, “Apossible mechanism [38] M. W. Radomski and S. Moncada, “Regulation of vascular of protection by polyamines against gastric damage induced homeostasis by nitric oxide,” Thrombosis and Haemostasis,vol. by acidified ethanol in rats: polyamine protection may depend 70, no. 1, pp. 36–41, 1993. on its antiperoxidative properties,” The Japanese Journal of [39]P.K.M.Kim,R.Zamora,P.Petrosko,andT.R.Billiar,“The Pharmacology,vol.44,no.1,pp.43–50,1987. regulatory role of nitric oxide in apoptosis,” International Im- [24] B. Djahanguiri, “The production of acute gastric ulceration by munopharmacology,vol.1,no.8,pp.1421–1441,2001. indomethacin in the rat,” Scandinavian Journal of Gastroenterol- [40] S. Ueki, K. Takeuchi, and S. Okabe, “Gastric motility is an ogy,vol.4,no.3,pp.265–267,1969. important factor in the pathogenesis of indomethacin-induced [25] H. Matsuda, Y. Li, and M. Yoshikawa, “Roles of capsaicin-sen- gastric mucosal lesions in rats,” Digestive Diseases and Sciences, sitive sensory nerves, endogenous nitric oxide, sulfhydryls, and vol. 33, no. 2, pp. 209–216, 1988. prostaglandins in gastroprotection by momordin Ic, an oleanolic acid oligoglycoside, on ethanol-induced gastric mucosal [41] B. J. R. Whittle, G. L. Kauffman, and S. Moncada, “Vasoconstric- lesions in rats,” Life Sciences,vol.65,no.2,pp.PL27–PL32,1999. tion with thromboxane A2 induces ulceration of the gastric mucosa,” Nature,vol.292,no.5822,pp.472–474,1981. [26]M.Rahgozar,H.Pazokitoroudi,A.Bakhtiarian,andB.Dja- [42] P. Kubes, M. Suzuki, and D. N. Granger, “Nitric oxide: an endo- hanguiri, “Diazoxide, a K𝐴𝑇𝑃 opener, accelerates restitution of ethanol or indomethacin-induced gastric ulceration in rats genous modulator of leukocyte adhesion,” Proceedings of the independent of polyamines,” Journal of Gastroenterology and National Academy of Sciences of the United States of America, Hepatology, vol. 16, no. 3, pp. 290–296, 2001. vol. 88, no. 11, pp. 4651–4655, 1991. [27] A. J. Lapa, C. Souccar, M. T. R. Lima-Landman, M. A. S. [43] J. L. Wallace and M. J. S. Miller, “Nitric oxide in mucosal defense: Castro, and T. C. M. Lima, Metodos´ de avaliaçao˜ da atividade alittlegoesalongway,”Gastroenterology,vol.119,no.2,pp.512– farmacologica´ de plantas medicinais, Sociedade Brasileira de 520, 2000. Plantas Medicinais, Sao˜ Paulo, Brazil, 2008. [44] F. F. G. Rodrigues, B. S. Cabral, H. D. M. Coutinho et al., “Anti- [28] E.M.RibeiroPrata,M.Q.Paulo,andA.R.M.SouzaBrito,“Iso- ulcer and antimicrobial activities of Stryphnodendron rotundi- lamentodoprinc´ıpio ativo de Croton campestris St. Hill. folium Mart,” Pharmacognosy Magazine,vol.4,no.15,pp.193– (Euphorbiaceae),” Revista Brasileira de Farmacologia,vol.74, 196, 2008. no. 2, pp. 36–41, 1993. [45] J. A. S. Zuanazzi and J. A. Montanha, “Flavonoides,”´ in Farma- [29] G. E. Samonina, G. N. Kopylova, G. V. Lukjanzeva et al., “Anti- cognosia: da planta ao medicamento,pp.577–614,Editorada ulcer effects of amylin: a review,” Pathophysiology, vol. 11, no. 1, UFRGS/Editora da UFSC, Porto Alegre, Brazil, 2004. pp. 1–6, 2004. [46] H. Sakai, E. Kumano, A. Ikari, and N. Takeguchi, “A gastric [30] R. S. Pandian, C. V.Anuradha, and P.Viswanathan, “Gastropro- housekeeping Cl-channel activated via prostaglandin EP3 2+ tective effect of fenugreek seedsTrigonella ( foenum graecum)on receptor-mediated Ca /nitric oxide/cGMP pathway,” The Jour- experimental gastric ulcer in rats,” Journal of Ethnopharmacol- nal of Biological Chemistry,vol.270,no.32,pp.18781–18785, ogy,vol.81,no.3,pp.393–397,2002. 1995. 10 Evidence-Based Complementary and Alternative Medicine

[47] B. Yelken, T. Dorman, S. Erkasap, E. Dundar, and B. Tanriverdi, “Clonidine pretreatment inhibits stress-induced gastric ulcer in rats,” Anesthesia & Analgesia,vol.89,no.1,pp.159–162,1999. [48] B. B. Hoffman, “Farmacos´ antagonistas dos adrenorreceptores,” in Farmacologia basica´ e cl´ınica,B.G.Katzung,Ed.,pp.127–141, McGraw-Hill, Sao˜ Paulo, Brazil, 10th edition, 2007. [49] N. B. Standen, J. M. Quayle, N. W. Davies, J. E. Brayden, Y. Huang, and M. T. Nelson, “Hyperpolarizing vasodilators + activate ATP-sensitive K channels in arterial smooth muscle,” Science,vol.245,no.4914,pp.177–180,1989. [50] M. T. Nelson and J. M. Quayle, “Physiological roles and properties of potassium channels in arterial smooth muscle,” The American Journal of Physiology,vol.268,no.4,part1,pp.C799– C822, 1995. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 823453, 8 pages http://dx.doi.org/10.1155/2013/823453

Research Article Bonellia albiflora: A Mayan Medicinal Plant That Induces Apoptosis in Cancer Cells

Rosa Moo-Puc, Juan Chale-Dzul, and Edgar Caamal-Fuentes

Unidad de Investigacion´ Medica´ Yucatan,´ Unidad MedicadeAltaEspecialidad,CentroM´ edico´ Ignacio Garc´ıa Tellez,´ Instituto Mexicano del Seguro Social, 41 No. 439 x 32 y 34, Colonia Industrial CP, 97150 Merida,´ YUC, Mexico

Correspondence should be addressed to Rosa Moo-Puc; [email protected]

Received 3 April 2013; Revised 31 May 2013; Accepted 1 June 2013

Academic Editor: Ulysses Paulino de Albuquerque

Copyright © 2013 Rosa Moo-Puc et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Few studies have been carried out on the medical flora of Mexico’s Yucatan Peninsula in search for new therapeutic agents, in particular against cancer. In this paper, we evaluated the cytotoxic potential of the extract of Bonellia albiflora,aplantutilizedinthe traditional Mayan medicine for treatment of chronic injuries of the mouth. We carried out the methanolic extracts of different parts of the plant by means of extraction with the Soxhlet equipment. We conducted liquid-liquid fractions on each extract with solvents of increasing polarity. All extracts and fractions were evaluated for cytotoxic activity versus four human cancer cell lines and one normal cell line through a tetrazolium dye reduction (MTT) assay in 96-well cell culture plates. The methanolic root-bark extract possessed much greater cytotoxic activity in the human oropharyngeal cancer cell line (KB); its hexanic fraction concentrated the active metabolites and induced apoptosis with the activation of caspases 3 and 8. The results demonstrate the cytotoxic potential of the B. albiflora hexanic fraction and substantiate the importance of the study of the traditional Mayan medicinal plants.

1. Introduction Mayan people knew of and treated distinct diseases, including those of infectious origin (intestinal infections, infec- Traditional medicine is a practice that has been carried out tious dermatitis, and respiratory infections), chronic dis- from antiquity to our present time by inhabitants of the eases (asthma, fatigue, nephritis, and hypertension), and indigenous pueblos of Mexico, among which the Mayan pop- psychological-type diseases (insomnia, nervousness, and ulation of the Yucatan Peninsula in Mexico is included. In the hysteria). In addition, they cured other illnesses such as the traditional Mayan medicine, plants are of great importance, following: abscesses; calluses; corns, hard protuberances; pol- which can be considered as evidence of their effectiveness for yps; tumors; and warts or sores, generally tangible or visible the control of many types of diseases. Likewise, they comprise on the skin [1, 4]. one of the most important alternatives for health care, above In the traditional Mayan medicine of the Yucatan Penin- all, in communities where primary health services are not sula, “cancer” is known as an illness or a set of illnesses that accessible. In addition, they can be taken advantage of widely as a natural renewable resource. Together with what was can manifest themselves as an affectation of the skin or sub- previously described, the traditional medicine of the indige- adjacent muscle mass, or an affectation in the form of pain in nous pueblos was recognized by the World Health Organi- some internal organ. The term alludes to a difficult-to-cure zation (WHO), which caused a powerful drive toward the illness or a one with a disagreeable aspect (when it affects the research of medicinal plants [1]. skin); if it is an internal cancer, the patient’s semblance reveals The Mayan ethnobotanical literature in its majority is the disease. The old inhabitants assigned names in the Mayan composed of historic or descriptive studies, in whose contents language to this set of symptoms; in the Mayan tongue, there predominates a compendium of diseases and treat- “cancer” is known as “tsunuz” or “tsunuztacan”,andhard ments known to Mayan healers of distinct eras [2, 3]. The protuberancesortumorsareknownas“chu’uchum” [3, 5]. 2 Evidence-Based Complementary and Alternative Medicine

Prior studies have demonstrated that the extracts of plants were kindly provided by Veronica Vallejo-Ru´ız from the East utilized in the traditional Mayan medicine for the treatment Biomedical Research Center-IMSS. The cells were cultured of the signs and symptoms suggestive of cancer possess in DMEM medium, containing 10% SFB supplemented with cytotoxic activity [6]. Similarly, two studies conducted on 100 units/mL penicillin G and 100 𝜇g/mL streptomycin in 5% ∘ two species of the genus Bonellia (Bonellia macrocarpa and CO2-95% humidified air at 37 C. Bonellia flammea) from the Yucatan Peninsula reveal the presence of novel compounds, such as active agents with anti- 2.5. Cytotoxicity Assay. The cytotoxicity was determined by carcinogenic activity [7, 8]. Within this context, the Yucatan the MTT assay according to the method described by Denizot 3 Peninsula has five species of the genus Bonellia,among and Lang [11] with some modifications. Briefly, 5×10 viable which the species B. macrocarpa, B. flammea,andB. albiflora cells from each cell line were seeded in a 96-well plate and are employed in the traditional Mayan medicine for the treat- incubated for 24–48 h. When cells reached >70% confluence, ment of the dermatological-type afflictions5 [ , 9, 10]. Of these the medium was replaced and the cells were treated with the three species, only B. albiflora has not been the object of any extract dissolved in DMSO (maximum concentration of phytochemical or biological activity study. B. albiflora is 0.05%) at 2.34 to 300 g/mL. After 48 h of incubation, 10 𝜇L denominated “Si’ik” in the traditional Mayan medicine and MTT (5 mg/mL) was added to each well and incubated at ∘ is used as an antitussive for the treatment of skin and mouth 37 Cfor4h.Themediumwasremoved,andtheformazan wounds and to relieve toothache pain [10]. In this work, we precipitate was dissolved in 100 𝜇L of acidified isopropanol proposed an evaluation of the cytotoxic potential of the (0.4 N HCl). The optical density was determined with a spec- organic extracts of B. albiflora. trophotometer at 540 nm. Cells treated with 0.05% DMSO and docetaxel were used as negative and positive controls, 2. Materials and Methods respectively. The concentration of the extract that killed 50% of the cells (CC50) was calculated by GraphPad Prism 4.00 2.1. Plant Material. Bonellia albiflora (Lundell) B. Stahl˚ software. All determinations were performed in triplicate. and Kallersj¨ o¨ was collected from different Localities of MDCKcelllinewasusedtoevaluatetheselectiveindex(SI) the State of Yucatan, Mexico, during the summer of 2010. of extracts. SI is defined as the ratio of cytotoxic activity from Plant material was identified and authenticated by tax- normal cell and cancer cell lines. onomists from the Department of Natural Resources of the Scientific Research Center of Yucatan (CICY). 2.6. GC-MS Analysis. The chromatographic separation was carried out by GC-MS analysis on an Agilent gas chromato- 2.2. Chemicals. Dulbecco’s modified Eagle’s medium graph, model 6890N, coupled to a mass selective detector, (DMEM), heat-inactivated fetal bovine serum (FBS), and model5975B.CompoundswereseparatedonaDB-5mscap- penicillin and streptomycin (PS) were purchased from illary column (30 m × 0.32 mm i.d., 0.25 𝜇mfilmthickness) Gibco, Carlsbad, CA, USA. The 3-(4-5-dimethylthiazol- (J&W Scientific, Folsom, CA, USA). One microliter of the 2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sample was injected into GC-MS using split mode (50 : 1). ∘ sulfoxide (DMSO), and etoposide were purchased from The injector temperature was 250 C. The column temperature ∘ Sigma,St.Louis,MO,USA.Caspaseassaykitsandapoptotic was programmed as follows: initial temperature at 160 Cfor ∘ ∘ ∘ ∘ ∘ DNA laddering kit were purchased from BioVision Research 3min,10 C/minto240 C, 240 Cfor2min,5 C/minto250 C, ∘ ∘ ∘ ∘ Products, Palo Alto, CA, USA. 250 Cfor10min,5C/minto300C, and 300 C for 10 min. Mass detector conditions were the following: electronic ∘ 2.3. Extraction and Fractionation. Each vegetal part was sep- impact (EI) mode at 70 eV; source temperature: 230 C; arated, dried, and pulverized. Dried powder of the separated scanning rate: 1 scan/s; mass acquisition range: 20–600 amu; plant material (100 g) was exhaustively extracted using a solvent delay, 4 min. Carrier gas was helium at 1 mL/min. ∘ Soxhlet apparatus at 60 C of temperature with methanol Volatile components were tentatively identified by comparing (500 mL). The supernatants were filtered and evaporated their mass spectra using NIST Standard Reference Database under vacuum by means of a rotaevaporator to obtain a dried Version NIST 05 for Windows. An authentic standard of extract. The methanol extract of each vegetal material (10 mg) bonediol compound was kindly provided by Dr. Peraza- was suspended in 20 mL methanol : water (1 : 3) and extracted Sanchez´ from CICY. successively using 50 mL of solvents of increasing polarity: hexane, dichloromethane, and ethyl acetate, such that the 2.7.Analysis of DNA Fragmentation. DNA fragmentation was final residue extract was an aqueous fraction. The finger- determined according to the method described by Tong et al. print of active hexane extract (5 mg) was obtained for gas [12]. Briefly, the cells were treated with the extract at 10 and chromatography-mass spectrometry (GC-MS). 50 𝜇g/mL and incubated for 6, 12, and 24 h. After incubation, the cells were harvested by centrifugation and washed twice 2.4. Cell Lines and Culture. Cell lines of the oropharyngeal in ice-cold PBS. An apoptotic DNA laddering kit (BioVision carcinoma (KB ATCC-CCL-17), laryngeal carcinoma (Hep- apoptotic DNA ladder extraction kit) was used to isolate 2), cervix adenocarcinoma (HeLa ATCC-CCL-2), and cervix DNA according to the manufacturer’s protocol; the DNA in squamous carcinoma (SiHa ATCC-CCL-35) as well as one the samples was separated on 1.5% agarose gel containing normal cell line, canine cell kidney (MDCK ATCC-CCL- 1 𝜇g/mL of ethidium bromide. DNA bands were visualized 34), from the American Type Culture Collection (ATCC) under ultraviolet illumination and were photographed. Evidence-Based Complementary and Alternative Medicine 3

Table1:Cytotoxicity(CC50)ofmethanolicextractsfromB. albiflora. Cell lines CC 𝜇g/mL (selective index) Extract 50 MDCK KB HeLa Hep-2 SiHa Leaves 91.39 23.85 (3.83) 47.05 (1.94) 35.20 (2.59) 47.45 (1.92) Stem bark 249.40 62.30 (4.00) NA 72.30 (3.45) NA Root bark 173.52 12.64 (13.72) 31.85 (5.44) 35.34 (4.91) 31.50 (5.50) Docetaxel 1.10 0.23 (4.78) 0.20 (5.50) 0.08 (13.75) 0.32 (3.43) NA: no activity > 200 𝜇g/mL.

2.8. Assays of Caspases Activities. Caspases 3, 8, and 9 presented superior cytotoxic effects compared to the original activities were performed using FLICE/Caspase Colorimetric extract, with a CC50 between 2 and 27 𝜇g/mL in the distinct assay kit, following the manufacturer’s protocols. Briefly, 5× cell lines (Table 2). The hexanic fraction’s SI also improved 6 10 cells treated with 10 or 50 𝜇g/mL extract for 6, 12, or compared to the original extract in the cell lines evaluated 24hwereharvested,washedwithPBS,andcentrifugedat (SI = 5–54). The methanolic fractions of the bark and leaves ∘ 800 ×gfor10minat4C. The cell pellets were resuspended extracts were not active at concentrations of >200 𝜇g/mL in 50 𝜇L lysis buffer and incubated on ice for 10 min before (data not shown). being centrifuged at 10,000 ×gfor1min.Thesupernatantwas Previously, we conducted a bioguided study to evaluate collectedina1.5mLtubeandkeptonice.Aftermeasuring the antiproliferative activity of B. macrocarpa, yielding the protein concentration, 200 𝜇g of protein was dissolved in isolation of the compound bonediol, which showed moderate 50 𝜇L cell lysis buffer. The reaction buffer with 10 mM DDT activity in cancer cell lines [8]. However, the present study did was added to each sample. Finally, a specific substrate for not show cytotoxic effects with HFBa comparable to the orig- each caspase (DEVD-𝜌NA, IETD-𝜌NA, and LEHD-𝜌NA) inal methanolic extract in the cell lines evaluated (Table 2). ∘ was added to the samples, incubated at 37 Cfor1h,andread An explanation to these results may be that bonediol inhibits at 405 nm. The enzyme activity was expressed as fold over some point of cellular proliferation (cycle cell or replication control sample. of DNA), while the effects that are observed in the cytotoxic assay are damage or general toxicity (apoptosis or necrosis) 3. Results and Discussion [14]. HFBa presented better cytotoxic effects compared to 3.1. Cytotoxic Activity of Methanolic Extracts. The cytotoxic- bonediolandwasmoreselectivetowardthetumorthan ity results of the methanolic extracts from different parts of B. toward normal cells; SI is considered an indicator of biologi- albiflora are summarized in Table 1.Therootbark’smethano- cal activity and is not related to cytotoxicity if the SI is >10 [15]. lic extract exhibited the most interesting cytotoxic activity In this regard, only HFBa satisfied these criteria and was more compared to extracts of B. albiflora leaves and stem bark, with potent in the KB cell line with a CC50 of 2.73 𝜇g/mL; this aCC50 of 12–31 𝜇g/mL on the four human cancer cell lines. cell line is related to oral cancer and is in agreement with the KB cell line showed a greater sensitivity to the extract with a plant’s use in the traditional Mayan medicine for chronic oral CC50 of 12.64 𝜇g/mL. The nontumor canine kidney cell line lesions [10], a term that could be related with cancer. MDCK was less sensitive to the effects of the extract with an SI of >5inthecelllinesevaluated(Table 1). The US National 3.3. GC-MS Analysis. Identification and chemical analysis of Cancer Institute (NCI) has proposed that crude extracts with bioactive hexane fraction by GC-MS is displayed in Table 3. potential cytotoxic activity are those presenting a CC50 of The chromatogram revealed a total of eight peaks, six of ≤30 𝜇g/mL; thus, this extract was identified as important for which were identified by the database: dodecanoic acid; future studies [13]. These data are similar to those obtained in tridecanoic acid; 2-nonyl-malonic acid, dimethyl ester; active B. macrocarpa-root methanolic extracts on human cell stigmasta-7,16-dien-3-ol; 9,19-Cyclo-lanost-24-en-3-ol; and lines: KB, prostate adenocarcinoma (PC3), cervix squamous bonediol. This last one was identified by retention time and carcinoma (SiHa), breast adenocarcinoma (MCF-7), cervix comparison of the mass spectrum of an authentic standard adenocarcinoma (HeLa), and laryngeal carcinoma (Hep-2) previously isolated from B. macrocarpa [8]. The major com- [6]. ponents found were the following: 2-nonyl-malonic acid, The extract of leaves was the second in greatest activity, dimethyl ester (37.39%), followed by stigmasta-7,16-dien-3-ol only on KB cell line with a CC50 of 23.85 𝜇g/mL according to (13.63%), dodecanoic acid (13.22%), 9,19-Cyclo-lanost-24- NCI criteria, followed by that of the stem bark’sextract, which en-3-ol (9.90%), and bonediol (8.98%). Unidentified com- was less cytotoxic to KB and Hep-2 cell lines. ponents with retention times of 8.092 (6.22%) and 14.207 (5.38%), as well as n-tridecanoic acid (5.25%), were minor 3.2. Cytotoxic Activity of Fractions. The methanolic extracts compounds in the HFBa (Figure 1). of different parts of the plant were fractionated with solvents Bonediol was isolated from the methanolic extract of of increasing polarity for later cytotoxicity studies in the cell B. macrocarpa roots as a bioactive component. In this lines. The hexanic fraction obtained from the liquid-liquid work, we detected the presence of this compound at a low partitioning of the methanolic extract of root bark (HFBa) concentration; thus, it could be referred to as a possible 4 Evidence-Based Complementary and Alternative Medicine

Table 2: Cytotoxicity of organic fractions from methanolic extract of B. albiflora root bark and bonediol.

Cell lines CC 𝜇g/mL (selective index) Extract 50 MDCK KB HeLa Hep-2 SiHa Hexane 148.48 2.73 (54.38) 14.29 (10.39) 15.48 (9.59) 27.02 (5.49) Dichloromethane NA NA NA NA NA Ethyl acetate NA NA NA NA NA Aqueous NA NA NA NA NA Bonediol 139.71 80.60 (1.73) 115.45 (1.21) 92.50 (1.51) 54.40 (2.56) Docetaxel 1.10 0.23 (4.78) 0.20 (5.50) 0.08 (13.75) 0.32 (3.43) NA: no activity > 200 𝜇g/mL.

Table 3: Chemical composition of hexane fraction of B. albiflora.

Peak Retention Peak relative 𝑚/𝑧 (relative abundance %) Component no. time (min) (%) 200 (10), 171 (10), 157 (30), 143 (10), 129 (40), 115 (20), 101 (15), 85 (30), 1 5.802 13.221 Dodecanoic acid 73 (100), 60 (85), 43 (70), 29 (40). 214 (10), 185 (10), 171 (35), 157 (5), 143 (5), 129 (40), 115 (25), 97 (15), 27.0125.256 Tridecanoic acid 85 (20), 73 (95), 60 (85), 43 (70), 29 (35). 208 (19), 166 (13), 152 (100), 137 (18), 121 (6), 107 (5), 91 (13), 77 (13), 3 8.091 6.222 Unidentified 55 (5), 41 (13), 28 (19). 259 (5), 156 (7), 145 (70), 132 (100), 113 (13), 100 (20), 87 (18), 69 (16), 2-Nonyl-malonic acid, dimethyl 4 9.826 37.396 55 (33), 41 (31), 29 (13). ester 350 (100), 209 (80), 195 (24), 179 (48), 164 (12), 151 (16), 136 (2), 5 14.1927 5.381 Unidentified 75 (8), 57 (8), 43 (20), 28 (26). 6 16.389 8.983 294 (70), 209 (13), 179 (10), 153 (100), 139 (5), 123 (20), 77 (9), 41 (20). Bonediol 412 (22), 369 (10), 341 (10), 300 (15), 271 (80), 246 (20), 207 (90), 737.54813.634 Stigmasta-7,16-dien-3-ol 173 (10), 147 (40), 107 (43), 81 (75), 55 (80), 43 (100), 28 (40). 426 (25), 411 (100), 393 (45), 259 (10), 215 (10), 187 (15), 173 (15), 8 39.632 9.907 9,19-Cyclo-lanost-24-en-3-ol 161 (15), 135 (25), 109 (40), 69 (90), 55 (40), 41 (45).

800000 In recent years, not only has the study of medically bioac-

700000 9.826 tive compounds from plants become more frequent, but also 600000 that of the plant extracts themselves or the mixture of compounds that together could yield better biological activity 500000 than that exhibited by a single compound has, moreover, 400000 37.548 become frequent [19]. In this work, the components are 300000 39.632 described as the fingerprinting of HFBa performed by GC- 5.802 Abundance

200000 16.389 MS for future standardizations. 14.192 8.091 100000 7.012 0 3.4. DNA Fragmentation. We observed that the methanol 313233343 extract of the roots of B. albiflora showed typical morphology Time (min) ofapoptosis(datanotshown)onKBcelllines.Similarly, HFBa was shown to induce apoptotic morphology on KB Figure 1: Gas chromatography of HFBa. cell lines. These results led us to evaluate whether the hexanic fraction that demonstrated the greatest cytotoxicity and apoptosis morphological characteristics in the KB chemotaxonomic marker. Additionally, in other species such cell line could induce this process; thus, we evaluated the as B. pungens,atriterpenehasbeenisolated[16], and from B. fragmentation of DNA, typical of the process of apoptosis. ruscifolia, two triterpenes have been isolated, without reports DNA fragmentation was registered from lesser to greater of biological activity [17, 18].Inthiswork,wehaveonlyfound magnitude within a treatment concentration range of 10 or evidence of the presence of a lanosterol-derived triterpene in 50 𝜇g/mLandanincubation-timerangeof6–24h.Figure 2 the active hexanic fraction. In addition, we detected a ubiq- shows typical DNA fragmentation in KB cells after treatment uitinated sterol, a derivative of stigmasterol. To our knowl- with 50 𝜇g/mL HFBa and an 18 h incubation period. Several edge,thisisthefirsttimethatbothcompoundshavebeen studies have shown the apoptotic effect of certain plant reported in this genus. methanolic extracts [20–24]. However, few studies have Evidence-Based Complementary and Alternative Medicine 5

1234 3.5 ∗ 3

2.5 ∗∗ +

1.5

Relative increase Relative 1 3000 0.5 2500 1500 0 1000 900 DMSO Control Etoposide HFBa 800 700 600 Figure 3: Treatment during six hours with Bonellia macrocarpa 500 400 hexane fraction induced caspase 8 activation. Treatments were the following: DMSO (0.05%), control (no treatment), etoposide 300 (50 𝜇g/mL), and HFBa (50 𝜇g/mL). Each symbol is the mean ± SD 200 relative caspase activation from three assays, normalized with the 100 control group. One-way ANOVA: 𝐹(3, 11) = 96.84, 𝑃 < 0.0001; ∗ Tukey’s post-hoc test: 𝑃 < 0.001 versus DMSO and control group; ∗∗𝑃 < 0.001 +𝑃 < 0.05 Figure 2: Effect of hexane root extract Bonellia macrocarpa on DNA versus DMSO and control group; versus fragmentation in KB cells. After the treatment of the cells with a HFBa. concentration of 50 𝜇g/mL of B. macrocarpa for 12 h, DNA was isolated and separated on 1.5% agarose gel. DNA was stained with 3.5 ∗∗ ethidium bromide and visualized under UV light. Lanes 1 to 4: lane 1 (negative control): DNA collected from untreated KB cells after 18 h; 3 lane 2 (positive control): DNA collected from KB cells treated with 50 𝜇g/mL of etoposide after 18 h; lane 3: DNA collected from KB cells 2.5 treatedwith50𝜇g/mL of extract after 18 h; lane 4: DNA molecular weight marker. 2 1.5

Relative increase Relative 1 investigated the chemical characteristics of the compounds 0.5 that may possess this activity. In those few studies, it was generally found that the low-polarity organic fractions are 0 responsible for the apoptotic effect on the cell lines, coincid- DMSO Control Etoposide HFBa ing with the results obtained in this study [25, 26]. It is unknown whether the compounds in HFBa are Figure 4: Treatment during 12 hours with Bonellia macrocarpa responsible for apoptosis induction, but it cannot be hexane fraction did not induce caspase 9 activation. Treatments were the following: DMSO (0.05%), control (no treatment), etoposide attributed to a single compound such as bonediol that, 𝜇 𝜇 ± although present in the extract, requires high concentrations (50 g/mL), and HFBa (50 g/mL). Each symbol is the mean SD relative caspase activation from three assays, normalized with the to induce apoptosis (data not shown), unlike HFBa, which control group. One-way ANOVA: 𝐹(3, 11) = 140.11, 𝑃 < 0.0001; 𝜇 ∗∗ induces DNA fragmentation at 10 g/mL. With regard to the Tukey’s post-hoc test: 𝑃 < 0.001 versus all groups. above, in addition to bonediol, we report the presence of dodecanoic acid and a derivative of stigmasterol as components of HFBa. These compounds have been associated with cytotoxic activity observed for the hexane fraction of Crocus from lanosterol, which have multiple activities against cancer sativus [27]. Furthermore, some authors have demonstrated cells including the induction of apoptosis [33, 34]. Possibly, that derivatives of stigmasterol showed significant cytotoxic the lanostane- and stigmasterol-type compounds reported in activity in cancer cell lines that depended on apoptosis [28– the active hexane fraction have a degree of cytotoxic activity 31]. In particular, spinasterol (stigmasta-7, 22-dien-3beta-ol) and an effect of inducing apoptosis. It is likely that several has demonstrated decreased incidence of skin tumors in vivo compounds present in the hexane fraction act synergistically [32]. In fact, spinasterol and the derivative reported in this to induce cytotoxicity and apoptosis. study differ in double enlace at position 22 in spinasterol and position 16 for the stigmasterol derivative. Perhaps, 3.5. Analysis of the Activity of Caspases. To know whether the stigmasta-7, 22-dien-3beta-ol could contribute to cytotoxic mechanism of activation of DNA fragmentation was induced activity observed in this study. In addition, it is known that by activation of apoptosis via the intrinsic or extrinsic path- lanostanes are a group of tetracyclic triterpenoids derived way, we evaluated the activity of caspases that is characteristic 6 Evidence-Based Complementary and Alternative Medicine

4.5 ∗∗ ∗ of the induction of apoptosis by activation of extrinsic signal- 4 ing mediated by natural and synthetic triterpenoids [46–48]. 3.5 Additionally, the lanostane-type triterpenoid, polyporenic acid C isolated from Poriacocos, induces caspase-8-mediated 3 apoptosis in human lung cancer [49]. Notably, 𝛽-sitosterol 2.5 (structural isomer of stigmasterol) has been shown to induce 2 apoptosis by activation of the extrinsic pathway, activating 1.5 Fas signaling in human breast cancer cells [50]. This might Relative increase Relative suggest that both the triterpene and sterol components of 1 the extract used in this study would have a major role in 0.5 the induction of apoptosis mediated by the activation of the 0 extrinsic pathway. DMSO Control Etoposide HFBa To our knowledge, this is the first time it has been demonstrated that the extract of a plant employed in the traditional Figure 5: Treatment during 12 hours with Bonellia macrocarpa Mayan medicine possesses an effect on apoptosis. Future hexane fraction induced caspase 3 activation. Treatments were studies will be directed toward standardizing the extract and the following: DMSO (0.05%), control (no treatment), etoposide evaluating the latter with in vivo models. Additional studies (50 𝜇g/mL), and HFBa (50 𝜇g/mL). Each symbol is the mean ± SD relative caspase activation from three assays, normalized with the are necessary for elucidating the compounds responsible for control group. One-way ANOVA: 𝐹(3, 11) = 363.2, 𝑃 < 0.0001; the observed cytotoxic activity and their exact mechanism of ∗∗ Tukey’s post-hoc test: 𝑃 < 0.001 versus DMSO and control group; apoptosis. ∗ 𝑃 < 0.001 versus DMSO and control group. 4. Conclusions The hexanic fraction of B. albiflora roots exerts cytotoxic of each. The incubation periods were of 2, 4, 6, and 12 h to effects and induces apoptosis via the extrinsic pathway, which obtain an activation profile. Caspase 8 was activated after 6h suggests its potential for the treatment of cancer. We suggest of treatment with 50 𝜇g/mL of HFBa; the increase was three the complete isolation of the components present in the times greater compared to control cells without treatment hexane fraction of B. albiflora for evaluation in the cytotoxic (negative control) (Figure 3). No increase was observed in the assay and induction of apoptosis, to elucidate which are the activation of caspase 8 in the 2, 4, and 12 h incubation periods. active compounds as well as to understand the mechanism of Caspase 9 was not activated in KB cells after treatment with action. 50 𝜇g/mL of HFBa during 2–12 h, which suggests a lack of apoptosis activation by the intrinsic pathway (Figure 4). Conflict of Interests Caspase 3 activity increased four folds compared to that of the control in HFBa-treated cells, which is in agreement with cas- The authors declare that they have no conflict of interests and pase 8 activation (Figure 5). The increase of caspase 8 activity no financial connection to any commercial entity mentioned is typical of extrinsic-pathway activation of apoptosis, which in the paper. in turn activates other procaspases, among these is caspase 3, which in turn leads to the degradation of nuclear proteins Acknowledgments such as laminin A, fodrin, actin, and gelsolin. It also leads to the release of the caspase-activated DNase protein inhibitor This paper was supported by SEP-CONACYTCB-2010- (ICAD),convertingitintothecaspase-activatedDNase 156755.TheauthorswouldliketothankL.Torres-Tapiafrom (CAD) enzyme, whose objective is DNA degradation [35], the Biotechnology Department of the Scientific Research as depicted in Figure 2. Given that caspase 9 was not acti- Center of Yucatan (CICY) for the technical assistance during vated, we conclude that B. albiflora induces apoptosis by the the GC-MS analysis. The authors are, moreover, grateful to extrinsic pathway. This evidences the great potential that this Dr. Glenn Jackson for the English language review of the fraction possesses as an alternative or adjunct therapy in the paper. treatment of cancer. In the literature are several studies of extracts from plants and their effects on the induction of the References intrinsic apoptosis pathway [36–42]. However, few studies [1] J. M. Spieler, “World Health Organization, the special pro- have shown the activation of the extrinsic apoptosis pathway gramme of research, development and research training in hu- by plant extracts; for example, the methanolic extract of man reproduction task force on indigenous plants for fertility Paeonia suffruticosa induces apoptosis in the human stomach regulation,” Korean Journal of Pharmacognosy,vol.12,pp.94– cancer cell line (AG3) [43], and dandelion root extract has the 97, 1981. capacity to induce apoptosis in the chronic myelomonocytic [2]R.M.Mendieta,Plantas Medicinales Del Estado De Yucatan´ , leukemia cell line (CMML) and in drug-resistant melanoma CECSA, Yucatan,´ Mexico,´ 1st edition, 1981. cells [44, 45]. It is not known which of the compounds present [3] G. Balam, Cosmogonia Y Uso Actual De Las Plantas Medicinales in the active fraction of B. albiflora may cause activation of the De Yucatan´ , Universidad Autonoma´ de Yucatan,´ Yucatan,´ extrinsic pathway of apoptosis. Regardlessly, there are reports Mexico,´ 1992. Evidence-Based Complementary and Alternative Medicine 7

[4] G. M. Cragg and D. J. Newman, “Plants as a source of anti- [21] J.-W. Chon, J.-H. Sung, E.-J. Hwang, and Y.-K. Park, “Chlorella cancer agents,” Journal of Ethnopharmacology,vol.100,no.1-2, methanol extract reduces lipid accumulation in and increases pp.72–79,2005. the number of apoptotic 3T3-L1 cells,” Annals of the New York [5] R. Osadao, El Libro del Jud´ıo o Medicina Domestica,´ Descripcion´ Academy of Sciences, vol. 1171, pp. 183–189, 2009. de las Virtudes de las Yerbas Medicinales de Yucatan´ ,Merida,´ [22] K.-J. Jo, M.-R. Cha, M.-R. Lee, M.-Y. Yoon, and H.-R. Park, Yucatan Mexico, 1834. “Methanolic extracts of Uncaria rhynchophylla induce cytotox- [6] E. Caamal-Fuentes, L. W. Torres-Tapia, P. Sima-Polanco,´ S. R. icity and apoptosis in HT-29 human colon carcinoma cells,” Peraza-Sanchez,´ and R. Moo-Puc, “Screening of plants used Plant Foods for Human Nutrition,vol.63,no.2,pp.77–82,2008. in Mayan traditional medicine to treat cancer-like symptoms,” [23] S. Nadova, E. Miadokova, L. Alfoldiova et al., “Potential antiox- Journal of Ethnopharmacology,vol.135,no.3,pp.719–724,2011. idant activity, cytotoxic and apoptosis-inducing effects of Cheli- [7] A. Sanchez-Medina,´ L. M. Pena-Rodr˜ ´ıguez, F. May-Pat et al., donium majus L. extract on leukemia cells,” Neuroendocrinology “Identification of sakurasosaponin as a cytotoxic principle from Letters,vol.29,no.5,pp.649–652,2008. Jacquinia flammea,” Natural Product Communications,vol.5, [24]T.N.Hasan,G.Shafi,N.A.Syedetal.,“Methanolicextractof no. 3, pp. 365–368, 2010. Nigella sativa seed inhibits SiHa human cervical cancer cell pro- [8] E. Caamal-Fuentes, L. W. Torres-Tapia, R. Cedillo-Rivera, R. liferation through apoptosis,” Natural Product Communications, Moo-Puc, and S. R. Peraza-Sanchez,´ “Bonediol, a new alkyl vol. 8, no. 2, pp. 213–216, 2013. catechol from Bonellia macrocarpa,” Phytochemistry Letters,vol. [25] C. P. Preethy, R. Padmapriya, V. S. Periasamy et al., “Antipro- 4, no. 3, pp. 345–347, 2011. liferative property of n-hexane and chloroform extracts of [9] B. Stahl˚ and M. Kallersj¨ o,¨ “Reinstatement of Bonellia (Theo- Anisomelesmalabarica (L). R. Br. in HPV16-positive human phrastaceae),” Novon,vol.14,no.1,pp.115–118,2004. cervical cancer cells,” Journal of Pharmacology and Pharma- [10] R. Arellano, G. Flores, and G. Tun, “Leguminosae,” in Nomen- cotherapeutics,vol.3,no.1,pp.26–34,2012. clatura, Forma de Vida, Uso, Manejo y Distribucion´ de las [26] A. Chicca, B. Adinolfi, E. Martinotti et al., “Cytotoxic effects Especies Vegetales de la Pen´ınsula de Yucatan´ ,pp.312–380, of Echinacea root hexanic extracts on human cancer cell lines,” Universidad Autonoma´ de Yucatan,´ Yucatan,´ Mexico,´ 2003. Journal of Ethnopharmacology,vol.110,no.1,pp.148–153,2007. [11] F. Denizot and R. Lang, “Rapid colorimetric assay for cell [27] C.-J. Zheng, L. Li, W.-H. Ma, T. Han, and L.-P. Qin, “Chemical growth and survival - Modifications to the tetrazolium dye constituents and bioactivities of the liposoluble fraction from procedure giving improved sensitivity and reliability,” Journal different medicinal parts of Crocus sativus,” Pharmaceutical of Immunological Methods,vol.89,no.2,pp.271–277,1986. Biology,vol.49,no.7,pp.756–763,2011. [12] X. Tong, S. Lin, M. Fujii, and D.-X. Hou, “Echinocystic acid [28] Y. Ahmed, M. H. Sohrab, S. M. Al-Reza, F. S. Tareq, C. M. induces apoptosis in HL-60 cells through mitochondria- Hasan, and M. A. Sattar, “Antimicrobial and cytotoxic con- mediated death pathway,” Cancer Letters,vol.212,no.1,pp.21– stituents from leaves of Sapium baccatum,” Food and Chemical 32, 2004. Toxicology,vol.48,no.2,pp.549–552,2010. [13]R.I.Geran,N.H.Greenberg,M.M.Macdonald,A.M. [29]R.Chowdhury,R.B.Rashid,M.H.Sohrab,andC.M.Hasan, Schumacher, and B. J. Abbott, “Protocols for screening chemical “12𝛼-hydroxystigmast-4-en-3-one: a new bioactive steroid from agents and natural products against animal tumors and other Toona ciliata (Meliaceae),” Pharmazie,vol.58,no.4,pp.272– biological systems,” Cancer Chemotherapy Reports,vol.3,pp. 273, 2003. 59–61, 1972. [30] K. H. Kim, S. U. Choi, C. S. Kim, and K. R. Lee, “Cytotoxic [14] R. Moo-Puc, D. Robledo, and Y. Freile-Pelegr´ın, “In vitro steroids from the trunk of Berberis koreana,” Bioscience, Biotech- cytotoxic and antiproliferative activities of marine macroalgae nology and Biochemistry,vol.76,no.4,pp.825–827,2012. from Yucatan,´ Mexico,” Ciencias Marinas,vol.35,no.4,pp.345– 358, 2009. [31] A. B. Awad and C. S. Fink, “Phytosterols as anticancer dietary [15] C. Vonthron-Sen´ echeau,´ B. Weniger, M. Ouattara et al., “In vitro components: evidence and mechanism of action,” Journal of antiplasmodial activity and cytotoxicity of ethnobotanically Nutrition,vol.130,no.9,pp.2127–2130,2000. selected Ivorian plants,” Journal of Ethnopharmacology,vol.87, [32] I. M. Villasenor and A. P.Domingo, “Anticarcinogenicity poten- no. 2-3, pp. 221–225, 2003. tial of spinasterol isolated from squash flowers,” Teratogenesis, [16] A. L. Okunade and D. F. Wiemer, “Jacquinonic acid, an ant- Carcinogenesis, and Mutagenesis,vol.20,pp.99–105,2000. repellent triterpenoid from Jacquinia pungens,” Phytochemistry, [33] J. L. R´ıos, I. Andujar,´ M. C. Recio, and R. M. Giner, “Lanos- vol. 24, no. 6, pp. 1203–1205, 1985. tanoids from fungi: a group of potential anticancer com- [17] K. Bhattacharyya, T. Kar, P. K. Dutta, G. Bocelli, and L. pounds,” Journal of Natural Products,vol.75,no.11,pp.2016– Righi, “Dimethyl oleana-13(18),15(16)-diene-3𝛽,28-diacetate,” 2044, 2012. Acta Crystallographica Section C,vol.55,no.6,pp.992–994, [34]L.Zhou,Y.Zhang,L.A.Gapter,H.Ling,R.Agarwal,andK.- 1999. Y. Ng, “Cytotoxic and anti-oxidant activities of lanostane-type [18] K. Bhattacharyya, T. Kar, P. Kumar, B. Achari, and G. Bocelli, triterpenes isolated from Poriacocos,” Chemical and Pharmaceu- “Oleana-12(13), 15(16)-diene-3𝛼, 28 diyl diacetate,” Acta Cristal- tical Bulletin, vol. 56, no. 10, pp. 1459–1462, 2008. lographica,vol.56,no.2,pp.60–61,2000. [35] J.F.R.Kerr,C.M.Winterford,andB.V.Harmon,“Apoptosis:its [19] H. J. Lee, H. Y. Kang, C. H. Kim et al., “Effect of new rotenoid significance in cancer and cancer therapy,” Cancer,vol.73,no. glycoside from the fruits of Amorpha fruticosa LINNE on the 8, pp. 2013–2026, 1994. growth of human immune cells,” Cytotechnology,vol.52,no.3, [36] R. R. Somasagara, M. Hegde, K. K. Chiruvella, A. Musini, B. pp.219–226,2006. Choudhary, and S. C. Raghavan, “Extracts of strawberry fruits [20] P. Bontempo, D. Rigano, A. Doto et al., “Genista sessilifolia DC induce intrinsic pathway of apoptosis in breast cancer cells and extracts induce apoptosis across a range of cancer cell lines,” Cell inhibits tumor progression in mice,” PLoS ONE,vol.7,no.10, Proliferation,vol.46,no.2,pp.183–192,2013. Article ID e47021, 2012. 8 Evidence-Based Complementary and Alternative Medicine

[37] J.-Q. Yu, Y. Yin, J.-C. Lei et al., “Activation of apoptosis by ethyl acetate fraction of ethanol extract of Dianthus superbus in HepG2 cell line,” Cancer Epidemiology,vol.36,no.1,pp.e40– e45, 2012. [38] A. C. Tan, I. Konczak, I. Ramzan, and D. M.-Y. Sze, “Native Australian fruit polyphenols inhibit cell viability and induce apoptosis in human cancer cell lines,” Nutrition and Cancer,vol. 63,no.3,pp.444–455,2011. [39] H. C. Pal, I. Sehar, S. Bhushan, B. D. Gupta, and A. K. Saxena, “Activation of caspases and poly (ADP-ribose) polymerase cleavage to induce apoptosis in leukemia HL-60 cells by Inula racemosa,” Toxicology in Vitro,vol.24,no.6,pp.1599–1609,2010. [40] Y.-C. Tien, J.-Y. Lin, C.-H. Lai et al., “Carthamus tinctorius L. prevents LPS-induced TNF𝛼 signaling activation and cell apoptosis through JNK1/2-NF𝜅B pathway inhibition in H9c2 cardiomyoblast cells,” Journal of Ethnopharmacology,vol.130, no. 3, pp. 505–513, 2010. [41] M. E. Juan, U. Wenzel, V. Ruiz-Gutierrez, H. Daniel, and J. M. Planas, “Olive fruit extracts inhibit proliferation and induce apoptosis in HT-29 human colon cancer cells,” Journal of Nutrition,vol.136,no.10,pp.2553–2557,2006. [42] M.-J. Liu, Z. Wang, H.-X. Li, R.-C. Wu, Y.-Z. Liu, and Q.-Y. Wu, “Mitochondrialdysfunctionasanearlyeventintheprocessof apoptosis induced by woodfordin I in human leukemia K562 cells,” Toxicology and Applied Pharmacology,vol.194,no.2,pp. 141–155, 2004. [43] H. S. Choi, H. S. Seo, J. H. Kim, J. Y. Um, Y. C. Shin, and S. G. Ko, “Ethanol extract of Paeonia suffruticosa Andrews (PSE) induced AGS human gastric cancer cell apoptosis via fas- dependent apoptosis and MDM2-p53 pathways,” Journal of Biomedical Science,vol.19,article82,2012. [44]S.Pandey,S.J.Chatterjee,P.Ovadje,M.Mousa,andC.Hamm, “The efficacy of dandelion root extract in inducing apoptosis in drug-resistant human melanoma cells,” Evidence-Based Complementary and Alternative Medicine, vol. 2011, Article ID 129045,11pages,2011. [45] P. Ovadje, C. Hamm, and S. Pandey, “Efficient induction of extrinsic cell death by dandelion root extract in human chronic Myelomonocytic Leukemia (CMML) cells,” PLoS ONE,vol.7, no. 2, Article ID e30604, 2012. [46] W.-S. Suh, Y. S. Kim, A. D. Schimmer et al., “Synthetic triterpenoids activate a pathway for apoptosis in AML cells involving downregulation of FLIP and sensitization to TRAIL,” Leukemia, vol. 17, no. 11, pp. 2122–2129, 2003. [47] K. H. Kim, H. S. Seo, H. S. Choi, I. H. Choi, Y. C. Shin, and S.-G. Ko, “Induction of apoptotic cell death by ursolic acid through mitochondrial death pathway and extrinsic death receptor pathway in MDA-MB-231 cells,” Archives of Pharmacal Research, vol. 34, no. 8, pp. 1363–1372, 2011. [48]Z.D.Nassar,A.F.A.Aisha,N.Idrisetal.,“Koetjapicacid,a natural triterpenoid, induces apoptosis in colon cancer cells,” Oncology Reports,vol.27,no.3,pp.727–733,2012. [49]H.Ling,L.Zhou,X.Jia,L.A.Gapter,R.Agarwal,andK.-Y.Ng, “Polyporenic acid C induces caspase-8-mediated apoptosis in human lung cancer A549 cells,” Molecular Carcinogenesis,vol. 48, no. 6, pp. 498–507, 2009. [50] A. B. Awad, M. Chinnam, C. S. Fink, and P. G. Bradford, “𝛽- Sitosterol activates Fas signaling in human breast cancer cells,” Phytomedicine,vol.14,no.11,pp.747–754,2007. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 164215, 7 pages http://dx.doi.org/10.1155/2013/164215

Research Article Biological Activities and Chemical Characterization of Cordia verbenacea DC. as Tool to Validate the Ethnobiological Usage

Edinardo Fagner Ferreira Matias,1,2,3 Erivânia Ferreira Alves,3 Beatriz Sousa Santos,3 Celestina Elba Sobral de Souza,4 João Victor de Alencar Ferreira,4 Anne Karyzia Lima Santos de Lavor,4 Fernando Gomes Figueredo,4 Luciene Ferreira de Lima,4 Francisco Antônio Vieira dos Santos,3 Flórido Sampaio Neves Peixoto,3 Aracélio Viana Colares,2,3,5 Aline Augusti Boligon,6 Rogério de Aquino Saraiva,6 Margareth Linde Athayde,6 João Batista Teixeira da Rocha,6 Irwin Rose Alencar Menezes,4 Henrique Douglas Melo Coutinho,7 and José Galberto Martins da Costa2,3,4

1 Universidade Estadual do Ceara-UECE-60740-000,´ Fortaleza, CE, Brazil 2 Rede Nordeste de Biotecnologia-RENORBIO-60740-000, Fortaleza, CE, Brazil 3 Faculdade Leao˜ Sampaio-CE-FALS-63180-000, Juazeiro do Norte, CE, Brazil 4 Universidade Regional do Cariri-URCA-63.100-000, Crato, CE, Brazil 5 Universidade Federal do Maranhao-UFMA-65085-580,˜ Sao˜ Lu´ıs, MA, Brazil 6 Universidade Federal de Santa Maria-UFSM-97105-900, Santa Maria, RS, Brazil 7 Laboratorio´ de Microbiologia e Biologia Molecular, Departamento de Qu´ımica Biologica,´ Universidade Regional do Cariri-URCA, Crato-CE, Brasil. Rua Cel. Antonio Luis 1161, Pimenta 63105-000, Brazil

Correspondence should be addressed to Henrique Douglas Melo Coutinho; [email protected]

Received 5 April 2013; Revised 1 May 2013; Accepted 4 May 2013

Academic Editor: Ulysses Paulino de Albuquerque

Copyright © 2013 Edinardo Fagner Ferreira Matias et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Knowledge of medicinal plants is often the only therapeutic resource of many communities and ethnic groups. “Erva-baleeira,” Cordia verbenacea DC., is one of the species of plants currently exploited for the purpose of producing a phytotherapeutic product extracted from its leaves. In Brazil, its major distribution is in the region of the Atlantic Forest and similar vegetation. The crude extract is utilized in popular cultures in the form of hydroalcoholic, decoctions, and infusions, mainly as antimicrobial, anti-inflammatory, and analgesic agents. The aim of the present study was to establish a chemical and comparative profile of the experimental antibacterial activity and resistance modifying activity with ethnopharmacological reports. Phytochemical prospecting and HPLC analysis of the extract and fractions were in agreement with the literature with regard to the presence of secondary metabolites (tannins and flavonoids). The extract and fraction tested did not show clinically relevant antibacterial activity, but a synergistic effect was observed when combined with antibiotic, potentiating the antibacterial effect of aminoglycosides. We conclude that tests of antibacterial activity and modulating the resistance presented in this work results confirm the ethnobotanical and ethnopharmacological information, serving as a parameter in the search for new alternatives for the treatment of diseases.

1. Introduction communities often depend on natural resources, including native plant species to fulfill or complement their therapeutic In many developing countries, various communities do not resources [2–4]. have sufficient resources to meet their needs with regard to Popular observations on the use of medicinal plants obtaining medicine to treat various diseases [1]. Thus, these contribute in a relevant way to spread awareness of the 2 Evidence-Based Complementary and Alternative Medicine therapeutic properties of plants often prescribed because of new drugs with antibacterial properties that are efficient in the medicinal effects they exhibit, despite that the chemical combating infections [27]. constituents of many are not known [5]. Therefore, the aim of this study was to justify, using In the last years, there has been great scientific interest in vitro experimental models, the utilization of the extract in chemicals and pharmacological investigations of the bio- and fraction obtained from the leaves of C. verbenacea logical properties of medicinal plants. Medicinal plants have DC. as an alternative therapeutic agent and source of new been the source of many medications that are now applied in isolated substances, correlating with information described clinical practice [6–10]. in ethnopharmaceutical studies. Brazil is the country with the greatest plant genetic diversity in the world, accounting for more than 55,000 cataloged species out of an estimated total of between 350,000 2. Materials and Methods and 550,000 species. Many of these species are endemic to a region and still have not been evaluated from a phytochemical 2.1. Plant Material. Leaves of Cordia verbenacea DC. were and pharmacological point of view [11]. collected in the municipality of Crato, Ceara,´ Brazil. The plant “Erva-baleeira,” Cordia verbenacea DC., is one of the material was identified and dried, and pressed specimens were deposited in the Herbario Prisco Bezerra of Universi- species of plants currently exploited in this sense, for the ∘ purpose of producing a phytotherapeutic product extracted dade Federal do Ceara(UFC),asN´ 044171. from the leaves. The genus Cordia belongs to the family Boraginaceae, which includes about 250 species, where the 2.2. Preparation of Methanolic Extracts and Fraction of Cordia majority have a bush or tree size. The species C. verbenacea verbenacea DC. For the preparation of the extracts, leaves DC. is native to Central and South America [12]. In Brazil, were collected which were kept submersed in methanol its greatest distribution is in the region of the Atlantic Forest separately for 72 h; afterward, the extract was filtered and and low areas of the Amazon [13]. The species can reach up concentrated using a rotary vacuum evaporator (model to three meters in height, but when grown as crops in this Q-344B-Quimis, Brazil) and ultrathermal bath (model Q- country, the plants are only one meter high [14]. 214M2-Quimis, Brazil). After obtaining the extract, vacuum The crude extract of the aerial parts of the herb (leaves fractionation was used to extract from fractions. We obtained and stems) is widely utilized in popular, medicine in the form 9,45 g of methanolic fraction of methanolic Extract of Cordia of hydroalcoholic extracts, decoctions, and infusions, mainly verbenacea DC. (MFMECV) from 44,33 g of methanolic as antimicrobial, anti-inflammatory, and analgesic agents. extract of Cordia verbenacea DC. (MECV). The solution Pharmacological studies have demonstrated that products utilized in the tests was prepared at a concentration of obtained from C. verbenacea have a pronounced anti- 10 mg/mL, dissolved in dimethyl sulfoxide (DMSO), and then inflammatory effect with topical and oral administration, diluted with distilled water to obtain a concentration of associated with low toxicity and an substantial protective 1024 𝜇g/mL, reducing the DMSO concentration lower than effect on the gastric mucosa of rodents [15]. 10%, to avoid the toxicity of DMSO. Staphylococcus aureus is distributed in nature, as well as being a part of the normal microbiota of the skin and 2.3. Phytochemical Prospecting. The phytochemical tests mucosa of animals, including birds. Some specimens of to detect the presence of heterosides, saponins, tannins, Staphylococcus are frequently recognized as etiological agents flavonoids, steroids, triterpenes, cumarins, quinones, organic of opportunistic infections in various animals and humans acids, and alkaloids were performed according to the method [16, 17]. Besides causing different types of poisoning, S. described by Matos [28]. The tests were based on the visual aureus is the most common etiological agent of purulent observation of a change in color or formation of precipitate infections (e.g., furuncles, carbuncles, abscess, myocarditis, after the addition of specific reagents, and the results for endocarditis, meningitis, pneumonia, and bacterial arthritis) the extract and fractions studied show presence of tannins [18]. Escherichia coli is one of the principal pathogens respon- Flobabens, flavonoids (flavones, flavonols, xanthones, chal- sibleforcausinginfectiousdiseasesinhumans.Thesebacteria cones, auron, Flavonones, leucoanthocyanidins, and cate- are known for producing enterotoxins, whose properties and chins), alkaloids, and terpenes. role in diarrheal disease have been widely investigated. The activity of cytotoxins and their role in human infections have The phytochemical tests were performed to detect the been identified [19–21], mainly in urinary tract infections presence of secondaries metabolics according to the method [22]. Pseudomonas aeruginosa is related to one of the main described by Matos [28](Table 1). The tests were based on causative agents of hospital infections, such as peritonitis, the visual observation of a change in color or formation of bacteremia, urinary tract infections, and surgical infections precipitate after the addition of specific reagents. in immunocompetent individuals [23]. Resistance to antibiotics is a growing and worrisome 2.4. Chemical, Apparatus, and General Procedures. All chem- problem in the treatment of many bacterial diseases [16, icals were of analytical grade. Methanol, acetic acid, gallic 24]. For patients with infections, antimicrobial resistance acid, chlorogenic acid, and caffeic acid, were purchased from increases morbidity and mortality, while there is a consider- Merck (Darmstadt, Germany). Quercetin and rutin were able increase in costs for the health institutions [25, 26]. In acquired from Sigma Chemical Co. (St. Louis, MO, USA). view of this situation, there is an increase in the need to obtain High performance liquid chromatography (HPLC-DAD) was Evidence-Based Complementary and Alternative Medicine 3

Table 1: Prospecting phytochemistry.

Extract fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 MECV −−+ −−+++++ + + + + − + MFMECV −−+ −−+++−−+ −−+ − + 1: phenols; 2: tannins pyrogallics; 3: tannins flobatenics; 4: anthocyanins; 5: anthocyanidins; 6: flavones; 7: flavonols; 8: xanthones; 9: chalcones; 10: auron; 11: flavonons; 12: leucoanthocyanidins; 13: catehins; 14: flavonones; 15: alkaloids; 16: terpenes; +: presence;−: absence; MECV: methanolic extract Cordia verbenacea; MFMECV: methanolic fraction methanolic extract Cordia verbenacea. performed with a Shimadzu Prominence Auto Sampler (SIL- (HIA, Difco). Prior to the assays, the cells were grown ∘ 20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with overnightat37C in Brain Heart Infusion (BHI, Difco). Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A 2.7. Drugs. Gentamicin, amikacin, and neomycin were diode array detector, and LC solution 1.22 SP1 software. obtained from Sigma Chemical Corp., St. Louis, MO, USA. All of the drugs were dissolved in sterile water before use. 2.5. Quantification of Compounds by HPLC-DAD. Reverse phase chromatographic analyses were carried out under 2.8. Antibacterial Test (MIC) and Modulation of Antibiotic × gradient conditions using C18 column (4.6 mm 150 mm) Activity. MIC (Minimal Inhibitory Concentration) was 𝜇 packed with 5 mdiameterparticles;themobilephasewas determined in a microdilution assay [30–32]utilizingan water containing 2% acetic acid (A) and methanol (B), and inoculum of 100 𝜇L of each strain, suspended in brain the composition gradient was 5% of B until 2min and heartinfusion(BHI)brothuptoafinalconcentrationof 5 changedtoobtain25%,40%,50%,60%,70%,and100% 10 CFU/mL in 96-well microtiter plates, using twofold serial B at 10, 20, 30, 40, 50, and 80 min, respectively, following dilutions. Each well received 100 𝜇L of each extract solution. the method described byLaghari et al. [29]withslight The final concentrations of the extracts varied 512–8 𝜇g/mL. modifications. The infusion of the leaves of Cordia verbenacea MICs were recorded as the lowest concentrations required to was analyzed at a concentration of 10 mg/mL. The presence inhibit growth. The minimal inhibitory concentration for the of five antioxidants compounds was investigated, namely, antibiotics was determined in BHI by the microdilution assay 5 gallic acid, chlorogenic acid, caffeic acid, quercetin, and utilizing suspensions of 10 CFU/mL and a drug concen- rutin. Identification of these compounds was performed by tration range of 2.500 to 2.4 𝜇g/mL (twofold serial dilutions) comparing their retention time and UV absorption spectrum [30–32]. MIC was defined as the lowest concentration with those of the commercial standards. The flow rate was at which no growth was observed. For the evaluation of 𝜇 0.8 mL/min, injection volume 40 L and the wavelength were the extracts as modulators of resistance to the antibiotics, 254 nm for gallic acid, 327 nm for caffeic and chlorogenic MIC of the antibiotics was determined in the presence or acids, and 365 nm for quercetin and rutin. The samples and absence of extract (MECV) and fraction (MFMECV) at 𝜇 mobile phase were filtered through 0.45 m membrane filter subinhibitory concentrations (128 𝜇g/mL), and the plates ∘ (Millipore) and then degassed by ultrasonic bath prior to were incubated for 24 h at 37 C. Each antibacterial assay for use. Stock solutions of standards references were prepared in MIC determination was carried out in triplicate [26, 30–32]. theHPLCmobilephaseataconcentrationrangeof0.020– 0.200 mg/mL for quercetin and rutin; 0.050–0.250 mg/mL for gallic, caffeic, and chlorogenic acids. The chromatography 2.9. Statistical Analysis of Microbiological Results. The results peaks were confirmed by comparing its retention time to of the tests were done in triplicate and expressed as geometric those of reference standards and by DAD spectra (200 to mean. Statistical analysis was applied to two-way ANOVA 500 nm). Calibration curve for gallic acid: 𝑌 = 12760𝑥 + followed by Bonferroni posttests using GraphPad Prism 5.0 1176.4 (𝑟 = 0.9997); chlorogenic acid: 𝑌 = 14158𝑥 + 1074.9 software. (𝑟 = 0.9995); caffeic acid: 𝑌 = 15734𝑥 + 1727.5 (𝑟 = 0.9999); rutin: 𝑌 = 13721 + 1268.4 (𝑟 = 0.9997); and quercetin: 3. Results and Discussion 𝑌 = 13795𝑥 + 1392.6 (𝑟 = 0.9991). All chromatography operations were carried out at ambient temperature and in The search for new drugs derived from natural products has triplicate. intensified in the last years33 [ ]. Harvey and collaborators [34] reported that drugs from 225 natural sources were in the 2.6. Strains. Experiments were performed with clinical iso- development phase, and of these, approximately 80% were lates of Escherichia coli (EC27), Staphylococcus aureus 358 extracted from plants. The search for medicines and genes (SA358), and Pseudomonas aeruginosa (PA03) resistant to from nature has been fostered as a nondestructive use of as well as to amikacin, neomycin, and gentamicin [26]. The habitats, which promote human health, as well as supporting EC-ATCC10536 strain of Escherichia coli,theSA-ATCC25923 economic development and conservation [35]. strain of Staphylococcus aureus, and the PA-ATCC15442 strain of Pseudomonas aeruginosa were used as positive 3.1. HPLC Analysis. HPLC fingerprinting of methanolic controls and were maintained on Heart Infusion Agar slants extract of leaves Cordia verbenacea revealed the presence of 4 Evidence-Based Complementary and Alternative Medicine

750 1000

750 2 500 3 500 (mAU) (mAU) 250 4 2 4 1 250 6 3 6 5 1 5 0 0

02550 02550 (min) (min) (a) (b)

Figure 1: (a) Representative high performance liquid chromatography profile of methanolic extract of leaves Cordia verbenacea. Gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), unidentified glycoside phenol (peak 4), rutin (peak 5), and quercetin (peak 6). Chromatographic conditions are described in Section 2. (b) Representative high performance liquid chromatography profile of methanolic fraction methanolic extract of leaves Cordia verbenacea. Gallic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), unidentified glycoside phenol (peak 4), rutin (peak 5), and quercetin (peak 6). Chromatographic conditions are described in Section 2.

the gallic acid (𝑡𝑅 = 13.27 min; peak 1), chlorogenic acid (𝑡𝑅 = Table 2: Phenolics and flavonoids composition of methanolic 20.54 min; peak 2), caffeic acid𝑡 ( 𝑅 = 28.02 min; peak 3), extract (MECV) and methanolic fraction of methanolic extract unidentified glycoside phenol (𝑡𝑅 = 39.91 min; peak 4), rutin (MFMECV) of leaves Cordia verbenacea. 𝑡 = 48.56 𝑡 = 60.15 ( 𝑅 min; peak 5), and quercetin ( 𝑅 min; peak Cordia verbenacea 6) (Figures 1(a) and 1(b) and Table 2). Compounds MeOH Phenolic compounds, including tannins and flavonoids, have demonstrated their therapeutic potential as anti- MECV% MFMECV% 1.14 ± 0.01 0.72 ± 0.05 inflammatory, antifungal, antimicrobial, antioxidant, and Gallic acid wound-healing agents [36]. Chlorogenic acid 1.59 ± 0.03 5.74 ± 0.02 Some investigators have reported synergism between Caffeic acid 3.85 ± 0.05 0.53 ± 0.02 ∗ flavonoids and conventional antibacterial agents against Glycoside phenol 1.43 ± 0.02 2.28 ± 0.01 resistant bacterial strains, and others have examined if the Rutin 0.38 ± 0.01 0.70 ± 0.03 activity of flavonoids is bacteriostatic or bactericidal37 [ ]. Quercetin 1.09 ± 0.06 0.77 ± 0.04 According to Cushnie and Lamb [37], the antibacterial Results are expressed as mean ± standard deviations (SD) of three determi- ∗ activity of flavonoids has been increasingly more docu- nations. quantified as caffeic acid. mented. Many researchers are a step further, where they have isolated and identified the structures of commer- cially available flavonoids, such as rutin, quercetin, 3-O- the geometric mean MIC, which are statistically significant methylquercetin, and various glycosides of quercetin [38– among themselves. The highest activity in relation to syn- 40]. ergistic action might be related to the higher concentration TheevaluationoftheantibacterialpotentialofMECVand of polar compounds in the fraction. However, analyzing MFMECV tested against standard and multiresistant strains the effect of P. ae r ug ino s a is not observed at the same of S. aureus, E. coli,andPseudomonas aeruginosa showed a level of modulating action, which could be related to the minimum inhibitory concentration of ≥1024 𝜇g/mL for all difference in chemical composition between this and MECV bacteria strains utilized, where the results were considered and MFMECV. clinically irrelevant. The use of extracts as antimicrobial agents presents alow Figures 2 and 3 showed the results of tests of the risk of increasing microbial resistance to its action, because modulation of bacterial resistance to aminoglycoside. MECV they are complex mixtures, providing greater difficulties for and MFMECV were found to potentiate the antibacterial microbialadaptability[41]. effect of the antibiotics tested against all the bacterial strains Despite the search for new substances from plant extracts used, except for MECV when combined with gentamicin and through their isolation and identification, some results tested against the strain EC27, where there was no statistically appear to be due to the combinationof compounds contained significant effect. in these complex mixtures, which characterize extracts. We The analysis of Figures 1 and 2 demonstrates that the S. see in some studies that when tested alone these substances aureus,E.coli, and MFMECV exhibit a better activity syner- demonstrated an antagonistic effect compared with the gistic combination (antibiotic natural product) compared to resultsofthisstudy,whichshowedtheirpresenceinthe Evidence-Based Complementary and Alternative Medicine 5

Methanolic extract Methanolic fraction methanolic extract 1250 1250 1125 1125 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 1000 1000 875 875 750 750 725 ∗∗∗ ∗∗∗ ∗∗∗ 725 ∗∗∗ ∗∗∗ ∗∗∗

g/mL) ∗∗∗ ∗∗∗ g/mL) ∗∗∗ ns ∗∗∗ ∗∗∗ 𝜇 𝜇 525 525 325 325 125 125 Value MIC ( MIC Value Value MIC ( MIC Value 40 40 30 30 20 20 10 10 0 0 S. aureas SA358 E. coli EC27 P. aeruginosa PA03 S. aureas SA358 E. coli EC27 P. aeruginosa PA03 Bactria Bactria

Amikacin Gentamicin + MECV Amikacin Gentamicin + MFMECV Amikacin + MECV Neomycin Amikacin + MFMECV Neomycin Gentamicin Neomycin + MECV Gentamicin Neomycin + MFMECV

Figure 2: Graph demonstrating the modulatory activity of bacterial Figure 3: Graph demonstrating the modulatory activity of bacterial resistance to aminoglycoside front of the methanolic extract of resistance to aminoglycoside front of the methanolic fraction of Cordia verbenacea (MECV). ∗∗∗value statistically significant with methanolic extract of Cordia verbenacea (MFMECV). ∗∗∗value 𝑃 < 0.0001. ns value statistically nonsignificant with 𝑃 > 0.05. statistically significant with 𝑃 < 0.0001. ns value statistically nonsignificant with 𝑃 > 0.05. extract and fraction by HPLC analysis, but they did not exert their effect when isolated42 [ ]. of popular knowledge in the development of new therapies Comparatively, natural products can differ and have obtained from phytotherapeutic products as utilized in pop- an antibacterial activity or resistance-modifying activity, ular medicine [50]. when considering the existence of differences in polarity and secondary metabolites, which are related to affinities 4. Conclusions for biological action [43, 44]. The mechanisms by which the extracts and fractions can interfere with the growth Our results indicate that the extract and fraction obtained of microorganisms are varied and can be due in part to from leaves of C. verbenacea do not possess antibacterial thechemicalnatureofsomecomponents.Asaresult,they activity that is clinically relevant, but when combined with an can demonstrate a greater interaction with the lipid bilayer antibiotic to evaluate their influence on bacterial resistance of the cell membrane, affecting the respiratory chain and to aminoglycosides, the extract and fraction demonstrated production of energy [45], or even make the cell more significant synergistic activity. The use and sale of products permeabletoantibiotics,leadingtotheinterruptionofvital derived from C. verbenacea may tend to exert pressure on cellular activity [46, 47]. These mechanisms of action can the populations of this species. Therefore, we recommend be due to the combination of antibiotic with extracts and the development of management plans for rational and fractions at a subinhibitory concentration added directly to sustainable use of the species, reducing the possible pressure the culture medium [9, 10]. on this species, and more studies with emphasis on the use of Thisstrategyiscalled“herbalshotgun”or“synergistic the extracts and fractions in the treatment of other diseases. multieffect targeting” and refers to the utilization of plants and drugs in an approach that utilizes combined mono- or Acknowledgments multiextracts, which can affect not only a single target but various targets, in which the different therapeutic compo- The authors acknowledge the support and cooperation nents act together in a synergistic or antagonistic way. This received from FUNCAP (Foundation for Research Sup- procedure is not only through the combinations of extracts, port Cearense), RENORBIO/UECE (Northeast Biotechnol- but also due to combinations between natural products or ogy Network/State University of Ceara),´ FALS (College extracts and synthetic products or antibiotics [48, 49]. Leao˜ Sampaio-CE), URCA/LPPN/LMBM/LFQM (Univer- The importance of the ethnic knowledge of traditional sity Regional Cariri-CE/Laboratory of Natural Products communities demonstrates that prior ethnobotanical and Research/Laboratory of Microbiology and Molecular Biol- ethnopharmacological information guide experimental stud- ogy/Laboratory of Pharmacology and Molecular Chemistry), ies in vitro and in vivo aimed at determining the applicability and UFSM (University Federal of Santa Maria-RS). 6 Evidence-Based Complementary and Alternative Medicine

References [16] A. Nostro, A. R. Blanco, M. A. Cannatelli et al., “Susceptibility of methicillin-resistant staphylococci to oregano essential oil, [1] Food and Agriculture Organization of the United Nations carvacrol and thymol,” FEMS Microbiology Letters,vol.230,no. (FAO), “The State of food insecurity in the world. Monitoring 2, pp. 191–195, 2004. the progress towards the world food summit and millennium [17]H.D.M.Coutinho,J.G.M.Costa,E.O.Lima,V.S. development goals,” Annual Report. Rome, Italy, 2004. Falcao-Silva,˜ and J. P. Siqueira, “Herbal therapy associated [2]B.M.Campbell,“Theimportanceofwildfruitsforpeasant with antibiotic therapy: potentiation of the antibiotic activity households in Zimbabwe,” Food and Nutrition,vol.12,no.1,pp. against methicillin—resistant Staphylococcus aureus by Turnera 38–44, 1986. ulmifolia L,” BMC Complementary and Alternative Medicine, [3] A. Zemede, “Indigenous African food crops and useful plants: vol. 9, article 13, 2009. survey of indigenous food crops, their preparations and home [18]J.Verhoef,D.Beaujean,H.Bloketal.,“ADutchapproachto gardens Nairobi,” The United Nation University Institute for methicillin-resistant Staphylococcus aureus,” European Journal Natural Resources in Africa,1997. of Clinical Microbiology and Infectious Diseases,vol.18,no.7,pp. [4] B. Becker, “Wild plants for human nutrition in the Sahelian 461–466, 1999. zone,” Journal of Arid Environments,vol.11,no.1,pp.61–64, [19] J.Konowalchuk,N.Dickie,S.Stavric,andJ.I.Speirs,“Properties 1986. of an Escherichia coli cytotoxin,” Infection and Immunity,vol.20, [5] N. M. Maciel, C. A. Schwartz, O. Rodrigues Pires et al., no. 2, pp. 575–577, 1978. “Composition of indolealkylamines of Bufo rubescens cutaneous secretions compared to six other Brazilian bufonids [20] J. Konowalchuk, J. I. Speirs, and S. Stavric, “Vero response to with phylogenetic implications,” Comparative Biochemistry and acytotoxinofEscherichia coli,” Infection and Immunity,vol.18, Physiology,vol.134,no.4,pp.641–649,2003. no. 3, pp. 775–779, 1977. [6] J. M. Barbosa-Filho, A. A. Alencar, X. P.Nunes et al., “Sources of [21] S. M. Scotland, N. P. Day, G. A. Willshaw, and B. Rowe, alpha-, beta-, gamma-, delta- and epsilon-carotenes: a twentieth “Cytotoxic enteropathogenic Escherichia coli,” Lancet,vol.1,no. century review,” Brazilian Journal of Pharmacognosy,vol.18,no. 8159,p.90,1980. 1,pp.135–154,2008. [22] C. Hughes, D. Mueller, J. Hacker, and W. Goebel, “Genetics and [7]J.M.Barbosa-Filho,F.A.DoNascimentoJunior,´ A. C. De pathogenic role of Escherichia coli haemolysin,” Toxicon,vol.20, Andrade Tomaz et al., “Natural products with antileprotic no. 1, pp. 247–252, 1982. activity,” Brazilian Journal of Pharmacognosy,vol.17,no.1,pp. [23]H.FerreiraandE.R.P.Lala,“Pseudomonas aeruginosa:um 141–148, 2007. alerta aos profissionais de saude,”´ Revista Panamericana de [8] M. W. Biavatti, V.Marensi, S. N. Leite, and A. Reis, “Ethnophar- Infectologia,vol.12,no.2,pp.44–50,2010. macognostic survey on botanical compendia for potential [24] N. H. Georgopapadakou, “Infectious disease 2001: drug resis- cosmeceutic species from Atlantic Forest,” Brazilian Journal of tance, new drugs,” Drug Resistance Updates,vol.5,no.5,pp.181– Pharmacognosy,vol.17,no.4,pp.640–653,2007. 191, 2002. [9]H.D.M.Coutinho,J.G.M.Costa,E.O.Lima,V.S.Falcao-˜ [25] S. J. Dancer, “The problem with cephalosporins,” Journal of Silva, and J. P.Siqueira, “In vitro interference of Hyptis martiusii Antimicrobial Chemotherapy,vol.48,no.4,pp.463–478,2001. Benth. & chlorpromazine against an aminoglycoside—resistant [26] H.D.M.Coutinho,L.N.Cordeiro,andK.P.Bringel,“Antibiotic Escherichia coli,” Indian Journal of Medical Research,vol.129,no. resitance of pathogenic bacteria isolated from the population of 5, pp. 566–568, 2009. Juazeiro do Norte-Ceara,”´ Revista Brasileira Cienciasˆ e Saude´ , [10]H.D.M.Coutinho,J.G.M.Costa,J.P.SiqueiraJr.,andE. vol.9,no.1,pp.127–138,2005. O. Lima, “In vitro anti-staphylococcal activity of Hyptis mar- [27] D. C. Michelin, P. E. Moreschi, A. C. Lima, G. G. F. Nasci- tiusii Benth against methicillin-resistant Staphylococcus aureus- mento, M. O. Paganelli, and M. V. Chaud, “Evaluation of the MRSA strains,” Brazilian Journal of Pharmacognosy,vol.18,pp. antimicrobial activity of vegetal extracts,” Revista Brasileira de 670–675, 2008. Farmacognosia,vol.15,no.4,pp.316–320,2005. [11] C. C. Simoes,˜ D. B. Araujo,andR.P.C.Ara´ ujo,´ “Estudo in vitro e ex vivo da açao˜ de diferentes concentraçoes˜ de extratos [28] F. J. A. Matos, Introduçao˜ aFitoqu` ´ımica Experimental, UFC, de propolis´ frente aos microrganismos presentes na saliva de Fortaleza, Brazil, 2nd edition, 1997. humanos,” Revista Brasileira de Farmacognosia,vol.18,no.1,pp. [29] A. H. Laghari, S. Memon, A. Nelofar, K. M. Khan, and A. 84–89, 2008. Yasmin, “Determination of free phenolic acids and antioxidant [12] I. C. E. Barroso, F. de Olivera, L. H. Z. Branco, E. T. M. activity of methanolic extracts obtained from fruits and leaves of Kato, and T. G. Dias, “O generoˆ Cordia L.: botanica,ˆ qu´ımica Chenopodium album,” Food Chemistry,vol.126,no.4,pp.1850– e farmacologia,” Revista Lecta,vol.20,no.1,pp.15–34,2002. 1855, 2011. [13] M. C. Bayeux, A. T. Fernandes, M. A. Foglio, and J. E. Carvalho, [30] M. M. Javadpour, M. M. Juban, W. C. J. Lo et al., “De “Evaluation of the antiedematogenic activity of artemetin iso- novo antimicrobial peptides with low mammalian cell toxicity,” lated from Cordia curassavica DC,” Brazilian Journal of Medical Journal of Medicinal Chemistry,vol.39,no.16,pp.3107–3113, and Biological Research,vol.35,no.10,pp.1229–1232,2002. 1996. [14] H. Lorenzi, H. M. Souza, M. A. Torres, and V. L. B. [31] National Comittee For Clinical Laboratory Standards (NCCLS), Bacher, “Arvores´ exoticas´ no Brasil: madeireiras, ornamentais “Methods for dilution antimicrobial susceptibility tests for bac- earomaticas,”´ Nova Odessa, Plantarum,p.384,2003. teria that grow aerobically,” 5th ed., NCCLS approved standard [15]J.A.A.Sertie,´ R. G. Woisky, G. Wiezel, and M. Rodrigues, M7-A5, Villanova, Pa, USA, 2000. “Pharmacological assay of Cordia verbenacea V: oral and topical [32] Clinical and Laboratory Standards Institute (CLSI), “Reference anti-inflammatory activity, analgesic effect and fetus toxicity of method for broth dilution antifungal susceptibility testing of a crude leaf extract,” Phytomedicine,vol.12,no.5,pp.338–344, filamentous fungi. Aproved standard M27-A2,” Clinical and 2005. Laboratory Standards Institute, Wayne, Pa, USA, 2002. Evidence-Based Complementary and Alternative Medicine 7

[33]D.X.Kong,X.J.Li,andH.Y.Zhang,“Whereisthehope [49] H. Wagner and G. Ulrich-Merzenich, “Synergy research: for drug discovery? Let history tell the future,” Drug Discovery approaching a new generation of phytopharmaceuticals,” Phy- Today,vol.14,no.3-4,pp.115–119,2009. tomedicine,vol.16,no.2-3,pp.97–110,2009. [34] A. L. Harvey, “Natural products in drug discovery,” Drug Dis- [50] V. Butterweck and A. Nahrstedt, “What is the best strategy for covery Today,vol.13,no.19-20,pp.894–901,2008. preclinical testing of botanicals? a critical perspective,” Planta [35]T.A.Kursar,C.C.Caballero-George,T.L.Capsonetal., Medica,vol.78,no.8,pp.747–754,2012. “Linking bioprospecting with sustainable development and conservation: the Panama case,” Biodiversity and Conservation, vol. 16, no. 10, pp. 2789–2800, 2007. [36] S. C. Santos and J. C. P. Mello, “Taninos,” in Farmacognosia: Da Planta ao Medicamento,C.M.O.Simoes,˜ E. P. Schenkel, G. Gosmann, J. C. P. Mello, L. A. Mentz, and P. R. Petrovick, Eds., pp.527–554,EditoradaUFRGS/EditoradaUFSC,PortoAlegre, Brazil, 2004. [37] T. P.T. Cushnie and A. J. Lamb, “Detection of galangin-induced cytoplasmic membrane damage in Staphylococcus aureus by measuring potassium loss,” Journal of Ethnopharmacology,vol. 101, no. 1–3, pp. 243–248, 2005. [38] J. P. Rauha, S. Remes, M. Heinonen et al., “Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds,” International Journal of Food Microbiology, vol.56,no.1,pp.3–12,2000. [39]A.Basile,S.Sorbo,S.Giordanoetal.,“Antibacterialand allelopathic activity of extract from Castanea sativa leaves,” Fitoterapia,vol.71,no.1,pp.S110–S116,2000. [40] H. Arima and G. I. Danno, “Isolation of antimicrobial compounds from guava (Psidium guajava L.) and their structural elucidation,” Bioscience,BiotechnologyandBiochemistry,vol.66, no. 8, pp. 1727–1730, 2002. [41]D.J.Daferera,B.N.Ziogas,andM.G.Polissiou,“The effectiveness of plant essential oils on the growth of Botrytis cinerea, Fusarium sp. and Clavibacter michiganensis subsp. michiganensis,” Crop Protection,vol.22,no.1,pp.39–44,2003. [42] H. N. H. Veras, I. J. M. Santos, A. C. B. Santos et al., “Comparative evaluation of antibiotic and antibiotic modifying activity of quercetin and isoquercetin in vitro,” Current Topics in Nutraceutical Research,vol.9,no.1-2,pp.25–30,2011. [43] E.F.F.Matias,K.K.A.Santos,T.S.Almeida,J.G.M.Costa,and H. D. M. Coutinho, “Atividade antibacteriana In vitro de Croton campestris A., Ocimum gratissimum L. e Cordia verbenacea DC,” Revista Brasileira de Biocienciasˆ ,vol.8,no.3,pp.294–298,2010. [44]E.F.F.Matias,K.K.A.Santos,T.S.Almeida,J.G.M.Costa, and H. D. M. Coutinho, “Enhancement of antibiotic activity by Cordia verbenacea DC,” Latin American Journal of Pharmacy, vol. 29, no. 6, pp. 1049–1052, 2010. [45] K. Nicolson, G. Evans, and P. W. O’Toole, “Potentiation of methicillin activity against methicillin-resistant Staphylococcus aureus by diterpenes,” FEMS Microbiology Letters,vol.179,no. 2, pp. 233–239, 1999. [46] S. Burt, “Essential oils: their antibacterial properties and potential applications in foods—a review,” International Journal of Food Microbiology,vol.94,no.3,pp.223–253,2004. [47] B. J. Juven, J. Kanner, F. Schved, and H. Weisslowicz, “Factors that interact with the antibacterial action of thyme essential oil and its active constituents,” JournalofAppliedBacteriology,vol. 76, no. 6, pp. 626–631, 1994. [48]H.D.M.Coutinho,J.G.M.Costa,E.O.Lima,andJ.P. Siqueira-Junior,´ “Additive effects of Hyptis martiusii Benth with aminoglycosides against Escherichia coli,” Indian Journal of Medical Research,vol.131,no.1,pp.106–108,2010. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 913671, 7 pages http://dx.doi.org/10.1155/2013/913671

Research Article Evaluations of the Antimicrobial Activities and Chemical Compositions of Body Fat from the Amphibians Leptodactylus macrosternum Miranda-Ribeiro (1926) and Leptodactylus vastus Adolf Lutz (1930) in Northeastern Brazil

Mario Eduardo Santos Cabral,1 Diógenes de Queiroz Dias,1 Débora Lima Sales,1 Olga Paiva Oliveira,1 Diego Alves Teles,1 João Antonio de Araujo Filho,1 José Guilherme Gonçalves de Sousa,1 Henrique Douglas Melo Coutinho,2 José Galberto Martins da Costa,3 Marta Regina Kerntopf,4 Rômulo Romeu da Nóbrega Alves,5 and Waltécio de Oliveira Almeida1

1 Laboratory of Zoology, Regional University of Cariri-URCA, Pimenta, 63105-000 Crato, CE, Brazil 2 Laboratory of Microbiology and Molecular Biology, Regional University of Cariri-URCA, Pimenta, 63105-000 Crato, CE, Brazil 3 Laboratory of Natural Products Research, Regional University of Cariri-URCA, Pimenta, 63105-000 Crato, CE, Brazil 4 Laboratory of Phamacology and Medicinal Chemistry, Regional University of Cariri-URCA, Pimenta, 63105-000 Crato, CE, Brazil 5 Department of Biology, Paraiba State University-UEPB, 58429-500 Joao˜ Pessoa, PB, Brazil

Correspondence should be addressed to Mario Eduardo Santos Cabral; [email protected]

Received 17 January 2013; Revised 18 March 2013; Accepted 4 April 2013

Academic Editor: Edwin L. Cooper

Copyright © 2013 Mario Eduardo Santos Cabral et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Leptodactylus macrosternum and L. vastus (family: Leptodactylidae) are commonly encountered in the “Caatinga” biome in northern Brazil. The body fat of L. vastus is used as a zootherapeutic for treating a number of human maladies. The aim of this work was to determine the chemical composition of the body fats of L. macrosternum and L. vastus and to evaluate their antimicrobial activities as well as the ecological implications of their use in traditional folk medicine. Oils were extracted from body fat located in the ventral region of L. macrosternum (OLM) and L. vastus (OLV) using hexane as a solvent. The fatty acids were identified by GC-MS. The antimicrobial activities of the oils, either alone or in combination with antibiotics and antifungal drugs, were tested on standard strains of microorganisms as well as on multiresistant strains of Escherichia coli and Staphylococcus. OLM contained 40% saturated and 60% unsaturated fatty acids, while OLV contained 58.33% saturated and 41.67% unsaturated fatty acids. Our results indicated that both OLM and OLV demonstrated relevant antimicrobial activities (with MIC 256 𝜇g/mL for both) against Pseudomonas aeruginosa and Candida krusei. However, no antimicrobial effects were observed when these oils were combined with antibiotics or antifungal drugs.

1. Introduction researchers have documented their open commercialization in public markets in that region [5–7]. Brazil is culturally diverse and has an extremely wide vari- Many natural products have been investigated as promis- ety of animal species that are used by humans in many ing sources of new drugs [8, 9] and increasing attention different ways [1–3]. Many animals and plants are used has been given to both vertebrate and invertebrate animals as ingredients of folk remedies in traditional medicinal as potential sources of these medicines [10]. These possible practices in northeastern Brazil [4], and ethnozoological products can represent secondary metabolites or proteins, 2 Evidence-Based Complementary and Alternative Medicine

(a) (b)

Figure 1: Species of frogs used in traditional medicine. (a) Leptodactylus macrosternum and (b) Leptodactylus vastus (Photos: (a), (b) Robson Waldemar Avila).´

as squalamine, magainins and others [11–13]. Hunt and [7, 21–23]. Ferreira et al. [23] noted that the body fat of L. Vincent [14] and Mayer and Gustafson [15]notedthatmany vastus is used to treat throat inflammations, coughs, asthma, faunal resources have been tested for extractable bioactive arthritis, and sore backs. compounds, and pharmaceutical companies have isolated While there are no available citations of the medicinal significant numbers of substances derived from animals in uses of L. macrosternum, its investigation in the present the search for new drugs [16]andmanyarenowused studyisjustifiedfollowingthechemotaxonomicmethod— to produce essential medicines [17]. Rashid et al. [18], for which takes into account phylogenetic relationships between example, isolated and purified a polysaccharide from the organisms and uses this information to initiate investigations sponge Celtodoryx girardae that demonstrated important of the pharmacological properties of related taxa [33]. antiviral activity against Herpes simplex; Stankevicins et al. The present study reports the identification of the chem- [19] evaluated the antimutagenic activity of extracts of the ical constituents of the fixed oils of L. macrosternum and L. sponge Arenosclera brasiliensis;andDeBarrosetal.[20] vastus and the evaluations of their antimicrobial activities isolated a type of heparin from the ascidia Styela plicata that (when administered individually or in combination with demonstrated significant anesthetic properties. antibiotics and antifungal drugs). Amphibians are generally only infrequently mentioned [4] among the vertebrates used in Brazilian folk medicine, although species of the genera Leptodactylus (Leptodactyli- 2. Materials and Methods dae) and Rhinella (Bufonidae) have received a fair number of citations [5–7, 17, 21–25]. 2.1. Collecting the Amphibian Specimens. Specimens of L. The family Leptodactylidae comprises four genera and macrosternum and L. vastus (Figure 1)werecollectedin ∘ 󸀠 ∘ 󸀠 ∘ 󸀠 100 species, and the genus Leptodactylus comprises 89 species the Aiuaba Biological Station (06 36 –06 44 Sand4007 – ∘ 󸀠 distributed throughout South America, principally in Brazil 40 19 W) in the Sertao˜ dos Inhamuns microregion, Ceara´ and the Antilles [26]. Leptodactylus macrosternum belongs State,Brazil.ThecollectionsaremadeinMay2011usingactive to the Leptodactylus ocellatus group(Linnaeus,1758),which collection techniques, as described by Auricchio and Salomao˜ is the smallest genus of the family (with only six species) [34]. The captured frogs were anesthetized with a combina- although there are various taxonomic problems associated tion of ketamine (60 mg/kg) and xylazine (6 mg/kg) [35]and with this taxon [27]. L. macrosternum is widely distributed subsequently sacrificed to remove their body fat. Testimonial throughout South America (east of the Andes Mountains) specimens were fixed with 10% formol and subsequently and occurs from Venezuela to Argentina, including Brazil deposited in the herpetological collection of the Universidade [28]; it is considered a generalist species that is well adapted Regional do Cariri/LZ-URCA (registry numbers LZ-1325 and to disturbed areas and its habitats vary from open, dry LZ-1309 for the species L. macrosternum and L. vastus, resp.). environments to humid tropical forests [29]. L. vastus is endemic to South America and is widely distributed throughout northeastern Brazil [30]whereitis 2.2. Extraction of the Fixed Oils of L. macrosternum (OLM) popularly known as “jia” or “Northeastern pepper frog.” and L. vastus (OLV). The fixed oils present in body fat It is a large animal that inhabits freshwater and terrestrial in the ventral regions of these frogs were extracted with ∘ environments [31] and deposits its foam egg masses in hexane (60 C) for 6 h in a Soxhlet apparatus. The hexane freshwater sources [32]. In spite of their ample distributions was subsequently decanted and filtered, and the solvent in northeastern Brazil, only a few ethnozoological surveys removed using a rotary evaporator under reduced pres- ∘ ∘ have mentioned the medicinal use of the body fat of L. vastus sure and controlled temperature conditions (70 C ±2C). Evidence-Based Complementary and Alternative Medicine 3

Collection of amphibians

Fixed oil + hexane Obtaining the body fat Distillation in route evaporator Crude oils Extraction in Soxhlet

Saponification and methylation

Biological Methyl esters of assays fatty acids

Analysis by CIM Modulation GG/MS

Results

OLM and OLV showed fatty acids saturated and unsaturated in varying proportions; the oils demonstrated significant antimicrobial activity at MIC against different microorganisms; no synergistic effect was observed when both oils were combined with antimicrobial drugs.

Figure 2: General diagram of the method of extraction of oils from L. macrosternum and L. vastus and results in biological assays.

Table 1: Data relating to obtaining fixed oils from the species indices as a preselection routine, and visual inspection of studied. the mass spectra from the literature for confirmation37 [ ], as Species (1) (2) (3) well as by visually comparing standard fragmentation to that reported in the literature [38, 39]. L. macrosternum (OLM) 11.56 4.10 36.55 L. vastus (OLV) 60.47 11.03 18.24 2.5. Microorganisms. Experiments were undertaken using (1) fat fresh weight (g); (2) Volume (mL) of the extracted oils; (3) Oil yields (%); OLM: fixed oil of L. macrosternum;OLV:fixedoilofL. vastus. clinical isolates of Escherichia coli (EC27) resistant to neomycin and gentamicin (low levels), tobramycin, amikacin, and kanamycin, Staphylococcus aureus 358 (SA358) resistant The quantities of fats and the oil volumes and yields are listed to various aminoglycosides, and Pseudomonas aeruginosa in Table 1. (PA22). E. coli ATCC 10536, S. aureus ATCC 25923, P. aeruginosa ATCC 15442, and Klebsiella pneumoniae ATCC 4362 were used as positive controls. To evaluate antifungal 2.3. Identification of the Fatty Acids. The fatty acids were activity, isolates of Candida albicans ICB 12 and C. krusei identified indirectly using their corresponding methyl esters. ATCClineage6258wereused.Allofthelineageswere The extracted oils (0.2 g) were saponified by refluxing for maintained in heart infusion agar slants (HIA, Difco). The 30 min. in a solution of potassium hydroxide and methanol, ∘ cells were cultivated during the night before the trials at 37 C following the methodology described by Hartman and Lago in a Brain Heart Infusion medium (BHI, Difco). [36]. The pHs of the extracts were adjusted, and the free fatty acids were subsequently methylated by acid catalysis to obtain their methyl esters. 2.6. Drugs. The antibiotics gentamicin, amikacin, and neo- mycinwereobtainedfromSigmaChemicalCorp.,St.Louis, 2.4. Gas Liquid Chromatography (GLC) Analysis. The anal- MO, USA. The antifungal drugs used were amphotericin B ysis of volatile constituents was carried out in a Hewlett- (Sigma Co., St. Louis, USA), Mebendazol (Lasa Pharmaceu- Packard GC/MS, model 5971, using the nonpolar fused silica tical Industries LTDA, Brazil), nystatin (Laboratorio´ Teuto column DB-1 (30 m × 0.25 mm i.d., 0.25 𝜇mfilm),elutedwith Brasileiro S/A, Brazil), and metronidazole (Prati, Donaduzzi helium gas at 8 mL/min with split mode. Injector and detector & Cia LTDA, Brazil). All of these compounds were dissolved ∘ ∘ temperatures were set to 250 Cand200C, respectively. The in sterile water before use. ∘ ∘ column temperature was programmed from 35 Cto180Cat ∘ ∘ ∘ ∘ 4 C/min and then from 180 Cto250Cat10C/min. Mass 2.7. Determination of the Minimum Inhibitory Concentrations spectra were recorded from 30 to 450 m/z,withanelectron (MICs) and Modulatory Activities. The MIC of the oils of beam energy of 70 eV. The individual components were L. macrosternum and L. vastus, antibiotics, and antifungal identified by computer MS library searches, using retention agents were determined in BHI by microdilution using 4 Evidence-Based Complementary and Alternative Medicine

Table 2: Methyl esters identified in the fixed oils in the body fat of Leptodactylus macrosternum (oil I) and Leptodactylus vastus (oil II), with their respective percentages.

Oil I Oil II Components RIa (%) RIa (%) Myristic acid methyl ester 1680 1.16 1680 1.42 Pentadecanoic acid methyl ester 1779 0.53 1779 1.74 Palmitoleic acid methyl ester 1878 31.52 1878 9.23 Palmitic acid methyl ester 1978 0.75 1886 20.01 Oleic acid methyl ester 2085 0.49 — — Linoleic acid methyl ester 2077 16.46 2093 17.02 Isoheptadecanoic acid methyl ester — — 1914 0.69 Heptadecanoic acid methyl ester — — 1978 0.66 Eicosanoic acid methyl ester — — 1986 0.93 Elaidic acid methyl ester — — 2085 27.87 Stearic acid methyl ester 2241 7.05 2241 10.01 5,8,11,14-eicosatetraenoic acid methyl ester 2499 0.65 2308 2.51 4,7,10,13,16,19-docosahexaenoic acid methyl ester — — 2523 0.27 (8E,11E,14E)-docosatrienoic acid methyl ester 2093 33.55 — — (E,E,Z)-1,3,12-nonadecadienoic acid methyl ester-5,14-diol 2308 6.82 — — Total identified 98.98 92.36 Saturated esters 40 58.33 Unsaturated esters 60 41.67

5 suspensions of 10 CFU/mL, with antibiotic concentrations Table 3: MIC values (𝜇g/mL) of the fixed oils of Leptodactylus varying from 2500 to 2.44 𝜇g/mL and antifungal drug con- macrosternum and Leptodactylus vastus applied to standard and centrations varying from 512 to 8 𝜇g/mL (double serial dilu- multiresistant microorganisms. tions) [40]. The MIC was defined as the lowest concentration MIC (𝜇g/mL) of a test compound that could inhibit bacterial growth. To Microorganisms evaluate the effects of the oils as modulators of antibiotic L. macrosternum L. vastus and antifungal activities, the MICs of the antibiotics at subin- OLM OLV hibitory concentrations were determined in the presence of E. coli ATCC 10532 ≥1024 ≥1024 the oils extracted from L. macrosternum and L. vastus (32 and S. aureus ATCC 25923 ≥1024 ≥1024 𝜇 64 g/mL, resp.), as were the MICs of the antifungal agents K. pneumonia ATCC 4362 ≥1024 ≥1024 (64 and 32 𝜇g/mL, resp.); the plates were incubated for 24 ∘ hours at 37 C(Figure 2). P. ae r ug inos a ATCC 15442 256 512 C. albicans ICB 12 ≥1024 ≥1024 C. krusei ATCC 6258 512 256 MIC: minimum inhibitory concentration; OLM: oil from L. macrosternum; 3. Results OLV: oil from L. vastus. The methyl esters of the fatty acids from the fixed oils of the two anuran species examined were analyzed using GC/MS. The in vitro antimicrobial tests indicated that both oils Ten chemical constituents were identified in the oil extracted had inhibitory effects on at least some of the microorganisms from L. macrosternum (representing fully 98.98% of the tested. The OLM demonstrated antibacterial activity against constituents) and 12 constituents (92.36%) in the oil extracted P. ae r ug ino s a ATCC 15442 (with an MIC of 256 𝜇g/mL). This from L. vastus (Table 2). MIC was similar to that observed when OLV was admin- The OLM contained 40% saturated fatty acids and 60% istered to C. krusei ATCC 6258 (Table 3). However, neither unsaturated fatty acids; the principal constituents were (8E, of the oils demonstrated antimicrobial activity at clinically 11E, 14E)-docosatrienoic acid methyl ester (33.55%), palmi- relevant concentrations against E. coli ATCC 10532, S. aureus toleic acid methyl ester (31.52%), linoleic acid methyl ester ATCC 25923, K. pneumoniae ATCC 4362, or C. albicans ICB (16.46%), and stearic acid methyl ester (7.05%).The OLV con- 12 (MIC ≥ 1024 𝜇g/mL). These results indicated that both oils tained 58.33% saturated fatty acids and 41.67% unsaturated were effective in inhibiting opportunist microorganisms and fatty acids; the principal constituents were elaidic acid methyl thattheycouldbeusedasalternativesourcesoftreatmentsfor ester (27.87%),palmitoleic acid methyl ester (20.01%), linoleic illnesses such as sore throats caused by bacterial infections. acid methyl (17.02), stearic acid methyl ester (10.01%), and Tests of the abilities of these oils to modify antibiotic palmitoleic acid methyl ester (9.23%). activities revealed the absence of any inhibitory activity Evidence-Based Complementary and Alternative Medicine 5

Table 4: Minimum inhibitory concentrations (𝜇g/mL) of the aminoglycosides alone and in association with the fixed oils of L. macrosternum and L. vastus. L. macrosternum Antibiotics MIC OLM (32 𝜇g/mL) MIC OLM (32 𝜇g/mL) MIC OLM (32 𝜇g/mL) SA 358 + antibiotic EC 27 + antibiotic PA 22 + antibiotic Amikacin 78.1 78.1 9.8 9.8 156.2 156.2 Neomycin 78.1 78.1 4.9 4.9 156.2 156.2 Gentamicin 9.8 9.8 2.4 2.4 39.1 39.1 L. vastus Antibiotics MIC OLV (64 𝜇g/mL) MIC OLV (64 𝜇g/mL) MIC OLV (64 𝜇g/mL) SA 358 + antibiotic EC 27 + antibiotic PA 22 + antibiotic Amikacin 39.1 39.1 9.8 9.8 156.2 156.2 Neomycin 9.8 78.1 2.4 2.4 39.1 156.2 Gentamicin 4.9 4.9 2.4 2.4 39.1 39.1

against any of the bacterial lineages when OLM was com- quantities of this oil. Zheng et al. [45] suggested that the bined with the aminoglycosides tested. Some antagonistic antibacterial activities of fatty acids (principally unsaturated effects were observed against S. aureus 358 and P. ae r ug ino s a varieties) may be due to their effects on bacterial synthesis of 22, however, when OLV was associated with neomycin endogenous fatty acids. (Table 4). Granowitz and Brown [46] reported antagonistic effects Neither oil demonstrated clinically relevant activity in from the combined use of antibiotics, which they attributed to modulating the effects of antifungal drugs, with MIC values mutual chelation. Similar antagonistic effects may be dimin- ≥1024 𝜇g/mL. These results indicated a lack of efficiency of L. ishing the activities of the aminoglycosides when combined macrosternum and L. vastus fat associated with antibiotics or with OLV in the present study. antifungal agents in treating illnesses caused by opportunist Relatively few studies have been undertaken to examine bacteria or fungi. the capacities of zootherapeutics to modify the actions of antibiotics or antifungal agents. Ferreira et al. [47]evaluated themodulatoryactivityoffatderivedfromTupinambis 4. Discussion merianae on aminoglycosides and determined that the body fat of this lizard did not increase their efficiency against The presence of large quantities of unsaturated fatty acids in the bacterial strains tested. Combinations of amikacin and both OLM and OLV was quite unexpected as these essential neomycin with OTM did not increase their effectiveness fatty acids are not synthesized by animals. The linoleic acid in against E. coli 27 or S. aureus 358, but this same oil demon- the oils extracted from both species may have been acquired strated antagonistic effects with kanamycin and gentamicin through their diets. The presence of a number of these fatty against these same bacteria. acidsinbothoftheoilsassayedherewassimilartothe The use of natural products in association with industri- results reported by Lopes et al. [41] for oils extracted from alized medicines has been well documented in the scientific the adipose tissue of the amphibian Rana catesbeiana SHAW literature. Calvet-Mir et al. [48]reportedtheuseoftraditional (including stearic, linoleic, myristic, palmitic, and palmitoleic folk medicines in association with western pharmaceuti- acids). The myristic acid content (1.8%) reported by these cals to treat diarrhea, vomiting, and stomachaches among same authors was essentially equal to that found in both of the individuals of the Tsimane ethnic group in the provinces oilsanalyzedinthepresentwork,althoughtheleveloflinoleic of Ballivian and Yacuma in Bolivia. Vandebroek et al. acid (25%) from R. catesbeiana was significantly greater than [49] reported that rural communities in Quechua, Bolivia, that found in both OLM (16.46%) and OLV (17.02%). used combinations of natural products and industrialized The fatty acids lauric, palmitic, linoleic, linolenic, stearic, medicines to treat illnesses of the respiratory and digestive myristic, and caprylic are known to have antibacterial and tracts—demonstrating that at least some communities utilize antifungal properties [42, 43]. Silva et al. [44] demonstrated natural and industrialized medicines simultaneously. the efficiency of oil extracted from Rana catesbeiana in The present report is the first investigation of the use of inhibiting different pathological organisms, and these authors natural products derived from L. macrosternum and L. vastus reported that this natural product was very active against all to modulate the effects of antibiotic and antifungal com- of the microorganisms tested (S. aureus, E. coli, P. ae r ug ino s a , pounds on standard and multiresistance microorganisms. C. albicans, C. tropicalis,andC. guilliermondii)—indicating Hunt and Vincent [14] warned that bioprospecting for that it is a promising antimicrobial agent, especially in light pharmaceuticals could result in the overexploitation of re- of the fact that it was relatively easy to obtain relatively large gional biodiversity, with strong direct and negative effects on 6 Evidence-Based Complementary and Alternative Medicine these living resources. As such, pharmacological testing of [5]R.R.N.Alves,I.L.Rosa,andG.G.Santana,“Theroleof products derived from animal species (whether threatened or animal-derived remedies as complementary medicine in not by extinction) will require proactive measures to guaran- Brazil,” BioScience,vol.57,no.11,pp.949–955,2007. tee their rational and sustainable use and the perpetuation of [6]R.R.N.Alves,H.N.Lima,M.C.Tavares,W.M.S.Souto,R. the species [50, 51]. R. D. Barboza, and A. Vasconcellos, “Animal-based remedies as OurresultsdemonstratedthatOLMandOLVareefficient complementary medicines in Santa Cruz do Capibaribe, Brazil,” antimicrobial agents—and it will therefore be necessary to BMC Complementary and Alternative Medicine,vol.8,article guarantee the rational harvesting of L. macrosternum and L. 44, 2008. vastus in order to avoid exerting excessive pressure on their [7] R.R.N.Alves,J.A.A.Barbosa,S.L.D.X.Santos,W.M.S.Souto, natural populations. The indiscriminate use of native species and R. R. D. Barboza, “Animal-based remedies as complemen- for medicinal purposes has been cited as one of probable tary medicines in the semi-arid region of northeastern Brazil,” Evidence-Based Complementary and Alternative Medicine,vol. causes of the population declines in a number of plant and 2011, Article ID 179876, 15 pages, 2011. animal species [1, 17, 52], even though there is no actual data [8] G. Appendino and L. Banfi, “Molecular diversity and natural availablethatcouldconfirmtheefficiencyorsafetyoftheuse products,” Molecular Diversity, vol. 15, no. 2, pp. 291–292, 2011. of products derived from those organisms. [9] A. Ganesan, “The impact of natural products upon modern drug discovery,” Current Opinion in Chemical Biology,vol.12, 5. Conclusions no.3,pp.306–317,2008. [10] E. Chivian, Biodiversity: Its Importance to Human Health Cen- The oils derived from the body fat of L. macrosternum ter for Health and the Global Environment, Harvard Medical and L. vastus demonstrated relevant antimicrobial activities School, Boston, Mass, USA, 2002. against P. ae r ug ino s a and C. krusei, respectively; they did [11] M. Zasloff, A. P. Adams, B. Beckerman et al., “Squalamine not, however, demonstrate clinically satisfactory inhibitory as a broad-spectrum systemic antiviral agent with therapeutic effects when combined with antibiotics or antifungal drugs. potential,” Proceedings of the National Academy of Sciences of the The use and sale of products derived from these amphibians United States of America, vol. 108, no. 38, pp. 15978–15983, 2011. could result in excessive harvesting pressure on natural wild [12] M. Zasloff, “Magainins, a class of antimicrobial peptides from populations. As such, we recommend (i) the elaboration of Xenopus skin: isolation, characterization of two active forms, proactive management plans for the rational and sustainable and partial cDNA sequence of a precursor,” Proceedings of the use of these species and (ii) undertaking additional studies National Academy of Sciences of the United States of America, on the usefulness of the body fat of L. macrosternum and L. vol.84,no.15,pp.5449–5453,1987. vastus in treating other human infirmities. [13]H.Coutinho,K.Lobo,D.Bezerra,andI.Lobo,“Peptides and proteins with antimicrobial activity,” Indian Journal of Pharmacology,vol.40,no.1,pp.3–9,2008. Acknowledgments [14] B. Hunt and A. C. J. Vincent, “Scale and sustainability of marine bioprospecting for pharmaceuticals,” AMBIO,vol.35,no.2,pp. The authors would like to thank the Coordenaçao˜ de 57–64, 2006. Aperfeiçoamento de Pessoal de N´ıvel Superior-CAPES for the studygrantsawardedtoMarioE.S.CabralandDiogenes´ Q. [15]A.M.S.MayerandK.R.Gustafson,“Marinepharmacology in 2005-2006: antitumour and cytotoxic compounds,” European Dias; the Fundaçao˜ Cearense de Apoio ao Desenvolvimento Journal of Cancer,vol.44,no.16,pp.2357–2387,2008. Cient´ıfico e Tecnologico-FUNCAP´ for the study grants [16] P. A. Cox, “Ethnopharmacology and the search for new drugs,” awarded to Debora´ L. Sales and Olga P. Oliveira, and for Ciba Foundation symposium,vol.154,pp.40–47,1990. supportprovidedtoWaltecio´ O. Almeida (process BPI-0112- [17] R. R. N. Alves and I. L. Rosa, “Zootherapy goes to town: the use 2.05/08); IBAMA for the collecting permits (SISBIO-IBAMA: of animal-based remedies in urban areas of NE and N Brazil,” 154/2007 no. 27542-2, process no. 96683918); and Robson W. Journal of Ethnopharmacology,vol.113,no.3,pp.541–555,2007. Avila for identifying the amphibians; CNPq for the grants to [18] Z. M. Rashid, E. Lahaye, D. Defer et al., “Isolation of a sulphated W.O. Almeida, R. R. N. Alves and J. G. M. da Costa. polysaccharide from a recently discovered sponge species (Celtodoryx girardae) and determination of its anti-herpetic References activity,” International Journal of Biological Macromolecules,vol. 44,no.3,pp.286–293,2009. [1] R. R. N. Alves and I. L. Rosa, “Why study the use of animal [19] L. Stankevicins, C. Aiub, L. C. D. S. Maria, G. Lobo-Hajdu, products in traditional medicines?” Journal of Ethnobiology and and I. Felzenszwalb, “Genotoxic and antigenotoxic evaluation Ethnomedicine,vol.1,article5,2005. of extracts from Arenosclera brasiliensis, a Brazilian marine [2] E. Rodrigues, “Plants and animals utilized as medicines in sponge,” Toxicology in Vitro,vol.22,no.8,pp.1869–1877,2008. the Jau´ National Park (JNP), Brazilian Amazon,” Phytotherapy [20] C. M. De Barros, L. R. Andrade, S. Allodi et al., “The hemo- Research,vol.20,no.5,pp.378–391,2006. lymph of the ascidian Styela plicata (Chordata-Tunicata)con- [3] R.R.N.Alves,W.M.S.Souto,andR.R.D.Barboza,“Primatesin tains heparin inside basophil-like cells and a unique sulfated traditional folk medicine: a world overview,” Mammal Review, galactoglucan in the plasma,” JournalofBiologicalChemistry, vol.40,no.2,pp.155–180,2010. vol. 282, no. 3, pp. 1615–1626, 2007. [4] R. R. N. Alves, “Fauna used in popular medicine in Northeast [21] R. R. N. Alves and H. N. Alves, “The faunal drugstore: animal- Brazil,” Journal of Ethnobiology and Ethnomedicine,vol.5,article based remedies used in traditional medicines in Latin America,” 1, 2009. Journal of Ethnobiology and Ethnomedicine,vol.7,article9,2011. Evidence-Based Complementary and Alternative Medicine 7

[22] J. A. A. Barbosa, V. A. Nobrega, and R. R. N. Alves, “Hunting [38] E. Stenhagen, S. Abrahamson, and F. W. Mclafferty, Registry of practices in the semiarid region of Brazil,” Indian Journal of Mass Spectra Data Base, Government Printing Office, Washing- Traditional Knowledge, vol. 10, no. 3, pp. 486–490, 2011. ton, DC, USA, 1974. [23]F.S.Ferreira,U.P.Albuquerque,H.D.M.Coutinhoetal.,“The [39] R. P. Adams, Identification of Essential Oil Components by trade in medicinal animals in northeastern Brazil,” Evidence- Gas Chromatography/Quadrupole Mass Spectroscopy,Allured Based Complementary and Alternative Medicine,vol.2012, Publishing Corporation, Carol Stream, Ill, USA, 2001. Article ID 126938, 20 pages, 2012. [40] M. M. Javadpour, M. M. Juban, W. C. J. Lo et al., “De [24]F.S.Ferreira,S.V.Brito,S.C.Ribeiro,W.O.Almeida, novo antimicrobial peptides with low mammalian cell toxicity,” and R. R. N. Alves, “Zootherapeutics utilized by residents Journal of Medicinal Chemistry,vol.39,no.16,pp.3107–3113, of the community Poço Dantas, Crato-CE, Brazil,” Journal of 1996. Ethnobiology and Ethnomedicine,vol.5,article21,2009. [41]V.S.Lopes,T.N.C.Dantas,A.F.Cunhaetal.,“Obtençao˜ de [25] W. M. S. Souto, J. S. Mourao,R.R.D.Barboza,andR.R.N.˜ um tensoativo anionicoˆ a partir de oleodeRanacatesbeiana´ Alves, “Parallels between zootherapeutic practices in ethnovet- Shaw,” Revista Universidade Rural. Serie´ Cienciasˆ Exatas e da erinary and human complementary medicine in northeastern Terra (UFRRJ),vol.30,no.2,pp.85–97,2010. Brazil,” Journal of Ethnopharmacology,vol.134,no.3,pp.753– [42] M. O. Nobre, P. S. Nascente, M. C. Meireles, and L. Ferreiro, 767, 2011. “Drogas antifungicas´ para pequenos e grandes animais,” Cienciaˆ [26] D. R. Frost, AmphibianSpeciesoftheWorld:AnOnlineRefer- Rural, Santa Maria,vol.32,no.1,pp.175–184,2002. ence. Version 5. 6, American Museum of Natural History, New [43] G. Agoramoorthy, M. Chandrasekaran, V. Venkatesalu, and York, NY, USA, 2013, http://research.amnh.org/herpetology/ M. J. Hsu, “Antibacterial and antifungal activities of fatty acid amphibia/index.html. methyl esters of the blind-your-eye mangrove from India,” [27] I. De La Riva and M. Maldonado, “First record of Leptodactylus Brazilian Journal of Microbiology,vol.38,no.4,pp.739–742, ocellatus (linnaeus, 1758) (Amphibia, Anura, Leptodactylidae) 2007. in Bolivia and comments on related species,” Graellsia,vol.55, [44] L. P. Silva, G. A. Joanitti, J. R. S. A. Leite, and R. B. Azevedo, pp.193–197,1999. “Comparative study of the antimicrobial activities and mam- [28] E. B. Andrade, T. B. Lima-Junior, J. M. A. Leite-Junior, and J. malian cytotoxicity of 10 fatty acid-rich oils and fats from animal R. S. A. Leite, “Predation by native fish and feeding by crab and vegetable,” The Natural Products Journal,vol.1,no.1,pp.40– species on Leptodactylus macrosternum Miranda-Ribeiro, 1926 46, 2011. (Anura: Leptodactylidae) in northeastern, Brazil,” Herpetology [45] C. J. Zheng, J. S. Yoo, T. G. Lee, H. Y. Cho, Y. H. Kim, and W. G. Notes,vol.5,pp.173–175,2012. Kim, “Fatty acid synthesis is a target for antibacterial activity of [29] R. Heyer, J. Langone, E. La Marca et al., Leptodactylus latrans. unsaturated fatty acids,” FEBS Letters,vol.579,no.23,pp.5157– In: IUCN, 2010. IUCN Red List of Threatened Species. Version 5162, 2005. 2012.2, 2010, www.iucnredlist.org . [46] E. V. Granowitz and R. B. Brown, “Antibiotic adverse reactions [30] W. R. Heyer, “Variation and taxonomic clarification of the and drug interactions,” Critical Care Clinics,vol.24,no.2,pp. large species of the Leptodactylus pentadactylus species group 421–442, 2008. (Amphibia: Leptodactylidae) from Middle America, northern [47]F.S.Ferreira,S.V.Brito,J.G.M.Costa,R.R.N.Alves,H.D.M. South America, and Amazonia,” Arquivos De Zoologia,vol.37, Coutinho, and W. D. O. Almeida, “Is the body fat of the lizard pp.269–348,2005. Tupinambis merianae effective against bacterial infections?” [31] R. Heyer, Leptodactylus vastus, IUCN, 2012. IUCN Red List of Journal of Ethnopharmacology,vol.126,no.2,pp.233–237,2009. Threatened Species. Version 2012.1, 2008, www.iucnredlist.org . [48] L. Calvet-Mir, V. Reyes-Garc´ıa, and S. Tanner, “Is there a divide [32] C. P. D. A. Prado, M. Uetanabaro, and C. F. B. Haddad, between local medicinal knowledge and Western medicine? A “Description of a new reproductive mode in Leptodactylus case study among native Amazonians in Bolivia,” Journal of (Anura, Leptodactylidae), with a review of the reproductive Ethnobiology and Ethnomedicine,vol.4,article18,2008. specialization toward terrestriality in the genus,” Copeia,no.4, [49] I. Vandebroek, E. Thomas, S. Sanca, P. Van Damme, L. V. Van, pp. 1128–1133, 2002. and N. De Kimpe, “Comparison of health conditions treated [33] A. Smelcerovic, V.Verma, M. Spiteller, S. M. Ahmad, S. C. Puri, with traditional and biomedical health care in a Quechua and G. N. Qazi, “Phytochemical analysis and genetic character- community in rural Bolivia,” Journal of Ethnobiology and ization of six Hypericum species from Serbia,” Phytochemistry, Ethnomedicine,vol.4,article1,2008. vol.67,no.2,pp.171–177,2006. [50] U. P. de Albuquerque, J. M. Monteiro, M. A. Ramos, and E. L. C. [34] P.Auricchio and M. G. Salomao,˜ Tecnicas´ de coleta e preparaçao˜ de Amorim, “Medicinal and magic plants from a public market de vertebrados para fins cient´ıficos e didaticos´ ,InstitutoPau in northeastern Brazil,” Journal of Ethnopharmacology,vol.110, Brasil de Historia´ Natural, Sao˜ Paulo, Brazil, 2002. no. 1, pp. 76–91, 2007. [35] F. A. B. Viana, Guia terapeuticoˆ veterinario´ ,Grafica´ e editora [51]R.R.N.Alves,N.A.L.Neto,S.E.Brooks,andU.P. CEM Ltda, Belo Horizonte, Brazil, 2003. Albuquerque, “Commercialization of animal-derived remedies [36] L. Hartman and R. C. A. Lago, “Rapid preparation of fatty acid as complementary medicine in the semi-arid region of North- methyl esters from lipids,” Laboratory Practice,vol.22,no.6,pp. eastern Brazil,” Journal of Ethnopharmacology,vol.124,no.3,pp. 475–477, 1973. 600–608, 2009. [37] J. W.Alencar, A. A. Craveiro, and F. J. A. Matos, “Kovats’ indices [52] R. R. N. Alves and I. L. Rosa, “Zootherapeutic practices as a preselection routine in mass spectra library searches of among fishing communities in North and Northeast Brazil: a volatiles,” JournalofNaturalProducts,vol.47,no.5,pp.890– comparison,” Journal of Ethnopharmacology, vol. 111, no. 1, pp. 892, 1984. 82–103, 2007. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 291592, 9 pages http://dx.doi.org/10.1155/2013/291592

Research Article Efficacy of Plectranthus amboinicus (Lour.) Spreng in a Murine Model of Methicillin-Resistant Staphylococcus aureus Skin Abscesses

Francisco Fábio Martins de Oliveira,1 Alba Fabiola Torres,1 Thially Braga Gonçalves,1 Gilvandete Maria Pinheiro Santiago,2 Cibele Barreto Mano de Carvalho,3 Milena Braga Aguiar,1 Lilia Maria Carneiro Camara,3 Silvia Helena Rabenhorst,3 Alice Maria Costa Martins,1 José Telmo Valença Junior,3 and Aparecida Tiemi Nagao-Dias1

1 Department of Clinical Analysis and Toxicology, Faculty of Pharmacy, Universidade Federal do Ceara(UFC),´ Rua Capitao˜ Francisco Pedro 1210, 60430-370 Fortaleza, CE, Brazil 2 Department of Pharmacy, Faculty of Pharmacy, UFC, Rua Capitao˜ Francisco Pedro 1210, 60430-370 Fortaleza, CE, Brazil 3 Department of Pathology and Legal Medicine, Faculty of Medicine, UFC, Rua Monsenhor Furtado S/N, 60430-350 Fortaleza, CE, Brazil

Correspondence should be addressed to Aparecida Tiemi Nagao-Dias; [email protected]

Received 17 September 2012; Accepted 17 January 2013

Academic Editor: Ulysses Paulino de Albuquerque

Copyright © 2013 Francisco Fabio´ Martins de Oliveira et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The present work aimed to evaluate the effectiveness of Plectranthus amboinicus (Lour.) Spreng against MRSA clinical isolates. The in vitro antimicrobial activity of the hydroalcoholic extract (HE), the ethyl acetate (EA) fraction and its subfractions were determined by broth microdilution and bioautography against MRSA clinical isolates. The microdilution checkerboard method was used to assess in vitro drug combination studies. To induce abscess formation, bacterial suspensions were added to Citodex and inoculated subcutaneously into male Swiss mice. The treatment protocol consisted of 2 doses of HE, the EA fraction or vancomycin introduced intraperitoneally into mice 3 and 12 h after infection. The EA fraction and its subfractions presented the lowest minimal inhibitory concentrations (MIC, 0.25 to 0.5 mg/mL). The plant samples were bacteriostatic at 2x and 4x MIC and bactericidal at 100 mg/mL. The EA fraction presented synergism with vancomycin and an additive effect with ciprofloxacin. A significant reduction ofabscess volume, bacterial cell counts in abscess slurries, and inflammatory scores was observed in the HE and EA fraction-treated groups. The samples were effective in treating the animals in a dose-dependent fashion. The present study proved the effectiveness of P. amboinicus fractions against MRSA using in vitro and in vivo assays.

1. Introduction in the community, thus leading to high morbimortality [3]. Recently, methicillin-resistant Staphylococcus aureus (MRSA) Methicillin resistance in staphylococcal infections is due has gained notoriety as an important human pathogen to the acquisition of the mecA gene, which encodes an altered worldwide. MRSA comprises more than 50% of nosocomial penicillin-binding protein (PBP2a). The 2-kb mecA gene is bacterial infections in some countries, including the United locatedonanelementofchromosomallyinsertedDNAthat States and Brazil [1]. The wide distribution of cases of MRSA is at least 32kb and may be greater than 60kb [4]. in Brazil demonstrates the need for new strategies to improve Several plants, including Curcuma longa [5], Ducrosia thecontrolandtreatmentofthesebacterialinfections,astheir anethifolia [6], Rhizophora mucronata [7], Acacia aroma [8], prevalence in health services has reached high levels, ranging Zataria multiflora [9], Ballota acetabulosa [10], and Tylosema from 54% to 93% [2]. MRSA can also affect individuals esculentum [11], possess antimicrobial activity against MRSA. 2 Evidence-Based Complementary and Alternative Medicine

Plectranthus amboinicus (Lour.) Spreng (Lamiaceae) is a Table 1: Partition of the ethanolic extract of Plectranthus amboinicus perennial plant distributed throughout tropical Africa, Asia, by using a column chromatography packed with silica gel 60 (70– Australia, and the Americas, including Brazil [12]. It is pop- 230 mesh). Elution procedure was done with solvents of increasing ularly known as “hortela-da-folha-grossa”˜ and “malvarisco.” polarities. The species is used in the treatment of several diseases, Eluent Volume (mL) Quantity (g) Fractions including skin, digestive, genitourinary, and respiratory dis- Hex 2000 2.3 F1 orders, infections, and pain. P. amb oini c u s and P. b arb atu s comprise approximately 68% of the traditional uses of the Ep 2000 1.2 F2 Plectranthus genus [12]. P. amb oini c u s has been a topic of DCM 5000 35.9 F3 intensive studies because it is rich in metabolites with poten- AcOEt 5000 29.6 F4 tial antimicrobial [13–15] and anti-inflammatory activities MeOH 2000 5.9 F5 [16–18]. The leaves of P. amb oini c u s secrete components such Hex: hexane; Ep: petroleum ether; DCM: dichloromethane; AcOEt: ethyl as essential oils, flavonoids, and terpenes, which exhibit good acetate; MeOH: methanol. antimicrobial activity [14, 15]. Few studies regarding the antimicrobial activity of P. Table 2: Partition of the ethyl acetate fraction F4 of Plectranthus amboinicus against S. aureus are available. Nogueira et al. [13] amboinicus by using a column chromatography packed with silica first demonstrated the inhibitory effect of the essential oil gel 60 (70–230 mesh). Elution procedure was done with different and hydroalcoholic extract of P. amb oini c u s on clinical strains ratios of hexane (Hex) and ethyl acetate (AcOEt). of methicillin-sensitive S. aureus (MSSA). Gurgel et al. [14] Eluent Ratio (%) Volume (mL) Quantity (g) Subfractions demonstrated the bacteriostatic and bactericidal effects of the hydroalcoholic extract of P. amb oini c u s against a standard Hex:AcOEt 90:10 2000 2.1 F4a laboratory strain of MRSA. Hex : AcOEt 80 : 20 2000 2.4 F4b We did not find any studies regarding the antimicrobial Hex:AcOEt 70:30 2000 3.2 F4c activity of P. amb oini c u s in animal models. For this reason, Hex:AcOEt 60:40 1000 1.5 F4d we proposed the present study to investigate the therapeutic Hex:AcOEt 50:50 1000 1.1 F4e effects of extracts from the leaves of P. amb oini c u s in MRSA- AcOEt 100 1000 0.8 F4f infected mice. MeOH 100 2000 17.3 F4g

2. Materials and Methods chromatography (22 × 6 cm) packed with silica gel 60 (70– 230 mesh), and elution was performed with different ratios 2.1. Plant Material. The leaves of P. amb oini c u s (Lour.) of hexane and ethyl acetate (Table 2). The subfractions (F4a Spreng were collected from the Horto de Plantas Medicinais to F4g) were tested using direct bioautography, and their Professor Francisco JosedeAbreuMatos(Fortaleza,Cear´ a,´ minimal inhibitory concentrations were determined. Brazil). A voucher specimen (EAC40008) was identified by Professor E. P. Nunes and deposited at the Herbario´ Prisco Bezerra (EAC), Universidade Federal do Ceara,´ Brazil. 2.3. Microbial Strains. The standard control strains used in these experiments were MSSA (ATCC 25923) and MRSA (ATCC 65398), obtained from the Instituto Nacional de 2.2. Preparation of the Extracts and Fractions. The hydroalco- Controle de Qualidade em Saude,´ Rio de Janeiro, Brazil. holic extract (HE) was obtained by macerating 3 kg of fresh Methicillin-resistant strains were isolated from 14 clinical leaves with 70% ethanol (3 litres) for 10 days. The material was specimens (2 from catheter tips, 10 from blood, and 2 from concentrated on a rotary evaporator under reduced pressure wound fluid from diabetic foot ulcers) that were biochem- ∘ at 40 C. ically identified by the Clinical Microbiology Department The ethanol extract was obtained by macerating 600 gof of the Hospital Universitario´ Walter Cantidio/UFC, Brazil. driedleavesinabsoluteethanolfor20days.Thesolventwas Screening for the bacterial resistance to antimicrobial drugs was performed using discs (Oxoid, UK) containing trimetho- changed every 5 days. The extract was filtered and evaporated 𝜇 𝜇 on a rotary evaporator under reduced pressure in a water prim/sulfamethoxazole (1.25/23.75 g), ciprofloxacin (5 g), ∘ gentamycin (10 𝜇g), ampicillin (30 𝜇g), chloramphenicol bath at 40 C. The ethanol extract (80 g) was fractionated bya 𝜇 𝜇 𝜇 liquid-liquid partition using a column chromatography (10 × (10 g), tetracycline (30 g), and erythromycin (15 g) [19]. 16cm)packedwithsilicagel60(70–230mesh).Theelution For vancomycin, bacterial resistance was evaluated using the procedurewasperformedwithsolventsofincreasingpolari- microbroth dilution assay. ties (i.e., hexane, petroleum ether, chloroform, methanol, and The molecular typing of MRSA was based on the detec- ethyl acetate). Solvents were completely evaporated under tion of the coagulase gene for species identification and on the ∘ reduced pressure at 40 C on a rotary evaporator. The fractions presence of the mecA gene confirming methicillin resistance, were identified as F1 to F5 (Table 1). according to the procedure described by Rallapalli et al. [20]. A bioassay-guided study of the fractions F1 to F5 was conducted. Because the ethyl acetate fraction F4 presented 2.4. Determination of the Minimal Inhibitory Concentrations the best inhibitory effect, it was submitted to a new column (MICs). To determine the MICs, microdilution tests [21] Evidence-Based Complementary and Alternative Medicine 3 were performed in 96-well microplates (Costar, USA). The 2.7. Bioautography. The bioautography technique23 [ ]was bacterial concentration of standard and clinical isolate strains employed to evaluate the antimicrobial activity of the sub- 6 (1–5 × 10 CFU/mL) was adjusted according to the turbidity of fractions F4a–d. Twenty milligrams of the plant samples were 0.5 McFarland scale. Each well contained 100 𝜇LMHIbroth, dissolved in 1 mL of methanol and applied in volumes of 10 𝜇L 5 𝜇Lofinoculum,and100𝜇L of hydroalcoholic, ethanolic to two glass plates coated with silica gel 60F254.Thesolvent extracts and fractions, and the F4 subfractions, in the range solution used for elution was hexane: ethyl acetate (6 : 4). of 0.062–16,000 𝜇g/mL in 5% DMSO. The plates were covered After the elution of the components and evaporation of the ∘ and incubated for 24 h at 35 C. solvents, a volume of 60 mL of warm overlay agar, previously 6 Vancomycin (Sigma-Aldrich, USA) was used as a positive seeded with a standard MRSA strain (10 CFU/mL), was control, in the range of 0.125–32 𝜇g/mL, and DMSO (Merck, poured onto the surface of one of the TLC plates. The Germany)wasusedasanegativecontrol,intherangeof TLC plate was then placed into a sterile Petri plate and ∘ ∘ 0.039–10% v/v. After the incubation period, 10 𝜇Lofsterile left for 2 h at 4 Candfor24hat35C. The TLC plate was 0.01% resazurin (Sigma, USA) in aqueous solution was added then stained with a solution of tetrazolium bromide dye to the plates. After 2 h of incubation, readings were per- MTT stain (Sigma-Aldrich, USA) at 5 mg/mL. Yellowish or formed. Resazurin is a blue dye used to visually determine the colourless halos indicated growth inhibition. In parallel, the absence (blue colour) or presence (pink colour) of bacterial other TLC plate was submitted to vanillin-perchloric acid growth. staining.

2.5. Bacteriostatic and Bactericidal Activities. Tubes contain- 2.8. Toxicity Test. For the in vitro cytotoxic activity assay, ing 4 mL of MHI broth were inoculated with 1 mL of the the macrophage cell line RAW 264.7, obtained from the MRSA standard strain (ATCC 65398) at a concentration 8 ∘ Rio de Janeiro Cell Bank (BCRJ, Brazil), was grown in of 10 CFU/mL and were incubated for 2 h at 35 C. A microculture plates containing Minimum Essential Media volume of 5 mL of each hydroalcoholic extract or acetyl medium supplemented with 10% v/v foetal bovine serum, acetatefractionF4at2xor4xMICand100mg/mLwas penicillin (100 U/mL), and streptomycin (100 𝜇g/mL). Cells added to the test tubes, or a volume of 4 mL of BHI ∘ were incubated at 37 Cin5%CO2 and 95% humidity. Before broth with 5% DMSO was added to the control tube. The ∘ each experiment, the cells were incubated in medium without tubes were incubated at 35 C, and aliquots of 30 𝜇Lwere foetalcalfserumfor24htoobtaincellsintheG0 phase of the collected at intervals of 0, 2, 4, 6, 8, and 24 h. Tenfold cell cycle. For each experiment, cells were removed from the serial dilutions of the suspensions were plated onto BHI agar culture medium and incubated with 0.25% trypsin and 0.02% to determine a viable cell count. The bactericidal (≥3-log- ∘ EDTA (v/v) for approximately 10 min at 37 C. Trypsin was unit reduction in log 10 CFU/mL) and bacteriostatic activities inactivatedbyaddingthesamevolumeofmediumcontaining (<3-log-unit reduction in log 10 CFU/mL) were determined foetal bovine serum. The suspension was centrifuged for [21]. 10 min at 1500 ×g. The supernatant was discarded, and the cells were resuspended in culture medium. The macrophages 2.6. Checkerboard Method. The microdilution checkerboard were quantified using a Neubauer chamber and subcultured 5 method is frequently used to assess in vitro antimicrobial (1 × 10 cells/mL) into a 96-well microplate for 24 h. The EH combination studies [22]. The hydroalcoholic extract and and ethyl acetate fraction F4 at final concentrations of 2x to the ethyl acetate fraction F4 were tested at 8x MIC. A 4x MICs were added to the microplates. After an incubation volume of 100 𝜇L of Mueller-Hinton broth was added to of 24 h, 100 𝜇L of the supernatant was discarded and 10 𝜇L each well of a 96-well microplate. Vancomycin (Sigma, USA) of MTT at 500 𝜇g/mL dissolved in phosphate-buffered saline or ciprofloxacin (Miles Inc., USA) was diluted along the (PBS), mL in phosphate buffered saline, pH 7.4, was added to ∘ horizontal axis, and the plant extracts were diluted along the wells after incubation for 4 h at 37 C, 10% sodium dodecyl the vertical axis. Five microlitres of the bacterial suspension sulphate in 0.01 N HCl was added to solubilise the formazan (MRSA standard strain or 3 MRSA clinical isolates) were crystals [24]. Plates were then incubated for 17 h, and readings adjustedtothe0.5McFarlandstandardandaddedtothe were performed at 570 nm using a microplate reader. Assays ∘ plates. After incubation for 24 h at 35 C under aerobic were performed in triplicate. conditions, 10 𝜇L of 0.01% sterile resazurin was added to the In vivo toxicity studies of P. amb oini c u s were performed plates, followed by 2h incubation. To evaluate the combina- according to the Organization for Economic Cooperation tory effects, fractional inhibitory concentrations (FICs) were and Development [25]. The protocol proposed by the OECD calculated. The FIC was defined according to the following has advantages over other protocols, as it uses very few formula: the MIC of the drug associated with the plant animals (𝑛=3) for each step. A single dose of 2000 mg/kg sample divided by the MIC of the drug or plant tested alone. oftheHEorthefractionF4wasadministeredtoSwiss The sum of the drug’s FIC and the plant extract’s FIC was mice by gavage. The highest starting dose was used when considered to be the FIC index (IFIC). The interaction was the test sample was likely to be nontoxic. Animals were determined to be synergistic when the IFIC ≤ 0.5, additive observed daily for 14 days. Once no death occurred during when 0.50 < IFIC ≤ 1.0, indifferent if 1 < IFIC < 4, and the evaluation period, the test was repeated with 3 more antagonistic when IFIC ≥ 4.0. The tests were performed in animals. No death was reported after the second experiment; triplicate. therefore, the toxicity of the test sample was categorised as 4 Evidence-Based Complementary and Alternative Medicine

Table 3: Resistance of Staphylococcus aureus clinical isolates to antimicrobial drugs. The tests were performed according to the CLSI recommendations [19]. The presence or absence of the genes mecA and CoA was verified by the polymerase chain reaction technique, according to the procedure described by Rallapalli et al. [20].

Clinical isolates Specimen mecA gene CoA gene Antibiotic resistance Strain01 BL + + Amp,Chlo,Cip,Ery,Gen,Pen,Sul Strain02 BL + + Amp,Chlo,Pen,Sul,Tet Strain03 BL + + Amp,Chlo,Cip,Ery,Gen,Pen,Sul,Tet Strain 04 BL + + Amp, Ery, Gen, Pen, Sul Strain 05 BL + + Chlo, Ery, Gen, Pen, Tet Strain 06 BL − + Amp, Chlo, Cip, Ery, Gen, Pen, Sul Strain 07 BL + + Amp, Ery, Gen, Pen, Sul Strain08 BL + + Amp,Chlo,Pen,Sul,Tet Strain 09 BL + + Amp, Gen, Pen, Sul, Tet Strain10 BL + + Amp,Chlo,Ery,Gen,Pen,Sul Strain11 CT + + Amp,Chlo,Ery,Gen,Pen,Tet Strain 12 WF + + Amp, Ery, Gen, Pen, Sul, Tet Strain13 CT + + Amp,Chlo,Cip,Ery,Gen,Pen,Sul,Tet Strain14 WF + + Amp,Chlo,Cip,Ery,Gen,Pen,Sul,Tet Catheter tip (CT); blood (BL); wound fluid (WF) from diabetic foot ulcers. Pen: penicillin; Amp: ampicillin; Ery: erythromycin; Cip: ciprofloxacin; Gen: gentamicin; Chlo: chloramphenicol; Tet: tetracycline; Sul: sulfamethoxazole/trimethoprim; Van: vancomycin. unclassified, and the LD50 was reported to be 5000 mg/kg, features, and Gram stain characteristics. A small fragment of according to the OECD [25]. the injured tissue was removed, fixed in 10% formalin, and submitted for histopathological analysis. 2.9. Cutaneous Abscess and Treatment. A mouse skin infection model was established according to the procedure 2.10. Statistical Analysis. The data underwent analysis of described elsewhere [26]. Animals were housed and cared variance (ANOVA) to compare the treatment and control for in compliance with regional regulations. The experiments groups, complemented by the Tukey-Kramer test for multiple were performed in accordance with the protocol approved comparisons. The tests were performed using GraphPad by the Ethics Committee on Animal Research of the Univer- Instat Version 3.01. The level of significance for a null sidade Federal do Ceara (process 28/09). A clinical isolate hypothesis was 5% (𝑃 < 0.05). (MRSA 08) was grown on a BHI agar plate for 16 h at ∘ 35 C. An isolated colony was inoculated into a 5 mL BHI ∘ 3. Results and Discussion broth tube and left for 6 h at 35 C. Before the experiment, the bacterial suspension turbidity was adjusted to match The use of medicinal plants has increased in Brazil because the0.5McFarlandstandard,dilutedto1:10inphosphate- they are considered to be a good source for alternative thera- buffered saline, and added to an equal volume of 2% Cytodex peutic purposes. We have recently shown that the essential oil (Sigma, USA). The final suspension (0.2 mL) was inoculated from P. amb oini c u s alters wall permeability and inhibits ure- subcutaneouslyintothebackofpreviouslyshavedmaleSwiss aseactivityandcapsuleexpressionofmultiresistantstrainsof mice (25–30 g). The control suspension consisted of BHI Klebsiella pneumoniae [15]. The present work aims to show, broth containing 2% Cytodex. Seven groups of 6 animals both in vivo and in vitro, the antimicrobial effectiveness in each group were tested. The control group consisted of 4 of hydroalcoholic extract and ethyl acetate fraction from P. animals. Two doses of the HE extract, the fraction F4 (250 amboinicus against multiresistant S. aureus. and 500 mg/kg/dose) or vancomycin (10 and 20 mg/kg/dose) Fourteen strains of MRSA isolated from clinical samples were given intraperitoneally at 3 and 12 h after infection. of hospitalised patients were identified using biochemical and Abscess diameters were measured after 72 h of infection molecular methods. After screening for oxacillin resistance using a Vernier calliper. The abscess volumes were calculated by disk diffusion, the strains were tested for their suscep- 2 using the following formula: 𝑉 = (𝜋/6)𝐿 ⋅𝑊 (𝐿,length; tibility to other antimicrobial drugs, as shown in Table 3. 𝑊, width). The animals were then sacrificed by cervical Allstrainswereresistanttoatleast5antibiotics.The dislocation, and subcutaneous abscesses were excised. After amplification of a 759 bp product by the polymerase chain mechanical teasing, abscess slurries were suspended in 0.9% reaction revealed the coagulase gene, which identified S. saline and stirred for 10 min. The supernatant was recovered, aureus strains. The amplification of a 533 bp product revealed and tenfold serial diluted aliquots were plated onto BHI agar the mecA gene, which encodes resistance to methicillin. All ∘ and incubated for 48 h at 35 C. The number of viable cells clinical isolates, except for one (strain 06), contained the was expressed as log CFU/mL. The presence of S. aureus was mecA gene. Strains with this type of multiresistance are determined by the aspects of bacterial growth, morphology commonly identified in nosocomial infections. When this Evidence-Based Complementary and Alternative Medicine 5

Table 4: Minimal inhibitory concentration of extracts, fractions, and subfractions of P. amb oini c u s against MRSA clinical isolates and ATCC.

Controls Extracts Fractions Subfractions Strain Van (mg/mL) DMSO (𝜇g/mL) Bact (% v/v) EH (mg/mL) ET (mg/mL) F1 F2 F3 F4 F5 F4a F4b F4c F4d F4e F4f F4g ATCC 25923 1 no + 4 8 no no no 0.5 no 2 0.5 0.5 0.5 no no no ATCC 65398 1 no + 4 4 no no no 0.5 no 1 0.5 0.5 0.5 no no no MRSA 01 2 no + 4 8 no no no 0.5 no 1 0.5 0.5 0.5 no no no MRSA 02 2 no + 2 4 no no no 0.5 no 2 0.5 0.5 0.5 no no no MRSA 03 1 no + 2 8 no no no 0.25 no 1 0.5 0.5 0.5 no no no MRSA 04 1 no + 2 8 no no no 0.5 no MRSA 05 1 no + 4 4 no no no 0.5 no MRSA 06 2 no + 4 8 no no no 0.25 no MRSA 07 2 no + 2 8 no no no 0.5 no MRSA 08 1 no + 2 4 no no no 0.5 no MRSA 09 2 no + 2 8 no no no 0.5 no MRSA 10 1 no + 2 4 no no no 0.5 no MRSA 11 2 no + 2 8 no no no 0.5 no MRSA 12 2 no + 2 8 no no no 0.5 no MRSA 13 2 no + 2 8 no no no 0.25 no MRSA 14 2 no + 2 8 no no no 0.5 no Bact: bacteria; CT: growth control; EH: hydroalcoholic extract; ET: ethanolic extract; F1: hexane fraction; F2: petroleum ether fraction; F3: dichloromethane fraction; F4: ethyl acetate fraction; F5: methanol fraction; F4a–g: subfractions of fraction F4; Van: vancomycin; DMSO: dimethyl sulfoxide; no: no activity.

type of infection occurs, vancomycin or ciprofloxacin is the Bhattacharjee [30], ethanolic extract from P. amb oini c u s chosen therapy. In our study, 5 of the 14 strains (35.7%) presents alkaloid, flavonoids, tannins, triterpenroids, and demonstrated resistance to ciprofloxacin. It is important to saponins. In the class of flavonoids, at least quercetin and be cautious when using either of these antibiotics because luteolin were identified [14, 30]. It has recently been demon- vancomycin is nephrotoxic [27],andciprofloxacinisnot strated that both flavonoids exhibit inhibitory activity against indicated for general use in children due to the risk of MRSA [31]. permanent injury [28]. Our present work demonstrated that The fraction F4 was subjected to a new partition chro- all S. aureus strains were sensitive to vancomycin. Strains matography, resulting in seven subfractions called F4a–g. of S. aureus with reduced susceptibility to vancomycin have Their MICs are also shown in Table 4. The subfractions F4a– previously been identified by other groups29 [ ]. d presented MICs similar to those of the fraction F4 (0.25 to Table 4 presents the inhibitory activity of the hydroalco- 0.5 mg/mL), except for the subfraction F4a. The MIC of this holic and ethanolic extracts and the fractions and subfrac- subfraction varied from 1 to 2 mg/mL. tions from P. amb oini c u s against MRSA and MSSA standard The bacteriostatic and bactericidal activities of the strains and MRSA clinical isolates. The hydroalcoholic and hydroalcoholic extract and the ethyl acetate fraction F4 ethanolic extracts showed activity against the MRSA strains, against the standard MRSA strain (ATCC 65398) were evalu- with MICs varying from 2 to 4 mg/mL and from 4 to ated, as shown in Figures 1(a) and 1(b),respectively.Theplant 8 mg/mL, respectively. The first report that demonstrated the samples presented bacteriostatic activity at concentrations plant activity against MRSA was published by Gurgel et al. of 2x and 4x MIC. Bactericidal activity was observed when [14]. The authors used the gel diffusion technique to verify the plant samples were tested at 100 mg/mL. Considering the that the hydroalcoholic extract of the plant presented MICs rate and extent of their bactericidal effects, it is necessary to of 9.3 mg/mL and 18.6 mg/mL against MSSA and MRSA evaluate their minimum bactericidal activity. standard strains, respectively. The antimicrobial activity of subfractions F4a-d against The low activity of the ethanolic extract in our work MRSA was tested using the bioautography technique. Bioau- might be due to the accumulation of substances with no tography revealed clear zones representing bacterial zone antimicrobial activity, such as proteins, fat, and carbohy- inhibition (Figure 2(a)). The results suggested that the drates. For this reason, to isolate the active compounds, antimicrobial activity of the subfractions was due to a set of the ethanolic extract was subjected to a thin-layer chro- substances with high or intermediate polarities (Figure 2(b)). matography. The ethyl acetate fraction F4 presented lower It is important to remember that the eluting solution con- MIC values (0.25 to 0.5 mg/mL) than the other fractions tained a mixture of hexane: ethyl acetate (6 : 4), so nonpolar and extracts. The antimicrobial activity of the ethanolic substances and molecules with low polarity would move to extract and its fraction F4 was likely related to the presence the top of the chamber. of flavonoids and terpenes. These compounds can damage A checkerboard method was used to investigate the the bacterial cell membrane. According to Hullatti and interactions between the plant samples and the antibiotics 6 Evidence-Based Complementary and Alternative Medicine

Hydroalcoholic extract Fraction ethyl acetate F4 10 10 9 9 8 8 7 7 6 6 5 5 4 4 log 10 (UFC/mL) log 10 (UFC/mL) 3 3 2 2 1 1 0 0 0246820 0246820 (h) (h) Controls 4xMIC Controls 4xMIC 2xMIC 100 mg/mL 2xMIC 100 mg/mL (a) (b)

Figure 1: Bacteriostatic and bactericidal activities of the hydroalcoholic (HE) extract (a) and the ethyl acetate fraction F4 (b) from P. amboinicus against a standard MRSA strain (ATCC 65398). The results are expressed as log 10 colony-forming units/mL.

(a) (b)

Figure 2: Bioautography of the ethyl acetate F4 subfractions from P. amb oini c u s . Two thin-layer chromatography plates were tested as follows. (a) A developed thin-layer chromatography (TLC) plate was dipped into a Petri dish containing Mueller-Hinton agar previously inoculated 6 with 10 CFU/mL of MRSA. After incubation, the plate was stained with thiazolyl blue tetrazolium MTT stain. (b) A developed TLC plate was directly stained with vanillin/perchloric acid. The arrows indicate the zones of bacterial growth inhibition. The solvent solution usedfor elution was hexane: ethyl acetate (6 : 4).

(vancomycin and ciprofloxacin). The results are presented associate the fraction F4 with antimicrobials such as van- in Table 5. The association between the EH extract and comycin and ciprofloxacin. The testing combination studies antibiotics was considered to be indifferent. Previous reports willbeconductedinanimalmodelstoconfirmtheseinvitro have shown a synergistic effect of the essential oil from observations. the leaves of P. amb oini c u s with cephalothin and ampi- A slight cytotoxicity was observed when the HE extract cillin as well as antagonistic activity with chloramphenicol and the ethyl acetate fraction F4 were incubated with and gentamicin [32]. The ethyl acetate fraction F4 showed RAW 264.7 macrophages. Their half-maximal inhibitory synergism with vancomycin and an additive effect with concentrations (IC50) were 817.0 𝜇g/mL and 99.6 𝜇g/mL, ciprofloxacin. These data may suggest that it is possible to respectively. Although the HE extract showed a relatively Evidence-Based Complementary and Alternative Medicine 7

Table 5: FIC values (FIC) and FIC index (IFIC) of hydroalcoholic extract (a) and the fraction F4 (b) in combination with vancomycin (Van) or ciprofloxacin (Cip) against a standard strain of S. aureus (ATCC 65398) and three randomly chosen MRSA strains. The interaction between the drugs and the plant was additive (A), synergic, (S) or indifferent (I).

(a) Hydroalcoholic extract (EH)

CIF CIF ICIF Interaction IFIC Interaction Eh Van Eh Cip ATCC (65398) 1 1 2I0.5 2 2.5 I MRSA 05 1 2 3I0.5 2 2.5 I MRSA 08 1 1 2I0.5 2 2.5 I MRSA 12 1 1 2I0.5 2 2.5 I (b) Ethyl acetate fraction (F4)

CIF CIF ICIF Interaction IFIC Interaction F4 Van F4 Cip ATCC (65398) 0.5 0.25 0.75 S 0.5 0.5 1A MRSA 05 0.5 0.25 0.75 S 0.5 0.5 1A MRSA 08 0.5 0.25 0.75 S 0.5 0.5 1A MRSA 12 0.5 0.25 0.75 S 0.5 0.5 1A CIF: fractional inhibitory concentration; ICIF: fractional inhibitory concentration index.

Table 6: Mice infected subcutaneously with an MRSA clinical isolate (strain #08) and treated intra-peritoneally with hydroalcoholic extract (HE) or fraction F4 from P. amb oini c u s or with vancomycin. The results were expressed by bacterial growth (log colony-forming unities (CFU) per site), abscess volume mean (mm3), and by indices of the histopathological analysis.

Abscess volume Bacterialcellcount Histopathological Dosage MIC Treatment mean 𝑃 Log CFU/site 𝑃 analysis 𝑃 (mg/kg) (mg/mL) (mm3 ± SD) (𝑥±SD) (index sum ± SD) Control 130.0 ± 29.1 7.2 ± 0.41 11.4 ± 0.78 HE 500 2.0 63.0 ± 26.7 <0.01 5.6 ± 0.51 <0.01 6.7 ± 2.9 <0.01 99 6.9 ± 0.48 < 9.3 ± 0.82 250 99.5 ± 37.3 N.S. 0.05 N.S. F4 500 0.5 26.9 ± 14.5 <0.001 4.7 ± 0.89 <0.001 5.4 ± 3.1 <0.001 250 55.1 ± 11.8 <0.001 5.3 ± 0.48 <0.001 6.7 ± 2.7 <0.01 Vancomycin 20 0.2 46.0 ± 22.9 <0.001 4.2 ± 0.91 <0.01 7.5 ± 1.6 <0.01 10 82.7 ± 27.9 <0.05 5.4 ± 0.86 <0.05 7.8 ± 2.3 <0.05 MIC: minimal inhibitory concentration; SD: standard deviation, N.S.: not significant. high cytotoxicity, this was not observed with any significant Thesampleswereeffectiveintreatinganimalsinadose- alterations in the in vivo toxicity studies. dependent fashion. The results are demonstrated in Table 6. AccordingtoShenoyetal.[33], rats were able to tolerate The average volume of the abscesses in animals treated 3 oral doses of up to 3,000 mg/kg of their body weight of the with the HE at 500 mg/kg/dose was 63.0 mm ,whichwas ethanolic extract of P. amb oini c u s without signs of toxicity. significantlylowerthanthoseintheanimalstreatedwith 3 A phase I clinical trial conducted with healthy volunteers saline (130.0 mm , 𝑃 < 0.01), but less effective than those in [34] to evaluate the safety of a commercial phytotherapic animals treated with vancomycin at 10 mg/kg/dose. There was formulation composed of Schinus terebinthifolius Raddi, Plec- no difference in abscess volume between the animals treated tranthus amboinicus Lour, and Eucalyptus globulus Labill did with the HE at 250 mg/kg/dose and those treated with saline. not demonstrate clinical and laboratory alterations during the Abscess volume was significantly reduced in animals treated period of study. with the ethyl acetate fraction F4 (Figure 3), both at 500 and Different models of experimental skin and soft-tissue 250 𝜇g/kg/dose, in comparison to the untreated animals (𝑃< infections have been described and vary widely according 0.001). to the animal model, bacterial strains, and the period of The HE extracts at 500 and 250 mg/kg significantly evaluation. We chose to test in the present work an animal reduced the number of viable bacterial cells in abscesses model of subcutaneous abscess using Cytodex with the compared to the untreated group (𝑃 < 0.01 and 𝑃< infecting inoculum [26]. 0.05, resp.). The fraction F4 was more effective than the HE 8 Evidence-Based Complementary and Alternative Medicine

(a) (b)

Figure 3: Volume of subcutaneous abscesses in mice infected with an MRSA clinical isolate strain (#08), 72 h after intraperitoneal treatment with 2 doses of ethyl acetate fraction F4 from P. amb oini c u s at 500 mg/kg/dose. (a) Untreated animal. (b) Animal treated with fraction F4. extract and vancomycin in reducing the bacterial cell counts, MSSA: Methicillin-sensitive Staphylococcus aureus compared to the untreated group (𝑃 < 0.001). TLC: Thin-layer chromatography. The parameters of inflammation (i.e., cellular infiltration, edema, angiogenesis, fibrosis, necrosis, and haemorrhage) Conflict of Interests were evaluated and graded on a 0–3 point scale. The sum of the scores was used to estimate the inflammatory status There is no conflict of interests. of each animal. Significant improvement in suppurative inflammation was observed in the groups treated with the Acknowledgments HE, vancomycin, and the fraction F4, as shown in Table 6. The HE at 500 mg/kg/dose and vancomycin at 20 mg/kg/dose This research was supported financially by the CNPq proved to be efficient in resolving the abscesses, compared (processes 579437/2008-6 and 554970/2010-4) and Fun- to the untreated animals (𝑃 < 0.05). The fraction F4 at cap/PPSUS (process 09100057-2). 500 mg/kg/dose proved to be the most effective treatment (𝑃 < 0.001). The rapid recovery of the animals treated with the plant References samples was most likely due to both the antimicrobial and [1] F. Rossi and D. B. AndreazzI, Resistenciaˆ Bacteriana: Interpre- anti-inflammatory activities of P. amb oini c u s .Someanti- tando o Antibiograma, Atheneu, Sao˜ Paulo, Brazil, 2005. inflammatory effects of P. amb oini c u s extracts are described [2] M. Guzman-Blanco,´ C. Mej´ıa, R. Isturiz et al., “Epidemiology in the literature, including the blockage of NF-𝜅Bactivation, of meticillin-resistant Staphylococcus aureus (MRSA) in Latin which would consequently reduce the production of proin- America,” International Journal of Antimicrobial Agents,vol.34, flammatory cytokines18 [ ]. no. 4, pp. 304–308, 2009. To the best of our knowledge, this is the first report [3] J. T. Weber, “Community-associated methicillin-resistant demonstrating the efficacy of P. amb oini c u s in treating a Staphylococcus aureus,” Clinical Infectious Diseases,vol.41,no. subcutaneous abscess model caused by MRSA. The present 4, pp. S269–S272, 2005. study not only corroborates the in vitro model but also [4] T. Ito, Y. Katayama, K. Asada et al., “Structural comparison demonstrates its efficacy in animal models. This highlights of three types of staphylococcal cassette chromosome mec theeffectivecapacityofP. amb oini c u s for use in association integrated in the chromosome in methicillin-resistant Staphy- with antibiotic therapy for MRSA cutaneous infections. lococcus aureus,” Antimicrobial Agents and Chemotherapy,vol. 45,no.5,pp.1323–1336,2001. [5] K.J.Kim,H.H.Yu,J.D.Cha,S.J.Seo,N.Y.Choi,andY.O.You, Abbreviations “Antibacterial activity of Curcuma longa L. against methicillin- resistant Staphylococcus aureus,” Phytotherapy Research,vol.19, BHI: Brain heart infusion no. 7, pp. 599–604, 2005. CFU: Colony-forming units [6] M. Mahboubi and M. M. Feizabadi, “Antimicrobial activ- DMSO: Dimethyl sulfoxide ity of Ducrosia anethifolia essentialoilandmaincompo- EH: Hydroalcoholic extract nent, decanal against methicillin-resistant and methicillin- FIC: Fractional inhibitory concentration susceptible Staphylococcus aureus,” Journal of Essential Oil- FICI: Fractional inhibitory concentration index Bearing Plants, vol. 12, no. 5, pp. 574–579, 2009. MHI: Mueller-Hinton infusion [7]R.K.RajaretinamandS.G.P.Vincent,“Isolationofanovel MIC: Minimal inhibitory concentration bioactive compound from Rhizophora mucronata for methi- MRSA: Methicillin-resistant Staphylococcus aureus cillin resistant Staphylococcus aureus (MRSA) and compound Evidence-Based Complementary and Alternative Medicine 9

toxicity assessment in zebra fish embryos,” Journal of Pharmacy [23] B. Moulari, Y. Pellequer, H. Lboutounne et al., “Isolation and Research, vol. 3, pp. 200–203, 2010. in vitro antibacterial activity of astilbin, the bioactive flavanone [8]C.M.Mattana,S.E.Satorres,A.Sosa,M.Fusco,andL.E. from the leaves of Harungana madagascariensis Lam. ex Poir. Alcaraz,´ “Antibacterial activity of extracts of Acacia aroma (Hypericaceae),” Journal of Ethnopharmacology,vol.106,no.2, against methicillin-resistant and methicllin-sensitive Staphylo- pp. 272–278, 2006. coccus,” Brazilian Journal of Microbiology,vol.41,no.3,pp.581– [24] T. Mosmann, “Rapid colorimetric assay for cellular growth and 587, 2010. survival: application to proliferation and cytotoxicity assays,” [9] M. Mahboubi and F. Ghazian Bidgoli, “Antistaphylococcal Journal of Immunological Methods,vol.65,no.1-2,pp.55–63, activity of Zataria multiflora essential oil and its synergy with 1983. vancomycin,” Phytomedicine,vol.17,no.7,pp.548–550,2010. [25] Organization for Economic Cooperation and Development [10] B. Dulger and M. A. Kilcik, “Antibacterial activity of Ballota (OECD), Guideline for Testing of Chemicals: Acute Oral Toxicity- acetabulosa against methicillin-resistant Stapylococcus aureus,” Acute Toxic Class Method, OEDC, Guideline, Paris, France, Asian Journal of Chemistry, vol. 23, no. 1, pp. 416–418, 2011. 2001. [11] W. Chingwaru, G. Duodu, Y. Van Zyl et al., “Antibacterial [26]C.L.Bunce,L.Wheeler,G.Reed,J.Musser,andN.Barg, and anticandidal activity of Tylosema esculentum (marama) “Murine model of cutaneous infection with gram-positive extracts,” South African Journal of Science, vol. 107, no. 3-4, pp. cocci,” Infection and Immunity,vol.60,no.7,pp.2636–2640, 79–89, 2011. 1992. [12] C. W.Lukhoba, M. S. J. Simmonds, and A. J. Paton, “Plectranthus [27]K.A.Hazlewood,S.D.Brouse,W.D.Pitcher,andR.G. a review of ethnobotanical uses,” Journal of Ethnopharmacology, Hall, “Vancomycin-associated nephrotoxicity: grave concern vol.103,no.1,pp.1–24,2006. or death by character assassination?” American Journal of [13] J. C. R. Nogueira, M. F. M. Diniz, and E. Lima, “Atividade Medicine,vol.123,no.2,pp.182–e1,2010. antimicrobiana in vitro de produtos vegetais em otite externa [28] S. I. Nwadioha, E. O. P. Nwokedi, E. Kashibu, M. S. Odimayo, aguda,” Revista Brasileira de Otorrinolaringologia,vol.74,no.1, andE.E.Okwori,“Areviewofbacterialisolatesinblood pp. 118–124, 2008. cultures of children with suspected septicemia in a nigerian [14]A.P.A.D.Gurgel,J.G.daSilva,A.R.S.Grangeiroetal., tertiary hospital,” African Journal of Microbiology Research,vol. “Antibacterial effects of Plectranthus amboinicus (Lour.) spreng 4, no. 4, pp. 222–225, 2010. (Lamiaceae) in methicillin resistant Staphylococcus aureus [29] H. K. Tiwari and M. R. Sen, “Emergence of vancomycin (MRSA),” Latin American Journal of Pharmacy,vol.28,no.3, resistant Staphylococcus aureus (VRSA) from a tertiary care pp.460–464,2009. hospital from northern part of India,” BMC Infectious Diseases, [15]T.B.Gonc¸alves,M.A.Braga,F.F.M.Oliveiraetal.,“Effect vol. 6, p. 156, 2006. of subinihibitory and inhibitory concentrations of Plectranthus [30] H.-X. Xu and S. F. Lee, “Activity of plant flavonoids against amboinicus (Lour.) Spreng essential oil on Klebsiella pneumo- antibiotic-resistant bacteria,” Phytotherapy Research,vol.15,no. niae,” Phytomedicine,vol.19,no.11,pp.962–968,2012. 1,pp.39–43,2001. [16]A.P.A.D.Gurgel,J.G.daSilva,A.R.S.Grangeiroet [31] K. K. Hullatti and P. Bhattacharjee, “Pharmacognostical evalu- al., “In vivo study of the anti-inflammatory and antitumor ation of different parts of Coleus amboinicus lour., Lamiaceae,” activities of leaves from Plectranthus amboinicus (Lour.) Spreng Pharmacognosy Journal,vol.3,pp.39–43,2011. (Lamiaceae),” JournalofEthnopharmacology,vol.125,no.2,pp. [32] R. A. G. Oliveira, E. O. Lim, W. L. Vieira et al., “Estudo da 361–363, 2009. interferenciaˆ de oleos´ essenciais sobre a atividade de alguns [17] J. M. Chang, C. M. Cheng, L. M. Hung, Y. S. Chung, and R. Y. antibioticos´ usados na cl´ınica,” Brazilian Journal of Pharmacog- Wu, “Potential use of plectranthus amboinicus in the treatment nosy,vol.16,no.1,pp.77–82,2006. of rheumatoid arthritis,” Evidence-Based Complementary and [33]S.Shenoy,H.Kumar,Thashmaetal.,“Hepatoprotectiveactivity Alternative Medicine,vol.7,no.1,pp.115–120,2010. of Plectranthus amboinicus against paracetamol induced hep- [18]Y.J.Chiu,T.H.Huang,C.S.Chiuetal.,“Analgesicandanti- atotoxicity in rats,” International Journal of Pharmacology and inflammatory activities of the aqueous extract from Plectran- Clinical Sciences,vol.2,pp.32–38,2012. thus amboinicus (Lour.) Spreng both in vitro and in vivo,” Evidence-Based Complementary and Alternative Medicine,vol. [34] P. T. C. Paulo, M. F. F. M. Diniz, I. A. Medeiros et al., “Ensaios 2012,ArticleID508137,11pages,2012. cl´ınicos toxicologicos,´ fase I, de um fitoterapico´ composto (Schinus terebinthifolius Raddi, Plectranthus amboinicus Lour [19] Clinical and Laboratory Sandards Institute (CLSI), Performance e Eucalyptus globulus Labill),” Revista Brasileira de Farmacog- Standards for Antimicrobial Disk Susceptibility Tests,vol.29 nosia,vol.19,no.1,pp.68–76,2009. of Approved standard M02-A10,CLSI,Wayne,Pa,USA,10th edition, 2009. [20] S.Rallapalli,S.Verghese,andR.Verma,“Validationofmultiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus,” Indian Journal of Medical Microbiology,vol.26,no.4,pp.361–364,2008. [21] Clinical and Laboratory Sandards Institute (CLSI), Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically,vol.29ofApproved standard M07-A8,CLSI,Wayne, Pa, USA, 8th edition, 2009. [22] F. C. Odds, “Synergy, antagonism, and what the chequerboard puts between them,” Journal of Antimicrobial Chemotherapy, vol. 52, no. 1, p. 1, 2003.