Evidence-Based Complementary and Alternative Medicine

Special Invertebrate Models and Integrative Medical Applications: Regulations, Mechanisms, and Therapies

Guest Editors: Chih-Yang Huang, Edwin L. Cooper, Catherine Fang-Yeu Poh, Wei-Wen Kuo, Tung-Sheng Chen, and Ronald Sherman Special Invertebrate Models and Integrative Medical Applications: Regulations, Mechanisms, and Therapies Evidence-Based Complementary and Alternative Medicine

Special Invertebrate Models and Integrative Medical Applications: Regulations, Mechanisms, and Therapies

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

Special Invertebrate Models and Integrative Medical Applications: Regulations, Mechanisms, and Therapies, Chih-Yang Huang, Edwin L. Cooper, Catherine Fang-Yeu Poh, Wei-Wen Kuo, Tung-Sheng Chen, and Ronald Sherman Volume 2014, Article ID 843961, 2 pages

Recent Advances in Developing Insect Natural Products as Potential Modern Day Medicines, Norman Ratcliffe, Patricia Azambuja, and Cicero Brasileiro Mello Volume2014,ArticleID904958,21pages

Corals and Their Potential Applications to Integrative Medicine, Edwin L. Cooper, Kyle Hirabayashi, KevinB.Strychar,andPaulW.Sammarco Volume 2014, Article ID 184959, 9 pages

Mechanisms of Maggot-Induced Wound Healing: What Do We Know, and Where Do We Go from Here?,RonaldA.Sherman Volume 2014, Article ID 592419, 13 pages

Millipedes as Food for Humans: Their Nutritional and Possible Antimalarial Value—A First Report, HenrikEnghoff,NicolaManno,Sev´ erin´ Tchibozo, Manuela List, Bettina Schwarzinger, Wolfgang Schoefberger, Clemens Schwarzinger, and Maurizio G. Paoletti Volume2014,ArticleID651768,9pages

Recommendations for the Use of Leeches in Reconstructive Plastic Surgery,KostaY.Mumcuoglu Volume 2014, Article ID 205929, 7 pages

Marine Invertebrate Natural Products for Anti-Inflammatory and Chronic Diseases, Kalimuthu Senthilkumar and Se-Kwon Kim Volume 2013, Article ID 572859, 10 pages

Recombinant Protein Production of Earthworm Lumbrokinase for Potential Antithrombotic Application, Kevin Yueju Wang, Lauren Tull, Edwin Cooper, Nan Wang, and Dehu Liu Volume 2013, Article ID 783971, 8 pages

Honey as a Potential Natural Anticancer Agent: A Review of Its Mechanisms, Sarfraz Ahmed and Nor Hayati Othman Volume 2013, Article ID 829070, 7 pages Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2014, Article ID 843961, 2 pages http://dx.doi.org/10.1155/2014/843961

Editorial Special Invertebrate Models and Integrative Medical Applications: Regulations, Mechanisms, and Therapies

Chih-Yang Huang,1,2 Edwin L. Cooper,3 Catherine Fang-Yeu Poh,4 Wei-Wen Kuo,5 Tung-Sheng Chen,1 and Ronald Sherman6

1 Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan 2 Department of Biotechnology, Asia University, Taichung 41354, Taiwan 3 Laboratory of Comparative Neuroimmunology, Department of Neurobiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA 90095-1763, USA 4 FacultyofDentistry,TheUniversityofBritishColumbia,2151WesbrookMall,Vancouver,BC,CanadaV6T1Z3 5 Department of Biological Science and Technology, China Medical University, Taichung 40402, Taiwan 6 Bio Therapeutics, Education & Research Foundation, Irvine, CA 92617, USA

Correspondence should be addressed to Chih-Yang Huang; [email protected]

Received 13 March 2014; Accepted 13 March 2014; Published 6 May 2014

Copyright © 2014 Chih-Yang Huang 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 special issue of eCAM has brought together several Others include corals, millipedes, maggots, and earthworms. unique investigators interested in invertebrate animal mod- Corals are marine invertebrates that live in compact colonies. els. Investigators would benefit by examining advances and Humans have learned that they are useful in the manufacture applications of findings derived from useful terrestrial and of jewelry and chemical compounds that are useful against marine invertebrate animal models. Results derived from cancer, AIDS, and pain and serve as skeletons during bone well-defined evidence-based approaches are usually relevant grafting. Millipedes are arthropods (the same group as to humans; the advantages are numerous, inexpensive, and shrimps, crabs, butterflies, and bees) which in some cultures noncontroversial and are not subject to rigid, ethical, or moral are used during pregnancy and as cures for fever, wounds, guidelines. earaches, and hemorrhoids and food among the Bobo people Inthehistoricalcontext,theseanimalshavebeencrucial of Burkina Faso when crushed; sometimes they are boiled and in many cultures before the advent of industrial medicines eaten in tomato sauce. thus providing the only source of aid. Now, there is a move Since the time of Napoleon and even centuries earlier, to return to or at least to include these practices lest they maggots have been used especially in patients with severe disappear since, in certain cultures, they represent the sole intractable wounds caused by nerve degeneration that results source of useful health remedies. However, evidence-based from diabetes. In fact, leeches and maggots are now valuable medicine emphasizes the importance of subjecting animal components of the emerging field of biotherapy, the ther- products to rigorous analysis. Results are then standardized apeutic use of living creatures to clean gangrenous tissue and serve to educate populations including those that use often found in ulcers, burns, and postoperative infections. exclusively western practices. Earthworms are experiencing growing popularity due to eCAM stands ready to accept the importance and con- their many applications including sources of high protein tributions of bees as valuable sources of beneficial activities as food and the production of an anticlotting agent known (pollination) and products (honey and propolis) that for as lumbrokinase that dissolves blood clots in patients. Aside centuries have enhanced human lives as food and medicine. from observations and usefulness by the layman, certain 2 Evidence-Based Complementary and Alternative Medicine techniquesarevaluableasmolecularbiologistsstriveto understand the minute structure of molecules in order to improve therapies. Let us not forget the contributions of earthworms as tillers of the soil which enhance agricultural output. Earthworms keep the soil aerated by their constant churning which makes for improved yield of plants regardless of the proposed use. Chih-Yang Huang Edwin L. Cooper Catherine Fang-Yeu Poh Wei-Wen Kuo Tung-Sheng Chen Ronald Sherman Review Article Recent Advances in Developing Insect Natural Products as Potential Modern Day Medicines

Norman Ratcliffe,1,2 Patricia Azambuja,3 and Cicero Brasileiro Mello1

1 Laboratorio´ de Biologia de Insetos, Departamento de Biologia Geral, Universidade Federal Fluminense, Niteroi,´ RJ, Brazil 2 Department of Biosciences, College of Science, Swansea University, Singleton Park, Swansea SA2 8PP, UK 3 Laboratorio´ de Bioqu´ımica e Fisiologia de Insetos, Instituto Oswaldo Cruz, Fundac¸ao˜ Oswaldo Cruz, Avenida Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil

Correspondence should be addressed to Patricia Azambuja; [email protected]

Received 1 December 2013; Accepted 28 January 2014; Published 6 May 2014

Academic Editor: Ronald Sherman

Copyright © 2014 Norman Ratcliffe 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.

Except for honey as food, and silk for clothing and pollination of plants, people give little thought to the benefits of insects in their lives. This overview briefly describes significant recent advances in developing insect natural products as potential new medicinal drugs. This is an exciting and rapidly expanding new field since insects are hugely variable and have utilised an enormous range of natural products to survive environmental perturbations for 100s of millions of years. There is thus a treasure chest of untapped resources waiting to be discovered. Insects products, such as silk and honey, have already been utilised for thousands of years, and extracts of insects have been produced for use in Folk Medicine around the world, but only with the development of modern molecular and biochemical techniques has it become feasible to manipulate and bioengineer insect natural products into modern medicines. Utilising knowledge gleaned from Insect Folk Medicines, this review describes modern research into bioengineering honey and venom from bees, silk, cantharidin, antimicrobial peptides, and maggot secretions and anticoagulants from blood-sucking insects into medicines. Problems and solutions encountered in these endeavours are described and indicate that the future is bright for new insect derived pharmaceuticals treatments and medicines.

1. Introduction from insects and only a few originated from invertebrates such as leeches, sponges, and cone snails. Difficulties in Previously, a number of overviews on insect natural products species identification, drug toxicity, development costs, and and their potential for development into drugs to treat human large scale production [3] partially explain the reason for diseases have been published [1–3].Recently,however,there theslowprogressindevelopinginsectproductsaspotential have been additional advances in this field. The present modern medicines. However, since modern genomics, in review therefore focuses on these as well as their implication for studying mammalian physiology and the immune reac- silico drug design and high throughput screening have failed tions to human pathogens. to yield new generations of novel drugs; there is now renewed Surprisingly, despite the success of insects in terms of interest in more traditional methods of screening using the numbers and diversity, the most successful drugs derived huge diversity of animals, plants, and microbes available [5]. from natural products, including artemisinin, quinine, Furthermore, more traditional biochemical screening tech- aspirin, cocodamol, simvastatin, and cyclosporine, have been niques have now resulted in notable progress in developing isolated from plants, marine organisms, and microbes [3, therapeutics from arthropods, including melittin from bees 4]. Altogether, 939 nature-derived approved drugs were [6], alloferon from blowflies [7], and anticoagulants from developed between 1961 and 2010 [4] but none of these were ticks [8]. 2 Evidence-Based Complementary and Alternative Medicine

Araneae (spiders)

Chrysidoidea

Hornet >500 M yrs ago ≈200 M yrs ago Social wasp Vespoidea Silk with 𝛽-sheet molecular structure Weaver ant Australian bulldog ant

≈280 M Ponerine ant yrs ago Indian jumping ant Coiled coil ∗ silk arose Honey bee Lepidoptera (including silkworms) Bumble bee Apoidea Stingless bee

Figure 1: Schematic showing approximate evolutionary relationship of spiders, silkworms, bees, wasps, and ants mentioned in this review. Figure from Tara D. Sutherland, Sarah Weisman, Andrew A. Walker, and Stephen T. Mudie, “The Coiled Coil Silk of Bees, Ants, and Hornets,” Biopolymers volume 97, Issue 6, pp. 446–454, 2012 (DOI 10.1002/bip.21702) published by John Wiley and Sons with permission.

2. Use of Insects in Folk Medicine Medicine to develop potential new medicines for treating intractable diseases such as cancer and the problems associ- Despite the fact that insects have not been a rich source of ated with newly emerging antibiotic-resistant bacteria. In the modern drugs, they have, for thousands of years, provided followingexamplesofthedevelopmentofinsectproductsas many invaluable natural substances, including silk and honey potential modern medicines, there is already a long history of products (royal jelly, beeswax, pollen, and propolis). Insect the use of these substances in Folk Medicine. secretions and ground-up bodies have commonly been used in Folklore Medicine not only in China and Bahia but also in India, Asia, Africa, and Mexico (e.g., [2, 9, 10]). Unfortunately, 3. Bee, Wasp, and Ant Products two of the most fascinating accounts of the use of insects in Folk Medicine have not been published in mainline scientific Bee, wasp, and ant products, including honey and venom, journals but are well worth reading [11, 12]. One of these have been used in Folk Medicine for thousands of years includesanunpublishedbookbyLockhart[11], while the for treating wounds, ulcers, inflammation, infections, pain, other is a blog describing the experiences of the author cancer, and allergies [3]. Studies of natural products from with the use of ants in the Bolivian Amazon [12]. A more hymenopterans (Figure 1)havemainlyconcentratedon recent published review on insects as medicines deserves honey bee compounds because of the ready availability mentioning too as it presents alluring accounts of Insect Folk of large numbers of these insects reared under relatively Medicine in India and Zaire as well as the use of insects as constant controlled conditions. food [13]. Insects and insect extracts have been used in Folk 3.1. Honey Products from Bees. Recently, the use of honey Medicine for a huge range of conditions including arthritis for wound healing has been extensively reviewed [15–17]. treatment with Pseudomyrmex ant venom which resulted in These studies demonstrated the efficacy of honey in wound US patent number 4, 247, 540 in 1981 [11, 12]. Amazonian repair and sterilization of infected wounds and generally Indians also diagnosed diabetes by watching to see whether supported the use of honey in clinical practice, but only ants swarmed over urine which in diabetics contains high with certain types of wounds and after additional clinical levels of sugar to attract the ants [11]. Particularly thought pro- trials [15]. The evidence available, for example, includes voking is the account of ants being used to cure lethargy [11]. 19 randomized controlled trials with 2554 patients which Altogether in China, 1,700 medicines have been produced suggested that honey improved healing times but only in from ca. 300 insect species while 42 species have been used mild-to-moderate superficial burns and not in full thickness as Folk Medicines in Bahia [14]. burns [18]. In addition, more recently, an analysis of 44 Only a few Insect Folk Medicines have undergone exhaus- Cochrane reviews also provided robust evidence that in some tive clinical trials to prove their efficacy. Scientists, however, wound care interventions topical honey application reduced are now using knowledge accrued from exponents of Folk healing times of burns [19]. Evidence-Based Complementary and Alternative Medicine 3

Honey is a complex mixture of substances and progress Table 1: Examples of phenols present in honey with anticancer ∗ is being made at the molecular level in understanding properties . the functions of the various components on cells and the Class of phenolic Examples of specific phenolic effectiveness of honey in treating a range of human ailments. compounds compounds researched For example, Tonks et al. [21]isolateda5.8kDahoney Quercetin, kaempferol, galangin, component which stimulated the production of the TNF- (1) Flavonols fisetin, and myricetin alpha cytokine via TLR4 in human monocyte cultures. TNF- alpha is involved in the repair and regeneration of tissues. (2) Flavanones Hesperidin Apigenin, acacetin, chrysin, luteolin The antimicrobial activity of honey is probably due to (3) Flavones genkwanin, wogonin, and tricetin a combination of low pH, high osmolality, and hydrogen peroxide generation together with defensin-1 and methylgly- (4) Phenolic acids Caffeic acid oxal, with the latter an aldehyde generated from pyruvic acid (6) Coumarins Coumarin [3, 22]. Interestingly, Kwakman et al. [22]recentlyshowed (7) Tannins Ellagic acid ∗ that Revamil and Manuka honeys have different antibac- Table modified from Abubakar et al. [20]. terial components, with the former containing defensin-1, hydrogen peroxide, and methylglyoxal, while the latter only had methylglyoxal at 44 times the concentration of Revamil. In addition, Manuka honey was also shown to contain jelly and the flavonoids in propolis responsible [3]. An excel- other unidentified antibacterial factors. Great variations in lent overview of the immunomodulatory and antitumour antimicrobialpropertieshavealsobeendiscoveredforarange activity of bee honey in experimental and clinical studies of honeys, limiting those suitable for use in medicine [23]. was published in 2009 [32]. Further recent progress has There is great recent interest in the antimicrobial activ- been made in understanding more details of the anticancer ity of honey against important antibiotic-resistant human properties of the mixture of polyphenols present in honey, pathogens (reviewed in [17]). These studies showed, for propolis, and royal jelly [20]. An indication of the complexity example, inhibition of Gram-positive MRSA (methicillin of the phenolic mixture in honey is given in Table 1. resistant Staphylococcus aureus), of vancomycin-sensitive Of these compounds, quercetin has been shown to and resistant Enterococci (VSE and VRE, e.g., [24]), and enhance the apoptotic ability of anti-CD95 and rTRAIL of Streptococcus species isolated from wounds [25]. Honey (recombinant tumor necrosis factor-related apoptosis induc- also impacts Gram-negative bacteria associated with wounds ing ligand) in acute lymphocytic leukemia [33]. In addition, such as Pseudomonas aeruginosa, Stenotrophomonas species, details of the ability of polyphenols isolated from propolis to and Acinetobacter baumannii (e.g., [17]). Manuka honey overcome the resistance of cancer cells to TRAIL-mediated appears to inhibit cell division in MRSA [26], while, with apoptosis have recently been reviewed [34]. The possible use P. ae r ug ino s a , the cell wall is destabilised and lysis occurs of propolis as a dietary supplement in a cancer preventative [27]. Bacterial DNA degradation in pathogens has also strategy was emphasized [34]. been reported with Buckwheat honey [28]. Finally, honey Other phenolic compounds in honey in Table 1,withanti- can not only inhibits planktonic bacteria but also prevents cancer properties, include apigenin and acacetin which not the formation of biofilms [17, 29] that form, for example, only induce caspase-dependent apoptosis in human leukemia on surgical implants, thus causing prosthesis failure and cells in vitro but the former also produced apoptosis- additional patient distress. A review has been published of mediated inhibition of U937 leukemic cell xenografts in recent patents resulting from all this work on antibiotics from mice [35]. Other phenolic compounds in Table 1 also have hives [30]. antileukemic cell growth inhibition in vitro mediated by apoptosis [20]. None of these researches has, to date, led to The above benefits of honey in wound healing and new chemotherapeutic agents but the information from in bacterial inhibition have resulted in the development of vitro studies on human cancer cells should provide clues to special dressings to treat different types of wounds. Some of help the future development of new medicines [20]. these are in the form of ointments or gels, while others are More promising, for the more immediate development actual dressings made from mixes of alginate with honey [17]. of new anticancer therapeutics from honey products, is Other honey products have also been shown to have the work of Fernandez-Cabezudo et al. [36]. Initially, they antimicrobial activity so that propolis and the apalbumins confirmed the killing properties of Manuka honey on three in royal jelly have been reported to inhibit bacteria [3, cancer cell lines via a caspase 9-dependent apoptotic pathway 31]. Propolis also has a synergistic effect with antimicrobial inducing caspase 3, reducing Bcl-2 expression, and leading drugs in the treatment of experimental S. aureus keratitis to DNA fragmentation and cell death. Subsequently, they and diminishes the resistance of the bacterial cell walls to injected Manuka honey alone or in combination with a antibiotics (reviewed in [31]). The effect of propolis on oral chemotherapeutic agent (taxol) into mice implanted with Streptococcus mutans also indicates the possible development syngeneic melanoma cells and recorded inhibition of tumour of this factor as a cariostatic agent to control caries and other growth and host survival. Controls injected solely with infectious diseases of the mouth [31]. Manuka honey showed 33% inhibition of tumour growth. Regarding the anticancer properties of honey products, The combination group of Manuka honey plus taxol showed thesehavebeenreportedpreviouslywithafattyacidinroyal no increase in tumour inhibition in comparison with the 4 Evidence-Based Complementary and Alternative Medicine

Table 2: Examples of strategies to overcome the cytolytic properties of melittin.

Strategy Target References (1) Cancer cells killed by dilutions of melittin not Lung cancer cells in vitro Zhu et al. [45] affecting normal cells (2) Point mutation and deletion of specific Reduced haemolysis of normal cells but Zhao et al. [46] melittin amino acids inhibition of bacteria (3) Synthetic melittin coupled to hecate-CGba as a Ovarian, testicular, and adrenocortical Vuorenoja et al. delivery vehicle tumours in vivo [47] (4) Melittin coupled to a specific homing peptide Hepatocellular carcinoma cells in vitro Zhao et al. [48] identified by phage display (5) Gene therapy and transfection of melittin gene Human bladder carcinoma cells in vitro Winder et al. [49] into tumours (6) Use of nanoparticle technology for delivery of Soman et al. [50] Melanomas in vivo melittin to tumours Huang et al. [51] aHecate-CGb: the beta chain of human chorionic gonadotropin. taxol group alone; however, what was remarkable was the (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) which in the highly significant improvement in survival of mice in the venom reservoir have a tetrameric structure (reviewed in combination group. This study indicates the potential of [3, 41]). Upon binding, melittin induces cytolysis of most Manuka honey in alleviating chemotherapeutic toxicity [36] membranes such as those of normal mammalian cells. Thus, and improving patient survival. melittin is cytotoxic in vivo which has hindered its therapeutic development,despitethefactthatitinhibitsorkillsarangeof 3.2. Bee, Wasp, and Ant Venoms. Bee venom therapy has cancer cell types, such as melanoma, osteosarcoma, leukemic, been used in Folk Medicine for many thousands of years ovarian, prostate, hepatic, renal, bladder, and mammary for treating a range of ailments from arthritis, rheumatism, gland cells [42]. skin diseases, multiple sclerosis, cancer, infections, and pain Theprecisemodeofactionofmelittininkillingcancer (reviewed in [1, 3]). Apart from bee venom, the venoms of cells is not fully understood although recent advances in many other stinging insects, such as wasps and ants, contain understanding its cytolytic effect have been made43 [ ]. At low a large range of practically unexplored compounds awaiting concentrations, melittin induces transient pore formation in discovery and development into the medicines of tomorrow. the cell membrane due to tension resulting from one-sided For example, some ant and parasitoid wasp venoms may bindingofmelittintotheoutercellmembraneleaflet.At contain75ormoredifferentcomponents[37, 38]. higher concentrations, melittin binding results in the forma- Although bee venom therapy has been widely used, it tion of stable pores in the cell membrane leading to cell lysis as has neither, as yet, been approved by drug safety authorities the melittin concentration increases and the pores coalesces. nor commonly adopted by conventional medicine. How- Melittinhasmultipleeffectsoncells(reviewedin[42]). ever, there are some crude formulations available, including These effects range from hormone induction, membrane Apiven in France, produced from the crude venom of honey protein aggregation, and changes in membrane potential bees [39]. to stimulation of G-protein enzymes and PLA2,aswellas Honey bee venom is a mixture of at least 20 compounds, aroleincellsignalinduction[42]. The possible effects including 1/. active peptides such as melittin, apamine, of melittin and other bee venom components on cancer mast cell degranulating peptide (MCD), and adolapin, 2/. cells and host immunity involve inhibition of calmodulin 𝜅 the enzymes phospholipase A2 (PLA2), and hyaluronidase, and NF- B. These effects, in turn, would inhibit cancer cell and3/.theactiveamines,histamine,serotonin,andcate- proliferation, invasion and metastasis, and angiogenesis and cholamine. Of these components, melittin and phospholipase induce apoptosis [42]. A2 make up 40–60% and 10–12% dry weight of venom, It appears that bee venom induces apoptosis, necrosis, respectively [3, 40]. and lysis of tumour cells and, at the same time, can produce Despite the multifunctional use of honey bee venom immunosuppressive and/or immunostimulation in the host in Folk Medicine, recent research has focused mainly on [42]. Melittin apparently induces apoptosis via activation melittin and its anticancer properties, although apamine of the PLA2 in cancer cells, especially those transformed and phospholipase A2 have also received some attention by the ras oncogene [44]. Excellent detailed reviews of [3, 40]. There is an extensive literature on melittin which the interaction of melittin and other bee components with probably reflects the great potential of this peptide for tumour cells and the therapeutic potential of bee venom have development as a therapeutic medicine for treating different been published by Gajski and Garaj-Vrhovac [40]andOrˇsolic´ types of cancers. Melittin is a water soluble molecule, with [42]. cationic and amphipathic properties which enhance the In efforts to overcome the cytolytic properties of melittin electrostatic binding to the anionic cell membranes of many and to harness its anticancer properties, scientists have bacteria and cancer cells. Melittin contains 26 amino acids adopted several strategies (Table 2). First, since cancer cells Evidence-Based Complementary and Alternative Medicine 5 have higher anionic surface charges and are more sensitive 500 to melittin than normal cells then melittin can be diluted 400 to levels able to kill lung cancer cells in vitro, while normal ) 3

cells are unaffected [45].Second,Zhaoetal.[46]modified n.s the melittin chain by mutating Val 5 to Arg, Ala 15 to 300 Arg and deleting Leu 15 which significantly reduces the ∗∗ haemolytic properties but maintains its inhibitory effects. 200 Third, an alternative strategy involves using a synthetic ∗∗ Injection melittin peptide coupled to a delivery vehicle such as the beta (mm volume Tumor 100 chain of human chorionic gonadotropin (hecate-CGb). Cells with upregulated expression of hormone receptors, such as 0 ovarian, testicular, and adrenocortical tumours in mice, can 5791113 then be specifically targeted in vivo (e.g., [47]). Fourth, is Day after implantation (d) similar to three (above) but uses melittin linked to a specific homing peptide for hepatocellular carcinoma cells in vitro PBS control [48]. The importance of this study is that it identified a 𝛼-Peptide-NP control 𝛼 specific homing peptide for the cancer cells using a phage -Melittin-NP display technique for screening and identification of the novel (a) peptide. Fifth, by using gene therapy in which expression constructs carry the gene for melittin into tumours and induce antitumour effects and increased tumour latency49 [ ]. PBS Manyofthepreviousbioconjugatetechniques,however, control still induce some haemolysis of normal cells. The sixth, andfinalstrategy,isprobablythemostpromisingforthe 𝛼 therapeutic use of melittin. It involves using nanoparticles -Peptide- NP control to deliver melittin specifically to kill melanomas and other cancers in vivo with no cytotoxicity towards normal cells [50]. The nanoparticles were targeted to the tumours by 𝛼-Melittin- incorporating an avb3 integrin-binding ligand [3, 50]. The NP resultant reduction of the tumour load in the experimental mice was quite startling. This technology has been taken a step further since the nanoparticles used in the Soman et al. (b) [50] study were quite large (ca. 270 nm) and probably failed to ∗∗ penetrate solid tumours efficiently [51]. Thus, Huang et al. [51] 800 designed an ultrasmall, neutral charged, lipid nanoparticle n.s ∗∗ (ca. 20 nm) containing a hybrid 𝛼-melittin which inhibited the growth of the melanoma cells in mice in vivo by 82.3% 600 compared with the PBS controls (Figure 2). Apart from the role of melittin in killing cancer cells, 400 PLA2 and apamine in bee venom also have anticancer activ- ities. For example, venom PLA2 acting synergistically with the cell membrane phospholipid, phosphatidylinositol-(3,4)- Tumor weight (mg) weight Tumor 200 bisphosphate, has been shown to be involved in inhibition of tumour cell growth and potent cell lysis (detailed in [42]). Apamine too could potentially be developed as an 0 anticancer therapeutic agent since it reactivates the p53 PBS control 𝛼-Peptide-NP control 𝛼-Melittin-NP tumour suppressor pathway and would trigger the rapid elimination of tumours (reviewed in [42]). (c) Finally, the antimicrobial properties of melittin are well 𝛼 known and activity in vitro has been recorded against a range Figure 2: Evaluation in vivo of the effect of -melittin nanoparticles on the inhibition of melanoma development. (a) Tumor volume of microbes including not only Escherichia coli and Staphy- over time showing that only the 𝛼-melittin nanoparticle group was lococcus aureus but also Borrelia burgdorferi and Candida significantly inhibited. (b) Comparative sizes and (c) volumes of albicans [3]. Again, the cytolytic activity of this molecule the excised tumors between different groups after 13 days growth. ∗∗ for mammalian cells has been a barrier to its development Means ± SD, 𝑛=5, 𝑃 < 0.01. Reprinted with permission from C. as a therapeutic drug [3]. The insect antimicrobial peptides Huang, H. Jin, and Y. Qian et al., “Hybrid melittin cytolytic peptide- are discussed later in this review (see Section 6,“Antimi- driven ultrasmall lipid nanoparticles block melanoma growth in crobial Peptides” (AMPs)). Recent developments in the use vivo,” ACS Nano,volume7,number7,5791-5800,2013.(DOI: of melittin as an AMP have reported a synergistic effect 10.1021/nn400683s). Copyright (2013) American Chemical Society. 6 Evidence-Based Complementary and Alternative Medicine when melittin was combined with antibiotics against Gram- structure and composition [62]. Finally, silk can be produced positive bacteria even at concentrations as low as 0.5× MIC in aqueous solutions in order to avoid inactivation of the [52]. In addition, melittin loaded nanoparticle constructs associated drug or gene and the rate of delivery of which can have been shown to inhibit HIV-1 infectivity of TZM-bl be modulated by controlling the speed of degradation of the reportercells(astrainofHaLacells)but,atthesametime, silk vehicle [59]. to be nontoxic to these and to VK2 vaginal epithelial cells. Scientists have been developing both kinds of spider and Thus, melittin nanoparticle constructs have the potential to silkworm silk for potential uses in medicine. Silkworms silk be developed for use as topical therapeutic vaginal virucides is available in large quantities without recombinant methods [53]. necessary. However, interest persists in spider silk, despite thefactthatitisimpossibletodeveloplarge-scalefarming of spiders, due to the fact that spider silk is extremely strong, 4. Silk flexible, and tough and therefore particularly promising for the production of biomaterials. The toughness of silk is due Silkhasbeenproducedforatleast5,000yearswithnearly75% tothepresenceofnumerousinterlockingpoly-alanineand now originating from China [3]. In Chinese medicine, silk glycine-alanine subunits which strengthen the silk proteins has been used for a variety of human conditions including [63]. However, the spider silk proteins are long and this the relief of spasms and flatulence. Interestingly, silkworm hascausedproblemsinrecombinanttechnologydueto,for larvae have also been prescribed for treating impotence [54] example, the repetitive sequences inducing genetic instability only for, subsequently, a vasodilator compound enhancing [56]. Some of these problems with spider silk have, how- NO production to be extracted from Bombyx mori larvae and ever, been resolved, by various strategies. Thus, recombinant to be a candidate for the therapeutic treatment of vascular technologyhasbeenusedtoproducespidersilkinE. coli, impotency [54]. yeast, plants, and mammalian cells, as well as in the milk Interest in the medical or industrial use of silk is not of mice and goats, all of which present unique problems in confined to the silk produced by silkworms since many other execution [56]. One study even reports the use of piggyBac insects such as the Hymenoptera (bees, wasps, hornets, and vectors to create transgenic silkworms producing chimera ants) and the Trichoptera (caddis flies) [55], as well as the silkworm/spider silk proteins in which the composite fibres Arachnida (spiders) [56], produce silk. The macromolecular are as tough as native spider dragline silk [64]. structure of the silks from different arthropods varies accord- The recent work of Numata et al. [58, 59, 62, 65–67] ing to their function in the life of the animal. Basically, the indicates that rapid progress is being made in the develop- main structure of silkworm silk consists of fibroin protein ment of silk for use in medicine (Table 3). They have used fibres held together by a sticky protein called serecin. Boiling recombinant synthesis of spider silk in E. coli to produce B. mori cocoons remove the serecin glue to release the silk polymers which were then used for the production of fibroin fibres for subsequent processing (3). In the larvae of microspheres/nanoparticles and block copolymers for the bees, ants, and hornets, the silk produced has a coiled coil targeted delivery of drugs to cancer cells or to act as gene molecular structure, in contrast to other hymenopterans, as vectors [58, 59]. For example, nanoparticles enclosing cur- well as spider draglines (safety lines) and B. mori cocoons, cumin have been shown to be promising for treating breast in which the silk proteins form extended ß-sheets [57]. The cancer [59]. The block copolymers are engineered containing coiled coil silk proteins are small and ideal as structural silk with polylysine, for example, and cell-binding motifs materials to strengthen the walls of the brood comb cells. The such as RGD for targeting cells together with a therapeutic silk also absorbs water and maintains the high humidity and drug.Avariationofthecopolymeristoincludeplasmid constant temperature necessary for pupal development [57]. DNA for transfecting target cells with specific genes58 [ , 59]. Silkisnotprescribedinmodernmedicine;however,it More recently, further improvements have been made in the was used previously for medical sutures but now has been specificity of the silk polymer delivery system by introducing replaced by synthetic polymers. The ingenuity of science con- cationic motifs and tumour specific homing peptides and tinues to amaze with silk recently produced as biomaterials reducing the size of the silk carrier and the pDNA [65–67]. for the transport and delivery of dugs around the human body There are also numerous recent studies of the use of [58, 59] and for tissue engineering [60].Thisprogressinthe silk in tissue engineering with an enormously active group useofsilkresultedfromthepublicationoftheB. mori genome based in the Department of Biomedical Engineering at in 2008 [61] which led to gene cloning and modification to Tufts University working on both spider and silkworm silks. allow the expression of silk in a variety of vectors. It was then The work under Drs. Kaplan and Omenetto has looked possible to produce synthetic silk in different conformations, at the use of silk polymers for tissue engineering, vaccine such as scaffolds, films, and nanoparticles, for use in medicine production without the need for refrigerated storage, and [60]. cosmetic surgery. A number of start-up companies have What are the properties of silk that make it so attractive been spawned and the future prospects have great potential for use in medicine and which have fuelled recent intensive (see http://www.techtransfer.tufts.edu/tufts-silk-portfolio/). study? Silk is slow to biodegrade and biocompatible with Recent research from this group has reviewed the strategies the human body, although inflammatory responses have to produce spider silk by recombinant DNA [75]. In addition, been recorded [56]. In addition, silk has good self-assembly they have looked at silk-heparin biomaterials for vascular tis- properties and high tensile strength with manipulatable sue engineering [69], silk hydrogels for treating breast cancer Evidence-Based Complementary and Alternative Medicine 7

Table 3: Examples of potential use of silk biopolymers in medicine.

Form of Silk Potential Use References Numata and Kaplan [59]and (1) Nanoparticles Delivery of drugs to cancer cells Nitta and Numata [62] Numata et al. [58]andNumata (2) Co-polymer blocks Transfection of target cancer cells and Kaplan [59] (3) Small, globular units with tumour Improved tumour cell-specific transfection Numata et al.66 [ ] homing peptides (THP) (4) Nano-scale silk-based ionic complexes Further improved tumour cell-specific transfection Numata et al.67 [ ] with THP Forrepairofcartilage,bone,ligaments,tendons, (5) B. mori porous materials Zhang et al. [68] vascular tissue, nerves, corneas and as wound dressings (6) Silk-heparin support Vascular tissue growth application Seib et al. [69] (7) Silk hydrogels Treatment of breast cancer Seib et al. [70] (8) Antibiotic-loaded silk hydrogels Prevention and treatment of infection Pritchard et al. [71] (9) Electrically stimulated silk films Enhancement of neural growth Hronik-Tupaj et al.72 [ ] (10) Silk protein matrices Thermostabilisation of vaccines Zhang et73 al.[ ] (11) Vitamin-E loaded silk nanofibrous mats Skin tissue regeneration Sheng et al.[74]

[70], antibiotic-releasing silk biomaterials for infections [71], Lytta vesicatoria, supposedly have aphrodisiac properties and electrical stimulation of silk films for enhancement of neural were sold as a powder called “Spanish Fly” [3]. In fact, the growth and silk containing dressings for increased wound male beetle produces cantharidin and offers it to the female as healing [72], and silk protein matrices which thermostabilize a precopulatory incentive and she uses it to protect her eggs. labile vaccines and antibiotics [73]. The latter development There is increasing interest in the use of cantharidin and is very exciting and could potentially solve the problem itsderivativesforthetreatmentofarangeofcancersincluding of transporting vaccines to remote parts of Africa when hepatic, colorectal, bladder, breast, melanomas, pancreatic, vaccines against malaria are finally producedTable ( 3). In and leukemia [3]. The anticancer properties of cantharidin many of these studies, growth stimulating factors or drugs are result in arrest of the cell cycle in G2/M phase, apoptosis, and incorporated into the polymers and slowly released into the oxygen radical damage to DNA [79]. However, the potential target tissues [69–71]. of this small molecule and its derivatives in medicine is not Finally, many other studies have described the potential confined to their anticancer properties as they have also been useofsilkpolymersinmedicine[74, 76]. For example, Sheng reported to have activity against parasites such as Plasmodium et al. [74]usingvitamin-Eloadedsilknanofibrousmats falciparum and Leishmania major [80, 81]. showed enhancement of skin fibroblast growth, and therefore Cantharidin is a monoterpene (exo,exo-2,3-dimethyl- this technique can be developed for skin regeneration in the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid anhydride), future (Table 3). The FDA approval of silkworm silk for use stored in the beetle haemolymph and making up about in the human body has no doubt stimulated interest in this 5% of body dry weight [78]. Organic chemists have been exciting research area. working to produce derivatives which are bioactive but less toxic. In consequence, the norcantharidins have been produced with anticancer activity but reduced toxicity [78]. 5. Cantharidin from Blister Beetles and Other In addition, a new class of anticancer compounds, the Small Molecules cantharimides, has been discovered from a Chinese blister beetle, Mylabris phalerate, closely related to cantharidin but Blister beetles belong to the Coleopteran Family Meloidae with improved water solubility and toxicity against human which contains ca. 2500 species [77]. Many of these insects hepatocellular carcinoma cell lines [82]. An excellent account produce toxic defensive secretions which upon contact with of the strategies adopted to produce improved cantharidin the skin cause blistering. One such toxin is cantharidin which and cantharimide analogues is given in a review by Galvis has been extracted from Mylabris caragnae, the dried bodies et al. [78] and many of the derivatives described have higher ofwhichhavebeenusedinChineseFolkMedicinesince bioactivity and less toxicity. the13thcenturyfortheremovalofwarts[78]andforover Despite the development of less toxic analogues, there 2000 years for the treatment of cancer. Other uses include is still concern about the use of cantharidin in the clinical thetreatmentofrabiesandimpotencealthoughitishighly situation with trials mainly limited to external use on warts toxic affecting the gut and kidneys [3, 78]. The fatal dose, [3]. However, scientists have continued their research and causing renal failure, is between 10 and 65 mg and this toxicity now much more is known about the mode of action of has hindered cantharidin development as an anticancer cantharidin so that new strategies for drug administration drug [78]. In addition, the dried bodies of another beetle, are being developed. A recent limited clinical trial involving 8 Evidence-Based Complementary and Alternative Medicine combining cantharidin with chemotherapy for the treatment 120 of gastric cancer has been completed. The results showed 100 the beneficial effects of the cantharidin by a reduction of the serious side effects usually associated with chemotherapy for 80 gastric cancer [83]. 60 Research has also shown that cantharidin is an inhibitor of phosphoprotein phosphatases 1 (PP1) and 2A (PP2A) 40 Cell viability (%) Cell viability which results in DNA damage and apoptosis [78, 84]. These 20 enzymes are involved in regulation of metabolism and the 0 initiation of signal transduction in cells resulting in cell 0 24 48 72 division.Thus,cantharidinmayrepresentasmallmolecule able to switch cancer cells division and carcinogenesis off/on Time (h) as well as to probe the key regulatory role of PPA2 in cell 0𝜇M 20 𝜇M metabolism [78]. A detailed account of the interaction of 5𝜇M 40 𝜇M cantharidin analogues with PP1 and PP2A is given in Galvis 10 𝜇M et al. [78]. (a) Recently, a number of papers have been published show- ing that cantharidin, apart from inhibiting PP1 and PP2A, has multiple effects on cancer cells. Huang et al. [85]showed that growth inhibition and killing of human colorectal cancer cells by cantharidin was both time- and dose-dependent (Figure 3). The cantharidin exposure reduced CDK1 kinase activity which led to failure of the cells to progress from G2 to M phases in the cell cycle. In addition, the colorectal Control Cantharidin cellswerekilledbyapoptosiswhichwasinducedthroughthe (20 𝜇m) mitochondrial and death receptor pathways and activation of (b) caspases 8, 9, and 3 (Table 4). Another study by Huang et al. [86]onmetastasisof Figure 3: Effects of cantharidin on cell viability and morphological human bladder carcinoma cells, showed that exposure to changes of human colorectal cancer cells. (a) Cells treated with 0, 𝜇 cantharidin blocked the gene expression, protein levels, 5, 10, 20, or 40 M cantharidin for 0, 24, 48, and 72 h and then and activities of the matrix metalloproteinase-2 (MMP-2) harvested for determination of cell viability. (b) Cells exposed to 20 𝜇M cantharidin for 24 h and then examined for morphological and/or MMP-9. These enzymes are associated with invasive changes under phase-contrast microscopy. Data represent the mean properties of many cancers so that cantharidin had an ± SD of three experiments. From Huang et al., “Cantharidin antimetastatic effect possibly by targeting the p38 and JNK1/2 induces G2/M phase arrest and apoptosis in human colorectal MAPKs pathway of the bladder cancer cells. Other effects of cancer colo 205 cells through inhibition of CDK1 activity and cantharidinhavebeenstudiedinhumanbreastcancercells caspase-dependent signaling pathways,” International Journal of by Shou et al. [87]. They reported that cantharidin resulted Oncology, volume 38, pp. 1067-1073, 2011. Reprinted with permission in apoptosis and reduced growth, adhesion, and migration of of Spandidos Publications 2013. the cancer cells. The reduced adhesion resulted from repres- sion of cell adhesion to platelets through downregulation of the 𝛼2integrinadhesionmoleculeonthesurfaceofthecancer 𝛼 form of the PP2A catalytic subunit. Finally, and most impor- cells. The repression of the 2integrinoccurredthroughthe tant for therapeutic use of cantharidin, Dang and Zhu [89] protein kinase C pathway probably due to PP2A inhibition have tackled the problems of toxicity, insolubility, and short (Table 4). half-life in circulation of this drug by designing cantharidin Three further studies indicate novel approaches in the solid lipid nanoparticles as drug carriers which can be given use of cantharidin. Lissina et al., [79]inachemical- orally (Table 4). genomicsstudy,showedthatcantharidinisaneffectivegene probe of transcriptional regulation of the CRG1 gene, an uncharacterised methyltransferase, during cantharidin stress. 6. Antimicrobial Peptides (AMPs) Therefore by using such small molecules the authors showed The dried bodies and secretions of insects have been widely how it was possible to elucidate unknown mechanisms used in Folk Medicine to treat numerous diseases and of therapeutic action in cells involving, for example, the illnesses including many different types of infections and methyltransferase. Li et al. [88]haveusedtheknowledge cancers [1–3]. In Chinese Medicine, numerous species of of the inhibition of PP1 and PP2A by cantharidin, and the insects have been used to treat cancer [2]. Considering that resulting apoptosis of cancer cells, to design a new gene many insects thrive in inhospitable environments teeming therapyapproachtokillhepatocellularcarcinomacells.They with microorganisms, such as dung or rotting corpses, it is inhibited PP2A using the 𝛼-fetoprotein promoter enhancer notsurprisingthattheyhaverobustimmunedefencesto linked to the pgk promoter to drive the dominant negative counter infection. These insect innate immune defences have Evidence-Based Complementary and Alternative Medicine 9

Table 4: Examples of recent studies on the use of cantharidin.

Cells treated Results References Reduced CDK1 kinase activity, apoptosis induction (1) Human colorectal cancer cells through mitochondrial and death receptor pathways, Huang et al. [85] and activation of caspases 8, 9, and 3 Blocked activities of matrix metalloproteinase-2 (2) Human bladder carcinoma cells (MMP-2) and/or MMP-9 resulting in an antimetastatic Huang et al. [86] effect Reduced adhesion and migration by repressed cell (3) Human breast cancer cells adhesion to platelets by downregulation of 𝛼2integrin Shou et al. [87] adhesion molecule Details of transcriptional regulation of the CRG1 gene (4) Yeast CRG1 (cantharidin resistance Lissina et al. [79] gene 1) for methyltransferase, during cantharidin stress A new gene therapy approach to kill hepatocellular carcinoma cells by inhibiting PP2A with the (5) Hepatocellular carcinoma cells 𝛼-fetoprotein promoter enhancer linked to the pgk Li et al. [88] promoter Design of cantharidin solid lipid nanoparticles as drug (6) Normal rats Dang and Zhu [89] carriers which can be given orally both cellular and humoral components [90, 91], but it is negatively charged bacteria and tumour cell surfaces, whilst the humoral antimicrobial peptides (AMPs) that are of most neutrally charged normal cells are unaffected [3]. They are interest for the development of new antibiotic drugs. also amphipathic in their folded state with hydrophilic and Insect AMPs have been actively researched for over 50 hydrophobic regions mediating their solubility in phospho- years and in 2011 work involving these molecules led to the lipid cell membranes. These interactions of the AMPs result Nobel Prize for Physiology and Medicine being awarded to in their membrane disruptive properties which characterise Jules Hoffmann and Bruce A. Beutler for their discovery of these molecules [99]. Most of the insect AMPs are freely the Toll receptors and mechanisms of activation of innate circulating or associated with the gut or other epithelia immunity. Their work did much to increase interest in AMPs and often placed strategically at external openings on the which have recently been the subject of extensive reviews [92– body to combat infection [3]. Some AMPs are constitutively 96]. This interest has also been fuelled by the urgent need to combat the ever increasing number of antibiotic-resistant expressed but the majority is rapidly induced following pathogens such as MRSA, TB, and gonorrhoea. Despite this exposure to would-be invaders. Any one insect can produce urgency, and the length of time AMPs have been studied, multiple AMPs which enable it to differentiate between very few of these molecules have undergone clinical trials invading organisms and to respond selectively. Many of the or,thosethathave,failedtocompletethetrials[97]. There venom proteins such as melittin, described in Section 3.2, are many reasons for the slow development of AMPs into above, are also polypeptides with amphipathic and cationic new therapeutic drugs and these are discussed in detail below properties but are highly toxic and confined to venom sacs to together with recent progress in this area. combat other predatory insects and animals. Insect AMPs can be classified into 3 groups3 [ ], although 6.1. Basic Characteristics of Insect AMPs. The LAMP 2013 4 or 5 groups have also been recognised [92, 93]. database, links information on AMPs and holds 5547 AMP (1) Linear 𝛼-helical AMPs, which in insects include the sequences of which 3904 are natural AMPs, while the other cecropins, moricin, sarcotoxin, and melittin, are present in a 1643 are synthetic peptides [97]. Interestingly, of the 5547 wide range of insect orders, including coleopterans, dipter- AMPs, 5362 have antibacterial activity, 1616 antiviral, 1579 ans, and lepidopterans. Cecropins are active against Gram- antifungal, 138 antitumour, and 14 antiparasitic activities. The positive and Gram-negative bacteria, viruses, protozoans, amino acids composing these AMPs range from 4 to 99 in fungi, nematodes, and tumour cells [3, 100]. Cecropins are number [97].InsectnaturalAMPspreviouslyidentifiedare promising anticancer drugs when combined with melittin estimated as 400–500 in number [3]. AMPs are produced by bacteria, fungi, numerous invertebrates, vertebrates and (see Section 3.2)orwithchemotherapyagentstoreduce plants, and are usually associated with killing microbes their toxic side effects101 [ ]. Also, overexpressed defensin A although they may also be involved in wound repair, inflam- andcecropinAgenesintransgenicAedes aegypti blocked mation, development, chemotaxis, and cytokine activity (e.g., the transmission of Plasmodium gallinaceum [102]. Recently, [95, 96, 98]). the potential development of engineered cecropin A-melittin Insect AMPs are mainly cationic (although anionic forms analogues and other AMPs as drugs against protozoan do exist) which facilitates their binding electrostatically to parasites such as Leishmania has been reviewed [103]. 10 Evidence-Based Complementary and Alternative Medicine

(2) Linear proline or glycine-rich AMPs include drosocin, models. These models have been extensively reviewed pre- apidaecin, formaecin, and pyrrhocoricin. These are short, viously (e.g., [92, 99, 108, 109]). In the barrel stave model, proline-rich with specific intracellular targets in bacteria, clusters of 𝛼-helicalAMPsareinsertedinthemembranelike while mammalian cells are unaffected [3]. They generally a barrel with the staves (strips of wood forming the wall) target Gram-negative bacteria such as Escherichia coli and forming a transmembrane pore. The hydrophilic side groups kill over several hours, in contrast to the other two groups of of the AMPs line the aqueous pore, while the hydrophobic AMPs which kill rapidly. The bacterial target of the proline- tails of the phospholipid membrane fatty acids interact with rich AMPs is believed to be the intracellular chaperone the nonpolar side groups of the AMP. In the carpet model, DnaK [104]. Ostorhazi et al. [104] synthesised a proline-rich, the AMPs attach parallel with the membrane, form a carpet designer peptide, A3-APO, and showed its efficacy against resulting in holes in the membrane which then, at a critical multidrug resistant bacterial infections in the wounds and concentration, collapses. With the toroidal model, the AMPs lungs of mice. A3-APO upregulated the expression of the areinsertedperpendicularintothemembranetoformpores antiinflammatory cytokines interleukin-4 and interleukin- lined by the AMPs and the lipid head groups. Finally, with the 10 so that wounds lacked pus [104]. A shortened version ion channel forming model, the AMPs bind to the polar head of apidaecin, Api88, has similar activity to A3-APO against groups, insert into the membrane, aggregate, and span the pathogenic E. coli butisunstableinserum.Simplysubstitut- membrane to form pores through which ions leak from the ing Arg-17 with l-ornithine increased, by more than 20-fold, bacterialcell[93]. The same membanolytic activities of AMPs the serum stability of Api88 [105]. would apply to their killing of anionic cancer cells [100]. (3) Cysteine-stabilised AMPs are small cationic peptides Evidence, however, is accumulating that the activity of with 33–46 amino acids, and are stabilised by cysteine AMPs is probably not confined to cell membrane lysis. residues forming disulphide bridges [3]. They are com- Thus, AMPs may disrupt mitochondrial membranes, inhibit mon in most insects and include defensins or defensin- cell wall synthesis, inhibit DNA synthesis, inhibit protein like compounds such as gallerimycin, heliomycin, sapecins, synthesis, interact with membrane receptors and heat-shock drosomycin, spodoptericin, and phormicins [106]. They are proteins, and have antiangiogenesis effects [92, 99, 100]. mainly active against Gram-positive bacteria and fungi but One example is apidaecin which kills Gram-negative bacteria are also antiparasitic [103]. The defensins, like the cecropins without forming pores and interferes with protein synthesis and analogues of pyrrhocoricin, have considerable potential [99]. for development as drugs since short synthetic forms of insect defensins inhibit MRSA and disrupt myeloma cancer cells [3]. 6.3. Therapeutic Use of AMPs. AMPs have great potential for Furthermore, in mammals, defensins have the dual activities development as new classes of antibiotics for a number of of killing bacteria as well as modulating the immune response reasons. by recruiting and activating immune cells [107]. Invertebrate defensins exhibit high affinity binding to the bacterial cell (a) There is a huge variety, targeting a broad range of wall precursor, lipid II, and inhibit its incorporation into the microorganism and cancer cells and lending them- peptidoglycan network [107]. selves to synthetic improvement. (b) They seem to have multiple targets and do not 6.2.KillingMechanismsofAMPs. Thisisconsideredbrieflyas generally rely on specific receptor binding so that it is relevant to understanding the therapeutic use of AMPs. development of resistance by bacteria is difficult. The cationic AMPs are amphipathic with a net positive charge (c) They generally kill rapidly and within minutes while with large numbers of cationic amino acids such as argi- conventional antibiotics usually take hours. nine, histidine, and/or lysine and also contain hydrophobic residues [95]. The prevalent negative net charge of bacterial (d) They can kill antibiotic-resistant bacteria such as membranes due to the composition of their phospholipids MRSA as well as cancer cells. (predominantly with negative charge) plays a major role (e) Their antimicrobial activity occurs even with low in the attraction of the cationic AMPs, while membranes micromolar, concentrations. of eukaryotic cells enriched in zwitterionic phospholipids (f)Theymayhavedualeffectstokillmicrobesandto andcholesterolarerefractorytotheAMPs.Thecationic modulate the immune system. AMPs bind to the anionic residues of the outer bacterial (g) They can destroy biofilms on medical devices even envelope, which include the lipopolysaccharides of Gram- when used at low concentrations. negative bacteria and the lipoteichoic acids of Gram-positive forms [93]. Binding to the outer bacterial cell membrane does Despite the advantages of AMPs, progress to date in not, in contrast to antibiotics, involve specific receptors for developing them for clinical use has been disappointing. The the AMPs so that it is more difficult for the bacteria to mutate main advance has been with vertebrate AMPs for use in topi- andevolveresistancetotheAMPs[93]. This binding results cal applications and a few AMPs have entered clinical trials in disruption and permabilisation of the outer bacterial cell [3, 92, 108, 110]. These AMPs were designed for a number membrane and eventually microbial death. of external uses such as skin care, acne, eye infections, and There are a number of models for the mechanisms catheter-related pathogens. involved in bacterial membrane disruption. These include the Therehavebeenanumberofreasonsforthisslow barrel stave, the carpet, the toroidal, and ion channel forming progress in AMPs becoming available for clinical use. Evidence-Based Complementary and Alternative Medicine 11

(1) Lack of interest by large pharmaceutical companies compounds against clinically isolated, antibiotic resistant for many years. Thus, between 1998 and 2004, of the 290 pathogens at low MICs of <10 𝜇g/mL [117]. Further advances new antibacterial drugs under development, only 4 involved willoccurinisolatingandsynthesisingactiveAMPswith the major pharmaceutical companies [3]. This attitude is the introduction of new discovery pipelines utilising in silico now changing for various reasons including the emergence, designed AMP-encoding oligonucleotide libraries [118]and inthelast10years,ofmoreandmoreantibiotic-resistant advanced quantitative structure-activity relationship (QSAR) bacteria and fewer and fewer antibiotics available to treat models [119]. these pathogens. In addition, research has revealed the dual Regarding development of resistance of bacteria to AMPs, function of some of these AMPs with the ability not only to this was thought to be less likely to occur than with con- killmicrobesbutalsotomodulatetheimmunesystem[111]. ventional antibiotics as AMPs may have multiple sites of No doubt this fact has not escaped the attention of the phar- action within the bacterial cell and involve fundamental maceutical companies with the potential of developing new changes in the membrane (e.g., [120]). This view, however, classes of drugs able to control immune reactivity. In addition, has been shown to be over optimistic as reports have much progress has also been made in understanding the appeared of resistance to AMPs [3], including resistance functioning of the AMPs which can be produced at much to insect melittin and cecropin [121, 122]. In addition, the lower costs than their natural counterparts [95]. The more evolution of resistance to a cationic AMP has been shown effective delivery of AMPs by gene therapy or nanoparticles through continual selection in the laboratory [123]although is also being developed and will enhance the therapeutic conditions in nature are very different. Thus, each host will potential of AMPs ([50, 51], see Section 3.2) and again raise contain a different range of AMPs in the various tissues of the interest in this rapidly evolving subject. body. This point is emphasised by Chernysh and Gordja124 [ ] (2) High production costs have always been a major who prepared a Calliphora vicina maggot peptide complex hurdle to development of the AMPs since they only occur called FLIP7 (from Fly Larvae Immune Peptides) containing at low concentrations naturally and the cost of solid phase cecropins, defensins, diptericins, and a proline-rich peptide synthesis is very high [3]. Advances, however, are rapidly and compared its ability with the antibiotic, meropenem, to being made to reduce manufacturing costs. Thus, numerous kill a multiresistant strain of Klebsiella pneumonia over many reports describe the synthesis of truncated synthetic ana- generations. The results showed that after 25 passages with logues with enhanced killing activities and with potential the meropenem, the resistance of the bacteria was increased for cheaper production costs. For example, Ausbacher et al. 128 times, while there was no change in resistance towards 2,2 [112] designed a series of small antimicrobial 𝛽 -amino acid the C. vicinia FLIP7 complex. Thus, although resistance to derivatives of Mw < 500, with potent activity against both AMPs can occur, this should not deter their development MRSA and cancer cells, and Gaspar et al. [100] also described as therapeutics but their widespread use should be carefully a number of short, synthetic peptides for use against different regulated [123]. The use of hybrid molecules constructed, types of solid tumours. These latter peptides included four for example, from cecropin and melittin or cecropin and enantiomeric AMP analogues (D-peptides A, B, C, and D) rifampicin should also help to solve this problem [125]. designed from beetle defensins [113]. An alternative strategy In conclusion, the future of the development of AMPs as to reduce costs of mass production is to use recombinant new classes of drugs for killing antibiotic resistant bacteria technology but this has been hindered by the antibacterial and cancer cells looks very bright. The versatility of these activity of the AMPs and their proteolytic degradation during potential drugs seems to increase daily with, for example, production [114]. Recent studies, however, have used cost recentreportsoftheuseofAMPstocoattitaniumbone effective, modified recombinant techniques with E.coli or implants to prevent infection [126]andtheinhibitionof with the methylotrophic yeast Pichia pastoris as vectors, to biofilm formation by these compounds [125]. produce fully functional insect cecropins capable of killing a range of bacteria, including MRSA [115, 116]. Finally, regulatory rules governing the required performance prior 7. Maggot Molecules to approval for the release of drugs in the USA (FDA) MaggotshavebeenusedforwoundhealinginFolkMedicine and Europe have all to be navigated at extra cost [3]. It is by the aborigines and Mayan Indians for thousands of noteworthy that much of the significant research on AMPs years. Maggots for cleaning wounds also occurred in the is now being conducted in China. Napoleonic and American Civil Wars [3, 127]. However, (3) There are also concerns about the stability and maggot therapy only obtained wider recognition for treating toxicity of the AMPs towards mammalian cells [93, 94] infected wounds after its introduction into USA hospitals as well as the development of bacterial resistance to these in the 1920s by Professor William Baer at John Hopkins molecules. However, as a result of better understanding University. By the 1930s and 1940s over 300 USA hospitals of the structural-functional relationships of AMPs and the usedthisprocedureandithadalsospreadtoEurope[3, introduction of computer modelling, it is now possible to 127].Inthe1940s,however,thenewlydiscoveredantibiotics design and synthesise AMPs with increased stability in soon dampened the enthusiasm for maggot therapy and serum and saline, no toxicity, and greater killing activities onlytheappearanceofantibiotic-resistantbacteriainthe [3, 117]. These AMPs have been produced by amino acid 1980s rekindled interest in this procedure. Maggot therapy substitutions, sequence splicing, and changes in ratios of is now commonly used for many types of infected wounds hydrophobic amino acids to produce truncated designer such as diabetic foot wounds, postoperative infections, bed 12 Evidence-Based Complementary and Alternative Medicine

Table 5: Summary of factors/processes involved in maggot therapy of infected wounds.

Factors/processes mediated by maggot Effect on wound References extracts and secretions Debridement (1) Maggot proteases Digest wound debris Chambers et al. [132] Digest DNA of debris and infecting (2) Maggot DNase Brown et al. [133] bacteria in biofilms (3) Maggot glycosidases Digest wound debris Telford et al. [134] Wound healing (4) Specific amino acids Induce mitosis in endothelial cells Bexfield et al.[135] (5) Maggot fatty acid extracts Activate angiogenesis Zhang et al. [136] (6) Neutrophil migration inhibition Resolves inflammation van der Plas et al.[137] (7) Macrophage migration inhibition Resolves inflammation helped by van der Plas et al. [138] and TNF-𝛼. increased IL-10 Resolves inflammation helped by (8) Anti-inflammatory macrophages bFGF and VEGF cytokines inducing van der Plas et al. [139] increased mitosis and angiogenesis Inhibits adaptive immunity to maggot (9) Lymphocyte activation suppressed Elkington et al. [140] proteins Inhibits complement action against (10) Reduced complement activation Cazander et al. [141] maggot proteins Wound disinfection Active against gram-positive bacteria, Ceˇ ˇrovskyetal.[´ 142] (11) Maggot lucifensin for example, MRSA Andersen et al. [143] (12) Maggot alloferons Antiviral and antitumour activities Chernysh et al. [144] Active against gram-positive and (13) Maggot seraticin Bexfield et al.145 [ ] gram-negative bacteria sores, and leg ulcers, in the USA, Israel, and Europe [3, 127].ItisestimatedtohavesavedtheNHS,UKover500 million pounds. The larvae of the blowfly, Lucilia sericata, arefrequentlyused(Figure 4)althoughotherspecieshave also been tried such as Lucilia cuprina, Phormia regina, and Calliphora vicina [127]. Thus, a wide spectrum of dipteran species has potential as sources of new medicinal drugs, especially since the larval stage of L. sericata can kill MRSA [128].Recentreviewsofmaggottherapyprovidemoredetails of the processes involved [129–131]. The use of L. sericata larvae for treating wounds has been recognisedbytheU.S.FoodandDrugAdministrationand the UK Prescription Pricing Authority. Sterile maggots can therefore be officially prescribedhttp://www.medicaledu ( .com/maggots.htm). Figure 4: Showing newly washed final instar Lucilia sericata larvae Maggot therapy can be divided into 3 processes: prior to incubation for production of extracorporeal secretions. Photograph by kind permission of Mr. I.F. Tew, Swansea University. (i) debridement of wounds; (ii) wound healing; (iii) disinfection of wounds. debris which the maggots then feed upon [3]. Initially, the main enzymes identified in the maggot excretions/secretion 7.1. Debridement of Wounds. Once maggots are applied to (ES) were chymotrypsin- and trypsin-like serine proteases, the wound then debridement or cleaning and removal of an aspartyl proteinase and a metalloproteinase [132]. The necrotic tissue and debris (eschar) occur so that granulation secretion of ammonia by the maggots increases the pH to and healing can begin. Maggots clean wounds by the extra- activatetheserineproteases.Themostactiveenzymesare corporeal production of enzymes (Table 5)thatdigestthe produced by first instar larvae [132]. Evidence-Based Complementary and Alternative Medicine 13

More recent work from the Pritchard group in Notting- of proinflammatory cytokines such as migration inhibitory ham University has revealed more information about the factor and TNF-𝛼. At the same time, the production of the maggot proteases and also detected other enzymes present in anti-inflammatory cytokine IL-10 is increased so that the ES the MS. First; the L. sericata chymotrypsin I is resistant to the appear to be reducing the inflammatory response [138]. In endogenous wound protease inhibitors, 𝛼1-antichymotrypsin addition, in the presence of ES, macrophages develop into and 𝛼1-antitrypsin, present in eschar and which could poten- anti-inflammatory rather than proinflammatory forms [139]. tially inhibit debridement [146]. In contrast, mammalian 𝛼- The anti-inflammatory macrophages suppress inflammation chymotrypsin is inhibited by these enzymes so that maggot and secrete basic fibroblast growth factor (bFGF) and vascu- chymotrypsin I can survive in the wound to undertake lar endothelial growth factor (VEGF) which mediate mitosis debridement, whilst the mammalian enzyme cannot. Second, and migration of endothelial cells resulting in angiogenesis theMShavealsobeenshowntocontainaDNaseableto and eventual healing of wounds [157]. Recently, these results degrade genomic bacterial DNA, the extracellular bacterial were confirmed by applying ES to acute skin wounds made in DNA in preformed biofilms from a Pseudomonas aeruginosa rats since levels of the acute inflammatory cytokines, TNF- clinical isolate, and DNA from the slough/eschar of a venous 𝛼 and IL-6, remained significantly lower than in the rats leg ulcer [133]. This DNase must make a valuable contri- with untreated wounds [157]. Lymphocytes activation too is bution to debridement and healing by clearing tissue DNA inhibited by the ES so that the wound site would be protected as well as that of biofilms, thus freeing tissue protein for from the induction of an adaptive response to the maggot digestion by the ES proteases [133]. Third, the ES also contain proteins [140]. glycosidases which would remove sugars from the wound Even more interesting is the study by Cazander et al. [141] debris and contribute to the debridement process [134]. All whohaveshownthatEScouldreducecomplementactivation these enzymes (Table 5) in the ES remove the extracellular by 99.99% in the sera of healthy and postoperatively immune- matrix debris, fibrin clots, and any biofilms associated with activated human patients. The ES break down complement infecting bacteria and allow healing to begin [147–150]. All components C3 and C4 which could explain, in part, the the above work was undertaken in L. sericata but recent work improved wound healing following maggot therapy (Table 5). investigating the potential of another calliphorid species, Sarconesiopsis magellanica,foruseinmaggottherapyhas shown that larval ES of this insect also contain trypsin-like 7.3. Disinfection of Wounds. There is good evidence that serine proteases [151]. ES can kill bacteria infecting wounds, including antibiotic- Pritchard and colleagues have applied their findings to resistant strains such as MRSA [3]. There are reports of many develop hydrogel bandages containing these enzymes in different antibacterial factors in dipterans, including a range order to accelerate debridement and healing processes [152]. of AMPs such as Sarcotoxin 1A, a cecropin-like molecule Recently,theyhavemadeadditionalprogressbyproducing from the flesh fly Sarcophaga peregrine, which is more active a recombinant chymotrypsin I, using good medical practice against Gram-negative bacteria than Gram-positive forms guidelines, which successfully digested wound debris and is [158]. However, focus is now on calliphorid flies used in now available for clinical trials [153, 154]. wound healing in which one AMP, lucifensin (Table 5), has received particular attention recently as it is active against 7.2. Wound Healing. There is no doubt about the benefits of clinically relevant bacteria such as Streptococcus species (e.g., maggots in debriding chronic wounds but the outcome of [143]). Most of the other antibacterial factors described from < clinical trials on their use in wound healing is more uncertain calliphorids are 1300 Da in size [3], although Altincicek and [155]. The ES enzymes or other constituents have been shown Vilcinskas [149], and Andersen et al. [143]haveshownthatL. to activate the fibroblasts [156] and evidence is accumulating sericata has 65 immune-inducible genes including lysozyme- for an active role for ES in wound healing. Thus, specific and transferrin-likegenes and 3 proline-rich AMPs. amino acids derivatives and fatty acids extracts (Table 5)from Lucifensin was first purified in 2010 from an extract of L. sericata ES induce mitosis in human endothelial cells and the gut of L. sericacta larvae by Ceˇ ˇrovskyetal.[´ 142]. They activate angiogenesis and wound healing [135, 136]. showed that the peptide contained 40 amino acid residues In addition, there is accumulating evidence that ES have and 3 disulphide bridges and was a typical 4 kDa dipteran an immunomodulatory role in the wound healing process defensin. Subsequently, Andersen et al. [143] published the (Table 5)andthishasbeenreviewedindetailpreviously[131]. primary sequence, and Ceˇ ˇrovskyetal.[´ 159]chemically In particular, neutrophils, macrophages, lymphocytes, and synthesised lucifensin to provide material for a structural- the complement system respond to exposure to the MS. With activity study. More recently, lucifensin II was discovered and neutrophils, the ES inhibit elastase, the respiratory burst, characterised from Lucilia cuprina and found to be identical hydrogen peroxide production, and migration of these cells. to the L. sercata lucifensin except for one amino acid residue Elastase breaks down the extracellular matrix and delays [160]. Thus, lucifensins are cationic AMP with main activity epithelial repair, while oxygen radicals would probably have against Gram-positive bacteria [143] so that, together with a similar effect. Concomitantly, the inhibition of neutrophil seraticin (see below), they make an important contribution migration would help resolve the prolonged inflammatory in the ES to cleaning infected wounds of MRSA and other response, to which they contribute, present in a chronic antibiotic-resistant bacteria. This antibacterial activity occurs wound [131, 137]. Macrophages are similarly affected by the even at physiological salt levels [138]. Lucifensin is present ES and show reduced migration and inhibited production in the gut, fat body, and haemolymph of L. sericata and 14 Evidence-Based Complementary and Alternative Medicine appears to be constitutively expressed [142, 160]. In addition, involved [145, 170]. Subsequently, further fractionation of in L. sericata orally challenged with bacterial isolates from maggot secretions revealed a fraction of <500 Da active wounds, only in the fat body is there an increase in lucifensin against S. aureus,10strainsofMRSA,andanumberofGram- expressionsothatlevelsintheESremainunchanged[161]. positive and Gram-negative bacteria [128]. This <500 Da Lucifensin has also been studied in a detailed structural fraction, named “seraticin,”has been the subject of additional analysis by NMR [162]. It seems possible that it has two research, due to its inhibition and killing of clinical strains mechanisms of antimicrobial activity against the bacterial cell of MRSA and Clostridium difficile, and has been isolated and and interacts both with the bacterial membrane and binds characterised, and an empirical formula was calculated. Mass to the cell wall precursor, lipid II [162]. Finally, interest has spectrometry and NMR studies have been carried out and a also increased in the antibacterial factors of the house fly, synthesis has produced fractions having similar antimicrobial Musca domestica, because of its possible role as a vector of properties to the native seraticin molecule. A <1000 Da pathogens such as MRSA [163, 164]. Results show that these molecule, active against MRSA, and from sterile L. sericata insects also produce a defensin that is upregulated upon larvae, has also been reported previously [171]. Unfortunately, bacterial ingestion and that this, and probably other factors, lack of funding seriously delays the development of research is responsible for the antibacterial activity against MRSA and and commercialization of such interesting and potentially VRE (vancomycin-resistant enterococci) recorded for solvent important new medicines, especially if derived from such extracts of maggots [164]. unfashionable sources as fly maggots. As far as the calliphorid low weight antibacterial factors are concerned, there are two sets of molecules for develop- 8. Insect Anticoagulants ment as new medicinal drugs, namely, the alloferons and seraticin (Table 5). The anticoagulants in the salivary glands of blood suck- Two alloferons were originally isolated from the ing ticks and insects such as the Hemiptera, Diptera, haemolymph of Calliphora vicinia by Chernysh and Siphonaptera, and Anoplura have tremendous potential for colleagues [144] and are peptides with amino acid development of new anticoagulants and immune modulating sequences of HGVSGHGQHGVHG (alloferon 1) and medicines [3]. In fact, extracts of the salivary glands of GVSGHGQHGVHG (alloferon 2). Synthetic alloferon in horseflies have been used for centuries in Eastern Medicine in vitro tests stimulated natural killer cells, while in vivo as anticlotting agents [3]. Progress, however, has been made interferon was induced in mice. There were also indications in identifying and commercialisation of such invertebrate of antiviral and antitumour activities [144] with alloferon also anticoagulants not from insects but from leeches and ticks showing moderate tumoristatic and tumoricidal activities [1, 3]. In leeches, recombinant derivatives of hirudin have in transplanted tumours in mice [165]. More recently, a been made available commercially for some years in Europe derivative of alloferon, allostatin, has been shown to have a and the USA [172] with the approval of the FDA. With ticks, significant adjuvant effect in vaccination experiments against intense research is underway on the bioactive substances tumour cells in mice [166]. Clinical studies by Ryu et al. [167] produced by their salivary glands and a variety of molecules subsequently showed that alloferon activates immune cells with diverse functions have been described with potential use through the NF-kappaB signalling pathway. The Allopharm as pharmaceuticals (reviewed in [173]). Attention has been Company was then formed in Russia and Allomedin was focusedonticksprobablyduetothevarietyofpathogens marketed in 2005 for the treatment of genital herpes, cold vectored by these animals [173]. sores, and gingivitis [3]. A number of detailed structural- Regarding insects, much less is known about the anti- functional studies have been undertaken of alloferon by coagulants in their salivary glands possibly due to the sythesising analogues with amino acid substitutions in sheer numbers of protein families produced in these glands position 1, for example, in the peptide chain [7, 168]. Some [174]. However, a recent thorough analysis of the structure of these analogues extended the antiviral properties of the and function of thrombin inhibition by anophelin in the native molecules so that they inhibited not only human salivary glands of Anopheles mosquitoes has been made and herpes virus 1 but also coxsackievirus multiplication in discovered a unique thrombin inhibition mechanism [175]. vitro. Another study has shown the therapeutic potential of Thrombin is an atypical (chymo) trypsin-like enzyme, with alloferon for the treatment of Kaposi’s sarcoma, caused by the a narrow active site cleft for specific substrate recognition Kaposi’s sarcoma-associated herpesvirus, and a characteristic and also has secondary recognition surfaces (exosites) [175] condition in HIV patients [169]. In contrast to other natural bivalent inhibitors of thrombin Seraticin is present in the MS of sterile L. sericata which bind to one of the thrombin exosites through their larvae [128, 145]. The MS has antibacterial activity against C-terminals, anophelin shows reverse binding to an exosite both Gram-positive and Gram-negative bacteria including S. by means of the N-terminal and the C-terminal binds to aureus,MRSA,Bacillus thuringiensis, E. coli, P. ae r ug ino s a , theactivesiteasshowninFigure 5 [175]. The significance and Enterobacter cloacae.Thefactthatmaggotsamplescol- of this finding is that it imparts anophelin with potent lected with the highest pH also had the highest antibacterial inhibitory properties as well as high resistance to proteolysis activity probably eliminates phenylacetic acid, produced by by thrombin and this may have implications for the design of the commensal, Proteus mirabilis, as the source of the factor novel antithrombotics [175]. Evidence-Based Complementary and Alternative Medicine 15

C-term

II II

I I

N-term

N-term C-term

PAR1 FV/FVIII (a) (b)

C-term N-term II II

I I

N-term

C-term

Hirudin Anophelins (c) (d)

Figure 5: Comparison of mechanism of thrombin recognition by anophelin with other substrates and inhibitors. Thrombin is represented by an orange ellipse, with exosites in blue and the active site in red. Anophelin inhibitor binds to thrombin in a reverse orientation relative to the other molecules, such that the N-terminal portion recognises exosite I, whereas the C-terminal acidic segment binds to the active site of thrombin. From original Figure 4 of Ana C. Figueiredo, Daniele de Sanctis, Ricardo Gutierrez-Gallego,´ Tatiana B. Cereija, Sandra Macedo- Ribeiro, Pablo Fuentes-Prior, and Pedro Jose´ Barbosa Pereira, “Unique thrombin inhibition mechanism by anophelin, an anticoagulant from the malaria vector” which appeared in Proceedings of the National Academy of Sciences of the United States of America.” Volume, 109, Issue 52, pages E3649 to E3658, 2013 (doi: 10.1073/pnas.1211614109). Reprinted with permission of PNAS.

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Edwin L. Cooper,1 Kyle Hirabayashi,1 Kevin B. Strychar,2 and Paul W. Sammarco3

1 Laboratory of Comparative Neuroimmunology, Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1763, USA 2 Annis Water Resources Institute, Grand Valley State University, Muskegon, MI 49441-1678, USA 3 Louisiana Universities Marine Consortium (LUMCON), 8124 Highway 56, Chauvin, LA 70344-2110, USA

Correspondence should be addressed to Edwin L. Cooper; [email protected]

Received 5 November 2013; Accepted 23 December 2013; Published 13 March 2014

Academic Editor: Tung-Sheng Chen

Copyright © 2014 Edwin L. Cooper 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.

Over the last few years, we have pursued the use and exploitation of invertebrate immune systems, most notably their humoral products, to determine what effects their complex molecules might exert on humans, specifically their potential for therapeutic applications. This endeavor, called “bioprospecting,”is an emerging necessity for biomedical research. In order to treat the currently “untreatable,” or to discover more efficient treatment modalities, all options and potential sources must be exhausted so that we can provide the best care to patients, that is, proceed from forest and ocean ecosystems through the laboratory to the bedside. Here, we review current research findings that have yielded therapeutic benefits, particularly as derived from soft and hard corals. Several applications have already been demonstrated, including anti-inflammatory properties, anticancer properties, bone repair, and neurological benefits.

1. What Are Corals? Through the extensive work of Metchnikoff, we now know of cellular and humoral immunity as well as the impor- Corals have served as an excellent target taxon for bio- tance of invertebrate organisms in immunologic research [8, prospecting [1–4]. The earth’s surface is covered by ∼70% 9]. The revelation of a division between innate and adaptive water and contains 80% of all life found on the planet [5]. immunity came much later. The innate immune system is It is no wonder then that the ocean has been and still natural, nonspecific, nonanticipatory, nonclonal, germ line, is a source of food, let alone a vast source of therapeutic and for which there is no evidence that such a system molecules. In our case, corals will be the subject of our possesses the capacity to build memory from past exposures. investigation. Corals (Phylum: Cnidaria, Class: Anthozoa) Conversely, the adaptive immune system is acquired, specific, can generally be categorized into hard, soft, or gorgonian- anticipatory, clonal, and somatic and possesses the capacity to type organisms. Hard corals, called Scleractinian corals, build memory from past exposures [10]. Adaptive or acquired are generally hermatypic, the types that build reefs, with immunity has always been associated with vertebrates while which most people are familiar. Soft corals or octocorals theinnateimmunesystemisgenerallythoughttohaveorig- are generally flexible and do not produce the rigid structure inated in the invertebrates [11]. We propose immune-related characteristic of hard corals [6]. Gorgonian corals are also products as sources of therapeutic molecules (Figure 1). flexible, but their skeletal systems consist of a horny substance The discipline of coral immunology has not been vastly called gorgonin. Some of these coral types contain symbiotic studied and is not completely understood. While many mech- microalgae, genus Symbiodinium,andaregenerallycalled anisms associated with other organisms have been identified zooxanthellae [7]. These coral types tend to reside in shallow and examined, the general system of coral immunology still waters of tropical and subtropical locales. requires further investigation [12]. One of the mechanisms 2 Evidence-Based Complementary and Alternative Medicine

Agnatha (jawless vertebrates) Gnathostomata (jawed vertebrates) Lectins, C’, LRR VLR, IgSF, cytokine like molecules, mesodermal TcR, Ig, lectins, AMPs, C’, RAG1-2, immune cells 525 myr (vertebrates) cytokines, PRR, mesodermal immune Cephalochordata cells, transplant rejection Lectins, C’, IgSF, VCBP, PPO?, RAG? Urochordata Arthropoda Lectins, AMPs, C’, IgSF, (insects, crustaceans) Mollusca cytokine like molecules, IgSf (hemolin, Dscam), FREPs, lectins, PRR, PPO, mesodermal immune cells, PRR, CTX, PPO, transplant rejection Nematoda SCR, lectins, PRR (Toll), C’ AMPs, conserved signalling AMPs, conserved signalling IgSf, lectins, PRR, pathways, mesodermal immune

Adaptive immunity Adaptive pathways, mesodermal apoptosis, AMP, cells Hemichordata immune cells Annelida Echinodermata conserved signalling Nemertea Lectins, PRR, Lectins, SCR, AMPs, C’, pathway Mesodermal immune cells, AMPs, mesodermal cytokine like molecules, transplant rejection immune cells, PPO, mesodermal immune cells, transplant rejection Sipunculida PPO, transplant rejection Mesodermal immune cells Platyhelminthes Ecdysozoa Lophotrochozoa Deuterostomes Innate immunity Innate Anthozoa Protostomes Bilateria Anti-inflammatory activity Cnidaria Cytotoxicity IgSf, lectins, tissue Neuroprotective qualities Porifera rejection, C’ Antinociceptive properties IgSf, lectins, tissue Bone and tooth regeneration rejection Metazoa

Applications Protozoa Rheumatoid arthritis treatment Parkinson’s disease treatment AMP: antimicrobial peptides LRR VLR: variable lymphocyte receptors Alternative cancer therapy C’: complement pathway elements with leucine rich repeats Pain medication CTX: cortical thymocyte marker in Xenopus PPO: prophenoloxidase cascade Hard tissue regrowth Dscam: drosophila homolog for PRR: pattern recognition receptors Down syndrome cell adhesion molecule RAG: recombination-activating genes FREPs: fibrinogen related peptides SCR: scavenger receptors IgSF: immunoglobulin superfamily VCBP: variable chitin binding proteins TCR: T cell receptor

Figure 1: An evolutionary map indicating where corals (Anthozoa) lie and some of the therapeutic benefits they exert. Adapted from [11].

that has been demonstrated extensively is alloimmune mem- coral, fucoside E, showed the same anti-inflammatory activity ory, or reaction to and rejection of cells and molecules as well as antimicrobial activity [16]. Sinularin, a molecule from members of the same species. For example, Montipora that we will discuss later in greater detail, blocks pathways verrucosa, a hard (Scleractinian) coral, actually displays a that increase the severity of neuroinflammation (see Table 1 limited form of memory during analyses of transplantation for a more extensive list of therapeutic molecules isolated responses [13, 14]. Alloimmune memory in this coral seems from corals) [17]. tobeduetovaryinghistocompatibilitymarkersthatserveas One particular clinical application that has proven to be identification badges of self/nonself. We will now turn to the especially promising in relation to coral natural products therapeutic benefits of certain coral species. is arthritis. Rheumatoid arthritis is a chronic inflammatory disease that causes joint destruction. In a recent article pub- lished in PLoS, 11-epi-sinulariolide acetate (Ya-s11), a known 2. Corals and Inflammation cembrane-type compound, was isolated from the soft coral, Sinularia querciformis, and evaluated in its anti-inflammatory Perhapsdisordersmostrelatedtotheimmunesystemare potency in vitro as well as in adjuvant-induced arthritis (AIA) those of inflammation. Inflammation is a physiological pro- in rats (Figure 2)[18]. AIA is the typical murine equivalent tective mechanism that is activated by the immune sys- to human rheumatoid arthritis used in experiments to test tem upon encountering foreign invaders in an attempt to treatment possibilities. Ya-s11 was shown to strongly inhibit remove the adverse stimuli and begin healing the organism. the production of proinflammatory proteins iNOS and Diterpenes are one specific class of molecules isolated from COX-2 in murine macrophages (Figure 3). In the AIA mice, corals that have been shown to exhibit therapeutic bene- Ya-s11 severely reduced the effects of arthritis by inhibiting fits, specifically anti-inflammatory. Three diterpenes isolated migration of inflammatory cells to joints and preventing from Eunicea fusca, a gorgonian coral native to Florida, were bone destruction (Figure 4). The authors concluded that this found to have superior anti-inflammatory effects to those of molecule may be a source of treatment for humans with indomethacin, a commonly used anti-inflammatory medica- rheumatoid arthritis. To draw a correlation here, there have tion [15]. Another diterpene isolated from the same species of also been other demonstrations of AIA attenuation in rats Evidence-Based Complementary and Alternative Medicine 3

Table 1: Bioactive compounds with medicinal qualities.

Genus Species Molecule Benefit Author Flexibiliquinone Lin et al. 2013 [47] Sinularia flexibilis Anti-inflammation Flexibilin D Hu et al. 2013 [48] Anti-inflammation and 11-epi-Sinulariolide anti-bone-loss Lin et al. 2013 [18] querciformis Acetate Antinociception and Huang et al. 2012 [17] Sinularin antineuroinflammation gaweli 5𝛼,8𝛼-Epidioxysterol Cytotoxicity Yen et al. 2013 [49] crassa Crassalone A Cytotoxicity Cheng et al. 2012 [50] Cytotoxicity and granosa 9,11-Secosterol Huang et al. 2012 [51] anti-inflammation 5-Epsinuleptolide sp. Cytotoxicity Huang et al. 2013 [24] acetate (5EPA) Cytotoxicity and Scleronephthya gracillimum Sclerosteroids Fang et al. 2013 [52] anti-inflammation Cytotoxicity and Cladiella krempfi Kremptielins Tai et al. 2013 [53] anti-inflammation Cytotoxicity and Paraminabea acronocephala Paraminabic acid Chao et al. 2013 [54] anti-inflammation Antineuroinflammation Capnella imbricata Capnellenes Jeanetal.2009[37] and antinociception Anti-inflammation and Lemnalia cervicomi tenuis Lemnalol Lee et al. 2013 [55] antinociception Cytotoxicity and Echinomuricea sp. Echinoclerodane A Cheng et al. 2012 [56] anti-inflammation Nebrosteroids Q, R, and Nephthea chabrolii Cytotoxicity Wang et al. 2013 [27] S

through the use of herbal products (Figure 5)[19]. Although The innate immune system’s property of phagocytosis the investigators used different methods for evaluating the gave rise to the well-understood common inflammatory sys- effectiveness, the point we are making is that there are various tem and its responses. Novel steroids isolated from Sinularia potential natural treatments, whether plant or animal, being crassa were found to downregulate expression of proin- tested for the treatment of arthritis. Next, we will examine flammatory proteins and showed cytotoxic activity against anticancer effects that some coral natural products exert. human liver cancer cells [22].Anothermoleculeisolatedfrom Sinularia sp., sinularin, demonstrated anticancer activity via proapoptotic factors (Figure 6)[23]. Apoptosis refers to 3. Corals and Cancer one type of programmed cell death leading to particular 3.1. The Role of Sinularia in Cancer. One particular genus of cell conformational changes and death. 5-episinuleptolide corals possesses a natural metabolite which has demonstrated acetate (5EPA), a norcembranoidal diterpene, isolated from significant anticancer ability. They are species of the soft Sinularia sp., has demonstrated cytotoxicity against many cell coral genus Sinularia. Goto et al. isolated an agglutinin called lines including K562, Molt 4, and HL 60, with HL 60 being sinularianfromthisspeciesandfoundthatitagglutinated the most sensitive to this treatment via Hsp90 inhibition experimental targets, for example, rabbit erythrocytes and (Figure 7)[24]. Hsp90 inhibition leads to apoptosis or cell murine leukemia cells, but not sheep or human erythrocytes death in the leukemia cells. [20]. Incidentally responses to foreign erythrocytes are a common approach used in analyzing the innate immune system of invertebrates. Moreover, Wright et al. isolated 3.2. Other Species of Soft Corals with Anticancer Properties. various compounds from corals of this genus, including a new Other species, besides those in the genus Sinularia,have nitrogenous diterpene, new and known lobanes, and known also demonstrated anticancer potential. One in particular, cembranes [21]. The lobanes and cembranes were tested for 13-acetoxysarcocrassolide, isolated from the soft coral Sarco- anticancer activity against three human cancer cell lines and phyton crassocaule exerted cytotoxic activity against bladder showed a 50% inhibition of tumor growth. Not only does this cancer cells [25].WangandDuhisolatedsixnovelcembra- coral’s product exhibit anticancer activity, but it also exerts nolides called michaolides and a known cembranolide called pronounced anti-inflammatory responses. lobomichaolide from the soft coral Lobophytum michaelae 4 Evidence-Based Complementary and Alternative Medicine

O

OAc

O Ya-s11 O (a) (b)

Figure 2: The chemical structure of 11-epi-sinulariolide acetate (Ya-s11) and the coral from which it is isolated, Sinularia querciformis.From [18].

iNOS

COX-2

𝛽-Actin

LPS (0.01 𝜇g/mL) − ++ + + + Ya-s11 (𝜇M) −−1102550 (a)

120 120

100 100 ∗ ∗ 80 80 ∗ 60 60 ∗ ∗ 40 (%) intensity relative 40 2 iNOS relative intensity (%) intensity relative iNOS

20 COX- 20 ∗ ∗ 0 0

LPS − +++++ LPS − +++++ Ya-s11 (𝜇M) −−1255010 Ya-s11 (𝜇M) −−1255010

(b) (c)

Figure 3: In vitro effects of Ya-s11 on production of iNOS and COX-2, proinflammatory proteins. From[18]. and determined that they possessed not only antitumor capa- cytotoxicity against the P-388 cell line (mouse lympho- bilities, but also activity against cytomegalovirus (HCMV), cytic leukemia) but did not exhibit antiviral activity against related to herpesviruses [26]. HCMV. Another genus of corals that has proven particularly generous with its demonstrations of marine natural products 4. A Focus on Hard Tissue Therapies is Nephthea [27].Wang,Puu,andDuhisolatedthreenew steroids, nebrosteroids Q, R, and S, from the soft coral 4.1. Bone Repair. In addition to the anti-inflammatory and Nephthea chabrolii and tested its anticancer and antiviral anticancer properties outlined above, corals and their prod- activity. They found that these three steroids did exhibit uctshavebeenshowntoexertcurativepotentialformetabolic Evidence-Based Complementary and Alternative Medicine 5

(a) (b) (c) (d) 140 ∗ 14 ∗ ∗ ∗ ∗ ∗ 12 ∗ ∗ ∗ 130 10

8 # 120 # # 6 # # 4 # Paw edema (%) Paw 110 # # 2 Mean arthritis score 100 0

0 7 9 11 13 15 17 19 21 23 25 28 0 7 9 111315171921232528 Day after immunization Day after immunization Naive (n=6) Naive (n=6) AIA (n=7) AIA (n=7) AIA + Ya-s11 (3 mg/kg, n=6) AIA + Ya-s11 (3 mg/kg, n=6) AIA + Ya-s11 (9 mg/kg, n=6) AIA + Ya-s11 (9 mg/kg, n=6) Ya-s11 (9 mg/kg, n=6) Ya-s11 (9 mg/kg, n=6) (e) (f)

Figure 4: Pictorial and graphical demonstrations of the attenuation of AIA symptoms in rats. From [18].

.) .c

Mtb (s S L

HLXL feeding

IJ

Cytokines Chemokines MMPs

IL-6 RANTES MMP-2 IL-17 MCP-1 MMP-9 MIP-1𝛼 GRO/KC

HLXL

Figure 5: A schematic of how herbal supplements were administered and evaluated as a treatment for AIA in rats. From [19]. 6 Evidence-Based Complementary and Alternative Medicine

13 C-NMR spectrum

OH O O

O Sinularin

7 6543210 (ppa)

13 C-NMR spectrum

160 140 120 100 80 60 40 20 (ppa)

13 Figure 6: The chemical structure and C-NMR spectra of sinularin, a potential anticancer molecule isolated from Sinularia.From[20].

100 90 conjunction with zeolite, a microporous mineral, assists in 80 protecting against and reversing bone loss in mice that have 70 60 been placed into an artificial, induced menopausal state [28] 50 (mice, like most mammals, have an estrous cycle). This same 40 30

Cell growth effect was observed in rabbits when used with human platelet-

(% of control) (% of 20 10 rich plasma [29]. Green et al. have highlighted the importance 0 of the skeletal matrices of marine invertebrates for bone 0 0.3125 0.625 1.25 2.5 5 10 regeneration [30]. Dose of 5-episinuleptolide acetate (𝜇g/mL)

K562 4.2. Restoration of Dental Deformities. Related to the poten- Molt 4 60 tial for bone repair discussed above is bioprospecting being HL performed within corals as it pertains to dental treatment. Figure 7: 5EPA activity against K562, Molt 4, and HL60 cell lines. Figueiredo et al. mention that their interest in this application From [24]. has been increasing due to the low supply and difficulty of using human-derived substitutes for certain products used in dental procedures [31], making this a necessary and promising area of research regarding this untapped deficits as well. The naturally occurring calcium within the resource. They assert, however, that the coral skeleton in aragonite found in scleractinian hard corals, and the calcite its unrefined form remains impractical because of its high found within the soft octocorals, when administered in degeneration (dissolution) rate. They argue that the potential Evidence-Based Complementary and Alternative Medicine 7 benefit of coral skeleton may reside in refined coral that is 6. Hypertensive Treatments modified (strengthened chemically) to increase its longevity and integrity. A particularly interesting experiment was per- While most of the research done pertaining to corals and formed which revealed that osteogenic bone marrow stromal therapeutic applications has yielded results in cancer treat- cells used with coral scaffolds could be effective in repairing mentandbonerepair,otherapplicationshavealsobeen mandibular defects in canines [32]. identified. For example, coral sand, which is biogenic, is generated by the erosion of scleractinian corals. It has been used as a silicon source to reduce blood pressure as well as to 5. The Nervous System: improve the expression of genes that increase cardiovascular Neuroprotective Compounds health in hypertensive rats [38].

One of the highly prospective treatment modalities that can 7. Comparing Corals with Therapeutic be added to the benefits of soft corals is the potential for abbreviating neurological deficits. Chen et al. extracted a Properties of Other Invertebrates neuroprotective compound, 11-dehydrosinularolide, from a The reason that we and others are interested in corals as coralthattheybelievecouldbeusedtotreatParkinson’s potential sources of medicinal molecules for humans is the disease [33]. Parkinson’s disease is a highly debilitating vast amount of progress already made with other invertebrate neurodegenerative disorder that leads to severe impairment organisms. Investigators have found an abundance of promis- in the central nervous system and the necessity for special ing evidence for earthworms specifically as treatment modal- care. Most of the treatments that exist for Parkinson’s disease ities for inflammation, cancer, and coagulatory disorders39 [ – only partially improve the symptoms and do not treat the 43]. Earthworms have also been used historically as a source source of the disease itself. Despite treatment using current of nutrition [44, 45]. Careful analyses do not seem to reveal pharmaceuticals, progression of the disease continues, with that corals, as nutritious sources, afford this same sustenance the patient losing motor skills and standard functions. Two as food. This is probably due to the small amount of tissue mechanisms which impact the pathogenesis of Parkinson’s available in relation to their skeletal materials, even in soft disease, however, are inflammation and apoptosis [34]. Proin- corals. Current results clearly indicate certain medicinal flammatory iNOS and COX-2 are two proteins that are well qualities. Moreover, cytotoxicity has been demonstrated by knowntobeassociatedwithinflammationandarecommon molecules isolated from tunicates, another invertebrate [46]. indices and markers when testing for anti-inflammatory With this large amount of evidence, it is definitely clear activity. Hoang et al. found that neuronal NOS and COX-2 we should be looking towards other invertebrates in our caused DNA damage in a mouse model of Parkinson’s disease bioprospecting endeavors. [35]. The next logical step in trying to find a treatment for this debilitating disease is to find an agent that will prevent theinflammationinducedbyiNOSandCOX-2.InChen Conflict of Interests et al.’s experiment, investigators found that administration of 11-dehydrosinulariolide significantly reduced expression of The authors declare that there is no conflict of interests iNOS and COX-2, with iNOS being almost entirely elimi- regarding the publication of this paper. nated. It was also demonstrated to halt apoptosis, another factorthoughttobeassociatedwithParkinson’sdisease. References This molecule provides highly promising potential for this severely incapacitating disease. [1] E. L. Cooper, “CAM, eCAM, bioprospecting: the 21st cen- Not only have corals yielded molecules that confer tury pyramid,” Evidence-Based Complementary and Alternative neuroprotection, but also that reduce neuropathic pain. Medicine,vol.2,no.2,pp.125–127,2005. Austrasulfone, which is isolated from the soft coral Cladiella [2] E.L.Cooper,“Bioprospecting:aCAMfrontier,”Evidence-Based australis, demonstrated neuroprotective qualities via anti- Complementary and Alternative Medicine,vol.2,no.1,pp.1–3, inflammatory pathways (reduction of iNOS and COX-2) 2005. [36]. It also showed potency in reducing neuropathic pain [3] E. L. Cooper, “eCAM: an emerging linkage with ethnophar- and slowing progression of atherosclerosis and multiple macology?” Evidence-Based Complementary and Alternative sclerosismodelsinrats.Anothermoleculethatexertsanti- Medicine,vol.5,no.4,pp.365–366,2008. inflammatory effects by reducing iNOS and COX-2 is capnel- [4]E.L.Cooper,“Drugdiscovery,CAMandnaturalproducts,” lene, which is isolated from the soft coral, Capnella imbricata Evidence-Based Complementary and Alternative Medicine,vol. [37]. In conjunction with the anti-inflammatory properties, 1, pp. 215–217, 2004. capnellene also exhibited antinociceptive properties. Noci- [5]A.W.Bruckner,“Life-savingproductsfromcoralreefs,”Issues ception refers to the neurological pathway by which the body in Science and Technology,vol.18,no.3,p.35,2002. perceives pain caused by tissue-damaging stimuli. Therefore, [6] H. Schuhmacher and H. Zibrowius, “What is hermatypic?—a this compound holds the potential not only to reduce inflam- redefinition of ecological groups in corals and other organisms,” mation causing or worsening neurodegenerative disorders, Coral Reefs,vol.4,no.1,pp.1–9,1985. butalsotoreduceanypainthatmaybeassociatedasa [7] http://animals.nationalgeographic.com/animals/invertebrates/ symptom. coral/. 8 Evidence-Based Complementary and Alternative Medicine

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Ronald A. Sherman

BioTherapeutics, Education & Research (BTER) Foundation, 36 Urey Court, Irvine, CA 92617, USA

Correspondence should be addressed to Ronald A. Sherman; [email protected]

Received 2 December 2013; Accepted 15 January 2014; Published 13 March 2014

Academic Editor: Edwin L. Cooper

Copyright © 2014 Ronald A. Sherman. 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.

Medicinal maggots are believed to have three major mechanisms of action on wounds, brought about chemically and through physical contact: debridement (cleaning of debris), disinfection, and hastened wound healing. Until recently, most of the evidence for these claims was anecdotal; but the past 25 years have seen an increase in the use and study of maggot therapy. Controlled clinical studies are now available, along with laboratory investigations that examine the interaction of maggot and host on a cellular and molecular level. This review was undertaken to extract the salient data, make sense, where possible, of seemingly conflicting evidence, and reexamine our paradigm for maggot-induced wound healing. Clinical and laboratory data strongly support claims of effective and efficient debridement. Clinical evidence for hastened wound healing is meager, but laboratory studies andsome small, replicated clinical studies strongly suggest that maggots do promote tissue growth and wound healing, though it is likely only during and shortly after the period when they are present on the wound. The best way to evaluate—and indeed realize—maggot- induced wound healing may be to use medicinal maggots as a “maintenance debridement” modality, applying them beyond the point of gross debridement.

1. Introduction killing (disinfection) and hastened wound healing (growth stimulation). Maggot therapy (sometimes called larval therapy) is the Scientific evidence for all three actions has been slow in applicationofliveflylarvaetowoundsinordertoaidin coming. The first controlled clinical trials were not begun wound debridement (cleaning), disinfection and/or healing. until 1990 [2], and it was not until just 10 years ago that A maggot infestation on a living vertebrate host is called theU.SFoodandDrugAdministration(FDA)firstgranted myiasis. When that infestation is limited to a wound, it is marketing clearance to medicinal maggots (Medical Maggots; called wound myiasis. Maggot therapy is basically a therapeu- Monarch Labs, Irvine, CA) as a medical device [3]. The tic wound myiasis, controlled in ways that optimize efficacy indications for that product were limited to debridement. and safety. We control the myiasis by carefully selecting the Clinical evidence of maggot-induced disinfection and growth species and strain of fly (the species most commonly used stimulation was not strong enough to convince regulators is Lucilia (Phaenicia) sericata), disinfecting the larvae, using at that time. But today, numerous clinical and laboratory special dressings to maintain the larvae on the wound, and studies demonstrate antimicrobial and/or growth-promoting integrating quality control measures throughout the process. activity. Some clinical studies do not demonstrate these The most noticeable change in maggot-treated wounds effects; instead, they leave us with doubts about the clinical is debridement: the dead (necrotic or gangrenous), infected significanceofthewoundhealingactivitiesthatweseein tissues and debris are removed from the wound, and the most other clinical and laboratory studies. wound bed is left looking clean and healthy. But ever since Several comprehensive reviews have been published over maggot therapy became a common practice [1], careful the past decade [4–6], and readers interested in a more observers also noted other effects on the wounds: microbial detailedorhistoricalperspectivewouldbeadvisedtoseekout 2 Evidence-Based Complementary and Alternative Medicine

Table 1: Publications identified and retrieved for review. 3. Results and Discussion Number of Number of Using the search terms of “maggot” (or “larva” or “larval”) Study design publications publications and “therapy” or “wound,” a total of 8,303 publications were retrieved and identified identifiedinPubMed,644inWileyOnlineLibrary,and8in reviewed the Cochrane Library. After deleting duplicate and irrelevant Randomized clinical trial (RCT) 3 3 articles and simple case reports or reviews, 97 articles met Nonrandomized, prospectively 44review requirements (Table 1). collected data, with control group The resulting body of literature provided both laboratory Nonrandomized, prospectively and clinical evidence to support all three actions associated collected data, without control 11with maggot therapy: debridement, disinfection, and growth group stimulation. Nonsupportive data also was retrieved, though Controlled retrospectively 11lesscommonly.Thebestwaytoconsidertheroleofmaggots collected data in wound healing may be to first review the wound healing Case series; no controls 20 18 process in general and then to separately summarize the Basic science 68 66 literature concerning each major wound healing effect of the Total 97 93 maggots.

3.1. Wound Healing and the Chronic Wound. Wound healing these references. This review differs from those earlier works is classically described as 4 distinct but overlapping phys- in that it was undertaken to examine the best clinical and iologicalphasesofrepairandrebuilding:(1)homeostasis; basic science evidence that exists today, so as to formulate (2) inflammation; (3) proliferation; and (4) remodeling and a course of future research that might answer some of the maturing [7]. With each phase, new cells are recruited into clinical questions that still remain. the area to perform the work, or cells already present alter their activity to secrete new cytokines or perform new duties, in response to changing conditions in the wound (bleed- 2. Methods ing, hypoxia, alterations in cytokine concentrations, etc.). When no longer needed, the cells undergo apoptosis and A thorough literature search was conducted, first using the are removed or engulfed by other cells (i.e., macrophages). National Library of Medicine search tool (“PubMed”) and Normally, these four waves in the healing process progress the Cochrane and Wiley Online Library databases, using the quickly and smoothly, one into the next. But occasionally terms: healing may stagnate, and the wound is said to be chronic. Wound healing may be trapped at any phase (or even while [“maggot” or “larva” or “larval”] and [“therapy” or undergoing a combination of phases), but typically it is within “wound”] the inflammatory phase: dead, infected debris may not be adequately removed from the wound bed, and/or it might notbepossibleforthebodytoeradicatethelocalinfec- Then, the holdings of the BTER Foundation’s Biotherapy tion, and/or the proteases and other destructive products library were searched for complete copies of these and of inflammation by clearing the newly formed cellular and any additional publications on maggot therapy. Articles not extracellular matrix as fast as it is being laid down. It is already in the library’s holdings were requested through in this context that debridement, disinfection, or cellular interlibrary loans or directly from the authors. Irrelevant proliferation and migration are so important, for they can publications (i.e., natural myiasis rather than maggot ther- push the stagnant wound into the next phase of healing. apy), nonquantitative case reports (fewer than 5 cases per publication) and simple reviews or news stories were then excluded from this working collection, along with articles 3.2. Debridement. Of the three described actions of maggot older than 20 years. This time frame was selected because therapy, debridement (physical and chemical) is the best the first controlled clinical trial of maggot therapy, published studied. Each maggot is capable of removing 25 mg of in a peer-reviewed journal, appeared 17 years ago. Three necrotic material from the wound within just 24 hrs [8]. abstracts were published prior to that, but they report on data The physical mechanics of maggot debridement [6, 9]are subsequently published in peer-reviewed journals within the readily apparent to anyone who has seen the larvae under time frame of our literature search, so the data was captured the microscope. Larvae are covered by minute spines which that way. scrape along the wound base as the maggots crawl about, From this working collection of data, and in the context loosening debris as does a surgeon’s rasper or file (Figure 1). of a larger body of literature and expert opinion going back The mandibles, in the form of “mouth hooks,” are used to 90 years, a cohesive scheme about maggot therapy was syn- help pull the maggot’s body forward as it crawls and to thesized. This made it possible to suggest clinical trial designs probe every nook and cranny for food or shelter. The maggot that might bring us substantially closer to understanding the does not “bite off” pieces of tissue, but it rather secretes clinical utility of maggot therapy. and excretes its digestive enzymes (alimentary secretions Evidence-Based Complementary and Alternative Medicine 3

could soon enter clinical trials as a purified debriding enzyme. Larval secretions also contain deoxyribonuclease (DNAse), able to degrade both microbial DNA and also human DNA in necrotic debris [17]. DNAse may play an important role not only in debridement but also in inhibiting microbial growth and biofilm. The wealth of case reports and case series in the literature suggests that most clinicians are impressed by the debride- ment efficacy of medicinal maggots. Controlled studies of maggot debridement are less common, but quite worthy of examination. In a prospective study of spinal cord injury patients with chronic, nonhealing pressure ulcers, patients were monitored for 3-4 weeks while receiving standard wound care (what- ever modality was prescribed by the surgically led wound Figure 1: Scanning electron micrograph of Lucilia (Phaenicia) sericata.FromFleischmannW.,GrassbergerM.,andSherman care team), followed by 3-4 weeks of maggot therapy [2]. RA Therapy. A Handbook of Maggot-Assisted Wound Healing. Tissue quality and wound size were assessed weekly. Maggot Stuttgart: Thieme, 2004:93 pg. debridement of necrotic tissue was achieved in less than 14 days (average of 10 days), but none of the control wounds were more than 50% debrided, even after 4 weeks of treatment. In a cohort of 63 patients with 92 pressure ulcers, followed and excretions or ASE), the consequence of which is that for at least 8 weeks while receiving either standard wound care digestion begins in the wound bed, outside of the maggot’s (as prescribed by the hospital’s wound care team), or maggot own body. The necrotic tissue liquefies, and the maggots can therapy (two 48- to 72-hour cycles per week), maggot-treated wounds were debrided four times faster than control wounds then easily imbibe it. The physical movement of the maggot 2 2 over the wound, plowing the tissue and spreading its ASE as it (0.8 cm /week versus 0.2 cm /week; 𝑃 = 0.001)[18]. goes, contributes significantly to the debridement effort. The In a similar cohort of 18 diabetic subjects with 20 physical action of the maggot over the wound is a primary nonhealing neuropathic and neuroischemic foot ulcers [19], reason given by the FDA for classifying medicinal maggots as maggot-treated wounds were 50% debrided within an average a medical device and not a simple drug. of 9 days, but control wounds did not achieve that level Hobson [10] was one of the first investigators to system- of debridement until an average of 29 days (𝑃 < 0.001). atically demonstrate proteolytic activity of L. sericata larval Within 2 weeks, maggot-treated wounds were left with only 2 digestive enzymes. Vistnes et al. [11]usedanimalmodels 7% necrotic tissue (0.9 cm ) compared to 39% necrotic tissue 2 to demonstrate that the maggots’ digestive enzymes were (3.1 cm ) in the control group (𝑃 < 0.01), and all maggot- capable of dissolving necrotic tissue and identified several treated wounds were completely debrided within 4 weeks, proteases. More recent studies of larval ASE help us see while most control wounds were still over 33% covered with just how these proteolytic enzymes fit into the context of necrotic tissue (𝑃 = 0.001). debridement and wound healing, for we now know that they Wayman and colleagues [20] randomized 12 venous stasis include a wide array of matrix metalloproteinases (MMPs), leg ulcer subjects to receive either maggot debridement including at least the trypsin-like and chymotrypsin-like therapy (MDT) or their standard of care (hydrogel). In serine proteases, an aspartyl proteinase, and an exopeptidase- this randomized controlled trial (RCT), the six wounds in like MMP, active across a wide pH range [12–14]. theMDTarmweredebridedfasterthanthesixwounds It is important to recognize that humans produce at in the control arm (𝑃 < 0.004), with all of the maggot- least 23 different MMPs which not only degrade extracellular debrided wounds completely debrided after just one 2-3- protein but also regulate a wide variety of cellular processes day treatment, compared to only 4 of the control wounds through activation (or deactivation) of signaling molecules completely debrided after a month of therapy. and/or their receptors [15]. MMPs play critical roles in In a larger clinical trial of maggot therapy for venous allphasesoftissuerepairandwoundhealing,including stasis ulcers, this time designed to look for maggot-associated hemostasis, thrombosis, inflammatory cell activation, colla- wound healing, Dumville and colleagues [21]enrolled263 gen degradation, fibroblast and keratinocyte migration, and subjectstoreceiveeitherstandard(“free-range”)mag- tissue remodeling. Disturbances in wound healing can occur got debridement, maggot debridement using “Biobags” (a when one group of proteases is deficient or out of balance with patented ravioli-like pouch containing the live larvae), or another. their standard of care, hydrogel, and compression dressings Telford et al. [14] demonstrated that some of the maggot’s (Figure 3). All subjects received compression dressings, proteases are resistant to human wound protease inhibitors. except during maggot debridement. Time to debridement At least one of these chymotrypsin-like proteases has now differed significantly between the three groups (25.38, df =2, been produced recombinantly in Escherichia coli [16]and log-rank test 𝑃 < 0.001). The median time to debridement 4 Evidence-Based Complementary and Alternative Medicine was 14 days with free-range larvae, 28 days with bagged versus contained maggots have suggested that maggots in larvae, and 72 days for the control arm. Healing results will direct contact with the wound are more effective, at least for be discussed later in this review. debridement, than maggots separated from the wound by Most other debridement studies are not as quantitative their containment dressings [9, 26]. To date, only one clinical in their data collection and assessments. Markevich and study was designed to compare the difference between these colleagues presented data from their RCT of maggot therapy two methods of maggot therapy. In this prospective clinical for neuropathic foot wounds at the 2000 Conference of trial, Steenvoorde and colleagues [27] enrolled 64 patients the European Association for the Study of Diabetes [22]. with 69 chronic, necrotic wounds. Patients were treated with Although never published as a full-length, peer-reviewed either free range or contained maggot debridement therapy, research paper, this abstract is often cited because it is the only depending on maggot availability and clinician preference. RCT of MDT in diabetic foot ulcers. Subjects were randomly The investigators monitored 8 specific outcome measures: assigned to receive maggot therapy (𝑁=70)orstandard (1) complete healing without any other intervention; (2) (hydrogel) therapy (𝑁=70). Wound dimensions and quality complete healing by secondary intervention (e.g., split-skin were then monitored every 3 days for 10 days. While the graft); (3) wound free from infection and less than one- authors did not quantify debridement per se, we know that third of the initial size; (4) wound clean but not decreased the maggot-treated patients were debrided more effectively in size; (5) no difference in wound size or character; (6) and efficiently because their necrotic wounds were ultimately wound worsened; (7) minor amputation was still required covered with more granulation tissue (𝑃 < 0.001)andwere (e.g., partial toe amputation); and (8) major amputation was smaller in size (𝑃 < 0.05)thanthewoundstreatedwith still required. Their analysis revealed better outcomes in the hydrogel. free range group compared to the contained maggots group In a retrospective case controlled study of lower extrem- (𝑃 = 0.028), despite the fact that the free range technique ity wounds in nonambulatory hospice patients (in whom required fewer maggot applications (𝑃 = 0.028)andfewer debridement was the goal, not wound healing) [23], Arm- totalnumberofmaggotspertreatment(𝑃 < 0.001). The strong and colleagues concluded that MDT was an effective authors concluded that containment of maggots reduced the debridement modality. Again, their objective measures were effectiveness and efficiency of maggot debridement therapy, not specifically changes in the amount of necrotic tissue but probably by preventing contact with, and/or complete access rather more clinically relevant surrogates: faster eradication to, the wound bed. of infection (127 versus 82 antibiotic-free days out of 6 Dumville et al.’s study [21]discussedaboveincludedfree months; 𝑃 = 0.001), two-thirds fewer amputations (10% range and contained maggots in two of the three study arms versus 33%; 𝑃 = 0.03), and significantly faster wound but was not specifically designed to detect differences in healing in the maggot-treated wounds (18 weeks, for those debridement between free range and contained maggots and that healed, versus 22 weeks; 𝑃 = 0.04). did not identify any significant differences. The median time Marineau and colleagues [24]publishedtheircase to debridement in this study was 14 days for the free range series of 23 complicated diabetic foot wounds (most with maggot therapy arm (95% confidence interval [CI] = 10–17) osteomyelitis) treated with MDT. There was no control group and28daysforthebaggedmaggots(95%CI=13to55; 2 and no analysis of individual wound changes, but the authors adjusted 𝜒 1.52, df = 1; 𝑃 = 0.22). As pointed out, this study did conclude that the 74% of success rate (debridement or was not powered to detect significant differences between complete limb salvage) was greater than expected, given that these two groups, so it is not possible to determine whether this group of patients had all failed prior conventional wound or not the twofold difference in debridement time is real. care. In their RCT of maggot therapy for chronic leg wounds, Opletalova´ and colleagues randomized 119 subjects to receive 3.3. Disinfection. The natural habitat of L. sericata larvae is either surgical debridement or bagged maggots (twice in rotting organic matter such as a corpse or excrement. weekly) for two weeks. Wounds were evaluated on days 8, Therefore, it should be no surprise that this maggot would 15, and 30 [25]. Wound slough was significantly less in the be well-protected from infection. Early on, scientists believed maggot-treated arm by day 8 (54.5% versus 66.5%; 𝑃= that ingestion was the primary method by which the maggots 0.04), but by day 15 that difference disappeared. The authors cleared the wounds of infection [8, 28], and subsequent concluded that, compared to surgical debridement, maggot researchers demonstrated that highly effective killing does therapy was more efficient and valuable for the first 2 weeks, indeedoccurinthegut[29, 30]. Greenberg hypothesized that though additional treatments provided no debridement ben- antimicrobial compounds might be produced in the gut by efit. symbiotic microbes such as Proteus mirabilis,and,in1986, This two-week limit to maggot debridement efficacy Erdmann and Khalil identified and isolated two antibacterial deserves comment and consideration, because it contrasts substances (phenylacetic acid and phenylacetaldehyde) from with what has been reported with free range maggots. the P. mirabi li s that they isolated from the gut of a related Unfortunately, very few studies have compared free range blowfly larva: Cochliomyia hominivorax [31]. with bagged maggots, though such a study could be a Antimicrobial killing also occurs outside the maggot’s valuable mechanism for evaluating the relative importance gut, and the extracorporeal secretion/excretion of antimi- of the maggot’s physical versus chemical activity. Most, crobial compounds may even be responsible for most of though not all, laboratory studies comparing free range the maggot’s antimicrobial activity [32, 33]. Some early Evidence-Based Complementary and Alternative Medicine 5 researchers believed that wound disinfection was largely due the evidence that maggots bring about clinically relevant to the physical “washing-out” (lavage) of microbes from the disinfection? Numerous case reports have purported wound wound bed during maggot therapy, by the fluid secreted by disinfection following maggot therapy, but controlled clinical both the maggots (ASE) and the host (“wound exudate”). evidence of maggot-induced antimicrobial activity has been They also pointed to the antimicrobial activity of ammonia- sparse, until recently. In a prospective clinical trial of maggot containing byproducts of the maggots’ digestion of tissue therapy for chronic leg ulcers, Contreras-Ruiz and colleagues proteins and the resulting alkalinized wound bed [1, 34, 35]. [56] randomized 19 subjects to either maggot therapy or With advanced molecular and biochemical methods now conventional debridement and compression therapy and at our disposal, many researchers over the past two decades found that maggot-treated wounds had significantly reduced have focused their attention on isolating antimicrobial pro- bacterial counts compared to control wounds. The maggot- teins and other biochemicals produced by L. sericata [36–47]. treatedgroupdisplayedmoreanxietyandwoundodorduring Often, the isolated molecules were more active against gram treatment, but no greater pain or other adverse events. positive bacteria than gram negatives, but sometimes this In Tantawi et al.’s case series [57], 13 diabetic ulcers was merely a matter of dose and potency [42]. Antimicrobial in 10 subjects similarly demonstrated significant decreases activity has been seen even against highly antibiotic-resistant in the number of microbial species and the colony counts bacteria [40, 43]andagainsttheprotozoanLeishmania after maggot therapy. In an observational study by Bowling parasite [44, 45]. Kawabata et al. [46] demonstrated that and colleagues [58], 13 sequentially enrolled stable diabetic the antimicrobial activity could be modified by exposure patients with MRSA-colonized ulcers, not already receiving to microbial challenges (as is the case with many innate MRSA-specific antibiotics, were debrided with maggot ther- immunodefense peptides). apy. Semiquantitative cultures were taken at baseline and By 2010, Cerovskyetal.[´ 47] completely sequenced the before each cycle of MDT. The mean duration of MDT was 40-residue defensin-like antimicrobial peptide now called: less than 3 weeks (one treatment per week), and the authors “lucifensin.” Altincicek and Vilcinskas [48]usedsuppres- noted that this was far less than the duration of conven- sion subtractive hybridization to show that 65 L. sericata tional antibiotic treatment for MRSA. By the end of maggot genes upregulated in response to septic challenge (cuticular debridement, MRSA colonization was eliminated from all but puncture) with lipopolysaccharide. Valachovaandcolleagues´ 1 of the 13 ulcers (efficacy = 92%); no complications or patient [49] demonstrated that lucifensin expression was increased complaints were encountered. in response to microbial ingestion only in the fat body; When reviewing their patients, Steenvoorde and Jukema lucifensin was expressed in the salivary glands throughout [59] also found decreased colony counts of gram posi- the larval period and not significantly affected by microbial ingestion. tive organisms following maggot therapy, but they found Even more antimicrobial molecules are likely to be increased counts of gram negatives. Their results may have discovered in the coming years. Numerous antimicrobial resulted from the decreased competition by gram positive molecules have already been isolated in other blow flies, microbes.Thestudyauthorsspeculatedthathigherdosesmay including the antibacterial peptide diptericin from Phormia be necessary for effective gram negative killing. terraenovae [50] and the antiviral alloferons from Calliphora Armstrong et al. [23] probably best addressed the clinical vicina [51], the latter of which has already been commercial- relevancy of maggot-induced disinfection by designing a ized. case-control study of maggot therapy for lower extremity Maggotsalsofightbacteriaintheirmoreresistantform: wounds in hospice patients and recording the antibiotics biofilm. In contrast to free living (“planktonic”) individual prescribed by the patients’ primary clinicians, as a measure bacteria, biofilm is a structured community of one or more of clinically significant infection. As described earlier in species of bacterial cells, living closely in an enclosed, protec- this review, this study revealed significantly fewer days of tive, self-produced polymeric matrix, and adherent to an inert antibiotics compared to controls, over a 6-month observation or living surface [52]. Antibiofilm activity is valuable because period, indicating that the patients were cleared of their biofilm is highly resistant to the penetration and successful infection faster and remained infection free longer. activity of the human immune system and antibiotics. Biofilm Not all clinical studies of maggot-induced disinfection is a particularly difficult problem in chronic wounds. One of have demonstrated such positive results. Dumville et al.’s 267- the most powerful tools we have against biofilm is physically eroding it (i.e., brushing our teeth to rid ourselves of dental subject RCT of maggot therapy for venous stasis wounds biofilm). Many therapists prescribe brushing to rid a wound [21] did not demonstrate any significant difference between of biofilm. It is reasonable to assume that the maggots are the time-dependent decreasing bacterial burden in maggot- helpingtoridawoundofbiofilmsimplybycrawlingover treated patients versus control patients, nor any significant it with their rough bodies. What was particularly surprising, difference in the number of MRSA-colonized wounds that though, was the discovery that maggot ASE is capable of were cleared. But then, as the authors pointed out, there were dissolving biofilm and inhibiting the growth of new biofilm so few patients with MRSA that the study was not adequately [53–55]. This has been shown at least for Staphylococcus powered to see any likely difference. What’s more, looking for aureus and Pseudomonas aeruginosa biofilm. significant population differences in colonizing bacteria may There should be no more doubt that maggots secrete not truly be an appropriate endpoint if we are really more and excrete potent antimicrobial compounds. But what is concerned with clinical infections. 6 Evidence-Based Complementary and Alternative Medicine

Table 2: Wound Healing results associated with selected published maggot therapy studies.

Pressure ulcer study1 Diabetic ulcer study2 Conventional therapy MDT Conventional therapy MDT Quality of wound base Initial granulation tissue as % of total area 31% 27% 18 19 ∗+ Granulation tissue at 4 weeks 29% 69% 15 56 Percentage of wounds developing ≥ 50% granulation tissue 18 51 Weeks until granulation tissue reached > 50% 4.7 2.1 ∗ Change in % of granulation tissue per week 3.30% 13% Wound size and healing ∗ Initial surface area in sq cm 14 22.1 6.3 13.3 ∗+ Change in surface area during treatment (sq cm) 6.3 −7. 3 5 −3.8 ∗+ Change in surface area per weeks 1.4 −1.5 1.15 −0.78 ∗ Percentage of wounds which decreased in size within 4 weeks 44% 79% ∗+ Healing rate at 4 weeks −0.038 0.101 −0.08 0.08 ∗+ Healing rate at 8 weeks −0.027 0.096 −0.02 0.07 Percentage of wounds completely healed 21% 39% 21 36 Average time to complete healing (weeks) 13.4 12 18 15 1 2 Sherman, 2002 [18](∗identifies significantly different results between the two arms of this study); Sherman, 2003 [19](+identifies significantly different results between the two arms of this study). The wound healing rate, based on studies by Gilman [69]andMargolisetal.[70],wasdefinedasthechangein surface area divided by the mean circumference over time. Study details provided in text.

3.4. Growth Stimulation. Evidence of maggot-induced tissue appear. These were small, due to a lack of funding and sup- growth or wound healing now comes from both labora- port; but they showed the promising results needed to propel tory and clinical studies and also suggests both mechanical maggot therapy into the scientific limelight and justified and biochemical pathways. Among the early theories about larger and more definitive studies. In a prospective study of maggot-induced wound healing were that the simple removal spinal cord injury patients with chronic, nonhealing pressure of debris and microbial killing [28] or the action of crawling ulcers,patientswerefollowedfor3-4weekswhilereceiving overthecleanwoundbed[60] might be enough to stimulate standard wound care (whatever modality was prescribed wound healing. We now know that both of these hypotheses bythesurgicallyledwoundcareteam),followedby3-4 likely contribute to wound healing: physical and electrical weeks of maggot therapy [2]. Tissue quality and wound stimulation of healthy cells can induce the release of host size were assessed and photographed weekly. The average 2 growth factors, and any meaningful reduction in debris woundsize(cm) increased weekly during control therapy and biofilm or microbial population likely decreases inflam- but decreased by over 20% per week with maggot therapy mation and promotes wound healing. Some investigators (𝑃 < 0.001). Debridement of necrotic tissue was achieved in believed that the alkalinity of maggot-treated wounds, along just 10 days with maggot therapy. None of the control wounds with the isolated allantoin and urea-containing compounds, were debrided by more than 50%, even with 4 weeks of was responsible for wound healing [61]. In fact, today, treatment. allantoin and urea are components of many cosmetics. Acohortof63patientswith92pressureulcerswas With recent advances in cellular biology and chemistry, prospectively followed for at least 8 weeks while receiving we now know that maggot ASE stimulates the prolifera- either standard wound care (as prescribed by the hospital’s tion of fibroblasts [62] and endothelial tissue (unpublished wound care team) or maggot therapy (two 48- to 72-hour data), increases angiogenesis [63], and enhances fibroblast cycles per week) [18]. In patients with bilateral wounds, only migration over model wound surfaces [64–66]. Biopsies of onewastreatedwithmaggottherapy,andpatientswere maggot-treated wounds reveal profound angiogenesis [67]. allowed to select that one. Therefore, maggot-treated wounds 2 2 Usingremittancespectroscopytoevaluatepatientsbeforeand tended to be larger (22 cm versus 14 cm ;P <0.05)and after maggot therapy, Wollina and colleagues [68]foundthat deeper (35% down to bone in the maggot therapy group; 8% vascular perfusion and tissue oxygenation surrounding the in the control group). Nevertheless, 4- and 8-week healing wound actually increased following maggot therapy. Zhang rates were significantly better for maggot-treated wounds and colleagues [69] are currently seeing evidence that maggot than control wounds, as was the weekly decrease in surface extracts may even stimulate the growth of neural tissue. area and the rate of granulation tissue growth over the base Earlyclinicalreportsofmaggot-inducedwoundhealing of the wound (see Table 2). were merely case studies or series; but beginning in the 1990’s, The wound healing rate, based on studies by Gilman [70] controlled comparative trials of maggot therapy began to and Margolis et al. [71], was defined as the change in surface Evidence-Based Complementary and Alternative Medicine 7 area divided by the mean circumference over time. Four and the difference, given that there were less than 100 subjects in eight-week healing rates have repeatedly been shown to be each of 3 arms. Some believe that the reason that no greater accurate surrogates for wound healing in general, although woundhealingwasseeninthemaggot-treatedarmswas they have not been accepted as substitutes for complete relatedtothestudydesign,whichuseda“maggotdebride- wound closure in clinical trials. ment” protocol rather than a “maggot growth promotion” Indeed, twice as many wounds in the maggot-treated protocol [72]. In this study, maggot therapy was stopped as group completely healed during the period of observation soon as wounds were debrided (treatment day number 15, (39% within an average of 12 weeks versus 21% within an on average, for the free range maggot therapy group) and average of 13.4 weeks). But most patients were not followed was never administered to those patients again, even if their more than 10 weeks, and this difference was not statistically wounds deteriorated over the subsequent 7 months that it significant. took, on average, to heal [73]. Inanothercohortof18diabeticsubjectswith20nonheal- Indeed, maggot-associated wound healing and antimi- ing neuropathic and neuroischemic foot ulcers, six wounds crobial activity is likely short-lived after the maggots are were treated with conventional therapy, six with maggot removed. Sherman and Shimoda [74] reported successful therapy, and eight with conventional therapy first and then wound healing without infection or dehiscence in patients maggot therapy [19]. As in the pressure ulcer patients, 4- and surgically closed 1–21 days following maggot debridement to 8-week healing rates were significantly better for maggot- be 100%, compared to wounds debrided without MDT or treated wounds than control wounds, as was the weekly those debrided with MDT more than 21 days before closure, change in surface area and the rate of granulation tissue which healed successfully only 68% of the time. growth over the base of the wound (Table 2). Repeated Many clinicians intuitively feel that faster debridement measures ANOVA indicated that treatment rendered was the brings faster wound healing. After all, the wound cannot only factor associated with these differences. heal if infected, necrotic tissue and debris are occupy- In Armstrong’s retrospective case-control study of lower ing the center of the wound. Yet, it has been difficult extremity wounds in nonambulatory hospice patients [23], to find any large RCT that demonstrates this to be true in which the researchers demonstrated significantly better [75]. Perhaps the problem has been that chronic wounds infection control and fewer amputations required in the often reacquire infection or biofilm; and additional tissue maggot-treated group, the difference in wound healing rates may die, requiring redebridement. Addressing the on-going between the maggot-treated group (57% healed) and the need for wound cleaning and disinfection is the paradigm control group (33% healed) was not statistically significant. behind “maintenance debridement,” and appears to be gain- In this study population, the probability of healing may have ing support as an important strategy for treating wounds had more do to with the patients’ underlying circulatory [76, 77]. compromise, malnutrition, and poor physiologic health than If this paradigm is correct, it would explain why maggot with the treatments rendered. For those wounds that did therapy continued beyond the point of gross debridement heal, wound healing was much faster in the maggot-treated has been associated with faster wound healing [2, 18, 19, 22]. wounds than in the control wounds (18 weeks versus 22 It may be true that no one single method of maintenance weeks; 𝑃 = 0.04). debridement is faster than another. But maggot therapy is one As previously discussed, in the 140-subject RCT by of the few highly effective methods of debridement which Markevich and colleagues [22], wounds treated with maggot can safely and inexpensively be continued throughout the therapy were ultimately covered with more granulation tissue healing process, which may explain why it remains one of the (𝑃 < 0.001)andweresmallerinsize(𝑃 < 0.05)than methods of maintenance debridement best associated with thewoundsinthecontrolstudyarm.This10-daylong faster wound healing. clinical trial failed to show any significant difference in wound healing between the MDT arm (60% healed by day 10) and thecontrolarm(34%healedbyday10),butitisgenerally 3.5. Miscellaneous Actions. Platelets, neutrophils, and mono- believed that the lack of any significant difference may be due cytes/macrophages are among the first cells recruited to the to the fact that this 10-day debridement study was much too young wound when they remain beyond their usefulness short to detect any meaningful wound healing. Indeed, 60% and contributed to an unending inflammatory phase that healingofdiabeticfootulcersinonly10daysinsteadof10 can interfere with or even prevent the wound from moving weeks is, by itself, quite impressive. forward in the healing process. Maggot secretions have Many in the wound care community looked with excite- recently been found to affect the activity of these cells in ways ment at the study by Dumville et al. [21], intended to evaluate that decrease inflammation. While this can be thought of as maggot-induced wound healing in venous stasis ulcers. This a subset of actions which promote wound healing, they are RCT demonstrated significantly faster debridement in the separatedoutforthepurposeofthisdiscussionbecausethese maggot therapy arms (as already discussed), but did not actions may also play important roles in disinfection, if not demonstrate any significantly faster healing in those subjects. also debridement. Several reasons may explain this, including the simple fact Exposing unstimulated human neutrophils to crude L. that the maggots may not expedite healing in any clini- sericata salivary gland extract, Pecivova and colleagues [78] cally meaningful way. Alternatively, as the authors pointed measured no effect on superoxide generation or myeloperoxi- out, their study may have been too small to demonstrate dase (MPO) release. But when opsonized zymosan stimulated 8 Evidence-Based Complementary and Alternative Medicine

Medicinal maggots (Lucilia sericata)

secretions and Physical contact with excretions host

Crawling over wound, Proteases rasping the Debridement tissue DNAse Directed movement to areas of greatest need

Release of antimicrobial peptides Ingestion Disinfection DNAse Scraping away biofilm Dissolving of biofilm

Production of allantoin and urea Elevation of host Fibroblast temperature proliferation and migration transmitted from maggot mass Angiogenesis Crawling Growth stimulation and over wound, Increased wound healing stimulating perfusion the release of Reduced growth factors inflammatory and inducing response changes in electrical Reduced potentials? compliment, C3 and C4

Figure 2: Schematic drawing of proven and postulated mechanisms by which medicinal maggots promote wound healing. neutrophils were exposed to high concentrations of the sali- Cazander and colleagues [82]recentlydiscoveredthat vary gland extract, superoxide generation and MPO release maggot ASE reduced complement activation in healthy and were significantly reduced. The researchers concluded that immune-activated (postoperative) human sera by as much as medicinal maggots might aid in wound healing by decreasing 99.9% by breaking down C3 and C4 proteins. the generation of proinflammatory factors in this way, while still maintaining normal phagocytosis or apoptosis. van der Plas et al. [79] monitored cyclic AMP (cAMP) 3.6. Integrated Conceptualization of Maggot Therapy Actions. in human neutrophils before and after exposure to L. seri- From clinical and laboratory studies to date, it is clear cata ASE and then again in human monocytes [80]. Their that maggot therapy contributes significantly to wound findings of elevated cAMP and suppressed proinflammatory care, both physically and biochemically. Figure 2 represents responses (without a measurable decrease in antimicrobial our current understanding of the mechanisms by which activity) led the authors to conclude that the larval secretions maggottherapyaffectswoundhealing.Thisschemaisa weremovingthemonocytesandneutrophilsforwardfrom work-in-progress, intended to be modified as additional theproinflammatoryphaseandintotheangiogenicphaseof research adds to our understanding of the maggot-wound wound healing [81]. interaction. Evidence-Based Complementary and Alternative Medicine 9

C DEadministered as a maintenance debridement modality, that is, during and/or after complete debridement has already been G F achieved? AB A randomized double cross-over study could address H I J these questions if subjects were randomized to receive either KLmaggot therapy or standard of care debridement, followed by standard of care or maggot therapy until wound closure. This MN O 4-armed RCT would consist of the following: (1) standard of care throughout; (2) Standard of care debridement following PQby maggot therapy; (3) maggot debridement followed by stan- dard of care thereafter; and (4) maggot debridement followed by maggot therapy maintenance (i.e., maggot therapy once 0 246 8 10 12 14 16 18 20 22 24 weekly, ala Sherman et al., 2007 [83]). Time (weeks) The addition of two more study arms (or alternatively a Figure 3: Schematic representation of a clinical trial proposed to separate study) could also address the advantages and disad- demonstrate the wound healing effects of maggot therapy. After vantages of free range versus contained (“bagged”) maggots. a 2-week baseline data collection (AB), nonhealing wounds are There is clear evidence that the long-touted benefits of maggot randomized either to receive the surgical and medical standard of therapy are due, in part, to the physical contact of the maggots care (CD), standard (confinement) maggot therapy dressings (HI), on the wound and the maggots’ ability to mobilize to the deep or containment (bagged) maggot dressings (MN) for debridement. recesses and other areas of need. A controlled comparative Maggot-debrided wounds would then receive either standard care trial between free range and contained maggots would allow for wound closure (IJ; NO) or maggot therapy (MDT maintenance us to assess the relative contribution of the maggot’s physical debridement, KL or PQ) to evaluate the presence of maggot- versus chemical contributions to wound healing. stimulated wound closure. To optimize enrollment and retention, Measurements of cost-efficacy, antimicrobial activity, and subjects randomized to standard care may cross over to maggot therapy if there has been no significant improvement after 12–24 relative safety should also be incorporated into such a weeks of therapy. study, in order to capture as much data and address as many perspectives as possible concerning the clinical utility of maggot therapy for nonhealing wounds. This could be accomplished by collecting cost data for materials, services, 3.7. Future Study Recommendations. Many questions remain and healthcare providers, collecting carefully selected and about wound healing, in general, and maggot therapy in performed microbial cultures over the course of treatment, particular. Several of these questions might be answered and monitoring a wide variety of health and quality-of-life by a single well-designed clinical study. This review was parameters. undertaken to help design the next study or at least offer an Such a large prospective study would be expensive and is initial proposal for what that study might look like. notlikelytobefundedwithinthenearfuture.Maggottherapy Evidence of maggots’ debridement efficacy is irrefutable. should not be withheld until such a study is completed, Clarity is still needed regarding maggot therapy’s role in for there exists, already, a wealth of data supporting the promoting wound closure. When maggot therapy has been efficacy and safety of maggot therapy in wound care. Smaller used for debridement alone, some studies have shown faster prospective studies and large registry studies may be able overall healing, others have not. Those studies that have to address many of the same issues as does the RCT just suggested or demonstrated significantly faster wound closure proposed. But for those with the will and resources to conduct have looked at short-term findings: healing that occurs alargeRCTofmaggottherapy—evenifsuchresourcesneed during or shortly after maggot therapy is administered. to be pooled together—this is the RCT that might most Studiesthathavelookedathealingratesmonthsaftermaggot efficiently yield the answers to the most pressing questions debridement was terminated have not demonstrated any remaining about the mechanisms of maggot-induced wound differenceinhealingrates.Thisislikelythekey,forwenow healing. understand that maintenance debridement and maintenance disinfectioncanpromotewoundhealing.Wealsonowrec- ognize that healthy-looking wounds can deteriorate quickly, 4. Conclusions especially when chronic, or when there are impediments to woundhealing.Thephysicaleffectsofmaggotsonthewound Maggot therapy has long been recognized as a safe and and the bioactive molecules that they secrete do not last effective treatment for wounds. It is associated with three long after therapy, so wounds that do not heal immediately broad actions: debridement, disinfection, and hastened tissue after maggot debridement will be at risk for recolonization, growth. We now know that these actions are the result of infection, stagnation, and necrosis. a large number of maggot-host interactions, some of them A single study might address these questions: Does chemicalandsomephysical.Essentially,themaggotscrawl maintenance debridement provide clinical benefits over sin- overthewound,plowingthebaseastheysecretetheir gle or episodic debridement, in terms of wound healing rich digestive enzymes, just as a farmer plows and fertilizes rates, and does maggot therapy enhance wound healing if the field. Plowing without fertilizing or fertilizing without 10 Evidence-Based Complementary and Alternative Medicine plowing, the farmer will produce a smaller yield and the (the effect on microbial flora and clinical infection over time), maggots will be less effective in their debridement. and the effect of maggot therapy on short- and long-term The maggots’ secretions may even induce the maturation qualityoflifecouldandshouldalsobepartofsuchastudy. of monocytes and neutrophils from proinflammatory cells By carefully planning our future clinical studies—pooling into their angiogenic phenotype, thereby lifting the wound multi-institutional resources if necessary—we can maximize outofitsinflammatoryrut,andthenforwardintothe the impact and clinical relevance of these studies, while proliferative, healing phase of wound healing. minimizing their overall expense. Until such studies are per- Today, the debridement efficacy and efficiency of medic- formed, clinicians can continue to use maggot therapy with inal maggots are beyond doubt. Debridement itself has been confidence, at least for wound debridement and maintenance associated with both infection control and faster wound debridement. We are now also confident in the maggot’s healing, yet the clinical utility of maggot-induced disin- capacity to push the infected or simply stagnant “clean” fection and growth stimulation activity remain suspect. wound to and through cellular proliferation and healing. Therapists can recount case after case of maggot-associated disinfection and wound healing, and most small clinical Conflict of Interests studies clearly demonstrate disinfection and/or growth stim- ulation alongside debridement. But the largest prospective The author declares that there is no conflict of interests clinical studies to date have demonstrated only maggot- regarding the publication of this paper. induced debridement, not disinfection or growth promotion. Laboratory studies demonstrating disinfection and growth- promoting properties abound. Are we imagining a clinical Acknowledgments effect that does not really exist? Or have we simply been The performance of this study received no commercial unable to perform the RCT that would adequately and support. Publication fees were subsidized by the BioThera- irrefutably demonstrate maggot-associated disinfection and peutics, Education and Research Foundation, whose mission wound healing? istoadvancehealthcarethrougheducationandresearch Thorough review of the literature suggests that the in biotherapy. The following individuals assisted in the debridement, antimicrobial, and growth-promoting activities searching and retrieving of some of the references reviewed maybeshort-lived,lastingnomorethanafewweeksafter for this study: Karin Thompson (Administrative Assistant), maggot therapy is terminated (not unlike the actions of most Dr. Tarek Tantawi (BTER Foundation Research Fellow), wound therapies). Debridement efficacy can be measured at Lynn Wang (BTER Foundation Communications Intern), the culmination of the maggot debridement treatment, but and Katherine Watt (BTER Foundation Research Intern). healing itself cannot be measured until the wound completely The author thanks and acknowledges the sacrifices of his closes, and, for some studies, this has not occurred until wife, Julie, and children, Rebecca and Hannah Sherman. This many months after maggot therapy was discontinued. The work would not have been possible had it not been for their full clinical benefits of maggot therapy may be best realized sacrifices. when treatments are continued as a method of maintenance debridement, that is, beyond the point of simple debride- ment. References Four- and six-arm clinical studies are proposed to test this hypothesis. As a multicenter study, it should be possible [1] W. S. Baer, “The treatment of chronic osteomyelitis with the maggot (larva of the blow fly),” Journal of Bone and Joint Surgery, to assemble, quickly, the large number of subjects needed vol. 13, pp. 438–475, 1931. to attain the necessary power. 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Springer Science+Business Media, Dordrecht, The Netherlands, [23] D. G. Armstrong, P. Salas, B. Short et al., “Maggot therapy in 2013. “lower-extremity hospice” wound care: fewer amputations and [7] J. Stechmiller and G. Schultz, “Bench science advances for more antibiotic-free days,” Journal of the American Podiatric chronic wound care,” in Chronic Wound Care: A Clinical Medical Association,vol.95,no.3,pp.254–257,2005. Source Book for Healthcare Professionals, D. L. Krasner, G. [24] M. L. Marineau, M. T. Herrington, K. M. Swenor, and L. J. Eron, T. Rodeheaver, and R. G. Sibbald, Eds., pp. 67–73, HMP “Maggot debridement therapy in the treatment of complex Communications, Malvern, Pa, USA, 4th edition, 2007. diabetic wounds,” Hawaii Medical Journal,vol.70,pp.121–124, [8] K. Y. Mumcuoglu, “Clinical applications for maggots in wound 2011. care,” The American Journal of Clinical Dermatology,vol.2,no. [25] K. Opletalova,´ X. Blaizot, B. 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Henrik Enghoff,1 Nicola Manno,2,3 Sévérin Tchibozo,4 Manuela List,5 Bettina Schwarzinger,5 Wolfgang Schoefberger,6 Clemens Schwarzinger,5 and Maurizio G. Paoletti2

1 Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen Ø, Denmark 2 Dipartimento di Biologia, Universita` di Padova, lab. Agroecology and Ethnobiology, Via U. Bassi, 58/b, 35121 Padova, Italy 3 Escuela de Postgrado en Ciencias Biologicas,´ Universidad Nacional de Trujillo, Peru 4 Centre de Recherche pour la Gestion de la Biodiversite,´ 04 BP 0385 Cotonou, Benin 5 Institute for Chemical Technology of Organic Materials, Johannes Kepler University Linz, Altenberger Strasse 69, 4040 Linz, Austria 6 Institute for Inorganic Chemistry, Johannes Kepler University Linz, Altenberger Strasse 69, 4040 Linz, Austria

Correspondence should be addressed to Maurizio G. Paoletti; [email protected]

Received 15 May 2013; Revised 22 October 2013; Accepted 23 October 2013; Published 12 February 2014

Academic Editor: Tung-Sheng Chen

Copyright © 2014 Henrik Enghoff 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 first record of millipedes (Diplopoda) being regularly used for food by humans (the Bobo people of Burkina Faso) is given, including information on how the millipedes are prepared. The species in question are Tymbodesmus falcatus (Karsch, 1881) and Sphenodesmus sheribongensis (Schiøtz, 1966) (Gomphodesmidae) and an unidentified species of Spirostreptidae. New information on the nutritional value of millipedes is provided; unsaturated fatty acids, calcium, and iron contents are particularly high. The millipedes’ defensive secretions, hydrogen cyanide and benzoquinones, present a severe challenge for the spread of millipedes as an everyday food source. On the other hand, the possibility that benzoquinones may act as insect-repellents, as known from studies on nonhuman primates, and that sublethal cyanide ingestion may enhance human innate resistance to malaria, suggests promising ethnomedical perspectives to our findings.

1. Introduction Millipedes(Diplopoda)havesofarnotbeeninfocus as minilivestock. Indeed, most orders of millipedes (Glom- Small vertebrates and invertebrates, especially insects, the erida, Polyzoniida, Siphonocryptida, Platydesmida, Siphono- so called minilivestock, have been considered a promising phorida, Callipodida, Julida, Spirobolida, Spirostreptida, and resource for Earth’s human population that will reach 9 Polydesmida) are known for their chemical defenses and, billion humans in 2050 [1–3] and are potential candidates unlike their relatives, the centipedes (Chilopoda), which in for reducing the higher and increasing impact on resources several cultures (China, Alto Orinoco in Venezuela, and represented by larger livestock and inland fish production [4]. Korea) have been used as medical remedies and/or food Information on traditional local use of these small animals items [9, 10], no information on millipedes as human food is an important starting point for studying minilivestock has been available until now. A wide spectrum of chemicals as potential food resources for humans [5–8]. In addition, has been identified from millipede defensive secretions11 [ ], local use of invertebrates may have unexpected ethnomedical the most widespread ones being benzoquinones (in most implications. cylindrical millipedes, superorder Juliformia) and hydrogen 2 Evidence-Based Complementary and Alternative Medicine cyanide derived from mandelonitrile and related compounds (in most flatbacked millipedes, superorder Merocheta). These toxic, smelly chemicals make millipedes unattractive for most predators, although there are some animals, vertebrates as well as invertebrates, which eat millipedes [12, 13], and some are even specialized on a millipede diet, for example, assassin bugs (family Reduviidae) of the subfamily Ectri- chodiinae [14] and beetle larvae of the family Phengodidae [15]. A few vertebrates are reported to eat millipedes as for instance banded mongoose (Mungos mungo)[16]. Some Figure 1: Live gomphodesmid millipede (Tymbodesmus falcatus) birds and nonhominid primates use toxic millipedes for from Kou. S. Tchibozo phot., 2011. “self-anointment,” presumably exploiting an insect-repellent effect of the millipedes’ defensive chemicals, especially ben- zoquinones [17–20]. We have, however, not been able to trace any record of millipedes being used as food in any human society, but now we can report the consumption of millipedes by the Bobo population of Burkina Faso, a region where entomophagy has been extensively described [21]. The millipedes which are used as human food by the Bobo belong to two families: Gomphodesmidae and Spirostrepti- dae. Gomphodesmidae (flatbacked millipedes of the order Polydesmida) (Figures 1 and 2)belongtothe“cyanogenic” millipedes; the family is endemic to the African continent south of the Sahara, includes 146 named species [22], and was monographed by Hoffman (2005)23 [ ]. The only gom- Figure 2: gomphodesmid millipede from Kou after boiling. S. Tchi- phodesmid species previously recorded from Burkina Faso bozo phot., 2012. is Tymbodesmus falcatus (Karsch, 1881), collected in Oua- gadougou [23]. One of the gomphodesmid species used for food at Kou village in Burkina Faso is indeed T. falcatus (D. Vandenspiegel det.), the other is Sphenodesmus sheribongensis (Schiøtz, 1966) (HE det.). T. falcatus is known from Mali, Burkina Faso, Nigeria, Sudan, and Central African Republic; S. sheribongensis was previously known from Ghana, Ivory Coast, and Nigeria [23]. Lewis [24]studiedthelifehistoryand ecology of both species in Zaria, Northern Nigeria. Both have a two-year life cycle. Juvenile stadia of S. sheribongensis live entirely in the soil, whereas adults can be extremely abundant on the soil surface during the first part of the rainy season (from May to July). T. falcatus is similar, except that the last juvenile (subadult) stadium is seasonally surface active like the adults, albeit in smaller numbers. Spirostreptidae (cylindrical millipedes of the order Spiro- streptida) (Figure 3) belong to the “quinone” millipedes: the Figure 3: Spirostreptid millipede from Kou. S. Tchibozo phot., 2012. family is near-endemic to the Afrotropical and Neotropical regions, includes 275 named species [22], and was mono- of millipede is small, they will constitute a poor source of graphed by Krabbe [25]. No species of Spirostreptidae have protein. Their guts mainly contain soil and litter remains24 [ ], been recorded from Burkina Faso, and the one occurring at but their calcified exoskeleton might constitute a considerable Kou has not yet been identified. source of calcium, which may constitute 13–17% of the dry We do not have any strong evidence that T. falcatus and weight [29–31]or9%ofthefreshweight[32], In fact, S. sheribongensis actually produce hydrogen cyanide, nor that millipedes are considered an essential source of calcium for the spirostreptid in question produces quinones. The general egg production in certain birds [30, 33, 34]. In this work occurrence of these substances in the respective higher taxa we present data based on Tymbodesmus falcatus, one of the to which the species belong, however, is strong circumstantial species eaten by the Bobo people. evidence that this is actually so, although recent studies have demonstrated a larger diversity in millipede defensive 2. Material and Methods chemicals than previously assumed [26–28]. Until this study, the nutritional value of millipedes has 2.1. Sampling and Ethnobiological Data Collection. Obser- never been assessed. Considering that the muscle volume vations and interviews with the Bobo people were made Evidence-Based Complementary and Alternative Medicine 3

∘ 󸀠 by ST in 2011 and 2012 in Burkina Faso (Kou, 11 10.88 N, Table 1: Tymbodesmus falcatus amino acid analysis (based on dry ∘ 󸀠 004 26.62 W, altitude 351 m, near Bobo-Dioulasso). Exem- matter). plars of the edible millipedes were collected and are now Amino acid mg/mg preserved in Musee´ Royale de l’Afrique Centrale (Tervuren, Belgium) and the Natural History Museum of Denmark Alanin 0.0676 (Copenhagen). Five Bobo people, especially women, were Asparagin 0.0137 interviewed for information on collection and preparation of Asparaginic acid 0.0164 the edible millipedes. Glutamin + lysin 0.0217 Glutamic acid 0.0000 2.2. Nutritional Analysis. In order to assess the nutritional Glycin 0.0000 value of this unconventional food we determined various Isoleucin + leucin 0.0283 nutritional parameters of a whole Tymbodesmus falcatus Methionin 0.0056 male specimen. The raw specimen was preserved in 70% Phenylalanin 0.0132 ethanol, subsequently dried and homogenized by cryomilling Prolin 0.0062 (SPEX Freezer/Mill 6770). The resulting powder was used Serin 0.0142 for analysis of chitin, fatty acids, amino acids, and metal Threonin 0.0251 content. No appropriately preserved specimens of the other species in question were available. Dry weight of millipedes Tryptophan 0.0053 wasmeasuredonoven-driedspecimensandkeptinalcohol Tyrosin 0.0151 for 45 years (collected in Nigeria by J.G.E. Lewis). Valin 0.0193 Total 0.2518 2.2.1. Pyrolysis-GC/MS for Fatty Acid Analysis. 100 𝜇gofthe sample was placed in a quartz tube, and 4.5 𝜇Lofadiluted, aqueous solution of tetramethylammonium hydroxide was for 2 minutes and in 17 minutes to 30% acetonitrile which is ∘ added. The samples were subsequently pyrolyzed at 450 C held for a further 3 minutes) on a Waters AccQ Tag column × for 10 s with a CDS 5250 pyrolysis autosampler attached to a (3.9 150 mm). Quantification was done using extracted ion Thermo Trace GC Ultra/MD 800 gas chromatography/mass chromatograms. With this procedure arginine, cysteine, and spectrometry system. Volatile products were separated on histidine could not be analyzed and leucine/isoleucine as well a Supelco SP 2330 column (30 m, ID 0.32 mm, 0.2 𝜇mfilm as glutamine/lysine could not be separated; therefore, the −1 thickness) with helium 4.6 as carrier gas (2 mL⋅min )and concentration of those amino acids is given as a sum. identified by comparison to reference compounds as well as interpretation of their EI mass spectra and comparison to 2.2.4. Metal Analysis. Metal content was analysed with NIST 2002, Wiley, and NBS electronic libraries. The pyrolysis inductively coupled plasma optical emission spectrometry ∘ ∘ interface was kept at 300 CandtheGC/MSinterfaceat280 C; (ICP-OES) according to EN ISO 11885 from a commercial ∘ ∘ the GC was programmed from 100 C(1min)to230 C(5min) laboratory. ∘ −1 at a rate of 10 Cmin . The mass spectrometer was operated ∘ in EI mode (70 eV) at a source temperature of 200 C[35]. 3. Results

2.2.2. Solid State NMR for Chitin. Chitin was determined by 3.1. Collection and Preparation of Millipedes at Kou Village. Solid State NMR; all spectra were recorded on a narrow-bore According to the interviewed villagers in Kou, millipedes 11.7 T instrument (500 MHz, 1 H Larmor frequency) at magic are collected under bricks around houses made of straw and angle spinning rates of 10.0 kHz at 300 K. 13C chemical shifts under decomposing wood. Once collected, the millipedes are are given in reference to tetramethylsilane TMS, using the placed in a pot with water filtered through firewood ashes, sharp resonance of TMS as external calibration. A basic cross for 3–5 minutes until boiling. Then they are removed and polarization experiment with total suppression of sidebands left to dry on a roof for 3 days. Such preparation is specific with a cross-polarization contact time of 2 ms was employed, for millipedes and different from those described for other with an effective acquisition time of 27.9 ms and a recycling arthropods in West Africa, especially for insect maggots and delay of 5 s. The magic angle was adjusted using the 79Br weevils, which are mainly roasted and fried [21]. The dried resonance of KBr, and the actual sample temperature was millipedes are placed in a tomato sauce to which is added the 207 determined using the Pb resonance of Pb(NO3)2 for calib- traditional African mustard known as soumbala (fermented ration [36]. seeds of the ner´ e´ tree, Parkia biglobosa,verywidelyconsumed in Burkina Faso and West Africa in general), shea butter oil, 2.2.3. Amino Acid Analysis. The powder obtained after cry- and toˆ (a paste made from maize or sorghum flour). For some omilling was hydrolysed by refluxing with hydrochloric acid meals, the millipedes replace meat. containing 5% phenol for 24 hours under exclusion of oxygen. The complete sample was then evaporated to dryness, 3.2. Nutritional Values of Tymbodesmus falcatus. Proteins redissolved in water, and analyzed with HPLC/MS using represent 25% of total dry weight (calculated as the sum of −1 0.5 mL⋅min of a water acetonitrile gradient (100% water amino acids); the amino acid profile (Table 1)issimilartothat 4 Evidence-Based Complementary and Alternative Medicine

5 Table 2: Fatty acid distribution as determined by pyrolysis-GC/MS 7.37 analysis. Unsaturated fatty acids constitute 40% of total fatty acids of 100 10 Tymbodesmus falcatus. 90 9.06 80 No. ID Name 𝑡𝑅 Percent 70 1 10:0 Caprylic acid 3 0.3 60 2 12:0 Lauric acid 4.33 0.5 50 3 14:0 Myristic acid 5.82 3.1 40 4 15:0 Pentadecanoic acid 6.56 1.0 9 30

Relative abundance Relative 8.73 5 16:0 Palmitic acid 7.37 43.1 3 20 N 6 16:1 n9 Sapienic acid 7.61 0.2 5.82 17.15 N 7 7 16:1 n7 Palmitoleic acid 7.67 1.5 10 4 1214 0 8 17:0 Margaric acid 8.01 0.8 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 9 18:0 Stearic acid 8.73 7.8 Time (min) 10 18:1 n9 Oleic acid 9.06 36.8 11 18:1 n7 Vaccenic acid 0.0 Figure 4: THM-GC/MS profile of Tymbodesmus falcatus; retention 𝑡 12 18:2 n6 Linoleic acid 9.5 1.6 times ( 𝑅) refer to compounds listed in Table 2: palmitic acid 18 : 1 n9 (𝑡𝑅: 7.37) (5) and oleic acid 16 : 0 (𝑡𝑅: 9.06) (10) constitute 80% of 13 18:3 n6 Gamma linolenic acid 0.0 total fatty acids. 14 20:0 Arachidic acid 10 1.1 15 18:3 n3 Alpha linolenic acid 10.08 0.1 16 18:2 x Octadecenoic acid 10.24 0.5 Table 3: Tymbodesmus falcatus metalcontents(basedondry 17 20:1 n9 Gadoleic acid 0.0 matter) and DRIs for a pregnant women of 19–30 yrs, by IOM 2004 18 18:2 x Octadecenoic acid 10.34 0.5 [39]. 19 18:2 x Octadecenoic acid 10.55 0.3 Metal mg/kg DRI(mg/day) 20 20:2 Eicosadienoic acid 0.0 Pb 6.2 — 21 20:3 Eicosatrienoic acid 0.0 Cd <0.5 — 22 22:0 Behenic acid 11.2 0.1 Ca 174,000 1,000 23 20:4 n6 Arachidonic acid 0.0 Fe 10,600 27 24 21:0 Heneicosylic acid 11.77 0.1 K 2,610 4,700 25 20:5 n3 Timnodonic acid 0.0 Cu 789 1 26 24:0 Lignoceric acid 12.33 0.4 Mg 4,990 350 27 22:5 n6 Docosapentaenoic acid 0.0 Na 1,630 1,500 28 22:6 n3 Docosahexaenoic acid 0.0 Zn 160 11 Total 100.0

Chitin constitutes around 5% of the total dry weight, a of insects and crustaceans, for example, crickets and shrimps lowamountcomparedtoCa,butitisonlylocatedinthe [37]. Unsaturated fatty acids constitute a relevant fraction exoskeleton (incl. legs). (40%) of total fatty acids (Table 2, Figure 4), however lower A trace level of dimethylcyanamide revealed in the GC/ than those described for widely consumed and appreciated MS (see Figure 4) is the only direct evidence of cyanogenic edible insects [38]. Calcium levels (Table 3)areveryhigh compounds in our sample. (17.4% of dry weight) which is higher than previously pub- lished values [29–32]. 4. Discussion The dry weight of individual Tymbodesmus falcatus was 0.42–0.54 g (mean 0.46, 𝑛=3)andofthesmallerSphen- The gomphodesmid millipedes utilized by the Bobo people odesmus sheribongensis, 0.08–0.11 g (mean 0.09, 𝑛=4). are quite small (max. 5 cm long), but gomphodesmids may Spirostreptidae vary very much in size; live weights of up occur in very large numbers at certain times of the year. to 80 g have been measured (HE unpublished). Considering Thus, Lewis [24]statedthat50specimensofS. sheribongensis that the Ca content of a single Tymbosdesmus f. is about could be collected in 10–15 minutes at the beginning of the 80–90 mg (174 mg/g × 0.46 g), consumption of around 12- rainy season in Zaria, Nigeria and even (pers. comm.) that 13 gomphodesmid individuals will provide 1000 mg/day, (the it was sometimes impossible to take a step without crushing Dietary Reference Intake (DRI), by IOM 2004 [39]). Also several individuals. Barbetta et al. [40]used1200specimens iron content (100,600 mg/kg) is important considering that of Haplogomphodesmus pavani (Demange, 1965) for their only 6 individuals provide the adequate amount for a women biochemical study, an indication that also this species can be during pregnancy (DRI: 27 mg/day) and only 2-3 for men quite abundant. Spirostreptids may occasionally form huge (DRI: 8 mg/day) [39]. swarms, for example, in Ghana and Zimbabwe [41]. They are Evidence-Based Complementary and Alternative Medicine 5

Table 4: Summary of the current knowledge about utilization of millipedes by mammals as anointing and/or food item.

Mammals Millipedes Chemicals Use Reference Marsupialia Leptodesmus dentellus (Chelodesmidae) Benzoquinones Consumption and Opossum (Didelphis albiventris) [47] Gymnostreptus olivaceus and Cyanogenics sniffing (Spirostreptidae) Carnivora Consumption after Orthoporus sp. White-nosed coatis (Nasua narica) Benzoquinones prey-rolling [13] (Spirostreptidae) treatment Consumption after Meerkat-mangoose (Suricata suricatta)?? [49, 51] treatment Striped skunk (Mephitis mephitis) ? ? Consumption [52] Primates Orthoporus dorsovittatus Capuchin monkeys (Cebus sp.) Benzoquinones Self-anointing [19] (Spirostreptidae) Orthoporus dorsovittatus Capuchin monkeys (C. olivaceus) Benzoquinones Self-anointing [18] (Spirostreptidae) Anadenobolus monilicornis Owl monkeys (Aotus sp.) Benzoquinones Self-anointing [55] (Rhinocricidae) Charactopygus sp. Black lemurs (Elemur macaco) Benzoquinones ? Self-anointing [17] (Spirostreptidae) Self-anointing and consumption after [50, 53, Lemurs (Varecia rubra and Eulemur fulvus albifrons)? ? handling and 72] salivating Tymbodesmus falcatus and Sphenodesmus sheribongensis Benzoquinones Consumption after This Humans, Bobo population (Gomphodesmidae) and Cyanogenics boiling and drying study unknown Spirostreptidae sometimes of considerable size, up to 30 cm long. Consid- as the majority of gomphodesmids, including A. laxus and H. ering the regular and high levels of some essential nutri- pavanii, that is, 11 pairs of glands opening along the sides of ents (Ca, Fe, PUFA) and seasonal abundance of millipedes, the body [23]. they likely represent a significant type of minilivestock for Benzoquinones have been detected in several spirostrep- the Bobo people. The ethnobiological/ethnopharmacological tid species [11, 28, 48]. In Figure 3 the defensive glands can uses of many unconventional species, that is, earthworms and be seen as a series of darker spots, one on each diplosegment insects, are documented in several cultures, being part of except the very first and last ones. a complex system of specific traditional knowledge adapted Some birds and mammals (see Table 4)havebeen to local resource availability and to very variable hygienic described to use “quinone” millipedes for self-anointing in standards [42, 43]thatmaynotfitwiththefood-quality/food- order to control ectoparasites and mosquito biting rate, and safety standards imposed in industrialized countries [44]. some mammals like opossum, coatis, skunk, mongoose, and As mentioned above, gomphodesmids belong to the lemurs are known to consume toxic millipedes—mainly “cyanogenic” millipedes [11]. Cyanogenesis in millipedes and Spirostreptidae—after different treatments such as prey- other arthropods has not been studied with state-of-art rolling, handling, and salivating [13, 49–52]. The complex methodology [41], but the presence of hydrogene cyanide and behaviours that precede ingestion require a considerable its precursors has been demonstrated in many species of the investment of time and energy and are evidently necessary, order Polydesmida [11, 26, 27, 45, 46], including two species of maybe in order to reduce toxicity of the millipedes to be Gomphodesmidae; HCN has been identified in the secretion eaten [53, 54]. The use of benzoquinonigenic plants for self- of Astrodesmus laxus (Gerstacker¨ 1873) from East Africa [47], anointing in orangutans [54]andowlmonkeys[55]supports and its precursor, mandelonitrile, in Haplogomphodesmus the hypothesis that benzoquinones are used by primates for pavani [40]. Although there is no direct information on their specific biochemical defensive properties. Contrarily, the secretions of T. falcatus and S. sheribongensis,thereis use and consumption of gomphodesmid cyanogenic milli- no reason to believe that they have lost the cyanogenic pedes seems to be rare even in most omnivorous mammals, function. They have the same complement of defense glands except opossum [56]. 6 Evidence-Based Complementary and Alternative Medicine

4.1. Cyanide in Traditional Foods. The use of millipedes as human food is absolutely exceptional, but the use of food items containing significant levels of cyanide is widespread [26]. Over 2000 plant species contain cyanide as a defense against insects and other herbivores [26]andthemost important cyanogenic crop is cassava (Manihot esculenta, Crantz), a staple food of hundreds of millions of humans in the tropics [57, 58]. In the Amazons—the centre of domestication of cassa- va—the more toxic bitter varieties named yuca amarga are the most intensively cultivated because of their resistance to pest insects and rodents. However, cassava domestication largely preceded malaria ingression in the New World [59] Figure 5: Scheme of the exceptional use of toxic millipedes: as and the indigenous preparation is aimed at avoiding the demonstrated for capuchin monkeys, that during the rainy season cyanogenic compounds, consisting of specific postharvest use millipedes’ secretions against mosquitoes, Bobo people in operations: grinding, squeezing, toasting, and fermentation Burkina Faso may consume millipedes for their antiparasite effect. [58, 60]. Boiling alone is not enough to avoid the toxicity of yuca amarga [58], and only the sweet varieties of yuca dulce, which are low in glycosides, can be consumed safely in sickle-cell anemia [60, 70], a genetic pathology affecting ery- soups or fried (MGP personal observation in Alto Orinoco, throcytes that confers protection against Plasmodium.Thus, Venezuela). after demonstrating that cyanide interacts with hemoglobins, The Bobo people subboil the millipedes, as part of partially compensating sickle-cell dysfunctionality [60, 71], the preparation for meals. This treatment may degrade the some authors proposed that the abundant consumption of cyanogenic compounds by releasing the hydrogen cyanide bitter foods could enhance biological fitness in West African gas, thereby detoxifying the gomphodesmids. populations exposed to malaria [62, 70]. Therefore, both ethological and bioanthropological evidences suggest that Nonetheless, the short (3–5 minutes) subboiling and nat- “toxic”millipedesconsumedbytheBobopeopletakepartin ural drying appear to be a specific treatment for millipedes, a complex biocultural mechanism for malaria control. maybe aimed at preserving part of their chemicals. Moreover, benzoquinones are not readily soluble in water and the most characteristic juliform benzoquinone, toluquinone, is 5. Concluding Remarks insoluble in water [61]; thus substantial amounts of them may remain in the millipede bodies even after cooking. KeytopicsofthispaperaresummarizedinFigure 5.Whether millipedes will ever become a major actor in minilivestock 4.2. Biocultural Perspective. African populations consume husbandry may be dubious, but the Bobo people have shown raw and subboiled bitter cassava [58, 60, 62, 63], as well as that they constitute a helpful food source for an ever-growing many other bitter food items that would not be tolerated human population, especially in rural Africa. In addition, the by other populations. It has been proposed that the reduced potential of millipede chemicals for deterring mosquitoes and sensitivity to bitterness is an ancient adaptation (70 kY BP) for influencing Plasmodium and other parasites constitutes a [64] of the human bitter-taste specific receptor to bitter promising field of research. antimalarial compounds (e.g., flavonoids), which are still abundant in the West African diet [62, 65]. Cassava contains Conflict of Interests the cyanogenic glucoside compounds linamarin and lotaus- tralin that, once ingested, are metabolized to thiocyanate The authors declare that there is no conflict of interests and cyanate. Notably, these metabolites are biologically active regarding the publication of this paper. although less toxic than cyanide and, at levels of expected dietary intake, cyanide-related compounds (e.g., cyanate) are Acknowledgments able to modify essential proteins of Plasmodium falciparum and inhibit parasite survival [66]. The authors are grateful to the Bobo people at Kou for Considering that malaria represents the main cause of sharing their knowledge with them, to The Fonds Franco- mortality in the adult sub-Saharan population and that Burk- phone des Inforoutes (FFI) and the Cooperation´ Belge au ina Faso is endemic for several Plasmodium species [67, 68], Developpement´ for funding the 2011 expedition in Burk- biocultural adaptations aimed at controlling this pathogen are ina Faso, to Bakary Sanou for help with field work, to strongly expected. Natural benzoquinones are known to exert Didier VandenSpiegel for identification of T. falcatus,and an antiplasmodic activity in vitro [69], and their metabolites to Mika Zagrobelny for her attempt—against all odds— might act as systemic repellents against mosquitoes or as anti- to isolate cyanogenic compounds from alcohol-preserved malarial prophylaxis in the Bobos. Moreover, studies on West millipede specimens. The NMR spectrometers were acquired African populations have demonstrated strong links between in collaboration with the University of South Bohemia (CZ) malaria, sub-lethal cyanide intake from bitter cassava, and with financial support from the European Union through Evidence-Based Complementary and Alternative Medicine 7

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Kosta Y. Mumcuoglu

Parasitology Unit, Department of Microbiology and Molecular Genetics, The Kuvin Center for the Study of Infectious and Tropical Diseases, The Hebrew University, Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel

Correspondence should be addressed to Kosta Y. Mumcuoglu; [email protected]

Received 20 October 2013; Revised 5 December 2013; Accepted 27 December 2013; Published 6 February 2014

Academic Editor: Ronald Sherman

Copyright © 2014 Kosta Y. Mumcuoglu. 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.

A written informed consent should be obtained from the patient before hirudotherapy is initiated. The patients should be treated each day of leech therapy with anti-Aeromonas antibiotics. Leeches should be applied on the darker spots of the reattached body parts or flaps. Usually 1–10 leeches are used for each treatment, while at the beginning, the patient might need two ormore treatmentsperday.Leechtherapyisuseduntilvenouscapillaryreturnisestablishedacrossthewoundborderbyangiogenesis. Usually the treatment with leeches lasts for 2–6 days. Hematologic evaluations should be performed every 4 hrs and the patient has to receive blood transfusions when the hemoglobin level is lower than 8 g/dL. Signs of regional lymphadenitis, slight swelling, and pain of regional lymph nodes on the side of leech application and subfebrile temperature can occur. Contraindications related to hirudotherapy include arterial insufficiency, hemophilia, hemorrhagic diathesis, hematological malignancies, anemia, hypotension, and sepsis. Leech therapy is not recommended in pregnancy and lactation and in patients with an unstable medical status, history of allergy to leeches or severe allergic diathesis, and disposition to keloid scar formation, as well as in those using anticoagulants and immunosuppressants.

1. Introduction are numerous studies showing the use of leech therapy for hematomas, penile and total scalp replantation, and pedicled Medicinal leeches have been used in the past 50 years skinflaps,aswellasforthesalvageoftheentirelowerlimp for the salvage of tissue with venous congestion. In 1960, [6–19]. Deganc and Zdravic [1] conducted the first treatment of congested flaps using leeches. Today, especially in the field Leech therapy is usually initiated after failure of more of reconstructive microsurgery, medicinal leech therapy is conventional treatment modalities such as warming, aspirin, enjoying a renaissance (for review see also [2, 3]). rheomacrodex (i.v.), immobilization and elevation of the Leeches are generally used during the critical post- injured area, and use of local heparin and vasodilators to operative period when venous outflow cannot match the improve venous status. Venous obstruction causes micro- arterial inflow, which can lead to venous congestion, clinically circulatory thrombosis, platelet trapping, and stasis. Thus, identified by the dusky purple appearance of the skin. If even after successful reanastomosis, secondary changes in the this complication is not corrected, cell death may result and microcirculation can persist and prevent adequate outflow the flap or finger may be lost. Therefore, medicinal leeches from being reestablished. Free flaps, pedicled flaps, and are used to salvage compromised microvascular free-tissue replanted tissues can survive arterial insufficiency for up to 13 transfers, replanted digits, ears, lips, and nasal tips until hours, but venous congestion can cause necrosis within three angiogenesis gradually improves the physiological venous hours. Medicinal leeches may be helpful in treating tissues drainage [4]. Frodel et al. [5] used medicinal leeches to sal- with venous insufficiency by establishing temporary venous vage soft tissue avulsion in key facial structures of 4 patients outflow, until graft neovascularization takes place [20]. involving avulsions of the ear, nose, lip, and scalp. In addition In July 2004, the FDA approved leeches as a medical to using leech therapy in head and neck reconstruction, there device in the field of plastic and reconstructive surgery. 2 Evidence-Based Complementary and Alternative Medicine

A survey of all 62 plastic surgery units in the United Kingdom andtheRepublicofIrelandshowedthatthemajorityofthese units uses leeches postoperatively [21]. Theaimoftheserecommendationsistoreviewthe practical use of medicinal leeches in reconstructive plastic surgery by reporting our experience with leeches in cases of reimplanted digits and free-tissue transfers.

2. Conditions for Treatment with Leeches

Once venous congestion has been identified and the patient Figure 1: Application of a leech to the area to be treated using a has agreed to undergo leech therapy, it is important that the syringe. patient is informed about the benefits and potential risks of the treatment. A written informed consent should be obtained from the patients, their parents, and/or the legal farms and fed artificially with animal blood. In addition, guardian before hirudotherapy is initiated. leeches purchased from recognized leech farms are suffi- There is a general consensus that antibiotic prophylaxis ciently starved prior to being sold and some of the leech farms for the Aeromonas bacteria, which are symbionts of leeches have been approved by regulatory agencies. Leeches, which andwhichcouldleadtocomplications,shouldbeiniti- were collected from a natural environment, should not be ated before leech therapy [22, 23]. Aeromonas species are used for hirudotherapy. sensitive to second- and third-generation cephalosporins, Leeches can survive one year or more without a blood fluoroquinolones, sulfamethoxazole-trimethoprim, tetracy- meal. Therefore, it is recommended to keep a large number cline, and aminoglycosides, while Aeromonas is resistant to of leeches in the laboratory by changing the water once or penicillin, ampicillin, first-generation cephalosporins, and twice weekly. For this purpose, the leeches can be transferred erythromycin [16, 24–27]. Patients should be treated each every week to a second container, which was previously day of leech therapy with anti-Aeromonas antibiotics such as cleaned and rinsed thoroughly to remove any remains of 500 mg of ciprofloxacin22 [ ]. However, out of 21 isolates of disinfectants and where the water was kept for at least 24 hrs Aeromonas species isolated from the water collected from the for dechlorination. The container should be filled up to 3/4 leech tanks, 71.4% were ciprofloxacin susceptible. All isolates with water and covered with a towel or netting, which would were sulfamethoxazole-trimethoprim susceptible, which was allow the leeches to have access to fresh air, without being also used as a prophylactic antibiotic regimen of choice for able to escape. A larger stone should be added into the leech therapy [28]. A regular surveillance to detect resistant container, which would help leeches during the process of Aeromonas species in medical leeches, by controlling the shedding their integument. The container with leeches should ∘ water in which they are kept, was suggested. Chepeha et be kept in a cool place (preferably 4–15 C). When leeches al. [29] used an antibiotic prophylaxis with double coverage are kept at room temperature, attention should be paid that ∘ during leech application and single coverage for 2 weeks after the ambient temperature does not exceed 25 C and that the leech therapy is discontinued. In the Iowa Head and Neck container with leeches is not exposed to direct sunlight. Protocol [30], levaquin is administered before the first leech When replacing leeches from one container to the other, the is applied to the skin and continued until 24 hours after leech replacement water must be at the same temperature as the therapy is discontinued. original. Approximately 8 leeches should be kept in 1 liter of Use of narcotics and benzodiazepines should be mini- water. mized, because they could negatively influence leech activity, Leeches should be handled gently by wearing nitrile or while the treated area should be photographed under the latex gloves, which would also prevent leeches from biting same light conditions using the same camera periodically to the health provider during maintenance of or treatment with follow the progress of decongestion [29]. leeches.

3. The Medicinal Leech 4. Application of Leeches to the Skin According to the literature, in most cases the medicinal Before application, leeches are thoroughly rinsed with deion- leech Hirudo medicinalis was used. In some cases, similar ized water. The area to be exposed to leeches should be results were also obtained with Hirudo verbana and Hirudo cleaned with sterile distilled water and ointments such as michaelseni [17, 31]. Using mitochondrial sequences and Doppler gel are removed. nuclear microsatellites, Siddall et al. [32] demonstrated that A plastic adhesive membrane or a thick layer of gauze can there are at least three species of European medicinal leech be applied around the leech(es) to prevent detached leeches and that many of the leeches marketed as H. medicinalis in from attaching themselves in other parts of the skin or even the USA, France, and Germany are actually H. verbana. under the flap, to fall inside the dressing around the wound Leeches should be purchased from recognized leech or other parts of the patient’s body or on the bed, which could producing companies, where they are kept in appropriate upset the patient and be unpleasant for other patients nearby. Evidence-Based Complementary and Alternative Medicine 3

Ingeneralleechesshouldbeappliedonthedarkerspotsof the reattached body parts or flaps. In the case of reimplanted fingers, leeches could be also placed on the region of the removed nail. The leeches are usually placed on a given spot of the skin using a 5 mL syringe. For this purpose, the nozzle of the syringe is removed using a scissor or scalpel. The leech is placed in the barrel of the syringe and the open end of the syringe is placed on the area to be treated. When the leech starts feeding, the syringe is removed gently (Figure 1). Leeches normally start feeding immediately, although in some cases the skin has to be punctured with a sterile Figure 2: Feeding leech: transparent liquid can be seen oozing from needle so that oozing blood will stimulate the leeches to feed. the body of the leech. Prickingthesitetobetreatedcouldalsodemonstratewhether there is sufficient blood-flow in the area. When the leech stops shortly after the leeches detach, or when leeches do not refuses to feed in a given place, the syringe is moved to the become fully engorged, a more aggressive treatment should neighboring area, until an appropriate place is found as close be followed by using a larger number of leeches and more as possible to the congested area. Depending on the intensity treatments per day. of blood-flow in the area, feeding can last for 30–90 min. During feeding drops of clear liquid can be seen oozing from Leech therapy is used until venous capillary return is the leech (Figure 2); this is the superfluous water in blood, established across the wound border by angiogenesis. Usually which the leeches remove to concentrate the red blood cells the treatment with leeches lasts for 2–6 days. In fact, the in their digestive tract. decision regarding the duration of the leech treatment is During hirudotherapy the patient should be under per- empiric, based on subjective appreciation of the color of the manent surveillance by a healthcare provider; leeches may skin, capillary refill, and the color of bleeding after pinprick seek other places to suck blood or after feeding may drop [12, 29, 34–36]. into the surrounding area. After autodetachment, the leeches Flap monitoring consists of listening to the Doppler signal arekilledin70%ethylalcoholandaredisposedinbagsfor and examining the flap every 1-2 hrs until the flap is stabilized biological waste. and thereafter every 2–4 hrs [29]. In cases of intraoral leeching, the path to the oropharynx Antithrombotic therapy could be administered with should be blocked with gauze to prevent leech migration aspirin, heparin sodium, and/or dextran 40 in parallel with into the more distal aerodigestive tract, and the perioperative hirudotherapy, although it should be noted that the leech tracheotomy is left in position to protect the airway29 [ ]. saliva also demonstrates antithrombotic activity. Speciallyshapedglasscontainerscanbeusedforthispurpose The patients may lose 5–15 mL of blood per leech, per [33]. session.However,thewoundmaycontinuetooozeupto24 Attentionshouldbepaidtoleeches,whichattachbutdo hours after the leech is removed. Accordingly, hematologic not change in size and have no visible gut peristalsis within evaluations should be performed every 4 hrs and consist of 30 min after their attachment. It could be assumed that they complete blood cell count, partial thromboplastin time, and are only attached but not feeding and should be replaced with serum chemistry studies. However, the number of hematocrit other leeches or they should be transferred to other parts checks depends on the number of leeches used, frequency of the treated area. The use of active (swimming) and larger of sessions, and total duration of therapy. In many cases, leeches could be of help. thepatientshavetoreceivebloodtransfusionswhenthe hemoglobin level is lower than 8 g/dL [29]. After detachment of the leech, the bite areas could be 5. Treatment Procedure cleaned hourly with gauze soaked in isotonic sodium chlo- ride solution or heparin solution (5,000 U/mL) to stimulate Dependingontheseverityandthesizeofthecongestion,1– hemorrhage from leech bite sites [22, 33]. 10 leeches are used for each treatment, although some authors recommend higher numbers of leeches. The degree of venous congestion is estimated from the percentage of violaceous 6. Assessment color of flap skin pedicle, testing capillary refill, and color ofthebloodoozingfromthebitesiteorafterhavingbeen Successful salvage of decongested tissues with leeching has pierced with a needle. At the beginning of the treatment, been reported in 70–80% of cases [10, 21, 37, 38]. In Israel, the patient might need two or more treatments per day. Mumcuoglu et al. [22] used hirudotherapy in 23 patients, Chepeha et al. [29]usedaprotocolaccordingtowhichleech 8–79 years old, presenting with venous congestion of revas- placement was continuous (3 leeches per hour) and tapered cularized or replanted fingers and free or local flaps. Of the slowly according to clinical assessment of inosculation. In the 15 fingers, 10 fingers were saved (66.7%) (4 out of 9 replanted Iowa Head and Neck Protocol [30], leeches are applied every fingers and 6 out of 6 revascularized fingers), while 17 outof 2 hours. The number of treatments per day depends also on 18 flaps (94.4%) were salvaged (3 out of 4 free flaps and all the bleeding of previous bite sites. In cases where the bleeding 14 island and random flaps) (Figure 3). Similar results were 4 Evidence-Based Complementary and Alternative Medicine

Figure 4: Y-shaped leech bite and the surrounding skin reaction. Figure 3: Treatment of a skin flap of the face, after removal of the skin with scars and coverage of the area with the enlarged, adjacent skin. application to a nearby mucosal surface could avoid these complications [33]. reported from countries such as the USA [15, 22], UK [17], Symbiotic bacteria such as Aeromonas hydrophila and Germany [39], and S. Africa [31]. Aeromonas veronii, living in the intestinal tract of the leech, The degree of venous congestion can be estimated by may cause infections in 4–20% of the patients, whose flaps describing the percentage of ruborous and violaceous color or replanted digits are treated with leeches [40]. Leech- of the flap skin pedicle, testing capillary refill, and observing borne Serratia marcescens infections were also reported color and amount of blood oozing from leech bite sites. [24]. Accordingly, prophylactic treatment with antibiotics is Serial photographs can help assess the intensity of venous necessary. Aeromonas infections can occur acutely (within congestion on a daily basis. The progress of treatment should 24 hours) or in a delayed fashion (up to 26 days) after be also documented. the beginning of leech therapy. Clinical manifestations of It should be kept in mind that venous obstruction Aeromonas infection vary from a minor wound infection causes microcirculatory thrombosis, platelet trapping, and to extensive tissue loss. It is important to stress that leech stasis. Thus, even after successful reanastomosis by leeches, related Aeromonas infections more frequently develop in very secondary changes in the microcirculation can persist and sickandimmunosuppressedpatients.Whenappliedonintact prevent adequate outflow from being reestablished. skin, for example, in patients treated for osteoarthritis, local pain, arterial hypertension, and different forms of spondy- 7. Side Effects and Contraindications losis and dorsopathies, Aeromonas infections are extremely rare. Modern leech therapy is generally recognized as a relatively The excess bleeding after leeching can be of concern safe and well-tolerated treatment modality. A patient who and transfusions may be needed, especially in patients with refuses to sign the consent form, refuses prophylactic treat- a tendency to hemorrhage, who suffer from anemia, or ment with antibiotics, or refuses blood transfusion should not for those taking anticoagulants or platelet-inhibiting drugs. be treated with leeches. Blood transfusions are given based on a hemoglobin level of Although the bite of a leech is felt as a slight pain on <8 g/dL. Usually, 3–6 units of packet blood cells are used to theintactskin,thisisnotrelevantinthecaseofrecently compensate for blood loss. reattached digits and flaps, where the skin is anesthetic. In very rare cases, thrombotic microangiopathy and renal Slight localized itching of the Y-shaped bite site (Figure 4) failurehavebeenreportedwhenleecheswereappliedin persisting for several hours and up to 3 days is the most patients with arterial insufficiency33 [ ]. common (37.3–75%) adverse effect of leech therapy. Use of Isolated reports describing the appearance of syncopal 5% potassium permanganate, cold compresses, 10% baking status or orthostatic hypotension (mainly in older persons soda paste, Golden Star balm, or Fenistil gel on the affected with initial sympathicotonia) at the start of or during leech skin areas can be for more pronounced cases. In severe therapy, have been reported. According to Michalsen et al. cases of generalized itching, topical corticosteroids and oral [41, 42]vasovagalattackscanoccurin0.1%ofpatients antihistamines should be prescribed. undergoing hirudotherapy, mainly in those with a history of Signs of regional lymphadenitis, slight swelling, and pain developing such attacks or syncope during invasive proce- of regional lymph nodes on the side of leech application dures such as venopunction. It is highly recommended that andsubfebriletemperaturecanoccurin6.4–13.4%ofthe patients drink plenty of fluids during hirudotherapy. In order treated patients and usually appears after 3-4 leech applica- to prevent those systemic side effects, hirudotherapy should tions. Apparently, such adverse reactions never appear when be performed in a calm atmosphere, under constant blood leeches are applied on oral, nasal, or vaginal mucosa. In very pressure monitoring, and with the patient lying down. rare cases, allergic skin reactions have been observed. When Hirudotherapy can cause some negative psychological or leeches are applied to the esthetically important areas with emotional reactions in patients, especially when detached thin skin and thin layers of subcutaneous tissue scarring after leeches fall inside the dressing or other parts of the patient’s aleechbitecouldbeacosmeticproblem.Insomecases, body. Less than 10% of patients undergoing leech therapy Evidence-Based Complementary and Alternative Medicine 5 for osteoarthritis had initial qualms before treatment, which Leeches may also secrete a vasodilative, histamine-like usually disappeared after the first treatment course. In fact substance, which increases the inflow of blood after a leech many patients treated with leeches and who had successful biteandreduceslocalswelling[45]. treatment changed their attitude towards hirudotherapy in a Hyaluronidase, which is known as the “spreading factor,” positive way. Nevertheless, it might be necessary to prepare can degrade tissue hyaluronic acid, thus facilitating the infil- the patients psychologically before the application of leeches tration and diffusion of the remaining ingredients of leech [41, 42]. saliva into the congested tissue. Tissue permeability, restored Contraindications related to hirudotherapy include arte- with the help of hyaluronidase, promotes the elimination of rial insufficiency, hemophilia, hemorrhagic diathesis, hema- tissue- and circulatory-hypoxia as well as local swelling [51]. tological malignancies, expressed and firm anemia, expressed The persistent bleeding largely potentiates tissue decon- and firm hypotension, sepsis, HIV-infection, decompensated gestionandleadstolossofblood,reliefofcapillary forms of hepatobiliary diseases, any form of cachexia, and net, decrease in venous congestion, decompression of the individual intolerance to leeches. Leech therapy is also not nerve trunks and endings, increase in lymph flow, positive recommended in pregnancy and lactation, in patients with changes of local hemodynamics, amelioration of hemorhe- an unstable medical status, history of allergy to leeches or ology, increase of oxygen supply, improvement of tissue severe allergic diathesis, disposition to keloid scar formation, metabolism, and elimination of tissue ischemia [33]. arterial insufficiency, and in those using anticoagulants, immunosuppressants, and some vasoactive drugs such as 9. Conclusions Ginkgo biloba products [33]. In summary, hirudotherapy is a safe, easy to use, beneficial, and cost-effective treatment modality to save reattached body 8. Mechanisms Involved parts and flaps in reconstructive plastic surgery. The early recognition of flap failure and initiation of leech therapy Hirudotherapy depends on the following main properties is of paramount importance. Prophylactic treatment with of medicinal leeches: the blood-letting action during active antibiotics and continuous monitoring of blood parameters suction of blood, passive oozing of the wound, and injection are necessary. of biologically active substances with the saliva into the host. The saliva of H. medicinalis contains more than 100 bioac- Conflict of Interests tive substances, including coagulation inhibitors, platelet aggregation inhibitors, vasodilators, and anaesthetizing, The author declares that there is no conflict of interests regar- antimicrobial and anti-inflammatory agents [43, 44]. ding the publication of this paper. One of the most important ingredients is hirudin, which is the principal anticoagulant responsible for enhanced bleed- ing and prevention of coagulation. In addition to hirudin, References leeches secrete two inhibitors of Factor Xa responsible for the [1] M. Deganc and F. 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Review Article Marine Invertebrate Natural Products for Anti-Inflammatory and Chronic Diseases

Kalimuthu Senthilkumar1 and Se-Kwon Kim1,2 1 Marine Biochemistry Laboratory, Department of Chemistry and Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea 2 Department of Chemistry, Pukyong National University, Daeyeon Campus, 599-1, Nam gu, Busan, 608-737, Republic of Korea

Correspondence should be addressed to Se-Kwon Kim; [email protected]

Received 11 September 2013; Accepted 10 December 2013

Academic Editor: Edwin L. Cooper

Copyright © 2013 K. Senthilkumar and S.-K. Kim. 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 marine environment represents a relatively available source of functional ingredients that can be applied to various aspects of food processing, storage, and fortification. Moreover, numerous marine invertebrates based compounds have biological activities and also interfere with the pathogenesis of diseases. Isolated compounds from marine invertebrates have been shown to pharmacological activities and are helpful for the invention and discovery of bioactive compounds, primarily for deadly diseases like cancer, acquired immunodeficiency syndrome (AIDS), osteoporosis, and so forth. Extensive research within the last decade has revealed that most chronic illnesses such as cancer, neurological diseases, diabetes, and autoimmune diseases exhibit dysregulation of multiple cell signaling pathways that have been linked to inflammation. On the basis of their bioactive properties, this review focuses on the potential use of marine invertebrate derived compounds on anti-inflammatory and some chronic diseases such as cardiovascular disease, osteoporosis, diabetes, HIV, and cancer.

1. Introduction also growing awareness that dietary source and form of food may affect overall health. Suitably, the role of food asan Chronic diseases constitute a major cause of mortality and agent for improving health has been recognized, initiating the World Health Organization (WHO) reports chronic the development of new classes of compound from marine noncommunicable conditions to be the leading cause of environment [4]. mortality in the world, representing 35 million deaths in The marine world, due to its phenomenal biodiversity, isa 2005 and over 60% of all deaths. Each year, more than 30 rich natural resource of many biologically active compounds million deaths (52% of all deaths) by chronic diseases include such as polyunsaturated fatty acids (PUFAs), sterols, proteins, cardiovascular disease (30%), cancer (13%), chronic respi- polysaccharides, antioxidants, and pigments. Many marine ratory disease (7%), and diabetes (2%). The global burden organisms live in complex habitats exposed to extreme con- of disease resulting from all noncommunicable conditions, ditions and, in adapting to new environmental surroundings, which includes premature death and disability, is 49%; 80% of they produce a wide variety of secondary (biologically active) these deaths occur in low and middle income countries [1, 2]. metabolites which cannot be found in other organisms. Increasing knowledge regarding the impact of diet on human Moreover, considering its great taxonomic diversity, inves- health along with the state-of-the-art technologies has led to tigation related to the search of new bioactive compounds significant nutritional discoveries, product innovations, and from the marine environment has seen in almost unlimited mass production on unprecedented scale [3]. In particular, field [5, 6].Marine-basedbioactivefoodingredientscan naturally occurring bioactive extracts or single compounds be derived from many sources, including marine plants, are believed to benefit human health and research resulting microorganisms, and sponges, all of which contain their in substantial advances in nutritional knowledge. There is own unique set of biomolecules [5]. However, these naturally 2 Evidence-Based Complementary and Alternative Medicine

Functions Marine invertebrates Isolation Antiinflammatory Sponge Cardiovascular diseases Secondary Diabetes Ascidian metabolites Arthritis Soft coral Osteoporosis Bioactive Cnidarians Neurodegenerative Gorgonian compounds diseases HIV Anticancer

Figure 1: Summary of marine invertebrate natural products with anti-inflammatory and some chronic diseases. occurring bioactive substances has defined health benefit on may be useful as an alternative medicine for various diseases. the human body significantly3 [ ]. Therefore, this review dis- Figure 1 provides an overview of this review. cusses the existing scientific knowledge which demonstrates the suitability of marine derived bioactive compounds for the prevention and treatment of anti-inflammatory and chronic diseases. 3. Anti-inflammatory Activity 2. Marine Environment and Availability of Natural Products Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, Marine biotechnology is the science in which marine organ- damagedcells,orirritants.Thesignsofacuteinflamma- ismsareusedinfullorpartiallytomakeormodifyproducts tion are pain, heat, redness, swelling, and loss of function. for specific uses. The different molecular and biotechno- Inflammations has different names in different parts of logical methods elucidated for isolating natural products the body asthma (inflammation of the airways), arthritis from aquatic and terrestrial organisms. A remarkable number (inflammation of the joints), dermatitis (inflammation of the of new natural products (NPs) have been isolated from skin), and so on. Inflammation is the crucial first step in various marine sources in the past decades [7]. In recent fighting infection and healing wounds. However, persistent years, many bioactive compounds have been extracted from inflammation on immune system is always activated, the marine invertebrates such as sponges, tunicates, bryozoans, condition known as chronic inflammation which leads to and mollusca, [8, 9]. The marine environment covers a wide chronic diseases [17]. However, if the response is exaggerated, thermal range including pressure range (1–1000 atm) and misdirected, or long term, the inflammation can adversely nutrient range (oligotrophic to eutrophic) and has extensive affect health and give rise to many conditions such as photic and nonphotic zones [10]. But with the development inflammatory bowel disease, arthritis, and asthma [18, 19]. of new diving techniques, remote operated machines, and so Anti-inflammatory refers to the property of a substance or forth, it is possible to collect marine organisms and during the treatment that reduces inflammation. Many steroids and past decade, over 5000 novel compounds have been isolated nonsteroidal anti-inflammatory drugs (NSAIDs) are widely from shallow waters to 900-m depths of the sea [11]. The used for the treatment for inflammation. Alternately herbal knowledge of the physiological and biochemical features of medicines also play a role in inflammation apart from marine organisms might contribute to the identification of that marine derived bioactive compounds showed anti- natural products of biomedical importance. Natural products inflammatory actions. Marine invertebrates are one of the havebeenthesourceofmostoftheactiveingredientsof major groups of biological organisms (Porifera, Cnidaria, medicines. This is widely accepted to be true when applied Mollusca, Arthropoda, Echinodermata, and so forth) that to drug discovery in “olden times” before the advent of high- gave until now significant number of natural products and throughput screening and the postgenomic era: more than secondary metabolites with pharmacological properties and 80% of drug substances were natural products or inspired lead in the formulation of novel drugs. These natural prod- by a natural compound [12]. However, comparisons of the ucts have a wide range of therapeutic properties, including information presented on sources of new drugs from 1981 to antimicrobial, antioxidant, antihypertensive, anticoagulant, 2007 and 2010 [13–15]indicatethatalmosthalfofthedrugs anticancer, anti-inflammatory, wound healing and immune approved since 1994 are based on natural products including modulator, and other medicinal effects [20]. marine invertebrates. Marine invertebrates synthesize pri- Alkaloid is a group of biological amine and cyclic mary and secondary metabolites that are ultimately screened compounds having nitrogen in the ring, naturally occur- and described by researchers as NPs. Primary metabolites ring in plant, microbes, animals, and marine organisms. include amino acids, simple sugars, nucleic acids, and lipids. Both halogenated and nonhalogenated forms have attracted Secondary metabolites, such as alkaloids, terpenoids, and researchers’ interest because of their pharmaceutical impor- other compounds, have known bioactivities and biological tance as bioactive compounds and as biological probes for functions [16]. Thus, marine invertebrate derived compounds physiological studies [21]. Indole alkaloids from marine Evidence-Based Complementary and Alternative Medicine 3

Table 1 Name of the compound Chemistry Name (species) Target effects References Anti-inflammatory action in human Ascidiathiazone Alkaloids Ascidian (Ascidian Aplidium) [27] neutrophils Cembranolides Cembranoids Soft coralLobophytum ( crassum) Inhibitors of COX-2 [29] Durumolides Cembranoids Soft coralLobophytum ( duru) Inhibitors of iNOS and COX-2 [30] Anti-inflammatory action in human Frajunolides Diterpenoids Gorgonian (Junceella fragilis) [31] neutrophils Sponge sp. Manzamine Alkaloids Inhibitors of thromboxane B2 [25]

Plakortide P Polyketide Sponge (P. ang u l ospi c u l atu s ) Antineuroinflammatory [32] Anti-inflammatory action in human Rubrolide O Halogenated furanone Ascidian (Synoicum sp.) [28] neutrophils Carteramine A Alkaloid Sponge (Stylissa carteri) Inhibit neutrophil chemotaxis [26]

invertebrates have reported to be anti-inflammatory poten- compounds that inhibit neutrophil chemotaxis, their finding tials; these include conicamin from tunicate [22], Lepad- provides a “novel platform to develop a new class of anti- iformines A and B from ascidian [23] and aplysinopsin- inflammatory agents”26 [ ]. Some of the anti-inflammatory type compound from sponge Hyrtios erecta [24], manzamine compounds from marine invertebrates are listed in Table 1. from sponge [25], carteramine A from sponge [26], and ascidiathiazones A and B [27, 28] from ascidan. The tricyclic 4. Chronic Diseases alkaloids ascidiathiazone is isolated from Ascidian aplid- ium species that affected superoxide production by human A chronic condition is a human health condition or disease ( = 0.44 1.55 𝜇 ) neutrophils in vitro IC50 – M ,aswellasex that is persistent or otherwise long lasting in its effects. The vivo and studies suggested that these two compounds might term chronic is usually applied when the course of the disease become “potential anti-inflammatory pharmaceutical” leads. lasts for more than three months. Data from the World Cyclooxygenase (COX) enzyme synthesizes prostaglandins, Health Organization show that chronic disease is also the creating inflammation. The new cembranoids, crassumolides major cause of premature death around the world. Common AandCfromthesoftcoralLobophytum crassum inhibited chronic diseases include asthma, arthritis, cancer, diabetes, 𝜇 the expression of iNOS and COX-2 (IC50 less than 10 M) heart diseases, and AIDS. Although chronic diseases are [29]. Also, another cembranolides durumolides A–C from among the most common and costly health problems, they the soft coral Lobophytum duru inhibited both iNOS and are also preventable and most can be effectively controlled. COX-2 proteins in LPS-activated RAW 264.7 cells in vitro, Apart from available treatment and intervention of drugs 𝛼 𝛼 suggested that the -methylene- -lactone moiety of these from various sources, marine derived compounds play a compounds was necessary for the activity [30]. major role in the health benefits. Therefore, here we discuss A new briarane-type diterpenoids frajunolides B and C, the role of marine invertebrate derived natural products for isolated from the Taiwanese gorgonian Junceella fragilis,sig- some of the chronic diseases. nificantly inhibited superoxide anion and elastase generation from human neutrophils in vitro (apparent IC50 greater than 4.1. Cardiovascular Disease. Cardiovascular disease (CVD) is 𝜇 10 g/mL) [31]. Manzamine A, (-)-8-hydroxymanzamine a class of diseases that affect the heart, blood vessels (arteries A, and hexahydro-8-hydroxymanzamine, potently inhibited and veins), and blood circulation and is one of the leading = 0.25 thromboxane (TXB2) generation (IC50 ,lessthan causes of mortality and morbidity worldwide. Examples of 0.1, and 1.97 𝜇M, resp.) in brain microglia [25]. Kossuga et CVD include atherosclerosis, CHD, stroke, heart failure, deep al. [32] demonstrated that the polyketide plakortide P iso- vein thrombosis, and peripheral arterial disease. The amount lated from the Brazilian sponge P. ang u l o spi c u l atu s ,potently of fat in the diet and the type of fatty acids consumed can inhibited thromboxane B2 release (IC50 = 0.93 𝜇M) from influence the likelihood of CVD and its risk factors [18]. activated rat brain microglia, appears to be a potentially High blood pressure (hypertension) is one of the major “novel anti-neuroinflammatory agent”. A halogenated fura- independent risk factors for CVD [33]. Significant discoveries none rubrolide O isolated from a New Zealand ascidian Syn- have resulted primarily from analyzing natural products. oicum sp., which inhibited superoxide anion production in Marine organisms such as invertebrates derived natural prod- human neutrophils (IC50 =35𝜇M) in vitro with low toxicity ucts have pharmacological properties [34]. Eryloside F from [28]. A novel dimeric oroidin (type of alkaloid) derivative sponge Erylus formosus was found to be a potent thrombin carteramine A in the marine sponge Stylissa carteri,showed receptor antagonist [35]. Thrombin receptor activation is that inhibited neutrophil chemotaxis (IC50 =5𝜇M).Because likely to play a key role not only in arterial thrombosis carteramine A has no structural resemblance to known but also in atherosclerosis [36]. Atherosclerosis starts with 4 Evidence-Based Complementary and Alternative Medicine damage to the endothelium and subsequent deposition of and Huntington’s disease occur as a result of neurodegenera- fats, cholesterol platelets, cellular waste products, calcium, tive processes. Current interventions for Alzheimer’s disease and other substances in the artery wall. These may stimulate (AD) include acetylcholinesterase inhibitors (AchI), which endothelial cells to produce a vascular cell adhesion molecule are indicated for patients with mild to moderate symptoms. that results in further buildup of cells and shrinkage of the A spectrum of alternative treatments for AD has also been arterial diameter [37]. Halichlorine from sponge Halichon- proposed and must be examined judiciously in preclinical, dria okadai is an inhibitor for the expression of vascular cell clinical, and evidence-based research (EBR) studies [54]. adhesion molecule 1 [38] and may thus impede atherogenesis Therefore, search for acetylcholinesterase inhibitors is useful [39]. for the treatment of Alzheimer’s disease. Pharmacological studies with marine compounds affecting the nervous system involved three areas of neuropharmacology: the stimulation 4.2. Diabetes. Diabetes mellitus is a most serious and chronic of neurogenesis, the targeting of receptors, and other miscel- disease whose incidence rates are increasing with increasing laneous activities on the nervous system. A new stigmastane levels of obesity and also with aging of the general population type steroidal alkaloid 4-acetoxy-plakinamine B isolated over the world. Currently, an estimated 150 million people from a Thai marine sponge Corticium sp.significantlyinhib- worldwide have diabetes and that this will increase to 300 ited acetylcholinesterase (IC50 = 3.75 𝜇M). This compound is million by 2025 [40]. Globally, type II diabetes (non-insulin reported to be the “first marine derived acetyl cholinesterase- dependent diabetes mellitus) accounts for greater than 90% inhibiting steroidal alkaloid” [55]. The inflammatory compo- of the cases [41, 42]. Research for novel anti-diabetic drugs nent to the pathology of neurodegeneration was most notably to complement those in present clinical use has intensified in Alzheimer’s disease but also in Parkinson’s disease and over the years [43]. Callyspongynic acid is a polyacetylenic motor neuron disease [56]. Hymenialdisine is an alkaloid acid isolated from sponge Callyspongia truncata inhibits 𝛼- 𝛼 isolated from marine sponges, such as Acanthella aurantianca glucosidase [44]. -Glucosidase inhibitors interfere with the and Stylissa massa [57]. Hymenialdisine inhibits phosphory- hydrolysis of glycogen, keeping the glucose concentration in lation of the protein tau (which is hyperphosphorylated in the blood at a lower level, and can be used to treat patients Alzheimer’s disease) with promising potential against human with diabetes l [45]. A polybromodiphenyl ether compound neurodegenerative diseases [58]. 11-Dehydrosinulariolide was isolated from an Indonesian marine sponge Lamellodysidea obtained from formosan soft coral, S. flexibilis, promoting herbacea inhibits protein tyrosine phosphatase 1B, an impor- neuroprotective properties as a promising candidate for the tant target for diabetes treatment [46]. treatment of Parkinson’s disease [59].

4.3. Arthritis and Osteoporosis. Arthritis describes a condi- tion involving inflammation of the joints and is a disease 4.5. HIV/AIDS. In order to combat the human immunodefi- affecting mostly the aged population. Preventing inflamma- ciency virus (HIV), diverse strategies have been developed to tionwithitsassociatedpainandreducedmobilitysymptoms research on compounds which can be developed as therapeu- is a primary requirement in arthritis treatment [47]. The tic agents. Complementary and alternative medicine (CAM) alkaloid hymenialdisine isolated from marine sponge Stylissa can be defined as any treatment used in conjunction (com- massa inhibits proteoglycan degradation in bovine articular plementary) or in place of (alternative) standard medical cartilage [48]. Osteoporosis is a multifactorial progressive treatment [60]. More research is also required on the harmful skeletal disorder characterized by reduced bone mass and and beneficial effects of concurrent CAM and treatment for deterioration of bone microarchitecture, predisposing to HIV/AIDS. However, for all other types of CAM, natural increased fracture risk [49]. Osteoporosis is called a “silent products may be the alternative medicinal use for HIV/AIDS. disease” because it progresses without symptoms until a Among them, marine derived natural products may be fracture occurs. Because of larger skeletons and no period of useful for the treatment of HIV. The peptides tachyplesin rapid hormonal change, osteoporosis progresses more slowly and polyphemusin, which are highly abundant in hemocyte in men than women [50]. In the recent days, much atten- debris of the horseshoe crabs Tachypleus tridentatus, Limulus tion has been paid for marine compounds for osteoporosis polyphemus and the sponge metabolites avarol, avarone, ili- treatment [51]. Norzoanthamine is one of the zoanthamine maquinone and several phloroglucinols has anti HIV activity classes of marine alkaloids isolated from a colonial zoan- [61, 62].AvarolinhibitsHIVbycompletelyblockingthe thid (cnidarians), Zoanthus sp.[52]. Norzoanthamine could synthesis of the natural UAG suppressor glutamine transfer protect skeletal proteins, such as collagen and elastin in tRNA. Avarol inhibits HIV by almost completely blocking the the host animal bodies from external stresses and possibly synthesis of the natural UAG suppressor glutamine transfer enhance survival as it may be a promising drug candidate for tRNA. Synthesis of this tRNA is upregulated after viral osteoporosis treatment and prevention [53]. infection and important for the synthesis of a viral protease, which is necessary for viral proliferation [63]. 4.4. Neurodegenerative Diseases. Neurodegeneration is the Clathsterol,anovelandactivesulfatedsterolfromtheRed term for the progressive loss of structure or function of neu- Sea sponge Clathria sp., has been shown that inhibits HIV-1 rons, including death of neurons. Many neurodegenerative RT at 10 𝜇M concentration [64]. An HIV-inhibitory cyclic diseases including Alzheimer’s disease, Parkinson’s disease, depsipeptide microspinosamide, isolated from the marine Evidence-Based Complementary and Alternative Medicine 5

Table 2: List of some marine invertebrate derived natural products for chronic diseases.

Name of the compound Chemistry Name (species) Target effects References Penasterol Eryloside F Sponge (Erylus formosus) Atherosclerosis [35] disaccharide Halichlorine Alkaloid Sponge (Halichondria okadai) Atherosclerosis [38] Callyspongynic acid Polyacetylenic acid Sponge (Callyspongia truncate)Diabetes [44] Hymenialdisine Alkaloid Sponge (Stylissa massa)Arthritis [48] Norzoanthamine Alkaloid Cnidarians, Zoanthus sp. Osteoporosis [53] Acetylcholinesterase 4-Acetoxy-plakinamine B Alkaloid Sponge (Corticium sp.) [55] inhibition Sponge Acanthella aurantiaca Hymenialdisine Alkaloid Alzheimer’s disease [57] and Stylissa massa

11-dehydrosinulariolide Cembranolide Soft coralS. ( flexibilis) Parkinson’s disease [59]

Sesquiterpene Avarol Sponge sp. HIV [63] hydroquinone Clathsterol Depsipeptide Sponge (Clathria sp.) HIV [65] Crambescidin 826 Alkaloid Sponge (Monanchora sp.) HIV [66] Sponge Dehydrofurodendin Furanoterpene HIV [67] (Madagascan Lendenfeldia) Neamphamide A Depsipeptide Sponge (Neamphius huxleyi)HIV [68] Lamellarins Alkaloid Mollusks (Lamellaria sp.) HIV [73] Horseshoe crabs Tachyplesins Peptide Tachypleus tridentatus and HIV [75] Limulus polyphemus

sponge Sidonops microspinosa, inhibits HIV-1 infection in horseshoe crabs Tachypleus tridentatus and Limulus polyphe- cell based in vitro assays [65]. A new polycyclic guanidine mus,respectively,werefoundtobeHIVcellfusioninhibitors alkaloid, crambescidin 826, was reported from the marine [74, 75]. Some of the marine natural products for chronic sponge Monanchora sp. and it inhibits HIV-1 envelope- diseases are listed in Table 2. mediated fusion in vitro (IC50 =1–3𝜇M);itsuggestedthat this compound might be the design of small molecule HIV- 1fusioninhibitors[66]. A new C22 furanoterpene (dehy- 4.6. Cancer. Many potent natural products which display drofurodendin) was isolated from the Madagascan Lenden- effective anticancer activities have been discovered in the feldia sponge, active against HIV-1 RT-associated RNA- and marine environment. Indeed, since the early 1990s, there has ( = 3.2 5.6 𝜇 ) DNA-directed DNA polymerase IC50 – M [67]. been a dramatic increase in the number of preclinical anti- Neamphamide A was isolated from the Papua New Guinea cancer compounds from marine sources that have entered marine sponge Neamphius huxleyi that inhibits the cytopathic = human clinical trials [14, 76]. Marine derived natural prod- effect of HIV-1 infection in cell based in vitro assays (EC50 ucts from marine invertebrates are the valuable sources for 28 nM) [68]. Two bisquinolizidine alkaloids, petrosin and anticancer drugs. Trabectidin (Yondelis), originally isolated petrosin A was isolated from the Indian marine sponge from the Caribbean marine tunicate Ecteinascidia turbinate, Petrosia similes inhibits HIV-1 replication, formation of giant hasbeenapprovedforuseasananticanceragentinEurope cells and recombinant reverse transcriptase in vitro [69]. Didemnaketals A and B were isolated from the ascidian [77, 78]. The compound was selected for clinical development Didemnum sp. and found to be inhibitors of HIV-1 protease on the basis of its novel chemical structure and its striking [70]. Bioassay-guided fractionation of extracts of the Palauan activity against tumor cell lines of different origins in vitro ascidian Didemnum guttatum led to the isolation of cyclo- and in vivo models. In 2007, trabectedin obtained market- didemniserinol trisulfate as an inhibitor of HIV-1 integrase ing authorization from the European Commission for the [71]. Lamellarins was first isolated from prosobranch mol- treatment of patients with advanced soft tissue sarcoma. In lusksofthegenusLamellariaandsubsequentlyobtainedfrom 2009, it received marketing authorization from the European Didemnid ascidians [72]. Lamellarin inhibits the integrase Commission in combination with pegylated liposomal dox- terminal cleavage activity and strands transfer activity [73]. orubicin for the treatment of patients with relapsed platinum The peptides Tachyplesins I–III and polyphemusins I and sensitive ovarian cancer. Clinical activity is currently being II, which are highly abundant in hemocyte debris of the evaluated in other neoplasms, including prostate and breast 6 Evidence-Based Complementary and Alternative Medicine

Table 3: List of some anticancer natural products from marine invertebrates.

Name of the Chemical class Source of organisms Targets Reference compound Tunicate (Ecteinascidia Anticancer effect in breast and prostate and Trabectidin Alkaloids [77, 78] turbinate) so forth. Kahalalide F Cyclic depsipeptide Mollusk (Elysia rufescens) Inhibits topoisomerase II in cancer [81] Heteronemin Sesterterpene Sponge (Hyrtios sp.) Inhibits Leukemia (K562 cells) cancer cells [82] Tyrindoleninone Inhibits ovary, granulosa, choriocarcinoma Indole derivative Mollusk (Dicathais orbita) [83] and 6-bromoisatin (OVCAR-3, KGN, Jar) cells Makaluvamine A Pyrroloquinoline Sponge (Zyzzya fuliginosa) Colon cancer (HCT-116 cells) inhibition [84] Mediterranean ascidian Inhibits leukemia (HL-60 and P388) cancer Ascididemin Alkaloid [85, 86] (Cystodytes dellechiajei) cells Prosobranch mollusc of the Lamellarin D Alkaloid Inhibits leukemia cancer cells [87, 88] genus (Lamellaria) Sponges Inhibits leukemia Spongistatin 1 Macrocyclic lactone (Spirastrella spinispirulifera [89] (Jurkat) cancer cells and Hyrtios erecta) Crabs and shrimp Inhibits and induces apoptosis of HeLa Chitosan Polysaccharides [90] (various species) cancer cell Inhibits various cancer types and currently Bryostatins Macrolides Bryozoan (Bugula neritina) [91] used in clinical trials Eribulin mesylate Halichondria okadai and Microtubule inhibitor and presently used in Macrolide [93] (E7389) Axinella family clinical trials Ascidian Currently in phase II/III clinical trials for Aplidin Cyclic depsipeptide [96] (Aplidium albicans) solid and hematologic malignancies

cancer. Trabectedin’s mechanism of action seems to be dif- different tumors. LAM-D potently stabilizes topoisomerase ferent from that of the available DNA damaging agents used I DNA covalent complexes to be promoting the formation in cancer chemotherapy to date. The cytoskeleton is also an of DNA single strand breaks. LAM-D also promotes nuclear interesting target for cancer therapy, as the microtubules and apoptosis in leukemia cells via the intrinsic apoptotic pathway microfilaments are involved in cellular organization during [87, 88]. Spongistatin 1 a macrocyclic lactone isolated from cell division. A number of compounds from marine sponges the marine sponges Spirastrella spinispirulifera and Hyrtios and ascidians are microtubule inhibition [79]. Kahalalide F, erecta induces apoptosis by interact with caspase dependent a cyclic depsipeptide from the herbivorous marine mollusk, pathway by the release of cytochrome c, Smac/DIABLO, and Elysia rufescens,hasshowntobeeffectiveagainstcancercell Omi/HtrA2 from the mitochondria to the cytosol, leading lines with strong multidrug resistance and against cell lines to apoptosis in Jurkat cells [89]. Chitosan is produced com- resistant to topoisomerase II inhibitors. In vivo models have mercially by deacetylation of chitin, which has the structural also confirmed anticancer activity in various solid tumor element in the exoskeleton of crustaceans (such as crabs models [80, 81]. and shrimp) and cell walls of fungi. Diethylaminoethyl The heteronemin, a marine sesterterpene isolated from chitosan induces apoptosis in HeLa cells via activation of the sponge Hyrtios sp., inhibits NF-𝜅B activation and acti- caspase-3 and p53 expression [90]. Bryostatins are a group vates both initiator caspase-8 and caspase-9, which are of macrolide lactones first isolated in the 1960s by George implicated in the extrinsic and intrinsic apoptotic pathway, Pettit from extracts of a species of bryozoan, Bugula neritina. respectively, in chronic myelogenous leukemia cells [82]. The structure of bryostatin 1 was determined in 1982. To date Tyrindoleninone and 6-bromoisatin are indole derivatives 20 different bryostatins has been isolated, those are currently from marine mollusk Dicathais orbita that induces apoptosis under investigation as anti-cancer agents [91, 92]. Bryostatin in female reproductive cancer cell lines ovary, granulosa, and 1 induces apoptosis in HL-60 chronic lymphocytic leukaemia choriocarcinoma (OVCAR-3, KGN, Jar), respectively [83]. and also acts synergistically in combination with other anti- Makaluvamine A is a pyrroloquinoline, principally isolated cancer drugs. from the sponge Zyzzya fuliginosa that has potent anticancer Eribulin mesylate (E7389) is a microtubule dynamics activity in HCT-116 cells [84]. Ascididemin (ASC), a aromatic inhibitor that is a simplified, synthetic analog of the marine alkaloid isolated from the Mediterranean ascidian Cystodytes natural macrolide halichondrin B, which was first isolated dellechiajei [85], which are strong inducer of apoptosis in from the Japanese sponge Halichondria okadai [93]and HL-60 and P388 leukemia cells [86]. An alkaloid Lamellarin subsequently from several unrelated sponges belonging to the D(LAM-D),initiallyisolatedfromaprosobranchmollusc Axinella family. 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Review Article Recombinant Protein Production of Earthworm Lumbrokinase for Potential Antithrombotic Application

Kevin Yueju Wang,1 Lauren Tull,1 Edwin Cooper,2 Nan Wang,3 and Dehu Liu3

1 Department of Natural Sciences, Northeastern State University, Broken Arrow, OK 74014, USA 2 Laboratory of Comparative Neuroimmunology, Department of Neurobiology, David Geffen School of Medicine at UCLA University of California Los Angles, Los Angeles, CA 90095-1763, USA 3 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Correspondence should be addressed to Kevin Yueju Wang; [email protected] and Dehu Liu; [email protected]

Received 26 August 2013; Revised 7 November 2013; Accepted 18 November 2013

Academic Editor: Ronald Sherman

Copyright © 2013 Kevin Yueju Wang 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.

Earthworms have been used as a traditional medicine in China, Japan, and other Far East countries for thousands of years. Oral administration of dry earthworm powder is considered as a potent and effective supplement for supporting healthy blood circulation. Lumbrokinases are a group of enzymes that were isolated and purified from different species of earthworms. These enzymes are recognized as fibrinolytic agents that can be used to treat various conditions associated with thrombosis. Many lumbrokinase (LK) genes have been cloned and characterized. Advances in genetic technology have provided the ability to produce recombinant LK and have made it feasible to purify a single lumbrokinase enzyme for potential antithrombotic application. In this review, we focus on expression systems that can be used for lumbrokinase production. In particular, the advantages of using a transgenic plant system to produce edible lumbrokinase are described.

1. Introduction plasminogen-rich and plasminogen-free fibrin. The enzymes were collectively named lumbrokinase (LK) after the genus Earthworms, which are also called Dilong (Earth Dragon) in name for earthworm, Lumbricus. Thrombolytic agents typi- Chinese, have been used as a traditional medicine and food cally used to dissolve clots are urokinase (u-PA), streptoki- resource in China, Japan, and other Far East countries for nase, and tissue plasminogen activator (t-PA). These drugs, thousands of years [1–3]. Earthworms contain many com- however, are not specific for fibrin and have adverse and pounds with potential medicinal properties and have been dangerous side effects including severe bleeding and heavy administrated to treat inflammatory, hematological, oxida- bloodlosswhichmayresultindeath[11, 12]. In contrast, LK is tive, and nerve disease [4–6]. Earthworms also have antimi- very specific to fibrin as a substrate and it does not cause crobial, antiviral, and anticancer properties [7]. Among many excessive bleeding [13, 14]. It can dissolve the fibrin itself or properties, earthworms also exhibit fibrinolytic activity [8– convert plasminogen to plasmin by inducing endogenous t- 11]. The pharyngeal region, crop, gizzard, clitellum, and intes- PA activity to dissolve fibrin clots10 [ , 14, 15](Figure2:LK tine secret an enzyme that plays a role in dissolving fibrin [9, mechanismofaction). 10](Figure1). Ground-up earthworm powder has been used LK has shown therapeutic promise for use in dissolving as oral administration to support circulatory health and treat clots, lowering whole blood viscosity, and reducing platelet blood diseases [9]. aggregation. It has not shown any adverse effects on the In 1991, Dr. Mihara and other scientists in Japan suc- functions of the nervous system, respiratory system, cardio- cessfully extracted and characterized a group of fibrinolytic vascular vessels, or the liver and kidney [2, 7]. Currently, LKs enzymes from the earthworm species, Lumbricus rubel- are widely used clinically as a thrombolytic agent in China to lus [10]. These enzymes are capable of degrading both treat cerebral infarction, coronary heart disease, pulmonary 2 Evidence-Based Complementary and Alternative Medicine

12 3411 56789 10 12 13 Lumbrokinase

Fibrinogen

Fibrin Fibrin degradation Mouth Anus t-PA (a) Plasmin

Plasminogen 2 1 3 4 Figure 2: Lumbrokinase mechanism of action. LK dissolves the fibrin itself or increases native t-PA activity to dissolve fibrin clots 6 7 5 [15,PLoSONE]. 8

13 12 11 10 molecular mass is between 20 to 35 kDa and the isoelectric 9 points (pI) range from 3 to 5. Some LKs are also resistant to ∘ high temperatures (up to 60 C) [9]. Since each fibrinolytic Water PBS 5 ut-PA enzyme was independently isolated and named by different research groups, the same enzyme may have multiple names. PBS Therefore, the total number of LKs is not clear [9]. LK nomenclature needs to be standardized based on proteinase function, property, and the source. Conventional methods of LK extraction and purification from earthworm are complicated and time consuming. The (b) process consists of multiple steps that include ammonium Figure 1: Earthworms exhibit strong fibrinolysis. (a) An earthworm, sulfate precipitation and filtration, ion exchange chromatog- Eisenia fetida, was collected locally (Tulsa, OK, USA) washed with raphy, hydrophobic interaction chromatography, and affinity deionized water and sectioned in 13 pieces. (b) The sections were chromatography [10, 19]. Since the molecular range of LK is applied directly to a petri dish plate containing artificial fibrin and relatively narrow (20–35 kDa), it is very hard to isolate and ∘ incubated at 37 C overnight. Human tissue plasminogen (t-PA) (5 purify a single LK protein with conventional methods. Thus, units) was used as a positive control. Deionized water and phosphate LK products usually contain multiple enzyme components. buffered saline (PBS) served as negative controls. Bigger lysis halos The use of different extraction and purification procedures (3–7) indicated higher fibrinolytic activity regions. This method was will also result in a final product that varies in LK compo- described by Mihara et al. [10]. sition.Thus,theleveloffibrinolyticactivitymayalsovary. The final product may also contain other earthworm contam- inants that can induce adverse side effects, such as an upset heart disease, deep vein thrombosis. angina pectoris, dia- stomach or vomiting [20]. Therefore, researchers have used betes, and cerebral infarction. In Japan, Korea and also in recombinant DNA technology for the expression and char- North American countries such as Canada and the United acterization of a single LK protein to assess its potential for States, LK has been used as oral supplement to support and clinical application. maintain healthy cardiovascular function. 3. LK Gene Cloning and Analysis 2. Extraction and Isolation of LKs To date, 24 lumbrokinase gene sequences are publicly avail- LKshavebeenmainlyisolatedfromL. rubellus and Eisenia able at NCBI GenBank (Table 1).Aminoacidsequencealign- fetida. Some reports refer to LKs as earthworm fibrinolytic ment indicates that some LK genes are highly related (Fig- enzymes (EFE) or earthworm powder enzymes (EPE). Some ure 3). For example, PI239 shares 99% amino acid similarity proteases have been named after the Latin binomial for the with 1T4, EFE-3, and lk-6 (F6) [9]. CST1 exhibits the highest earthworm species from which it was derived [9]. For level of sequence identity (99%) with the enzyme, PV242, example, a protease obtained from E. fetida is called E. andAF109648aswellasTFc,a1,andAY438625[15]. The fetida protease (Efp) [9]. Mihara et al. [10] extracted six fib- high level of similarity in amino acid sequence among LKs rinolytic enzymes (F-1-0, F-1-1, F-I-2, F-II, F-III-1, and F-III- indicates that some LKs have a comparatively recent common 2) from L. rubellus. Seven fibrinolytic enzymes were purified ancestor [9, 15]. The phylogenetic analysis also indicates that from E. fetida by Zhou et al. in 1988 [16]. Isozymes of LK most LK genes are closely related to each other [9, 15]. The have also been isolated from L. bimastus [17]andE. Andrei differences in LK protein sequences between species may be [18]. LKs can maintain activity under both acidic and basic the result of the diverse habitats occupied and food resources conditions. They have a wide pH range (1-11). Their protein utilized by the different species of earthworm [9, 21]. Current Evidence-Based Complementary and Alternative Medicine 3

Table 1: Lumbrokinase gene clones, expression, and characterization.

Earthworm Lumbrokinase GenBank no. Expression host(s) Characterization references P. p a stor i s Ge et al., 2005 [22] PI239 AF433650 E. coli Xu et al., 2010 [23] L. bimastus PM246 AY187629 P. p a stor i s Hu et al., 2005 [21] PV242 AF109648 E. coli Xu et al., 2002 [17] F-III-1 AB045720 P. p a stor i s Sugimoto and Nakajima 2001 [24] F-III-2 AB045719 E. coli Li et al., 2008 [25] L. rubellus 1T4 U25643 lk-6 (F6) AF304199 E. coli Cho et al., 2004 [26] ∗ Goat mammary glands Hu et al., 2004 [20] EFE3-1 U25648 (AY327442) Sunflower plant Guan et al., 2013 [27] CST1 AY840996 E. coli Li et al., 2012 [15] CST2-2 AY684712 CST2-1 AY684711 TFe EU167737 Tfd EU167736 Tfc EU167735 Tf2 EU167734 E. fetida F238 DQ202401 P. p a stor i s Zhao et al., 2006 [28] efp-1 DQ418454 efp-0 DQ836917 a1 AF393512 E. coli Dong et al., 2004 [29] EFE-3 AY438622 P. p a stor i s Yuan et al., 2006 [30] AF432224 AY438624 AY438623 AY438625 ∗ (AY327442) was codon optimized from U25648. evidence suggests that mostly Eisenia possesses lumbroki- catalytic amino acid residues of LKs are very conserved. For nase (Table 1). Their habitats are different from Lumbricus example, the catalytic triad, three catalytic subsites, and the terrestris. The amino acid sequence of efp-0 has the lowest primary substrate specificity determinants of t-PA and u-PA identity (25–41%) with all other reported LKs, indicating that are conserved in LK PI239 [22]. Our own analysis found that it may have evolved independently. CST1 contains a catalytic triad, pocket, and substrate recogni- An analysis of the N-terminal amino acid sequence of tion sites similar to tPA, uPA, and DSPA𝛼1. The amino acids, 209 210 LK proteins reveals a high degree of identity with only one Ser and Trp , of CST1 match the S1 and S2 subsites of tPA, or two differences in amino acid (Figure 4). The conserved uPA, and DSPA𝛼1[15]. These sites play an important role in region at the N-terminal end of LKs suggests that it plays the catalysis and cleavage of peptide bonds and degradation an important role in the activity of this enzyme in targeting of fibrin clots [15, 22]. Thus, the conserved features of LKs and degrading fibrin clots [15, 22]. Interestingly, even efp- explain the mechanism of fibrinolytic activity. 0, which has less identity to the other LKs, shares the same amino acid sequence, S-H-S-C-G-A-S-L-I, in the N-terminal 4. Engineering LKs for Potential region of the protein, which further suggests that this region Medical Application may be very critical for the fibrinolytic properties of LKs. Further molecular studies, utilizing deletion or mutation of Since it is easier and more straightforward to obtain repeat- specific bases in this part of the gene, are needed to clarify the able biological results when evaluating single compounds biological function of this region. LKs share common features rather than complex mixtures, most of the drugs approved by to other fibrinolytic proteases, such as t-PA, u-PA, or vampire FDA are single chemical entities. Similar to most traditional bat plasminogen activator 𝛼1(DSPA𝛼1) [9, 15, 22]. They medicines, LKs present challenges in the design of proto- also show similarity to mammalian serine proteases. The cols to meet FDA regulations. As mentioned, conventional 4 Evidence-Based Complementary and Alternative Medicine

Figure 3: Alignment of 24 LK amino acid sequences. All sequences were obtained from GenBank. Identical residues are highlighted with the same color.

extraction and purification methods are tedious and have protease was able to dissolve artificial fibrin. The F-III-2 limitations in their ability to isolate and purify a single LK cDNA, with or without a native signal peptide sequence, was protein from earthworms [10, 16, 19]. Current LK products further studied using an E. coli expression system. Results do not produce consistent thrombolytic results. Therefore, indicated that E. coli could not recognize the native signal recombinant technology that would allow for the expression peptide of F-III-2 [25]. Therefore, no significant fibrinolytic ofasingleLKgenewouldgreatlyassisttheabilityto activity was observed, even though the gene was expressed obtain data for evaluating pharmaceutical safety and efficacy [25]. Ko et al. [31] cloned and expressed a fibrinolytic enzyme standards. gene from E. andrei tobacco chloroplasts. The biological Sequences for 24 LK genes have been deposited in function of recombinant enzyme, however, was not reported. GenBank (Table 1). Only a few of these genes have been The development of an animal cell system for lumbroki- successfully expressed and characterized in E. coli [15, 17, 23, nase expression was led by Hu and colleagues [20]. Both the 25, 26, 29], goat mammary glands [20], yeast Pichia pastoris wild-type and codon-optimized EFE3-1 gene were expressed [21, 22, 24, 28, 30], or plants [27]. Over the past decade, in lactating goat mammary glands and characterized. The researchers have tried to produce LKs via recombinant tech- fibrinolytic activity (550,000 ± 21,600 tPA u/L) of the codon- nology; however, the majority of studies have reported that, optimized gene was twice that of the wild-type gene (215,000 for undetermined reasons, recombinant LKs are either not ± 13,200 tPA u/L). These results indicate that codon usage bias expressed or do not exhibit fibrinolytic activity. In 2001, in different species is very important for LK expression. When Sugimoto and Nakajima [24] cloned two genes encoding the the same vectors were transiently transfected in other mam- LKs, F-III-2, and F-III-1. Only F-III-2 was expressed in P. malian cells, such as baby hamster kidney (BHK)-21, Chinese pastoris and characterized. The secreted recombinant F-III-2 hamster ovary (CH), Vero cells, Madin-Derby canine kidney Evidence-Based Complementary and Alternative Medicine 5

PI239 EFE-3 1T4 1k-6 AF432224 EFE3-1 F-III-1 Group 1 PM246 Tf2 F238 F-III-2 CST2-1 CST2-2 Tfc a1 PV242 Group 2 AY438625 CST1 Tfe AY438623 * Tfd Group 3 efp-1 AY438624 ** efp-0

Figure 4: N-terminal sequence alignment of LKs. Except efp-0, the other 23 LKs were placed in three groups based on their N-terminal ∗ amino acid sequence. The conserved residues, S-H-S-C-G-A-S-L-L, are boxed. indicates an exception.

(MGCK), and COS-7, no lumbrokinase activity was detected. stability, and proteolysis resistance. Optimized high density The reason for the lack of fibrinolytic activity is unclear and fermentation of engineered P. p a stor i s produced 0.1784 g/L of warrants further investigation. lumbrokinase PI239 in the supernatant [36]. However, the use Due to its ease of handling and rapid growth, the E. coli of methanol to activate lumbrokinase gene expression in the expression system has the potential to produce high yields of yeast represents a safety concern. Therefore, the production LK protein at a low cost. The recombinant LKs produced in of functional LKs in transgenic plants could represent an E. coli,however,arepackagedasaninclusionbody[15, 17, 23, attractive alternative. 25, 26, 29].Asaresult,arenaturationprocessisnecessaryto recover and reconstitute enzyme activity. Since prokaryotic 5. Plant-Derived LK Proteins cells are not capable of performing posttranslational modi- fications32 [ ], E. coli may not be able to express eukaryotic Genetically modified plants have been developed commer- LK proteins with proper folding, processing, and glycosy- cially for the past twenty years [37]. Plants have emerged as lation. Yeast expression systems utilizing P. p a stor i s may be a convenient and economic alternative to expression systems a better option for producing LK proteins since yeast can that utilize bacteria, yeast, or cultured mammalian cells for accomplish eukaryotic posttranslational modification of the production of pharmaceuticals [37–40]. Plants have the recombinant proteins [33]. Even though several LKs have machinery necessary for posttranslational modifications that been successfully expressed and characterized with yeast are necessary to achieve protein stability and bioactivity. The system [21, 22, 24, 28, 30], the glycosylated pattern of the protein synthesis pathway in plants is also very similar to ani- recombinant LK protein has not been investigated. Studies mal cells. The cost of producing pharmaceutical protein in a have shown that LKs contain glycan chains [34, 35]. Wu and transgenic plant system is estimated to be much cheaper than colleagues [34]isolatedeightLKsfromE. fetida.Glycanmea- using mammalian cell cultures and microbial fermentation surement showed that all eight proteases were glycoprotein systems. For example, computed plant-derived single-dose with different carbohydrate contents. The glycosylation of Hepatitis B vaccine (HBV) will save 62% to 90% depending LKs might play important role in LKs’ fibrinolytic activity, on the locations of facilities (the United State, Korea, or 6 Evidence-Based Complementary and Alternative Medicine

1 2 demonstrates that plant systems represent an attractive and promising option for the production of therapeutic LKs. Earthworm LK gene Plant expression vector Transgenic plant 6. Conclusions

3 LKs, in the form of dry earthworm powder, have been widely 5 used clinically in China as an antithrombotic agent. LK cap- LK protein sules have also been used as a health supplement in various purification 4 countries, including Japan, Korea, Canada, and United States, LK for injection for supporting circulatory health. The multiple components found in LK products derived from earthworm powders, however, represent a significant barrier to the approval and 6 Plantation use of LKs as a pharmaceutical product. Twenty-four LK 7 genes have been cloned and sequenced and it is likely that additional LK genes will be identified and cloned in the Edible LK Harvest future. Since it is very difficult to isolate and purify a single LK protein from earthworms, the production of LKs using Figure 5: Using transgenic plants for pharmaceutical protein pro- recombinant technologies is essential. Optimization of LK duction. A candidate LK gene is introduced into a plant expression gene codons may be a good strategy to increase LK protein. vector (1) and integrated into a transgenic plant (2). Transgenic Additional studies are needed to clarify why some LK genes plants are grown (3) and then harvested for protein purification are capable of being expressed in cell culture systems but (4) which can then be administered by injection (5) or used as a vegetable or fruit (6) and eaten as a food with therapeutic properties without fibrinolytic activity. Research is also required to (7). investigate the structure and function of different LKs. Since plants provide a convenient and inexpensive transformation platform for the production of recombinant proteins, we India) comparing to the yeast-derived vaccine [41]. More anticipate that more LKs will be expressed and tested in trans- importantly, plants are not the host of human pathogens. genic plants. In addition to the limited yield and slow process Therefore, recombinant protein from plants is less likely to of the stably transformed plant system, transient expression transmit disease causing agents to human beings [42]. in plants using replicating viral vectors can provide high- LKs are very good candidates for recombinant production yield production capacity for pharmaceutical proteins within in a plant expression system. Unlike most proteins, LKs several days [46–48]. We transient express several fibrinolytic can resist low pH acidic conditions [9, 10]. They can also enzymesinplantsbyasingle-vectorDNArepliconsystem be absorbed in the gastrointestinal tract intact and retain (kindly provided by Dr. Hugh Mason, Arizona State Univer- activity [43–45]. Even though the intact LKs were detected sity) and the results are promising (data in preparation). In after the intestinal absorption, the mechanism of how LKs conclusion, plant expression systems represent a promising could transport into blood is still uncertain. Authors [43, alternativefortheproductionofLKsforbothoralingestion 45] suggested that LKs might cross the cell membrane by and injection. exocytosis and enter the blood stream to dissolve clots. N- terminal sequences of the majority LKs are rich in hydropho- Authors’ Contribution bic amino acid residues, which may play an important role in the resistance to degradation in the gastrointestinal tract [43]. K. Y. Wang wrote the review and provided funding resources. In a plant expression system, LKs could be expressed in a veg- L.Tullworkedontheprojectandprovidedpictures.E. etable or fruit and consumed as pharmaceutical agent directly Cooper provided ideas and assistance with writing and as food, which would eliminate the need for extraction and analysis of data. N. Wang performed data analysis. D. Liu purification. LKs could also be expressed in specific tissues provided funding and major conceptual contributions. like seeds that would allow for downstream processing and purification for oral administration or injection (Figure 5). Acknowledgments To date, only two studies have reported the expression of LKs in plant systems. In 2009, Ko et al. [31]introduced This work was supported by the National Institute of General a lumbrokinase gene into tobacco chloroplasts. Their results Medical Sciences of the National Institutes of Health through indicated a stable integration of the LK gene into the tobacco Grant no. 8P20GM103447 and the National Biotechnol- plastid genome and the expression level of the recombinant ogy Development Plan (2013ZX08005-004), the Researcher protein was confirmed by Western blot analysis. Even though Foundation of the Chinese Academy of Agricultural Sci- fibrinolytic activity was not assessed in this study, it demon- ences. strated that overexpression of LKs in a plant system was feasible. In 2013, Guan et al. [27] produced biologically active References EFE3-1 in sunflower seed using the seed-specific promoter, napA. A significant antithrombus effect was observed in [1] E. L. Cooper, M. Balamurugan, C. H. Huang et al., “Earthworms mice that were fed kernels of transgenic seed. This study dilong: ancient, inexpensive, noncontroversial models may Evidence-Based Complementary and Alternative Medicine 7

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Review Article Honey as a Potential Natural Anticancer Agent: A Review of Its Mechanisms

Sarfraz Ahmed and Nor Hayati Othman

Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

Correspondence should be addressed to Nor Hayati Othman; [email protected]

Received 9 July 2013; Revised 8 October 2013; Accepted 21 October 2013

Academic Editor: Tung-Sheng Chen

Copyright © 2013 S. Ahmed and N. H. Othman. 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 main treatment for cancer is by using chemotherapy and radiotherapy which themselves are toxic to other viable cells ofthe body. Recently, there are many studies focusing on the use of natural products for cancer prevention and treatment. Of these natural products, honey has been extensively researched. The mechanism of the anti-cancer activity of honey as chemopreventive and therapeutic agent has not been completely understood. The possible mechanisms are due to its apoptotic, antiproliferative, antitumor necrosis factor (anti-TNF), antioxidant, anti-inflammatory, estrogenic and immunomodulatory activities. We collate the findings of several studies published in the literature in order to understand the mechanism of its action.

1. Introduction activity against different types of leukemic cell lines15 [ ]. Its anticancer activity has been proved against various cancer cell Annually cancer is diagnosed in approximately 11 million lines and tissues, such as breasts [14, 16–19], colorectal [20], people causing 7.6 million deaths worldwide [1]. Cancer renal [21], prostate [17], endometrial [17], cervical [19]and is a multistep process. It starts as an onset from a single oral cancer [22]. transformed cell. Its genesis is characterized by the swift Honey potentiates the antitumor activity of chemother- proliferation, invasion, and metastasis [2]. This dynamic apeutic drugs such as 5-fluorouracil and cyclophosphamide process is activated by various carcinogens, tumor promot- [18]. Studies exhibiting anticancer effect of honey range from ers, and inflammatory agents. The whole modulation is tissue cultures [17, 19, 20, 22, 23] and animal models [14, 16, 18, controlled through the transcription factors, proapoptotic 24]toclinicaltrials[25]. Polyphenols in honey are considered proteins, antiapoptotic proteins, protein kinases, cell cycle as one of the main factors responsible for the anticancer proteins, cell-adhesion molecules, cyclooxygenase-2 (COX- activity of honey [15, 26]. 2), and other molecular targets [2–4]. This review presents current progress in understanding The standard treatments against cancer are surgery, radio- the mechanism of anticancer activity of honey. therapy, and chemotherapy. These modalities are beset with serioussideeffects[5]. New targets for cancer treatment focus on interfering with specific targeted molecules needed in 2. Honey and Its Apoptotic Activity carcinogenesis [6]. Natural products such as honey have potential anticancer Two characteristics of cancer cells are uncontrolled cellular effect [7]. Honey is composed of various sugars, flavonoids, proliferation and inadequate apoptotic turnover [27]. Drugs phenolic acids, enzymes, amino acids, proteins, and miscella- which are commonly used for cancer treatment are apoptosis neous compounds (Table 1). Its composition varies according inducers [28]. Programmed cell death or apoptosis is catego- to floral sources and origin [8]. It has been shown to have rized into three phases: (a) an induction phase, (b) an effector anti-inflammatory [9], antimicrobial [10], antimutagenic [11], phase, and (c) a degradation phase [29]. The induction antioxidant [12], and antitumor [7, 13, 14]effects.Thephenolic phase stimulates proapoptotic signal transduction cascades contentsofhoneyhavebeenreportedtohaveantileukemic through death-inducing signals. Effector phase is committed 2 Evidence-Based Complementary and Alternative Medicine

Table 1: Average composition of honey-source reference, [82, 83]. a possible natural substance as antic-cancer agent as many chemotherapeutics currently used are apoptosis inducers. Component Value/100 g Total carbohydrates 82.4 g Fructose 38.5 g 3. Honey and Its Antiproliferative Activity Glucose 31.28 g Epithelial cell divides throughout life. The cell cycle comprises Sucrose 1.31 g three distinguished phases known as G0,G1,S,andG2 M. Maltose 7.31 g Alltheeventsinthecellcycleareregulatedandmonitored Total acid as gluconic 0.57 g by several different proteins. The control panel of cell cycle Moisture content 17.1 g comprises cyclins and cyclin-dependent kinases. The G1/S Ash 0.169 g phase transition is a vital regulatory point where cell’s fate Amino acids 0.3 g is destined for quiescence, proliferation, differentiation and Nitrogen 0.041 g apoptosis [32]. Overexpression and dysregulation of cell Iron 0.42 mg cycle growth factors such as cyclin D1 and cyclin-dependent Potassium 52 mg kinases (CDK) are linked with tumorigenesis [32]. The loss of this regulation is the hallmark of cancer [32]. The nuclear Calcium 6.00 mg protein Ki-67 is a novel marker to probe the “growth fraction” Phosphorous 4.00 mg of cell proliferation. It is absent in the resting phase (G0)but Magnesium 2.00 mg expressed during the cell cycle in all the proliferation phases Calcium 6.00 mg (G1,S,G2, and mitosis) [33]. pH 3.9 Honey has been shown to affect cell cycle arrest. Admin- istration of honey mixed with Aloe vera solution showed a marked decrease in expression of Ki67-LI in tumor cells in rats [14]. It suggests that honey therapy could lead to lowering tumor cell proliferation by arresting cell cycle [14].Honey to bring cell death via a key regulator, mitochondrion. The last and its several components (like flavonoids and phenolics) degradation phase comprises nuclear and cytoplasmic events. arereportedtoblockthecellcycleofcolon[20], glioma Nuclear change includes chromatin and nuclear conden- [34], and melanoma [35] cancer cell lines in G0/G1 phase. sation, cell shrinkage, DNA fragmentation, and membrane This inhibitory effect on tumor cell proliferation follows blebbing [28, 29]. In the cytoplasm, a complex cascade of the downregulation of many cellular pathways via tyrosine protein cleaving enzymes called caspases is activated. The cell cyclooxygenase, ornithine decarboxylase, and kinase [20, 34– is finally destined into fragmented apoptotic bodies which 36]. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl are phagocytosed by macrophages or other surrounding cells tetrazolium bromide (MTT) and the trypan blue exclusion [28, 29]. assays have confirmed that anti-proliferative effect of honey The apoptosis usually follows two pathways: the caspase is a dose- and time-dependent manner [35]. Honey or its 8ordeath-receptorpathwayandcaspase9ormitochondrial components mediate inhibition of cell growth due to its pathway [30](Figure 1). perturbation of cell cycle [35, 36]. Cell cycle is also regulated Honey induces apoptosis in various types of cancer cells by p53 which is involved in tumor suppression. Honey is via depolarization of mitochondrial membrane [19]. Honey reported to be involved in modulation of p53 regulation [20]. elevates caspase 3 activation level and poly (ADP-ribose) polymerase (PARP) cleavage in human colon cancer cell lines [20] which is attributed to its high tryptophan and phenolic 4. Honey and Its Effect on Tumor content [20]. It also induces apoptosis by upregulating and Necrosis Factor (TNF) modulating the expression of pro- and antiapoptotic pro- teins in colon cancer cell lines [23]. Honey increases the Tumor necrosis factor (TNF), have been shown to medi- expression of caspase 3, p53, and proapoptotic protein Bax ate tumor initiation, promotion, and progression [37]. The and downregulates the expression of antiapoptotic protein proinflammatory effect of TNF is linked to many diseases Bcl2 [23](Figure 2). Honey generates ROS (reactive oxygen duetoitsabilitytoactivateNF-kB[38]. It activates NF- species) resulting in the activation of p53 and p53 in turn kB, leading to the expression of inflammatory genes like modulates the expression of pro- and antiapoptotic proteins lipoxygenase-2 (LOX-2), cyclooxygenase-2 (COX-2), cell- like Bax and Bcl-2 [23]. Honey as an adjuvant therapy with adhesion molecules, chemokines, inducible nitric oxide syn- Aloe vera boosts the expression of proapoptotic protein Bax thase (iNOS), and inflammatory cytokines [38]. It is consid- and decreases the antiapoptotic protein Bcl-2 expression in ered as a growth factor for many of the tumor cells [38]. In Wistar rats [14]. Manuka honey exerts its apoptotic effect contradiction, TNF-𝛼 has also been shown to be involved in on cancer cells through the induction of the caspase 9 host defense mechanisms as a key cytokine [39]. It has been which in turn activates the caspase-3, the executor protein. shown to play a dual role, beneficial and deleterious for the Apoptosis induced by Manuka also involves induction of promotion or inhibition of infectious diseases [39, 40]. DNA fragmentation, activation of PARP, and loss of Bcl-2 Royal jelly (RJ) proteins (apalbumin-1 and apalbumin-2) expression [31]. The apoptotic property of honey makes it in honey have antitumor properties. These proteins stimulate Evidence-Based Complementary and Alternative Medicine 3

External apoptotic stimuli

p53 Mitochondrion Bcl-2

Cytochrome c + APAF-1 BID Death legend + death receptor Caspase 9 Caspase 3 Caspase 8

IAP Cell death/apoptosis

Caspase 9 pathway Caspase 8 pathway

Figure 1: Apoptosis following caspase 8 and caspase 9 pathways; source [30]. Bcl-2:Bcelllymphoma2protein;Bid:Bcl-2associatedXproteins; Cyt. C: cytochrome C; Apaf-1: apoptotic protease activating factor; IAP: inhibitor of apoptosis proteins; Caspase 3-caspase protein that interacts with caspase 8 and caspase 9.

Extrinsic Intrinsic TRAIL Honey TNF TRADD Bcl-2 Bax p53

Mitochondrion Honey Caspase 8 Cyt. c + APAF-1

Caspase 9 Caspase 3

Apoptosis

Inhibition Supposed stimulation Stimulation

Figure 2: The effect of honey on apoptotic pathway. Honey exerts apoptotic effect through upregulation and modulation of proapoptotic proteins (p53, Bax, caspase 3, and caspase 9) and downregulation of antiapoptotic proteins (Bcl-2) [14, 23, 31]. Bcl-2: B cell lymphoma 2 protein; Cyt. C: cytochrome C; Apaf-1—apoptotic protease activating factor 1; TNF: tumor necrosis factor; TRAIL: TNF related apoptosis-inducing ligand; TRADD: TNFR associated death domain protein. macrophagestoreleasecytokinesTNF-𝛼, interleukin-1 (IL- important cellular processes such as apoptosis, cell prolifera- 1) and interlueken-6 (IL-6) [41, 42]. Pasture, jelly bush, tion, and inflammation [41, 45]. and Manuka honeys (at concentrations of 1% w/v) stimu- late monocytes to release tumor necrosis factor-alpha and 5. Honey and Its Anti-Inflammatory 𝛽 interleukin- (IL-) 1 and IL-6 [43, 44]. The possible mecha- and Immunomodulatory Activities nism involves the binding of TNF-R to TNF-𝛼 and adaptor protein such as TNFR associated death domain protein Chronic inflammation is linked to cancer formation. Exces- (TRADD), TNF receptor associated factor (TRAF), and sive or prolonged inflammation can prevent healing by dam- receptor-interacting protein (RIP) to regulate apoptosis and aging tissues. Honey exhibits anti-inflammatory response inflammation through these cytokines45 [ ]. This TNF-𝛼 [46]. The literature shows that it reduces inflammation release can play a pivotal role as a key cytokine to regulate when applied in cell cultures [47], animal models [48, 49], 4 Evidence-Based Complementary and Alternative Medicine and clinical trials [46, 50]. The inflammatory process is inflammatory disorders [73], cardiovascular diseases [74], induced by various types of chemicals and/or biological and cancer [75, 76]. The phenolic acids and flavonoids are agents including proinflammatory enzymes and cytokines responsible for the well-established antioxidant activity of [51]. The enzyme cyclooxygenase-2 (COX-2) in inflam- honey [77]. matory process catalyses the metabolism of arachidonic The antitumor effect of honey may be attributed to acid to prostaglandin [52, 53]. Anomalous arachidonic acid its antioxidant activity [75, 76]. An enhanced antioxidant metabolism is involved in carcinogenesis and inflammation status with apoptosis has been observed in hepatocellular [54]. COX-2 is overexpressed in premalignant and malignant carcinoma cells [75]. Daily consumption of 1.2 g/kg body conditions [54]. Phenolic compounds in honey are respon- weight of honey has been shown to elevate the amount sible for anti-inflammatory activity [55]. The mechanism and the activity of antioxidant agents such as beta-carotene, involves the suppression of the proinflammatory activities vitamin C, glutathione reductase, and uric acid [60]. of COX-2 and/or inducible nitric oxide synthase (iNOS) through these phenolic compounds or flavonoids [53]. Honey 7. Honey and Its Antimutagenic Activity and its components have been documented to be involved in regulation of proteins such as ornithine decarboxylase, Mutagenicity, the ability to induce genetic mutation, is inter- tyrosine kinase, iNOS, and COX-2 [56, 57]. linked with carcinogenicity [78]. Honey is shown to have a Manuka, Pasture, Nigerian Jungle, and royal jelly honeys strong antimutagenic agent and hence has anticarcinogenic are found to increase IL-1𝛽,IL-6,andTNF-𝛼 production property [79]. The effect of honey on radiation (UVor 𝛾) [16, 44, 58]. This immunomodulatory and immunoprotective exposed Escherichia coli cells shows SOS response (SOS is activity of honey is often linked to anticancer action [16, 59]. an error prone repair pathway contributing to mutagenicity) Honey stimulates antibodies, B and T lymphocytes, neu- [79]. A study was performed to knock out some impor- trophils, monocytes, eosinophils, and natural killer cells (NK- tant genes such as umuC, recA, and umuD involved in cells) production during primary and secondary immune SOS mediated mutagenesis. These changes are significantly responses in tissue culture [59–62]. It has been shown inhibited in the presence of honey confirming its strong thathoneystimulatesmacrophages,T-cells,andB-cellsto antimutagenic effect [79]. Honeys from different floral origins provoke antitumor effect [59]. exhibit inhibition of Trp-p-1 mutagenicity [11]. Sugars when ingested are slowly absorbed resulting in the formation of short chain fatty acid (SCFA) fermentation 8. Honey and Its Estrogenic products [63]. It is a probable mechanism that the ingestion of honey may result in SCFA formation [64]. Research has Modulatory Activity established that, either directly or indirectly, SCFA have Estrogen is involved in number of cancers [80]. Honey immunomodulatory actions [65]. Thus, honey may stimu- modulates estrogen by its antagonistic action. It may be late the immune system via these fermentable sugars [66]. useful in estrogen-dependent cancers such as breasts and A sugar, nigerooligosaccharides (NOS), present in honey endometrial cancers [17]. Estrogen receptors tie to estrogens has been found to have immunopotentiating activity [67]. to dimerize and then translocate into the nuclei. These com- Nonsugar components of honey may also be responsible for plexes then bind to the specific DNA base sequences called immunomodulation [66]. estrogen-response elements (EREs) resulting in transcription andtranslationoftheestrogeniceffectinthetargetedtissue 6. Honey and Its Antioxidant Activity [80]. This signaling cascade induced by estrogens may be modulated at any stage [80]. Honeys from various floral The role of oxidative stress involving free radicals in the sources are reported to mediate estrogenic effects via the carcinogenic process is well established [68]. Reactive oxygen modulation of estrogen receptor activity [17, 81]. This effect is species (ROS) and reactive nitrogen species (RNS), such as attributed to its phenolic content [17]. Greek honey extracts ∙ ∙− hydroxyl radical ( OH) superoxide (O2 ), hydrogen per- exert estrogen agonistic effect at high concentrations (20– ∙ − oxide (H2O2), nitric oxide (NO ), peroxynitrite (ONOO ), 100 lg/mL) and antagonistic effect at low concentrations (0.2– 𝜇 and others, are oxidative stress agents which damage lipids, 5 g/mL) [17]. proteins, and DNA in cells [69]. Cells exhibit defense system against oxidative damage. This defense system consists of 9. Conclusion antioxidants or oxidative protective agents such as catalase, superoxide dismutase, peroxidase, ascorbic acid, tocopherol, Evidenceisgrowingthathoneymayhavethepotentialto and polyphenols [70]. Antioxidants acting as free radical be anticancer agent through several mechanisms (Figure 3). scavengers may inhibit the cancer process in vivo [70]. Though the full mechanism is yet to be fully understood, The exact antioxidant mechanism is unknown, but the studies have shown that honey has anticancer effect through proposed mechanism is through hydrogen donation, free its interference with multiple cell-signaling pathways, such as radical sequestration, metallic ion chelation, flavonoids sub- inducing apoptosis, antiproliferative, anti-inflammatory, and strates for hydroxyl and superoxide radical actions [71]. The antimutagenic pathways. Honey modulates the body immune antioxidant capacity of honey contributes to the prevention system. There are still many unanswered questions; why sugar of several acute and chronic disorders such as diabetes [72], is carcinogenic, while honey which is basically sugar has Evidence-Based Complementary and Alternative Medicine 5

Honey

Antioxidant activity Anti-inflammatory activity p53 regulation Immunomodulatory activity Cell cycle arrest Anticancer response Cyclooxygenase-2 (COX-2) modulation Antimutagenic activity Tumor necrosis factor (TNF) modulation Estrogen modulation

Figure 3: Schematic presentation of anticancer activity of honey.

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