Journal of Medicinal Research Volume 11 Number 3, 17 January, 2017 ISSN 1996-0875

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Prof. Akah Peter Achunike Prof. Parveen Bansal Editor-in-chief Department of Biochemistry Department of Pharmacology & Toxicology Postgraduate Institute of Medical Education and University of Nigeria, Nsukka Research Nigeria Chandigarh India.

Associate Editors Dr. Ravichandran Veerasamy AIMST University Dr. Ugur Cakilcioglu Faculty of Pharmacy, AIMST University, Semeling - Elazıg Directorate of National Education 08100, Turkey. Kedah, Malaysia.

Dr. Jianxin Chen Dr. Sayeed Ahmad Information Center, Herbal Medicine Laboratory, Department of Beijing University of Chinese Medicine, Pharmacognosy and Phytochemistry, Beijing, China Faculty of Pharmacy, Jamia Hamdard (Hamdard 100029, University), Hamdard Nagar, New Delhi, 110062, China. India.

Dr. Hassan Sher Dr. Cheng Tan Department of Botany and Microbiology, Department of Dermatology, first Affiliated Hospital College of Science, of Nanjing Univeristy of King Saud University, Riyadh Traditional Chinese Medicine. Kingdom of Saudi Arabia. 155 Hanzhong Road, Nanjing, Jiangsu Province, China. 210029 Dr. Jin Tao Professor and Dong-Wu Scholar, Dr. Naseem Ahmad Department of Neurobiology, Young Scientist (DST, FAST TRACK Scheme) Medical College of Soochow University, Plant Biotechnology Laboratory 199 Ren-Ai Road, Dushu Lake Campus, Department of Botany Suzhou Industrial Park, Aligarh Muslim University Suzhou 215123, Aligarh- 202 002,(UP) P.R.China. India.

Dr. Pongsak Rattanachaikunsopon Dr. Isiaka A. Ogunwande Department of Biological Science, Dept. Of Chemistry, Faculty of Science, Lagos State University, Ojo, Lagos, Ubon Ratchathani University, Nigeria. Ubon Ratchathani 34190, Thailand.

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Prof Hatil Hashim EL-Kamali Dr. Arash Kheradmand Omdurman Islamic University, Botany Department, Lorestan University, Sudan. Iran.

Prof. Dr. Muradiye Nacak Prof Dr Cemşit Karakurt Department of Pharmacology, Faculty of Medicine, Pediatrics and Pediatric Cardiology Gaziantep University, Inonu University Faculty of Medicine, Turkey. Turkey.

Dr. Sadiq Azam Samuel Adelani Babarinde Department of Biotechnology, Department of Crop and Environmental Protection, Abdul Wali Khan University Mardan, Ladoke Akintola University of Technology, Pakistan. Ogbomoso Nigeria. Kongyun Wu Department of Biology and Environment Engineering, Dr.Wafaa Ibrahim Rasheed Guiyang College, Professor of Medical Biochemistry National Research Center China. Cairo Egypt. Prof Swati Sen Mandi Division of plant Biology, Bose Institute India.

Dr. Ujjwal Kumar De Indian Vetreinary Research Institute, Izatnagar, Bareilly, UP-243122 Veterinary Medicine, India.

Journal of Medicinal Plants Research

Table of Contents: Volume 11 Number 3 17 January, 2017

ARTICLES

The genus L. (): A review of their antimicrobial activity 43 Antonio Carlos Nogueira Sobrinho, Selene Maia de Morais, Elnatan Bezerra de Souza and Raquel Oliveira dos Santos Fontenelle

Hypoglycaemic effect of fractions and crude methanolic leaf extract of Phyllanthus fraternus in streptozotocin -induced diabetic and normal rats 58 Nadro, M. S. and Elkanah, G.

Vol. 11(3), pp. 43-57, 17 January, 2017 DOI: 10.5897/JMPR2016.6313 Article Number: 4F30B6962396 ISSN 1996-0875 Journal of Medicinal Plants Research Copyright © 2017 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR

Review

The genus Eupatorium L. (Asteraceae): A review of their antimicrobial activity

Antonio Carlos Nogueira Sobrinho1*, Selene Maia de Morais1, Elnatan Bezerra de Souza2 and Raquel Oliveira dos Santos Fontenelle2

1Postgraduate Program in Biotechnology-Renorbio, Center for Science and Technology, State University of Ceará, Fortaleza, Brazil. 2Course of Biological Sciences, Center for Agricultural Sciences and Biological Sciences, State University of Vale do Acaraú, Sobral, Brazil.

Received 30 November, 2016; Accepted 6 January, 2017

In recent years, the number of infectious diseases linked to the occurrence of bacterial and fungal resistance has increased, leading to extensive search for new drugs to treat these infections. Species of the Asteraceae family and the genus Eupatorium, have high biological potential and are used in folk medicine to treat various diseases. This review article presents the main phytochemical and biological characteristics of the Asteraceae family and the genus Eupatorium s.l., whose antimicrobial activity is promising, especially antibacterial and antifungal activity. The current review was achieved using an organized search of the scientific data published on antimicrobial activity and phytochemical of the species of the genus Eupatorium using various databases, including PubMed, ScienceDirect, Scopus, Scielo, SciFinder and Google Scholar. The species of Eupatorium are rich in terpenes, phytosterols and sesquiterpene lactones, the latter being chemotaxonomic markers of the group, with broad anticancer, antiplasmodial and antimicrobial activity, making them promising for the development of new drugs. Various species of Eupatorium seems to hold great potential for in-depth investigation for antimicrobial activities. Many species have broad folk use, with scientific confirmation of its antimicrobial properties making these plants potential sources of safer and more effective treatments.

Key words: Compositae, Eupatorium s.l., antimicrobial potential, ethnopharmacology, ethnobotany.

INTRODUCTION

The family Asteraceae, belonging to the class of their anti-inflammatory, antimicrobial, anticancer, eudicotyledons, is one of the largest families of antisyphilitic, antigonorrheal and insecticidal properties angiosperm plants. Its species are known for their (Carrillo-Hormaza et al., 2015). Biological studies of its therapeutic, cosmetic and aromatic properties and have extracts, oils and constituents for production of great importance both from the environmental and phytochemicals have attracting growing interdisciplinary economic standpoints. They have been widely studied for interest from the scientific community (Garcia-Sanchez et

*Corresponding author. E-mail: [email protected].

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 44 J. Med. Plants Res.

al., 2011; Yang et al., 2004; Souza et al., 2007). (Alviano and Alviano, 2009). The relevance of these Among several genera of Asteraceae studied, studies is based on the fact that infections of bacterial Eupatorium L. sensu lato stands out. Numerous studies etiology, and in particular fungal infections, are of its species have been performed and the majority aggravated in immunosuppressed individuals, due to showed the presence of terpenes, phytosterols and therapy against cancer and AIDS and suppression of the sesquiterpene lactones (Liu et al., 2015), the latter immune response to prevent rejection of transplanted identified as promising sources for development of new organs (Miceli et al., 2011). drugs due to the antimicrobial, anticancer and For a comprehensive literature overview, the current antiplasmodial action (Albuquerque et al., 2010; Lang et review was achieved by using an organized search of the al., 2002). scientific data published on antimicrobial activity and In a broader context, ethnobotanical and phytochemical, focusing on the antibacterial and ethnopharmacological studies are very useful for antifungal activities of the species of the genus investigation of new bioactive plant sources Eupatorium. The search was conducted using the (Silambarasan and Ayyanar, 2015). Many traditional keyword search term "Eupatorium antimicrobial activity". indigenous communities have longstanding contact with The searches were carried out using various databases, plant species having medicinal properties, with including PubMed (http://www.ncbi.nlm.nih.gov/pubmed), information being transmitted from generation to Science Direct (http://www.sciencedirect.com/), Scopus generation. This information is useful to the scientific (http://www.scopus.com/), Scielo (http://www.scielo.org/), community in studies of natural resources, as occurred in SciFinder (https://scifinder.cas.org/) and Google Scholar studies of Eupatorium s.l. (Silva et al., 2012; Paredes- (http://www.scholar.google.com/). Flores et al., 2007). Based on these considerations, in the present paper, Chemical compounds with biological activity isolated the antibacterial and antifungal potential of species of the from plants and used by the population through extracts genus Eupatorium s.l., were analyzed highlighting also are promising sources to prospect for new antimicrobial biological properties of the Asteraceae family. drugs. These substances may also act synergistically with other drugs, potentially improving the antibiotic action (Al-Fatimi et al., 2007). However, it is necessary to ASTERACEAE FAMILY establish whether the traditional use of plants for the treatment of infectious diseases is supported by Asteraceae (Compositae) includes about 1600 genera pharmacological action or merely based on folklore and 25000 species (Petacci et al., 2012) belonging to 17 (Pessini et al., 2003). tribes and three subfamilies. It is considered by some One obstacle to the treatment of bacterial infections is authors as the largest family of eudicotyledonous the phenomena of resistance to antibacterial drugs, angiosperm plants (Nakajima and Semir, 2001; Hattori caused by the selection of resistant bacteria from and Nakajima, 2008). The order is formed by exposure to chemotherapeutic agents, causing transfer of the Calyceraceae, Menyanthaceae, Goodeniaceae, resistant gene fragments between bacterial strains and Campanulaceae and Asteraceae (Judd et al., 2009). This clonal spread of resistant strains among hospitalized family has been studied intensely over the past 25 years patients (Spellberg et al., 2013). The exposure of regarding morphology, anatomy, ontogeny and ecology antimicrobial compound can increase bacterial resistance as well as phytochemical, cytogenetic and also by selecting for mutation in genes that help microbes macromolecular aspects (Hind and Beentje, 1996). detoxify antibiotics. As for antibiotics, inadequate Asteraceae are for the most part herbs, subshrubs or knowledge of the pharmacokinetic and pharmacodynamic shrubs, trees or vines (Roque and Bautista, 2008). Its properties, in addition to factors inherent to the patient, representatives of greatest importance are Baccharis such as immune status and non-adherence to treatment, trimera (Less) DC. (gorse), Matricaria chamomilla L. affect the efficiency of chemotherapy against pathogenic (camomile), Cynara scolimus L. (artichoke), Vernonia bacteria (Muller et al., 2015; Udy et al., 2013). condensata Baker (“boldo-da-bahia”) and Arnica The use of natural extracts in the treatment of infectious montana L. (true arnica). These species are widely used diseases is promising, but studies on antimicrobial and marketed as herbal products, suggesting species of activity of different plant extracts should be expanded, the family Asteraceae have significant potential as a including analysis of essential oils and their constituents source of bioactive compounds (Maia et al., 2010; Mello in order to maximize the effect studied and to discover et al., 2008). They are also sources of edible oils which chemical compound plays the main role in (Helianthus annuus L., sunflower), pesticides and latex, antimicrobial action (Danielli et al., 2013). In relation to as well as being eaten as leafy vegetables (e.g.,Lactuca antifungal drugs, natural products are widelyused and the sativa L., lettuce) and used as ornamental plants (Wu et results are promising (Cragg and Newman, 2013). Due to al., 2006). Phytochemical studies of Eupatorium have the resistance of many pathogenic microorganisms, the identified the presence of sesquiterpene lactones, search for new antimicrobial agents from plants is intense flavonoids, triterpenes, benzofuran compounds, Sobrinho et al. 45

pyrrolizidine alkaloids, chromene and steroids (Liu et al., diverse in tropical and subtropical areas of the Americas 2015; Zhang et al., 2008; Albuquerque et al., 2004; Lang (Zhang et al., 2008). et al., 2001). For antibacterial action, tests have been conducted on activity against a wide range of microorganisms, both Gram-positive and negative bacteria. For investigations GENUS EUPATORIUM of plant extracts, the most commonly used solvents are methanol, ethanol, ethyl acetate and n-hexane, used in The genus Eupatorium s.l. (tribe , subtribe studies the aerial parts, branches, twigs and leaves. Eupatoriinae) was one of the most representative of the Nevertheless, there are reports of the use of roots to family Asteraceae, containing around 1200 species, obtain the essential oil of E. adenophorum Spreng. however a detailed survey conducted by King and (Ahluwalia et al., 2014) and the aqueous extract of E. Robinson (1987) found that about 1010 species would be purpureum L. (Carlson et al., 1948; Pates Madsen, reclassified to different new genera. When interpreted in 1955), in both cases having antibacterial action. its traditional concept, this genus form a polyphyletic Referring to antifungal bioassays, the studies focused group. Eupatorium s.l. have been a highly artificial on Eupatorium generally also have involved tests against concept, tending to include all members of the tribe with a bacteria. The tested fungi include filamentous fungi, pappus of numerous capillary bristles, five ribs on the dermatophytes and yeasts. Tests with dermatophytes, achene, and another appendage as long as or longer that filamentous fungi that cause dermatophytosis are wide (King and Robinson, 1987). This genus has been promising for the genus, with reports of activity for restructured and was fragmented into 80 genera. Eupatorium arnottianum Griseb. (Muschietti et al., 2005), Currently, the genus Eupatorium sensu stricto containing Eupatorium aschenbornianum S. Schauer (García et al., about 45 species, distributed primarily by arctic-tertiary 2003 Rios et al., 2003), Eupatorium buniifolium Hook. region (King and Robinson,1987; Bremer, 1994). and Arn. (Muschietti et al., 2005), Eupatorium japonicum The species of this genus are used in folk medicine in Thunb. (Inouye et al., 2006), Eupatorium laciniatum different parts of the world (Albuquerque et al., 2010), like Kitam. (Inouye et al., 2006) and Eupatorium odoratum L. Mexico, where over 1000 medicinal plants have been (Taylor et al., 1996). In all these studies, extracts of plant used for over 400 years in folk medicine, many are in the material were used. There are few reports in the literature genus Eupatorium (Garcia-Sanchez et al., 2011). on the use of essential oils for susceptibility testing. As Ethnopharmacological studies of medicinal plants are for the fungi tested, of the three genera of dermatophytes important for the development of new drugs (Trichophyton, Microsporum and Epidermophyton), only (Silambarasan and Ayyanar, 2015). The main goal is to two showed sensitivity and growth inhibition find new bioactive substances for disease control with (Trichophyton and Microsporum), highlighting species T. minimal side effects (Rodríguez and Carlini, 2003). In this rubrum and T. mentagrophytes in the genus context, studies have been conducted of biological Trichophyton. activities such as toxic activity of essential oils (Albuquerque et al., 2004) and plant extracts (Rozo et al., 2008) against the larvae of Aedes aegypti and Eupatorium adenophorum Spreng. antiplasmodial activity against Plasmodium falciparum, a protozoan that causes malaria (Lang et al., 2002). The species E. adenophorum [syn. Ageratina adenophora In particular, the literature contains numerous studies of (Spreng.) R.M.King & H.Rob.], native to Mexico, is now the genus Eupatorium with use of plant extracts and widely distributed in the world (King and Robinson, 1970) essential oils against strains of bacteria and fungi that and is used in folk medicine as an antimicrobial, infect animals and plants, for development of new drugs antiseptic, analgesic and antipyretic (Bhattarai and for veterinary and human use and new herbicides. Shrestha, 2009). It has also been shown to have Studies were found from 1948 to 2016 with Eupatorium molluscicide potential against Oncomelania hupensis, the species. E. adenophorum, E. odoratum and E. triplinerve intermediate host of Schistosoma japonicum (Zou et al., are the species with the most work reporting their 2009). The essential oil extracted from the flowers, and antimicrobial potential (Table 1). roots was tested and showed inhibitory activity for the strains of Xanthomonas oryzae, Erwinia chrysanthemi, Staphylococcus aureus, Pseudomonas aeruginosa, Antimicrobial activity of Eupatorium s.l. Klebsiella pneumoniae. Assays were performed using the disk diffusion and agar dilution methods (Ahluwalia et al., Most studies of the antimicrobial activity involved species 2014). The essential oil extracted from the aerial parts of Asia and Europe, with fewer studies of South American was also investigated against strains of Arthrobacter plants. This is counterintuitive, given that although protophormiae, Escherichia coli, Micrococcus luteus, countries like China, Malaysia, India, Zaire, Indonesia and Rhodococcus rhodochrous and Staphylococcus aureus. Madagascar can be considered megadiverse (Sandes Assays were performed by broth dilution and the results and Di Blasi, 2000), species of Eupatorium are particularly showed growth inhibition for all strains tested (Kurade et 46 J. Med. Plants Res.

Table 1. Species of the genus Eupatorium s.l. with antimicrobial activity.

Biological Bioactive Scientific name Part used Biological activity Reference preparation compounds Bhattarai and Shrestha, Antibacterial activity: Gram-positive and 2009; Kurade et al., 2010; Eupatorium adenophorum I, L, St, R EO, OsE Terpenoids Gram-negative bacteria Kundu et al., 2013; Ahluwalia Spreng. Antifungal activity: filamentous fungi et al., 2014; Chauhan et al., 2015 Antibacterial activity: Gram-positive and Carlson et al., 1948; Cantrell Eupatorium altissimum L. AP DE - Gram-negative bacteria et al., 1998 Eupatorium arnottianum AP ME - Antifungal activity: filamentous fungi MuschietTI et al., 2005 Griseb. Eupatorium aromaticum L. L AqE - Antibacterial activity: Gram-positive bacteria Pates and Madsen, 1955 Benzofurans Eupatorium aschenbornianum Antifungal activity: yeast and filamentous García et al., 2003, Rios et AP HE, ME compounds and S. Schauer. fungi al., 2003 phytosterols Steroids, Antibacterial activity: Gram-positive and Eupatorium Vent. L PE Gupta et al., 2002 coumarins, tannins Gram-negative bacteria and saponins Eupatorium ballotifolium Kunth AP EO Terpenoids Antifungal activity: filamentous fungi Sobrinho et al., 2016 Eupatorium buniifolium Hook. AP ME - Antifungal activity: filamentous fungi MuschietTI et al., 2005 ex Hook. & Arn. Antibacterial activity: Gram-positive and Freerksen and Bönicke, Eupatorium cannabinum L. AP OsE - Gram-negative bacteria 1951; Senatore et al., 2001 Eupatorium capillifolium Senatore et al., 2001; AP EO Terpenoids Antifungal activity: filamentous fungi (Lam.) Small. Tabanca et al., 2010 Eupatorium fortunei Turcz. AP AqE - Antibacterial activity: Gram-negative bacteria Li et al., 2010 HE, ME Antibacterial activity: Gram-positive and Lopez et al., 2001; El-Seedi Eupatorium glutinosum Lam. AP Terpenoids Gram-negative bacteria et al., 2002 Eupatorium havanense Kunth AP DE - Antibacterial activity: Gram-positive bacteria Cantrell et al., 1998 Antibacterial activity: Gram-positive and Eupatorium intermedium F EO Terpenoids Czaikoski et al.. 2015 Gram-negative bactéria Pérez and Anesini, 1994; Euparorium inulaefolium Antibacterial activity: Gram-positive and L AqE, EE - Sanabria-Galindo et al., Kunth. Gram-negative bacteria 1998; Álvarez et al., 2005 Eupatorium japonicum Thunb. L, F EA Terpenoids Antifungal activity: filamentous fungi Inouye et al., 2006 Terpenoids and Eupatorium laciniatum Kitam. L, F EA Antifungal activity: filamentous fungi Inouye et al., 2006 coumarin Sobrinho et al. 47

Table 1. Cont’d

Alkaloids, steroids, Eupatorium laevigatum Lam. HE, EE, Antibacterial activity: Gram-positive and Schmidt et al., 2009; Fabri et al., L phenols, tannins and ME Gram-negative bacteria 2011 flavonoids Eupatorium Antibacterial activity: Gram-positive and AP EE, ME, EA - Ji et al., 2008 lindleyanum DC Gram-negative bacteria Bishop and Macdonald, 1951; Eupatorium maculatum L. AP, S EE - Antibacterial activity: Gram-positive bacteria Borchardt et al., 2008 Cáceres et al., 1994; Taylor et al., 1996; Suksamrarn et al., 2004; Antibacterial activity: Gram-positive and Eupatorium odoratum L. Chomnawang et al., 2005; Molina- L, AP, F EE, ME Polyphenols Gram-negative bacteria Salinas et al., 2007; Gautam et al.,

Antifungal activity: filamentous fungi 2007; Chomnawang et al., 2009; Sharma et al., 2013; Patil and Kamble, 2015 Eupatorium patens D. Don ex L EO Terpenoids Antibacterial activity: Gram-positive bacteria Bailac et al., 2000 Hook. & Arn. Antibacterial activity: Gram-positive and Carlson et al., 1948; Bishop and Macdonald, 1951; Madsen and Eupatorium perfoliatum L. L, F EE, PE - Gram-negative bacteria Pates, 1952; Liegey, 1953; Frisby Antifungal activity: yeast et al., 1953; Borchardt et al., 2008 Antibacterial activity: Gram-positive and Carlson et al., 1948; Pates and Eupatorium purpureum L. R, St, L AqE, EE - Gram-negative bacteria Madsen, 1955 Antibacterial activity: Gram-positive and Eupatorium rugosum Houtt. L, S AqE - Frisby et al., 1954 Gram-negative bacteria Eupatorium salvia Colla AP RE Resinous exudate Antibacterial activity: Gram-positive bacteria Urzua et al., 1998 Eupatorium serratum Spreng. Tannins, flavonoids, L ME, EA steroids and Antibacterial activity: Gram-positive bacteria Desoti et al., 2011 alkaloids Eupatorium tashiroi Hayata. AP GE - Antibacterial activity: Gram-positive bacteria Chen et al., 1989 Antibacterial activity: Gram-positive and Rahman and Junaid, 2008; Begum Eupatorium triplinerve Vahl St, L, AP EO, CE Terpenoids Gram-negative bacteria et al., 2010; Unnikrishnan et al., Antifungal activity: filamentous fungi 2014; Sugumar et al., 2015 Eupatorium urticaefolium Antibacterial activity: Gram-positive and AP AE - Carlson et al., 1948 Reichard Gram-negative bacteria

I- inflorescence; L- leaves; R- root; AP- aerial part; F- flower; St- stems; S- seed; ME- methanol extract; CE- chloroform extract; HE- hexane extract; EE- ethanol extract; PE- petroleum ether extract; DE- dichloromethane extract; EA- ethyl acetate extract; AqE- aqueous extract; AE- acid extract; GE- gross extract; OsE- organic solvents extract; RE- resinous exudate; EO- essential oil.

al., 2010). solvents were tested against 15 bacterial strains showed antibacterial activity against Salmonella

Extracts from fresh leaves using organic by the agar diffusion method. The organic extracts paratyphi, Salmonella spp., Staphylococcus aureus, 48 J. Med. Plants Res.

Staphylococcus spp., Bacillus cereus, Bacillus subtilis, colds and topically in plasters for bone fractures and Bacillus thuringiensis, Enterobacter aerogenes, Proteus dislocations by healers from the bolivian altiplano mirabilis and Proteus spp. while the aqueous extract (Giraoult, 1987), in addition to having anti-inflammatory showed activity against P. aeruginosa, E. coli, S. aureus, and analgesic properties (Muschietti et al., 2005). Staphylococcus spp., Citrobacter freundii, Proteus spp., A methanol extract of the aerial parts showed B. subtilis, E. aerogenes, Salmonella spp., S. paratyphi antifungal activity against the dermatophytes Microsporum and B. thuringiensis (Bhattarai and Shrestha, 2009). gypseum, Trichophyton rubrum and Trichophyton Cadinene derivatives extracted from leaves of E. mentagrophytes according to broth microdilution tests adenophorum showed antifungal activity against four (Muschietti et al., 2005). plant pathogenic fungi strains: Rhizoctonia solani, Sclerotium rolfsii, Fusarium oxysporum and Macrophomina phaseolina. Cadinene sesquiterpenes Eupatorium aromaticum L. were isolated by column chromatography and preparative thin layer chromatography. Less polar cadinene E. aromaticum [syn. Ageratina aromatica (L.) Spach.] is a sesquiterpenes were active against S. rolfsii and polar perennial herb, medium-sized, native to America, cadinene derivatives with mono or dihydroxy groups were common in the southeastern United States, known as more inhibitory towards R. solani (Kundu et al., 2013). “Small-leaved white snakeroot” or “Lesser snakeroot” Ahluwalia et al. (2014) analyzed the antifungal action of (Blackwell and Mcmillan, 2013). The ethyl extract of the the essential oil of E. adenophorum against five leaves of E. aromaticum showed antibacterial activity pathogenic fungi: Sclerotium rolfsii, Macrophomina against Gram-positive bacteria (Pates and Madsen, phaseolina, Rhizoctonia solani, Pythium debaryanum and 1955). Fusarium oxysporum. They found the strongest inhibitory activity against S. rolfsii. Regarding toxicity, they evaluated the phytotoxic action of the essential oil, which Eupatorium aschenbornianum S. Schauer. inhibited the growth and seed germination of Phalaris minor Retz. and Triticum aestivum L., used for E. aschenbornianum [syn. Ageratina pichinchensis phytotoxicity assays in a dose-dependent concentration. (Kunth) R.M. King & H. Rob.] is a plant traditionally used In another study, the essential oil from aerial parts in Mexico to treat skin lesions, mouth ulcers and tumors exhibited moderated antifungal activity against Fusarium (Rios et al., 2003). Studies of hexane and methanol oxysporum by disk diffusion method (Chauhan et al., extracts obtained from the aerial parts point to the 2015). Twenty-two components were identified in the antifungal potential of E. aschenbornianum against the composition with an abundance of monoterpenes p- dermatophytes, T. mentagrophytes, T. rubrum, the yeast cymene (14.56%) followed by phellandrene (12.25%), C. albicans and the filamentous fungus Aspergillus niger camphene (10.42%), bornyl acetate (9.76%) and δ-2- (Rios et al., 2003; García et al., 2003). carene (5.39%). Yeasts belonging to the genus Candida are involved in the etiology of candidiasis (Netea et al., 2015). Studies on the species E. aschenbornianum showed activity Eupatorium altissimum L. against C. albicans, using hexane and methanol extracts of the aerial parts (Figure 1). Benzofurans compounds (5- In a study on the species collected in the state of acetyl-3β-angeloyloxy-2β-(1-hydroxyisopropyl)-2,3- Louisiana in the United States, a dichloromethane extract dihydrobenzofurane and 5-acetyl-3β-angeloyloxy-2β-(1- was tested against Mycobacterium tuberculosis hydroxyisopropyl)-6-methoxy-2,3-dihy-drobenzofurane) bacterium, causative agent of most cases of tuberculosis and phytosterols were isolated (Rios et al., 2003; García (Cantrell et al., 1998). In a bioprospecting study of 550 et al., 2003). plants collected in the states of Ohio and Oregon in the United States, action was reported on the extract of aerial parts of E. altissimum against strains of S. aureus and E. Eupatorium ayapana Vent. coli (Carlson et al., 1948). The leaves of E. ayapana [syn. (Vahl) R.M. King & H. Rob.] are used in folk medicine as Eupatorium arnottianum Griseb. a heart stimulant, laxative and anticoagulant (Gupta et al., 2002). In rainforests of South America, its leaves are E. arnottianum [syn. Chromolaena arnottiana (Griseb.) used in decoctions, infusions, tea and baths as tonic, R.M. King & H. Rob.] is an herb native to Latin America, stimulant, sedative, febrifuge and anti-inflammatory, common in the North-East and Centre of Argentina and especially in Brazilian folk medicine (Melo et al., 2013). South of Bolivia, traditionally used to treat stomach The first preliminary studies of this species were ailments (Clavin et al., 2007), against asthma, bronchitis, performed by Oswaldo Gonçalves de Lima, then director Sobrinho et al. 49

Figure 1. Structures of two benzofurans compounds from aerial parts of Eupatorium aschenbornianum. A. 5-acetyl-3β- angeloyloxy-2β-(1-hydroxyisopropyl)-2,3-dihydrobenzofurane. B. 5-acetyl-3β-angeloyloxy-2β-(1-hydroxyisopropyl)-6- methoxy-2,3-dihy-drobenzofurane.

of the Antibiotics Institute of the University at Recife, combination of ketoconazole with essential oil reduced Brazil (Lima, 1963). the MICs for both strains of T. rubrum indicating a The phytochemical study of petroleum ether extract synergistic effect (Sobrinho et al., 2016). showed presence of steroids, coumarins, tannins and saponins. For the species E. ayapana, the petroleum ether extract inhibited growth of bacteria B. subtilis, Eupatorium buniifolium Hook. ex Hook. & Arn. Staphylococcus epidermidis, S. aureus, M. luteus, E. coli, P. aeruginosa, Salmonella typhi, Shigella sp., Vibrio E. buniifolium [syn. Acanthostyles buniifolius (Hook. ex cholerae and Vibrio parahaemolyticus. The extract also Hook. & Arn.) R.M. King & H. Rob.] is a shrubby species inhibited the growth of A. niger, A. flavus, Alternaria widely used in folk medicine, such as to treat rheumatic solani and Fusarium solani, pathogenic fungi causing pains and liver problems with tea obtained by decoction. black mold in fruits and vegetables, deterioration by Preparations from this plant are also used as mycotoxins in grains, alternaria spot in solanaceous disinfectants (Ríos et al., 1993). plants and root and stem rot, respectively (Gupta et al., The methanol extract obtained from the aerial parts 2002). was used to investigate antifungal activity against strains of the dermatophytes, M. gypseum (MIC = 250 µg/mL), T. rubrum (MIC = 100 µg/mL) and T. mentagrophytes Eupatorium ballotifolium Kunth (MIC = 250 µg/mL). The assays were performed by the broth microdilution method (Muschietti et al., 2005). Eupatorium ballotifolium Kunth (syn. Lourteigia ballotaefolia (Kunth) R.M. King & H. Rob.) popularly known in Brazil by many names, such as “maria-preta”, Eupatorium cannabinum L. “maria-preta-verdadeira” and “picão-roxo” is a perennial herbaceous species whose vertical growth ranges from E. cannabinum is a perennial herbaceous species 40 to 80 cm (Cardoso et al., 2013). distributed in the northern hemisphere, especially in The essential oil extracted from the aerial parts, was Europe and North America (Senatore et al., 2001). A tested by broth microdilution method and showed study of the essential oil of E. cannabinum subsp. antifungal activity against strains fungal dermatophytes, cannabinum L. obtained from the aerial parts identified Trichophyton rubrum. The chromatographic analysis high percentage of terpenoids, such as germacrene, showed 25 components, accounting for 93.84%, among monocyclic sesquiterpene, found in large amounts. The them mono- and sesquiterpenes, with prevalence of oil showed antibacterial activity against strains of Gram- sesquiterpenes (Sobrinho et al., 2016). positive bacteria (Senatore et al., 2001). The modulatory activity assay was performed to In another investigation, the extract of the aerial parts determine the combined effect of the essential oil with the showed antimicrobial activity against strains of Gram- ketoconazole, standard antifungal drug, against strains of positive and negative bacteria, the main ones tested T. rubrum by the checkerboard technique. The being S. typhi, Micrococcus aureus, B. subtilis and 50 J. Med. Plants Res.

Figure 2. Structures of two isolated compounds from leaves of E. glutinosum. A. 15-hydroxy-7-labden-17-oic acid: R = H; B. 15-acetoxy-7-labden-17-oic acid: R = Ac.

Proteus spp. (Freerksen and Bönicke, 1951). (Lam.) R.M. King & H. Rob.] is widely used in folk medicine, with preparations obtained by decoction used as antimicrobial, antirheumatic and antidiarrheal agents Eupatorium capillifolium (Lam.) Small. ex Porter & and to heal stomach ulcers (El-Seedi et al., 2002). Britton The methanol extract of leaves of E. glutinosum presented antibacterial activity against strains of E. capillifolium is a perennial herbaceous species found Mycobacterium phlei, B. subtilis and S. aureus, by the in North America. It is used as food for livestock in the disk diffusion method (Lopez et al., 2001). The southeastern United States when other fodder is lacking, antibacterial activity of the hexane extract of leaves and but it is generally considered a weed (Sellers et al., stems and of two isolated compounds (15-hydroxy-7- 2009). labden-17-oic acid and 15-acetoxy-7-labden-17-oic acid) Studies of the effect of plant extracts against were investigated by diffusion in agar (Figure 2). The pathogenic fungi are important to find new agents against tests showed activity against S. aureus, B. cereus, E. coli fungi causing plant diseases in agricultural crops. and P. aeruginosa (El-Seedi et al., 2002). Tabanca et al. (2010) investigated, by contact bioautography, the antifungal activity of the essential oil of E. capillifolium, which inhibited growth of the fungus Eupatorium havanense Kunth Colletotrichum acutatum, the cause of strawberry anthracnose. The main constituents were thymol methyl E. havanense [syn. Ageratina havanensis (Kunth) R.M. ether (36.3%), 2,5-dimethoxy-p-cymene (20.8%) and King & H. Rob.] is a native shrub found in North America myrcene (15.7%). (Zamudio and Villanueva, 2011) and Central America (Córdoba et al., 1995) known as “Havana snakeroot”. Previous study identified the flavone sakuranetin Eupatorium fortunei Turcz. (Wollenweber and Dietz, 1981) and flavonols, flavones and flavonoid glucosides (Yu et al., 1987). E. fortunei is a traditional Chinese medicinal herb, In an antimicrobial prospecting study of plants of the common in the northeastern of China, used in folk Americas, the dichloromethane extract of leaves of E. medicine to treat many diseases (Liu et al., 1992). In a havanense, collected in the state of Tamaulipas, Mexico, study of bacteria that produce volatile sulfur compounds inhibited the growth of the pathogenic microorganisms M. (VSCs) that cause halitosis, Li et al. (2010) investigated tuberculosis and M. avium, the latter an opportunistic extracts of 40 plants found in China, of which 14 inhibited pathogen related to infections in immunocompromised bacterial growth and production of VSCs. The main individuals (Cantrell et al., 1998). compounds found were hydrogen sulfide, dimethyl sulfide and methylmercaptan. Among the 14 plants that were active in the screening was E. fortunei, which was used Eupatorium intermedium DC. to obtain the aqueous extract. E. intermedium [syn. Grazielia intermedia (DC.) R.M. King and H. Rob.] is a branchy shrub, native to southern Eupatorium glutinosum Lam. Brazil with upright growth between 1.0 to 1.5 m high and it is densely leafy until close to the inflorescences, which This species E. glutinosum [syn. glutinosa are composed of white flowers (Czaikoski et al., 2015). Sobrinho et al. 51

The extracts obtained by supercritical extraction from Eupatorium laciniatum Kitam. the flowers with scCO2, propane and petroleum ether as solvents were tested and showed antibacterial activity E. laciniatum [syn. L.] an annual against two Gram-positive bacterial strains, S. aureus herbal plant, native to Asia is distributed in the south of and L. monocytogenes, and two Gram-negative bacterial China, used in Chinese folk medicine for the treatment of strains, E. coli and S. typhimurium. The assays were cold, snakebite and inflammation (Yang et al., 2005). performed using the disk diffusion method (Czaikoski et The antidermatophytic activity of this plant species was al., 2015). The extracts were effective against all bacteria tested against the fungus, T. mentagrophytes, using the strains, particularly the extract obtained with scCO2 and ethyl acetate extract of the leaves and flowers at a compressed propane. concentration of 0.25 mg/mL. The extract caused an The essential oil was tested against Gram-negative inhibition zone of 12 mm (Inouye et al., 2006). bacterial strains using the agar well method. The main constituents of essential oil were α-pinene, sabinene, β- Eupatorium laevigatum Lam. pinene, limonene, (E)-caryophyllene, germacrene D, spathulenol and caryophyllene oxide (Czaikoski et al., E. laevigatum [syn. Chromolaena laevigata (Lam.) R.M. 2016). King & H. Rob.], native to South America is widely distributed in the South part of Brazil and Argentina, whose common name in Brazil is “erva-de-santa-maria” Eupatorium inulaefolium Kunth. or “camara”, is used in folk medicine for wound healing and as an antifungal (Maia et al., 2002). In Argentina, it is E. inulaefolium [syn. Austroeupatorium inulaefolium known as “caá-hú” or “doctorcito” and used in folk (Kunth) R.M. King & H. Rob.] is a species found in Latin medicine as an anticefalalgic, analgesic, cathartic, America, whose common name in Argentina is emenagogue and purgative agent (Clavin et al., 2000). “sanalotodo” or “yerba de Santa María”. This species is The ethanol and hexane extracts of the leaves were used in folk medicine for treatment of skin infections due tested against B. subtilis, by the agar diffusion method, to its antimicrobial properties (Lancelle et al., 2009) and with moderate results for inhibition of bacterial growth by throat diseases (Álvarez et al., 2005). both solvents (Schmidt et al., 2009). In a chemical and In a study of 122 plant species from 54 families, Pérez antimicrobial prospecting study, the methanol extract of and Anesini (1994) investigated the antibacterial activity the leaves of E. laevigatum showed the presence of against the microorganism S. typhi, causative agent of alkaloids, steroids, phenols, tannins and flavonoids. The typhoid fever. Of the plants tested in the initial screening, antimicrobial activity was performed by the broth E. inulaefolium showed inhibitory activity against the microdilution method against strains of Gram-positive and growth of S. typhi. In an investigation of the in vitro negative bacteria, showing inhibitory activity against P. antimicrobial activity of Colombian angiosperms, the aeruginosa, B. cereus, Shigella sonnei and Salmonella inhibitory activity against the microorganisms S. aureus enterica sorovar typhimurium (Fabri et al., 2011). and B. subtilis, was investigated at a concentration of 3 mg/mL, by the agar diffusion method (Sanabria-Galindo et al., 1998). In a third study, the ethyl extract of the Eupatorium lindleyanum DC. leaves of E. inulaefolium were tested against the strains of S. aureus, E. coli and P. aeruginosa. The authors only E. lindleyanum is a perennial herbaceous plant, native to observed growth inhibition against S. aureus, at a China, known as “Yemazhui” and used in folk medicine to concentration of 50 mg/mL (Álvarez et al., 2005). relieve fever, remove toxic substances, treat coughs, promote urination, and lower blood pressure (Xia et al., 2004). In traditional Chinese medicine, its aerial part is Eupatorium japonicum Thunb. used as an antipyretic and detoxicant (Yang et al., 2010). The extracts of the aerial parts obtained by use of E. japonicum [syn. Eupatorium chinense L.] is a perennial ethanol, methanol and ethyl acetate were tested by the herb, native to Japan, Korea and northeastern area of broth dilution method against eight bacterial strains: four China. It is a medicinal herb used in folk medicine as a Gram-positive (B. subtilis, S. aureus, B. cereus and E. decoction for the treatment of measles, rheumatic bone faecium) and four Gram-negative (E. coli, S. typhimurium, pains, colds and cough (Roeder, 2000). P. vulgaris and Pseudomonas fluorescens). The extracts The ethyl acetate extract of the leaves and flowers of E. inhibited growth of all strains tested, with the best results japonicum was used to investigate the antifungal activity for B. subtilis, S. aureus and B. cereus (Ji et al., 2008). against the dermatophyte T. mentagrophytes, the most common cause of Tinea pedis. At the concentration of Eupatorium maculatum L. 0.25 mg/mL, the authors observed an inhibition zone of 11 mm (Inouye et al., 2006). Native to United States, E. maculatum known as “Joe- 52 J. Med. Plants Res.

pye weed” is widely found in moist places, especially in both Gram-positive and negative bacterial strains calcareous soils (Goebel et al., 2012). The first report of (Sharma et al., 2013). The major components analyzed antimicrobial activity of E. maculatum dates back to the by gas chromatography-mass spectrometry (GC-MS) work of Bishop and MacDonald (1951), who reported that were geijerene (25.10%), germacrene D (20.27%), trans- the ethanol extract of the aerial parts showed activity β-caryophyllene (10.04%), α-pinene (9.64%) and β- against strains of Gram-positive bacteria. pinene, (4.85%). In prospective study with 336 native species of the In a screening study, the aqueous leaf extract of E. states of Minnesota and Wisconsin in the United States, odoratum was tested for their fungitoxicity against spores ethanol extracts of leaves and stems of E. maculatum of Uromyces vignae and showed the better result for the were tested against strains of S. aureus, E. coli, P. inhibition of spore germination (Patil and Kamble, 2015). aeruginosa and C. albicans, presenting significant action only against S. aureus. The assays were performed using the disk diffusion method (Borchardt et al., 2008). Eupatorium patens D. Don ex Hook. & Arn.

E. patens [syn. Austrobrickellia patens (D.Don ex Hook. Eupatorium odoratum L. & Arn.) R.M. King & H. Rob.] is a densely branched shrub that is distributed from Bolivia and Paraguay to Patagonia E. odoratum [syn. Chromolaena odorata (L.) R.M. King & in Argentina, known by the common names of “acancio” H. Rob.] is a free standing shrub which is mostly or “bejuco” (Cabrera and Correa, 1971). distributes in America, tropical Asia and West Africa. It is The essential oil of the leaves was tested against the widely used in traditional medicine as immunemodulator, microorganisms S. aureus, E. coli, B. subtillis, C. albicans antispasmodic, hepatoprotective, antiprotozoal, and A. niger by the agar diffusion method. Only the antidiabetic, antihypertensive, anti-inflammatory strains of B. subtilis showed significant growth inhibition (Umukoro and Ashorobi, 2006). E. odoratum also can be zones after incubation for 24 h (Bailac et al., 2000). used in treatment of skin diseases, dysentery, malaria fever, tooth ache and diabetes (Omoregie et al., 2014). In Mexican folk medicine, it is used for chest complaints and Eupatorium perfoliatum L. pulmonary affections, known as “crucita” (Molina-Salinas et al., 2007). Native to North America, E. perfoliatum is a medicinal Various studies of E. odoratum have investigated the herb, known as “boneset” or “thoroughwort”, used in folk plant’s antimicrobial activity against bacteria that cause medicine by the native inhabitants for the treatment of various infectious diseases. Cáceres et al. (1995) fever and flu (Maas et al., 2008). The first record in the observed activity against the bacterium Neisseria literature on the antimicrobial potential of E. perfoliatum gonorrhoeae, which causes gonorrhea, with an excellent was performed by Carlson et al. (1948). The authors growth inhibition zone. found that the ether extract obtained from the aerial parts Four flavanones extracted from flowers of E. odoratum inhibited the growth of S. aureus. Later studies exhibited antibacterial activity against Mycobacterium investigated extracts from leaves and stems using tuberculosis. Flavanones were isolated by silica gel organic solvents (ethanol, ether and acetone) and water, chromatography and analyzed with infrared spectroscopy demonstrating antimicrobial activity against C. albicans, and nuclear magnetic resonance (Suksamrarn et al., Gram-positive and Gram-negative bacteria and 2004). Chomnawang et al. (2005) tested the antiacne Mycobacterium spp. (Bishop and MacDonald, 1951; activity (crude extracts with chloroform and ethylacetate) Madsen and Pates, 1952; Liegey, 1953; Frisby et al., against strains of Propionibacterium acnes, S. 1953). epidermidis, while Molina-Salinas et al. (2007) In prospective study, the ethanol extract of the leaves investigated the antibacterial activity (methanol extract; and flowers of E. perfoliatum was tested against strains leaves, flowers and roots) against Streptococcus of S. aureus, E. coli, P. aeruginosa and C. albicans. The pneumonia and Chomnawang et al. (2009) tested it extract only had significant action against S. aureus, with against methicillin-resistant S. aureus. inhibition zones of 11 and 7 mm for extracts of the leaves Methanol extract of the aerial parts of E. odoratum and inflorescences, respectively. The assays were inhibited the growth of M. phlei by disk diffusion method, performed using the disk diffusion method (Borchardt et with inhibitory activity at concentrations of 300 mg/mL al., 2008). (Gautam et al., 2007). In another study using the aerial parts, the methanol extract inhibited microbial growth of B. subtilis, methicillin-resistant S. aureus, methicillin- Eupatorium purpureum L. sensitive S. aureus, T. mentagrophytes and M. phlei, in the last case only when exposed to UV radiation (Taylor E. purpureum is a wildflower perennial plant, native to the et al., 1996). The essential oil from the dried leaves of E. eastern United States, known as “joe-pye weed”, “sweet odoratum showed in vitro antibacterial activity against joe-pye weed”, and “sweetscented joe-pye weed” Sobrinho et al. 53

(Sabanadzovic et al., 2010). It is used in traditional used in a folk medicine for treating edema and medicine for rheumatoid arthritis and several other hemoptysis in Taiwan (Wu et al., 1985). The aqueous disease conditions (Habtemariam, 1998). extract of the entire plant was tested by the agar diffusion The saline extract of the stems of E. purpureum L. method against two serotypes of S. mutans, bacteria showed antibacterial activity against S. aureus and E. associated with the development of dental caries. The coli, by the agar diffusion method (Carlson et al., 1948). extracts inhibited bacterial growth of both serotypes The saline extract of the leaves was also investigated (Chen et al., 1989). against S. aureus and E. coli, with positive results by the agar diffusion method (Pates and Madsen, 1955). Eupatorium triplinerve Vahl

Eupatorium rugosum Houtt. E. triplinerve [syn. Ayapana triplinervis (Vahl) R.M. King & H. Rob.] commonly found in Asia is an erect perennial E. rugosum [syn. Ageratina altissima (L.) R.M. King & H. herb with narrow lanceolate leaves and large number of Rob.] is a perennial herb commonly found in the pedicelled flower-heads at the top of the branch, known midwestern and eastern United States, known as “white as “ayapana” (Selvamangai and Bhaskar, 2012). The snakeroot” (Lee et al., 2012). The saline extract of the species was introduced in Indian as ornamental species. stems of E. rugosum showed antibacterial activity against In Indian, folk medicine is used as a stimulant, tonic, strains of Gram-positive bacteria by the agar diffusion laxative and for the treatment of yellow fever method (Frisby et al., 1954). (Unnikrishnan et al., 2014). The antimicrobial activities of the essential oil obtained from the leaves and the stem and thymohydroquinone Eupatorium salvia Colla dimethylether were evaluated using disk diffusion assay against strains of Gram-positive and negative bacteria, E. salvia [syn. Aristeguietia salvia (Colla) R.M. King & H. showing inhibitory activity against P. aeruginosa, S. Rob.] is a species used in folk Chilean medicine, known aureus, K. pneumoniae and E. coli. The essential oil as “sálvia macho”, “pegajosa” or “pega-pega” (Hoffmann, showed antifungal activity against fungal strains of 1995). The resinous exudate, 7-hydroxy-8(17)-labden-15- Penicillium chrysogenum and C. albicans; however oic acid (salvic acid) and 7-acetoxy-8(17)-labden-15-oic thymohydroquinone dimethylether only inhibited the acid (salvic acid acetate), isolated from E. salvia, were growth of P. chrysogenum (Unnikrishnan et al., 2014). tested against five bacterial strains, with activity against The chloroform extract of leaves exhibited antibacterial all tested microorganisms: S. aureus, B. cereus, B. activity by the broth microdilution and disk diffusion subtilis, M. luteus and Clavibacter michiganensis subsp. methods against strains of Gram-positive and negative Michiganensis. These diterpenoids do not affect Gram- bacteria Bacillus megaterium, B. subtilis, B. cereus, S. negative bacteria and the activity of these compounds on aureus, E. coli, S. dysenteriae, S. sonnei, S. typhi, S. filamentous fungi has not been reported (Urzua et al., paratyphi and P. aeruginosa, that exhibited the largest 1998). zone of inhibition against Vibrio (22 mm in diameter with 1000 µg/disc extract). The antifungal activity was performed with filamentous fungi Alternaria alternata, Eupatorium serratum Spreng. Curvularia lunata, Colletotrichum corchori, Fusarium equiseti, Macrophomina phaseolina and Botryodiplodia E. serratum [syn. Grazielia serrata (Spreng.) R.M. King & theobromae, with highest inhibition of fungal radial H. Rob.], popularly known as “erva-milagrosa” or mycelial growth against C. corchori, 73.5% with 100 µg (“miraculous herb”) in Brazil, is used in folk medicine to extract/ml medium (Rahman and Junaid, 2008). treat snake bites, as an analgesic, healing agent, In others studies, the essential oil from aerial parts and antimicrobial agent and for the treatment of stomach fresh leaves exhibited moderate antibacterial activity problems, liver disorders, diabetes, cancer and bronchial against B. megaterium, B. subtilis, B. cereus, S. aureus, asthma (Desoti et al., 2011). E. coli, V. cholerae, S. dysenteriae, S. sonnei, S. typhi, The hexane, methanol and ethyl acetate extracts Pseudomonas sp. and S. paratyphi. The essential oil obtained from fresh leaves of E. serratum were tested demonstrated strong antifungal activities against A. against S. aureus, M. luteus, E. coli and S. typhi. The alternata, C. lunata, B. theobromae, C. corchori, F. best inhibitory halo results were produced by the equiseti and M. phaseolina (Begum et al., 2010; Sugumar methanol extract against M. luteus (Desoti et al., 2011). et al., 2015).

Eupatorium tashiroi Hayata Eupatorium urticaefolium Reichard

E. tashiroi, native to Asia is a wild herb which has been E. urticaefolium [syn. Ageratina altissima (L.) R.M. King & 54 J. Med. Plants Res.

H. Rob.] is a species native to North America, it is their antimicrobial, antioxidant and phytotoxic properties. J. Pest. Sci. commonly found throughout the eastern half of continent. 87:341-349. Albuquerque MRJR, Silveira ER, Uchôa DEA, Lemos TLG, Souza EB, Ingestion of the plant causes a disease called trembles in Santiago GMP, Pessoa ODL (2004). Chemical Composition and livestock (Lee et al., 2010). The ethyl acetate extract of Larvicidal Activity of the Essential Oils from Eupatorium the aerial parts of E. urticaefolium inhibited the growth of betonicaeforme (DC.) Baker (Asteraceae). J. Agric. Food Chem. S. aureus and E. coli, by the agar diffusion method 52:6708-6711. Albuquerque MRJR, Santos HS, Souza EB, Silva RM, Pessoa ODL, (Carlson et al., 1948). Braz-Filho R, Costa SMO (2010). Composição química volátil e não- volátil de Eupatorium ballotifolium Kunth, Asteraceae. Rev. Bras. Farmacogn. 20:615-620. Conclusion Al-fatimi M, Wurster M, Schröder G, Lindequist U (2007). Antioxidant, antimicrobial and cytotoxic activities of selected medicinal plants from Yemen. J. Ethnopharmacol. 111:657-666. Thirty Eupatorium species were identified, some already Álvarez M, Isaza G, Echeverry HM (2005). Efecto antibacteriano in vitro reclassified to other genera. The aerial parts are the part de Austroeupatorium inulaefolium HBK (Salvia amarga) y Ludwigia of plant with more studies and so far the best source for polygonoides HBK (Clavo de laguna). Biosalud, 14:46-55. Alviano DS, Alviano CS (2009). Plant extracts: search for new anti-microbial product, being the essential oils and fixed alternatives to treat microbial diseases. Curr. Pharm. Biotechnol. compounds the main natural products. 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Cardoso AD, Viana AES, Barbosa RP, Teixeira PRG, Júnior NSC, Fogaça JJNL (2013). Levantamento fitossociológico de plantas daninhas na cultura da mandioca em Vitória da Conquista, Bahia. Conflict of interest Biosci. J. 29:1130-1140. Carlson HJ, Douglas HG, Robertson J (1948). Antibacterial substances The authors declare that there is no conflict of interest. separated from plants. J. Bacteriol. 5:241-248. Carrillo-Hormaza L, Mora C, Alvarez R, Alzate F, Osorio E (2015). Chemical composition and antibacterial activity against Enterobacter ACKNOWLEDGEMENTS cloacae of essential oils from Asteraceae species growing in the Páramos of Colombia. Ind. Crops Prod. 77:108-115.

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Vol. 11(3), pp. 58-65, 17 January, 2017 DOI: 10.5897/JMPR2016.6300 Article Number: 03CF87362400 ISSN 1996-0875 Journal of Medicinal Plants Research Copyright © 2017 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR

Full Length Research Paper

Hypoglycaemic effect of fractions and crude methanolic leaf extract of Phyllanthus fraternus in streptozotocin - induced diabetic and normal rats

Nadro, M. S.1* and Elkanah, G.2

1Department of Biochemistry,Gombe State University, Gombe, Nigeria. 2Department of Biochemistry, Modibbo Adama University of Technology, PMB 2076, Yola, Nigeria.

Received 14 November, 2016; Accepted 15 December, 2016

Treatments of diabetes with available agents come with one or more side effects, hence, the need for continual search of alternative treatment agents from medicinal plants. This study was designed to analyse qualitatively and quantitatively some phytochemicals in methanolic extract in Phyllanthus fraternus and evaluate their hypoglycaemic activity in both diabetic and normal rats. Sixty-six rats were used of which forty-two were diabetic. Diabetes was induced by intraperitoneal administration of 60 mg/kg body weight of streptozotocin (STZ). Thirty male rats of which twenty-four were diabetic were divided into five (5) groups of six rats each were used for prolonged treatment: Normal, diabetic control, standard control, and two treatments that were orally administered at a dose of 200 and 300 mg/kg body weight of crude methanolic leaf extracts of P. fraternus for 28 days. Thirty six (36) rats were used for oral glucose tolerance test (OGTT) which was divided into six groups of three rats each for both normal and diabetic rat. A single dose of 200 mg/kg body weight of crude and fractions (I, II and III) of methanolic leaf extracts of P. fraternus were orally administered to diabetic and normal rats before they were loaded with 2 g/kg body weight glucose. The results of phytochemical screening of the crude extract showed the presence of compounds like alkaloids, flavonoids, terpenoids, phenols and saponins. Fraction I contained only flavonoid, fraction II and III contained more than three phytochemicals. Oral administration of 200 and 300 mg/kg body weight of methanolic extracts to diabetic rats significantly reduced (p < 0.05) serum glucose levels in all the treatment groups. The results of OGGT showed that fraction I and metformin groups significantly (p<0.05) lowered blood glucose level 30 min after glucose load in both diabetic and normal rats when compared with their controls and other treatments groups. These results suggest P. fraternus methanolic leaf extract have phytochemicals with glucose lowering ability especially fraction I that competes favorably with metformin.

Key words: Streptozotocin, diabetes, Phyllanthus fraternus, phytochemicals, oral glucose tolerance test, (OGTT).

INTRODUCTION

Diabetes mellitus is described by world health levels arises either because insulin production is organization (WHO) as a group of metabolic diseases in insufficient, or because the body’s cell do not respond which there are high blood sugar levels over a prolonged properly to insulin, or both. Patients with high blood sugar period (WHO, 2014). This prolonged high blood sugar will typically experience frequent urination (polyuria), Nadro and Elkanah 59

increasingly thirsty (polydipsia) and distinctly hungry and to evaluate the hypoglycaemic efficacy of fractions of (polyphagia) (Hayat et al., 2010). methanolic leaf extract of P. fraternus on streptozotocin – Diabetes can cause many complications if left induced diabetic rats. untreated (WHO, 2013). Acute complications include diabetic ketoacidosis and non ketotic hyperosmolar coma (Kitabchi et al., 2009). Serious long-term complications MATERIALS AND METHODS include cardiovascular disease, stroke, kidney failure, foot ulcers and damage to the eyes (WHO, 2013). Plant material

Management concentrates on keeping blood sugar levels The plant material of P. fraternus Webster (Leaves) was collected in as close to normal ("euglycemia") as possible, without the month of May, around 6 am at Hayin gada, in Girei local causing hypoglycemia. This can usually be accomplished Government area of Adamawa State which lies on geographical with a healthful diet, exercise, and use of appropriate location 9° 21’53.19’’North and 12° 33’28.33’’East Google earth medications (insulin in the case of type 1 diabetes; oral (2014). It was authenticated by a botanist in the Department of Biological Sciences, Modibbo Adama University of Technology, medications, as well as possibly insulin, in type 2 Yola, Adamawa State. diabetes).In spite of these advances and effort made towards treating, managing, and perhaps preventing the health, economic and social effects of diabetes mellitus, Experimental animals the prevalence of the disease globally is on the increase. As of 2014, an estimated 387 million people have Male albino rats (5 - 6 weeks) weighing 100 to 130 g numbering 66 were obtained from Veterinary Research Institute VOM, Jos, diabetes worldwide as contained in the report of Plateau State and kept in plastic cages with 12 h dark/light cycle, International Diabetes Federation (IDF, 2014), with type 2 fed with pelletized grower diet (Vital Feeds, UACN) and given water diabetes making up about 90% of the cases. This is equal ad libitum. 30 rats were used for prolonged treatment (28 days) to 8.3% of the adult population, with equal rates in both while 36 rats were used for OGTT (18 each for diabetic and normal women and men (Vos et al., 2013). In the years 2012 to rats). 2014, diabetes was estimated to have resulted in 1.5 to

4.9 million deaths per year respectively (WHO, 2013; IDF, Equipments 2014). The number of people with diabetes is expected to rise to 592 million by 2035 (IDF, 2014). The global The following equipments were used; electronic balance (Golden economic cost of diabetes in 2014 was estimated to be mettle-2G2-USA), ACCU-CHEK Glucometer (GC-Roche $612 billion USD (IDF, 2013). Hence, there is an urgent Diagnostic-Germany) , ACCU-CHEK test strips (Roche Diagnostic- Germany), spectrophotometer (Vis spectrophotometer 721- PEC need for new therapeutic drug with high efficacy, low cost, Medical USA), water bath (HH-2 B-Scientific), column (5cm little or no side effects and wider availability if this trend diameter), silica gel/TLC- cards (Fluka- Germany). must be reversed. Many plants have been studied in search for antidiabetic activity, some components isolated, but with respect to P. fraternus, there has been Chemicals little scientific record to support its anti diabetic activity Hexane, dichloromethane and methanol (Sigma-Aldrich Chemie and to some extent, its active components. GMbH, Germany), streptozotocin (Tocris Bioscience London), P. fraternus belongs to the family Phyllanthaceae. It has metformin, chloroform, silica gel (Sigma Aldrich-Germany). All other been used in folk medicine for the treatment of liver, chemicals used were of analytical grade. kidney and bladder problem, intestinal parasites and diabetes (The Wealth of India, 1995). Particularly P. fraternus herb is bitter in taste and reported to possess Preparation of plant material diuretic, hypotensive, hypoglycemic effect, The fresh plant material (leaf) was washed with tap water and antihyperlipemic, antihepatotoxic and anti oxidant activity shade dried for seven days. It was made into powder using mortar (Calixto et al., 1998). An aqueous extract of the leaves and pestle. The powdered plant material was used for the lowers blood sugar level in normal and alloxan diabetic preparation of methanolic extract. rabbits (Ramkrishnan et al., 1982). Different fractions of alcoholic extracts of aerial parts and root of P. fraternus were screened for antihepatotoxic activity on carbon Preparation of methanolic extract tetrachioride (CCL4) induced liver damage (Ahmed et al., Methanolic extract was prepared by suspending 200 g of 1998). The aim of this study is to analyse qualitatively thepowdered sample in 2 L of methanol for 24 h with vigorous and quantitatively some phytochemical components of shaken intermittently, after which it was filtered and then the P. fraternus methanolic leaf extract and its fractions concentrated at 55°C using a water bath (Ugwu et al., 2011).

*Corresponding author. E-mail: [email protected]. Tel: +2348053472090.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 60 J. Med. Plants Res.

Table 1. OGTT following single administration of crude and fractions of methanolic leaf extract of Phyllanthus fraternus in diabetic rats.

Groups Treatment Group-A (Diabetic control) No treatment Group-B (Standard control) Metformin 5 mg/kg body weight Group-C (Fraction I) Fraction I (200 mg/kg body weight) Group-D (Fraction II) Fraction II (200 mg/kg body weight) Group-E (Fraction III) Fraction III (200 mg/kg body weight) Group-F (Crude) Crude extract 200 mg/kg body weight

Table 2. OGTT following single administration of crude and fractions of methanolic leaf extract of Phyllanthus fraternus in normal rats.

Groups Treatments Group -A (Normal control) No treatment Group- B (Standard control) Metformin 5 mg/kg body weight Group-C (Fraction I) Fraction I (200 mg/kg body weight) Group-D (Fraction II) Fraction II (200 mg/kg body weight) Group-E (Fraction III) Fraction III (200 mg/kg body weight) Group-F (Crude) Crude Extract 200 mg/kg body weight

Fractionation of the extract by column chromatography Experimental design

Twenty gram (20 g) of the methanolic leaf extract was subjected to Evaluation of hypoglycaemic activity following long term treatment: column chromatography for the isolation of the phytoconstituents. 30 rats (6 normal and 24 diabetic) were divided into 5 groups of six Slurry was prepared by dissolving 200 g silica gel in 600 ml hexane rats each: (Sarah and Ayesha, 2003). The fractions were collected and labeled accordingly. Six Group 1: Normal control fractions were obtained, but on subjecting them to thin layer Group 2: Diabetic control chromatography (TLC), they were pulled together into three broad Group 3: Metformin 5 mg/kgb.w. fractions based on the number of components and retention factor Group 4: Diabetic rats treated orally with 200 mg/kg body weight of (RF) values of the components in each fraction. The resultant methanolic leaf extract of P. fraternus. fractions were concentrated by placing them in an oven at a Group 5: Diabetic rats treated orally with 300mg/kg body weight regulated temperature of 40°C. The dried fractions obtained were methanolic leaf extract of P. fraternus kept in air tight containers which were later used for OGTT. Estimation of fasting blood glucose level was done on a weekly base for four weeks using a glucometer. Qualitative phytochemical analysis The effect of crude and fractions of methanolic leaf extract of P. fraternus on oral glucose tolerance test (OGTT) in diabetic rats is The qualitative phytochemical screening of the extract was carried determined as shown in Table 1 out as described by Harborne (1973), Nweze et al. (2004) and The effect of crude and fractions of methanolic leaf extract of P. Senthilkumar and Reetha (2009). The plant extract was screened fraternus on oral glucose tolerance test (OGTT) in normal rats is for carbohydrates, alkaloids, flavonoids, steroids, phenols, tannins, determined as shown in Table 2. saponins, terpenoids, glycosides, and proteins. After 30 min of fractions, metformin and crude extract administrations, the rats in all groups were given glucose (2 g/kg body weight). Glucose of blood sample from tail vein was estimated by using glucometer at 0, 30, 60, 90, 120 and 150 min. Induction of diabetes mellitus in rats

All the rats were fasted overnight before the administration of Administration of extracts Streptozotocin. Diabetes was induced in rats by intra-peritoneal injection of streptozotocin dissolved in distilled water at a dose of 60 The extract was administered orally using gastric tube on daily mg/kg body weight (Al-Hariri et al., 2011). After the injection, the basis for 28 days (long term treatment) while in OGTT, it was a rats were allowed free access to food and water. To prevent fatal single administration. hypoglycemia due to massive pancreatic insulin release, rats were given 5% glucose solution water for next 24 h (Barry et al., 1997). The animals were tested after 72 h of streptozotocin administration. Statistical analysis The rats with fasting blood glucose more than 300 mg/dl were considered diabetic and were used for the experiment Values obtained were expressed as mean ± SEM and data were (Akbarazadeh et al., 2007, Parthasarthy and Ilavarasan, 2009). analysed using analysis of variance (ANOVA) with Bonferroni Post Nadro and Elkanah 61

Table 3. Some phytochemicals detected in fractions/crude methanolic leaf extract of Phyllanthus fraternus.

Phytochemical Crude Fraction I Fraction II Fraction III Saponins + - + -

Tannins + - + + Terpenoids + - + + Flavonoids + + - - Alkaloids + - + - Glycosides - - - - Steroids + - - - Phenols + - + + Protein + - - +

+ = Present; - = absent.

Table 4. Concentrations of some phytochemicals in methanolic leaf extract of P. fraternus.

Phytochemical Concentrations (mg/g) Saponins 136.00 ± 0.02 Tannins 37.20 ± 0.02 Terpenoids 6.60 ± 0.04 Flavonoids 159.20 ± 0.05 Alkaloids 96.50 ± 0.01 Phenols 152.00 ± 0.03

Values are mean ± SEM (n=3).

hoc test multiple comparison versus control groups with help of blood glucose for all the treatments group (except normal Statistical Package for the Social Sciences (SPSS) software version control) were all slightly above 350 mg/dl on the initial 21. The values p< 0.05 were considered significant (Duncan et al., day (day 0). However, the curve for diabetic untreated 1977). group was shown to be at relatively steady level throughout the treatment period. Other treatment groups RESULTS AND DISCUSSION have shown decrease in fasting blood glucose level from day 7 of treatments with metformin showing the most Qualitative phytochemical screening hypoglycaemic effect. Treatment with Methanolic extract at 300 mg/kg body weight was shown to have the same Results of the qualitative phytochemical screening of the hypoglycaemic effect with metformin at day 21 of the crude/fractions of methanolic leaf extract of P. fraternus treatment. On the final day of treatment (day 28), there are presented in Table 3. was no significant difference among all the treatment groups with the fasting blood glucose level of all groups below 150 mg/dl. Quantitative phytochemical estimation

Results of quantitative estimation of some of the Hypoglycaemic effects of fractions of methanolic leaf phytochemicals in P. fraternus methanolic leaf extract extract of P. fraternus in Streptozotocin induced (Table 4). diabetic rats

The hypoglycaemic effects of the three fractions (F I, F II Effects of P. fraternus methanolic leaf extract on and F III) obtained were carried out in diabetic rats blood glucose level in streptozotocin-induced following a glucose load and from the result obtained diabetic rats (Figure 2), the blood glucose level increases rapidly in all the groups 30 min after commencement of the glucose In prolonged treatments (28 days) (Figure 1), the fasting tolerance test, but fraction one (FI) and the standard 62 J. Med. Plants Res.

450

400

350

300 Normal Control 250 Diabetic Control

200 Metformin ME 200mg/kg b.w 150 ME 300mg/kg b.w

Blood glucose (mg/dl) glucose Blood 100

50

0 Day 0 Day 7 Day 14 Day 21 Day 28 Time (Days)

Figure 1. Effects of methanolic leaf extract of P. fraternus on fasting blood glucose level (mg/dl) in Streptozotocin - induced diabetic rats.

/dl)

mg

(

Blood glucose Blood

Time (Minutes)

Figure 2. Effect of pre-treatment with 200 mg/kgbw of methanolic leaf extract of P. fraternus on oral glucose tolerance test in STZ-induced diabetic rats.

control (metformin) were significantly lower at p<0.05 sharp fall in the glucose level in both metformin and FI when compared against the diabetic control. There was treated groups with FI treated group giving the most Nadro and Elkanah 63

/dl)

mg

(

Blood glucose Blood

Time (Minutes)

Figure 3. Effect of pre-treatment with 200 mg/Kgbw of methanolic leaf extract of P. fraternus on oral glucose tolerance test in normal rats.

glucose tolerance all through the test period followed by showing gradual decrease at 90 min and finally going metformin. Fraction three (FIII) showed the least back almost to the initial value at 150 min. hypoglycaemic effect all through the period of the test, at Treatment with metformin (Standard control) and 150 min, FIII and diabetic control showed no difference. Fraction I prevented glucose induced hyperglycaemia 30 On the other hand, crude extract and FII had similar min after commencement of the glucose tolerance test. effect at 90 min, but FII maintained a relatively steady Treatment with metformin have significantly (p<0.05) level through the remaining period while the crude extract lowered the blood glucose level at 30, 60 and 90 min of further decreases the glucose level. The crude extracts the tolerance test when compared with other treatments showed maximum hypoglycaemic effects at 150 min except for fraction I which in all cases showed similar where it exerts similar hypoglycaemic effect with hypoglycaemic effect with the standard control metformin and FI. (metformin). More so, fraction I had significantly decreased blood glucose level ahead of the crude extract at 30, 60 and 90 min. All the treatment groups showed Hypoglycaemic effects of fractions of P. fraternus similar hypoglycaemic effect at 120 min differing extract in normal rats significantly (p<0.05) against the normal control.

Treatments with different fractions of P. fraternus extract prior to glucose load in normal rats have shown positive DISCUSSION hypoglycaemic effects after glucose load Figure 3. The control (untreated group) has shown a rapid increase in Phytochemical screening of P. fraternus methanolic blood glucose in the first 60 min after glucose load before extract revealed the presence of alkaloids, flavonoids, 64 J. Med. Plants Res.

tannins, saponins, steroids, phenols, carbohydrates, that required time to be freed up, hence the maximum terpenoids and proteins. This finding is similar to the effect coming after 2 h, probably, after digestion had research findings of Matur et al. (2009) and Okokon et al. taken place to free up the hypoglycaemic agent. (2005). Plants are considered as biosynthetic laboratory for a multitude of compounds that exert physiological effects (Garg et al., 2010). Earlier reported studies have Conclusion already confirmed that flavonoids and tannins are the class of compounds which are responsible for several From this study, it can be deduced that flavonoids from therapeutic activities (Garg et al., 2010; Iwu, 1983). Fraction 1 of methanolic leaf extract of P. fraternus is a Several Authors also reported flavonoids, sterols, potent hypoglycaemic agent. Similarly, repeated oral alkaloids and phenolics as bioactive antidiabetic administration of methanolic leaf extract of P. fraternus principles (Nadro and Onoagbe, 2012). Fortunately the was shown to evoke hypoglycaemic effect on the fasting leaf of P. fraternus contains all these bioactive blood glucose profile of streptozotocin induced diabetic antidiabetic principles in reasonable quantities. rats. These results support the traditional usage of P. Streptozotocin induced diabetes has been described as fraternus in the treatment of diabetes mellitus. a useful experimental model to study the activity of hypoglycaemic agents (Paul et al., 2006). Blood glucose level was increased consistently and significantly in the Conflicts of interests diabetic untreated groups with relative stability after seven days. This rapid increment may be due to The authors have not declared any conflict of interests. decreased glucose clearance as a consequence of a defect in glucose transport (Wi et al., 1998). Prolonged treatment (28 days) with methanolic extract REFERENCES of P. fraternus (MEP) (200 and 300 mg/kg body weight) Ahmed I, Adeghate E, Sharma AK, Pallot DJ, Singh J (1998). Effects of and metformin (5 mg/kg body weight) showed continual Momordica charantia fruit juice on islet morphology in the pancreas of decrease of blood glucose, suggesting long term the streptozotocin-diabetic rat. Diab. Res. Clin. Pract. 40(3):145-151. maintenance of blood glucose level in diabetic rats. Akbarazadeh A, Norouzian D, jamshidi SH, Farhangi A, AllaVerdia A, Several medicinal plants have been reported to restore Motidan SMA, Lame R (2007). Induction of diabetes by streptozotocin in rats. Indian J. Clin. 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