Approach to Identify Medicinal Plants A

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 308980 9 pageshttpdxdoiorg1011552013308980

Research ArticleWhich Approach Is More Effective in the Selection ofPlants with Antimicrobial Activity

Ana Carolina Oliveira Silva12 Elidiane Fonseca Santana3 Antonio Marcos Saraiva4

Felipe Neves Coutinho4 Ricardo Henrique Acre Castro4 Maria Nelly Caetano Pisciottano4

Elba Luacutecia Cavalcanti Amorim5 and Ulysses Paulino Albuquerque2

1 Rede Nordeste de Biotecnologia (RENORBIO) Programa de Pos-Graduacao Universidade Federal Rural de PernambucoRua DomManoel de Medeiros sn Dois Irmaos 52171-900 Recife PE Brazil

2 Departamento de Biologia Laboratorio de Etnobotanica Aplicada Universidade Federal Rural de PernambucoRua DomManoel de Medeiros sn Dois Irmaos 52171-900 Recife PE Brazil

3 Departamento de Biologia Universidade de Pernambuco Avenida Agamenon Magalhaes snSanto Amaro 50100-010 Recife PE Brazil

4Departamento de Farmacia Laboratorio de Analises Microbiologicas Universidade Federal de PernambucoAvenida Professor Moraes Rego 1235 Cidade Universitaria 50670-901 Recife PE Brazil

5 Departamento de Farmacia Laboratorio de Produtos Naturais Universidade Federal de PernambucoAvenida Professor Moraes Rego 1235 Cidade Universitaria 50670-901 Recife PE Brazil

Correspondence should be addressed to Ana Carolina Oliveira Silva acarolinaleshotmailcom and Ulysses Paulino Albuquerqueupa677hotmailcom

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

Academic Editor Romulo Romeu da Nobrega Alves

Copyright copy 2013 Ana Carolina Oliveira Silva et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The development of the present study was based on selections using random direct ethnopharmacological and indirect ethnophar-macological approaches aiming to evaluate which method is the best for bioprospecting new antimicrobial plant drugs A crudeextract of 53 species of herbaceous plants collected in the semiarid region of Northeast Brazil was tested against 11 microorganismsWell-agar diffusion andminimum inhibitory concentration (MIC) techniqueswere used Ten extracts fromdirect six from randomand three from indirect ethnopharmacological selections exhibited activities that ranged from weak to very active against theorganisms tested The strain most susceptible to the evaluated extracts was Staphylococcus aureus The MIC analysis revealed thebest result for the direct ethnopharmacological approach considering that some species yielded extracts classified as active ormoderately active (MICs between 250 and 1000 120583gmL) Furthermore one species from this approach inhibited the growth of thethree Candida strains Thus it was concluded that the direct ethnopharmacological approach is the most effective when selectingspecies for bioprospecting new plant drugs with antimicrobial activities

1 Introduction

The search for new natural compounds is growing mainlydue to the acquired resistance of microorganisms to com-monly used drugs [1ndash3] and because nosocomial infectionscaused by these microorganisms have increasingly resultedin public health problems Although several antibiotics areavailable on the market microbial resistance to them has

increased significantly mainly due to the extensive use ofdrugs and the rapid genetic transfer of resistance It is esti-mated that microbial resistance develops within seven toeight years of regular antibiotic use [2 4] Therefore thedevelopment of new drugs that are capable of overcomingmicrobial resistance is critical

Given these circumstances bioprospection studies havebeen developed aiming to identify plants from which new

2 Evidence-Based Complementary and Alternative Medicine

drugs may be produced either using crude plant extracts orby isolating and characterizing the isolated active compo-nents moreover the goal of these investigations is to under-stand the role of these components as the basis for the devel-opment of new drugs (both natural and synthetic) fromplants [5] It is estimated that 30 to 40 of the most recentantimicrobial drugs available on the market are derived fromnatural products However these resources have been poorlyexplored to produce additional antimicrobial drugs fromplants [6]

It is known that plants provide an unlimited range ofcompounds because of their high chemical diversity andbecause they have been used for centuries by several differentpeoples For exampleArctostaphylos uva-ursi (L) Spreng andVaccinium macrocarpon Aiton are used to treat urinary tractinfections and Melissa officinalis L Allium sativum L andMelaleuca alternifolia Cheel are well-known broad-spectrumantimicrobial agents [7]

Currently one of the major challenges is the selectionof plants for a bioprospecting study because this processis the first research step The various methods include theselection approach which is used to choose plants based oncertain criteria random selection which involves the arbi-trary collection of the species without consideration of forinstance taxonomic affinities and ethnobotanical informa-tion ethnodirected selection which includes ethnobotanicaland ethnopharmacological approaches and applies informa-tion about the traditional use of plants to treat specific dis-eases and the chemotaxonomic approach which is based onthe study of plants from the same family or genus of a speciesfromwhich active compounds have been already isolated [8]

Several studies have been focused on selecting plantsthrough the ethnodirected approach [1 9 10] corroboratingthe folk use of many species that are traditionally used Sev-eral authors also suggest that random selection should beavoided in favor of ethnodirected selection because the latterappears to be a more efficient method of selecting species forbioprospecting [7]

Despite the large body of published data (namely thestudies cited previously) suggesting that ethnodirected selec-tion is more efficient few studies have compared the differentselection approaches (eg see Melo [11] and Svetaz et al [12])to provide a more realistic scenario for bioprospecting stud-ies

The main aim of the present study was to assess wheth-er the probability of discovering plants with antimicro-bial potentials is greater when they already have indicationsof ethnopharmacological use for infectious and parasiticdiseases (direct ethnopharmacological selection) than whenthey are randomly selected (random selection) or do not havedirect indications of use for infectious and parasitic diseases(indirect ethnopharmacological selection) Furthermore weaimed to determine the selection group to which the plantsbelonged with the lowest minimum inhibitory concentration(MIC) that is to assesswhether plantswith higher antimicro-bial activities are among those with a history of folk use in thetreatment of infectious diseases Finally we aimed to definewhich species should be selected for future investigations

involving compound isolation and identification for the pro-duction of antimicrobial phytopharmaceuticals

11 Selection and Collection of Species For species selection adatabase was created from 10 floristic andor phytosociologi-cal studies conducted in the ldquoCaatingardquo (savanna-like vegeta-tion) in the semiarid region of Northeast Brazil Six hundredforty-five species belonging to 81 families and 319 generawere included Subsequently the folk use for each specieswas assessed in the literature totaling 147 species with indi-cations for use For the random selection all species withoutmedicinal uses were considered For the direct ethnopharma-cological selection plants with indications of use for infec-tious and parasitic diseases such as pleuritis helminthiasisgeneral inflammation urinary disorders prostate infectionand uterine inflammation were considered For the indirectethnopharmacological selection all of the species with anyindications of medicinal use were selected excluding thosespecies that were related to the direct ethnopharmacologicalapproach Thereafter the Biostat 50 software was used forspecies selection Initially 20 species were randomly selectedfor each type of approach However due to the unavailabilityof certain species during the collection period 19 speciesof herbs were selected for the random approach 20 for thedirect ethnopharmacological selection and 14 for the indirectethnopharmacological selection

In total 53 species of plants were collected from Aprilto July 2011 at the Experimental Station of the AgriculturalResearch Company of Pernambuco Agronomic Institute ofPernambuco (Instituto Agronomico de Pernambuco (IPA))(8∘141015840S and 35∘551015840W 537m altitude) in Caruaru Agresteregion of Pernambuco Northeast Brazil Voucher specimensare deposited in the Herbarium of Professor VasconcelosSobrinho of the Federal Rural University of Pernambuco(PEUFR) The area was selected because it is well studied interms of floristics and provides easy access for obtaining thevarious species

12 Extract Preparation Because the species were herba-ceous all of the extracts were prepared from the leavesTwenty grams of dried and powdered leaves wasmacerated inabsolute methanol for 24 hours The process was extensivelyrepeated The extract obtained was then evaporated underreduced pressure at 45∘Cuntil driedThe samples were placedin a desiccator for a one-week period The extracts werediluted in 20 dimethyl sulfoxide (DMSO) and used atconcentrations of 100mgmL and 50mgmL As there is nostandard regarding the maximum concentration of crudeextract of plants for screening the protocol of the Laboratoryof Microbiological Analyses from Federal University of Per-nambuco was adopted where the assays were performed

13 Antimicrobial Assay The antimicrobial activity was eval-uated in two trials First the antimicrobial activity wasdetermined by the agar diffusion method [13] Next for thespecies that displayed inhibition halo greater than 13mm theMIC [13] was determined

The crude extracts were tested against standard strains ofStaphylococcus aureus (ATCC 6538) S epidermidis (sperm)

Evidence-Based Complementary and Alternative Medicine 3

S saprophyticus (LACEN 07) Bacillus subtilis (ATCC 6633)Enterococcus faecalis (ATCC 51299) Klebsiella pneumo-niae (secretion) Pseudomonas aeruginosa (ATCC 14502)Escherichia coli (ATCC 35218) Candida albicans (urine) Ckrusei (blood) and C tropicalis (rectal swab) These strainswere selected because of their clinical relevance

Inoculumswere prepared 24 hours in advance and kept inMueller-Hinton agar (bacteria) and Sabouraud agar (yeast)The inoculumswere suspended in sterile saline solution usinga 05 McFarland standard (108UFCmL) [14]

For the agar diffusion method sterile swabs were used toinoculate sterile petri plates (20 times 100mm) containing 20mLof Mueller-Hinton agar for bacteria and 20mL Sabouraudagar for yeast On each plate four wells (6mmdiameter) werecreated to which 100 120583L of the extracts at concentrations of100mgmL and 50mgmL was added as well as the positivecontrols tetracycline (30mgmL) for bacteria and ketocona-zole (50mgmL) for yeast Twenty percentDMSOwas used asthe negative control The plates were incubated aerobically at37 plusmn 1∘C for 24 hoursThe antimicrobial activity was assessedby measuring the inhibition halo of microbial growth aroundthe well and the results were classified according to thefollowing scale inhibition zones down to 9mm inactive 9ndash12mm moderately active 13ndash18mm active above 18mmvery active [15] The G-test was performed (119875 lt 005)to determine significant differences between the selectionapproaches and the antimicrobial activity All of the assayswere performed in triplicate

The MIC of the extracts and the reference antibiotic(tetracycline) were determined using the Mueller-Hintonbroth microdilution technique following the protocol estab-lished by the Clinical and Laboratory Standards Institute [16]for bacteria Inoculums were prepared in the same mediumandwere adjusted to a 05McFarland standard (108UFCmL)and diluted 1 10 for the broth dilution method The micro-plates were incubated at 37∘C and the MIC was read aftera 24-hour incubation period The MIC was defined as thelowest compound concentration at which themicroorganismtested showed no visible growth

The MIC for yeasts was performed by the broth microdi-lution technique according to theCLSI [17] and ketoconazolewas used as the positive control The inoculum was used at aconcentration of 10times 106UFCmLTheMICwas determinedin Roswell Park Memorial Institute (RPMI) 1640 medium(Gibco Invitrogen Co New York USA) with MOPS bufferpH 70 The plates were incubated at 37∘C and the readingswere obtained after a 24-hour incubation period All of theassays were performed in duplicate

Plant extracts with MICs lt100120583gmL were consideredhighly active antimicrobial agents MICs ranging from 100 to500120583gmL were classified as active MICs ranging between500 and 1000 120583gmL were considered moderately activeMICs ranging from 1000 to 2000120583gmL were considered tohave low activity and MICs gt2000120583gmL were classified asinactive [14] The G-test was conducted (119875 lt 0005) to eval-uate significant differences between the selection approachesand the MIC

2 Results and Discussion

Of the 20 plant extracts obtained by direct ethnopharma-cological selection 10 (50) exhibited activity against themicroorganisms tested Of the 19 plant extracts obtainedby random selection seven (3684) showed antimicrobialactivity against at least one strain Of the 14 plant extractsobtained by indirect ethnopharmacological selection two(1428) exhibited antimicrobial activity (Table 1)The activeextracts that were tested exhibited antimicrobial activity onlyagainst the Gram-positive bacteria and the Candida strains

Of the 10 extracts obtained by direct ethnopharmacolog-ical selection 40 exhibited moderate antimicrobial activity30 were active and 30 were highly active Of the extractsobtained by random selection 3333 exhibited moderateactivity and 6666 were highly active Of the extractsobtained by indirect ethnopharmacological selection 70were moderately active and 30 were active Based on theG-test (119866 = 1271860) significant differences (119875 lt 0005)existed between the proportions of active species among thethree approaches indicating that direct ethnopharmacolog-ical selection is the most effective for the selection of plantswith greater antimicrobial activities

The plant species obtained by direct ethnopharmacolog-ical selection were more effective in terms of the number ofstrains with inhibited growth Four of the plants Acanthos-permum hispidum Euphorbia hyssopifolia Hyptis suaveolensand Indigofera suffruticosa displayed antimicrobial activityagainst four microorganisms Two of the plants Ludwigiaoctovalvis and Momordica charantia were active againstfive microorganisms Centratherum punctatum which wasobtained by random selection inhibited the growth offive microorganisms whereas Blainvillea acmella inhibitedfour microorganisms Only Tillandsia recurvata which wasobtained by indirect ethnopharmacological selection dis-played antimicrobial activity against five microorganisms

Although the plant species that were selected by the threeapproaches exhibited the same versatility in the inhibition ofvarious strains (ie fivemicroorganisms from the 11 that weretested) the selection approaches were distinguished by theantimicrobial activities presented by the species this activitywas greater in the species obtained by direct ethnopharma-cological selection resulting in the largest inhibition halo(ranging from 22 to 30mm) Furthermore of the 53 plantspecies studied only four of the extracts could inhibit fivemicroorganisms two of these plant species were obtainedby direct ethnopharmacological selection The findings ofthe present study indicate greater success when plant speciesare selected based on their direct indications of use forinfectious and parasitic diseases which is similar to thefindings of Phongpaichit et al [18] who investigated theuse of plants to treat fungal infections in AIDS patientsBy selecting species based on folk use the authors achieveda success rate of 40 Cruz et al [9] analyzing Brazilianplants traditionally used to treat mycoses achieved a successrate of 50 confirming the results of the present study vanVuuren and Naidoo [10] analyzed plants (selected from theethnobotanical literature) used in the treatment of sexuallytransmitted diseases and found that 90 of the extracts


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

Species 119862mgmL Sa Se Ss Bs Ef Ec Kp Pa Ca Ck Ct

Random approach

Astraea lobata (L) Klotzsch 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Blainvillea acmella (L) Philipson 10050

2318

2013

2016

2220 mdash mdash mdash mdash mdash mdash mdash

Centratherum punctatum Cass 10050

3025

2017

2214

2116

2214 mdash mdash mdash mdash mdash mdash

Croton hirtus LrsquoHer 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Cyperus uncinulatus Schrad ex Nees 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Delilia biflora (L) Kuntze 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Drymaria cordata (L) Willd ex Roem amp Schult 10050

7 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Euphorbia heterophylla L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Lepidium ruderale L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Melanthera latifolia (Gardner) Cabr 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Mollugo verticillata L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Parthenium hysterophorus L 10050

9 10 77


Ruellia asperula (Mart amp Nees) Benth amp Hook 10050 10 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Ruellia geminiflora Kunth 10050 mdash mdash mdash 8 mdash mdash mdash mdash mdash mdash mdash

Sida urens L 10050

2020

2020

1815 mdash mdash mdash mdash mdash mdash mdash mdash

Spermacoce verticillata L 10050

1010

86

107 mdash 6 mdash mdash mdash mdash mdash mdash

Stylosanthes scabra Vogel 10050


Talinum triangulare (Jacq) Willd 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Species 1 (Malvaceae) mdash mdash mdash mdash mdash mdash mdash mdash 30 30 30Indirect ethnopharmacological approach

Alternanthera brasiliana (L) Kuntze 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Alternanthera tenella Colla 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Commelina erecta L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Commelina obliqua Vahl 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Hypenia brachystachys (Pohl ex Benth) Harley 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash


Table 1 Continued


Hypenia salzmannii (Benth) Harley 10050

1412 mdash 12


Polygala paniculata L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Polygala violacea Aubl 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Portulaca elatior Mart ex Rohrb 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Portulaca oleracea L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Solanum agrarium Sendtn 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Solanum americanumMill 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Tillandsia recurvata (L) L 10050

1715

1817

1816

1313


Tillandsia usneoides (L) L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Direct ethnopharmacological approach

Acalypha multicaulisMull Arg 10050

1210

1010


Acanthospermum hispidum DC 10050

2220

1915

1915


Ageratum conyzoides L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Aosa rupestris Gardner 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Argemone mexicana L 10050

87

77

77 mdash mdash mdash mdash mdash mdash 15

15 mdash

Boerhavia diffusa L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Euphorbia hyssopifolia L 10050

2016

1613

1815

1916


Conocliniopsis prasiifolia (DC) RM King amp H Rob 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Heliotropium indicum L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Hyptis suaveolens (L) Poit 10050

1816

1513

1816


Indigofera suffruticosaMill 10050

3030

2222

2220 mdash 30


Leonotis nepetifolia (L) R Br6 10050


Ludwigia octovalvis (Jacq) PH Raven 10050

1311


151312

1312

Melochia tomentosa L 10050

1010

1411

87

87


Momordica charantia L 10050

2017

1515

1514

1815



Table 1 Continued


Physalis angulata L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Rhaphiodon echinus Schauer 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Richardia grandiflora (Cham amp Schltdl) Steud 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

Scoparia dulcis L 10050

1212 mdash mdash 12


Waltheria indica L 10050 mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash mdash

119862 concentration Sa Staphylococcus aureus Se S epidermidis Ss S saprophyticus Bs Bacillus subtilis Ef Enterococcus faecalis Ec Escherichia coli PaPseudomonas aeruginosa Kp Klebsiella pneumonia Ca Candida albicans Ck C krusei e Ct C tropicalis mdash no inhibition

exhibited significant antimicrobial activity against the testedmicroorganisms thereby validating the ethnomedicinal useof species Comparing the results obtained from the differentselection approaches (Table 1) direct ethnopharmacologicalselection yielded a greater number of species that were activeagainst the tested microorganismsmdashplants such as Acanthos-permum hispidum Euphorbia hyssopifolia and I suffruticosawhich caused inhibition halo ranging from 17 to 30mm forthe S aureus S epidermidis S saprophyticus and B subtilisstrains Another example of the efficiency of plant speciesobtained by direct ethnopharmacological selection was theiraction against E faecalis Whereas three of the plant specieswere able to inhibit the growth of E faecalis only one speciesobtained by random selection and one species obtainedby indirect ethnopharmacological selection inhibited thismicroorganism

The MIC was determined for four species obtained byrandom selection nine species obtained by direct ethnophar-macological selection and two species obtained by indirectethnopharmacological selection Blainvillea acmella Cen-tratherum punctatum Sida urens and species 1 that is inprocess of patent registration and so its name cannot bedisclosed (random selection) Acanthospermum hispidumArgemone mexicana E hyssopifolia Hyptis suaveolens I suf-fruticosa Leonotis nepetifolia Ludwigia octovalvis Melochiatomentosa and Momordica charantia (direct ethnopharma-cological selection) and Tillandsia recurvata and Hypeniasalzmannii (indirect ethnopharmacological selection) TheMICs for all of the analyzed species and strains are presentedin Table 2 Based on the G-test (119866 = 765443) a significantdifference (119875 lt 0005) existed among the MICs of thethree selection approaches with direct ethnopharmacolog-ical selection again being distinct because extracts of plantspecies belonging to this group exhibited superior MICsTheextracts were tested only against the strain to which theyshowed activity up to 13mm

All of the species obtained by indirect ethnopharma-cological selection were considered to have low activitiesbecause they displayed MICs above 1000 120583gmL In the ran-dom selection only Sida urens was considered moderatelyactive presenting an MIC of 500120583gmL against S aureus

For species obtained by direct ethnopharmacological selec-tion four extracts were classified as moderately active Ahispidum E hyssopifolia I suffruticosa and Momordicacharantia Ludwigia octovalvis from the direct ethnopharma-cological selection and species 1 from the random selectionwere the only species with MICs that were classified as activeand highly active forC albicans (125 120583gmL and 3125 120583gmLresp) L octovalvis also exhibited a moderately active extractagainst C krusei and C tropicalis (1000120583gmL) while theextract from species 1 was highly active against C krusei(625 120583gmL) and C tropicalis (625120583gmL) According toFabry et al [19] plants with MICs below 8mgmL are con-sidered to display some antimicrobial activity Moreover ourfindings indicate that these species are potential candidatesfor further investigations (of the isolation and identificationof compounds with antimicrobial activities) because theMICs were below 1mgmL confirming other studies indi-cating that plant extracts and natural products with MICsbelow 1mgmL deserve special attention and must thereforebe carefully analyzed [7 20]

The results obtained in the present study indicate thatdirect ethnopharmacological selection is an effective bio-prospecting tool for antimicrobial activity Svetaz et al [12]evaluated ethnomedical information on the discovery ofplantswith antifungal activity and determined that the proba-bility of finding plants with this activity is significantly higherwhen reports exist of their use as antifungal agents comparedwith the absence of such reports The authors categoricallyaffirmed that the ethnopharmacological approach is usefulin detecting plants with antifungal activity Furthermorein studies on the potential antimalarial effect of Nigerianplants Adebayo and Krettli [21] discussed the difficulty highcost and low efficacy of the random selection approachwhich was the selection method that had been used in thatcountry decades priorThe current method is ethnobotanicalselection based on information regarding indigenous uses ofthe species which has reduced costs and time compared withrandom selection

The low probability of finding promising plants forbioprospection through random selection suggests that thisapproach is not recommended for the discovery of new


Table 2 Minimal inhibitory concentration (120583gmL) of herbaceous species from the semiarid region Northeast of Brazil based on randomdirect and indirect ethnopharmacological approaches

Species Sa Se Ss Bs Ef Ca Ck CtRandom approach

Blainvillea acmella (L) Philipson gt1000 gt1000 gt1000 gt1000 NT NT NT NTCentratherum punctatum Cass gt1000 gt1000 gt1000 gt1000 gt1000 NT NT NTSida urens L 500 1000 1000 NT NT NT NT NTSpecies 1 (Malvaceae) NT NT NT NT NT 3125 625 625

Indirect ethnopharmacological approachTillandsia recurvata (L) L gt1000 gt1000 gt1000 gt1000 gt1000 NT NT NTHypenia salzmannii (Benth) Harley gt1000 NT NT NT NT NT NT NT

Direct ethnopharmacological approachAcanthospermum hispidum DC 1000 gt1000 gt1000 gt1000 NT NT NT NTArgemone mexicana L NT NT NT NT NT NT gt1000 NTEuphorbia hyssopifolia L 1000 gt1000 gt1000 1000 NT NT NT NTHyptis suaveolens (L) Poit gt1000 gt1000 gt1000 NT NT NT NT NTIndigofera suffruticosaMill 500 gt1000 gt1000 1000 gt1000 NT NT NTLeonotis nepetifolia (L) R Br gt1000 NT NT NT NT NT NT NTLudwigia octovalvis (Jacq) PH Raven 250 500 NT NT NT 125 1000 1000Melochia tomentosa L gt1000 gt1000 NT gt1000 NT NT NT NTMomordica charantia L 1000 gt1000 gt1000 1000 gt1000 NT NT NTSa Staphylococcus aureus Se S epidermidis Ss S saprophyticus Bs Bacillus subtilis Ef Enterococcus faecalis Ca Candida albicans Ck C krusei e Ct Ctropicalis NT extract not tested for the strain

antimicrobial agents Although this approachwas responsiblefor the discovery of taxol [22 23] it is currently knownthat only one in 10000 plants will be a promising source ofnew drugs while the ethnodirected selection is responsiblefor 74 of all the drugs of plant origin [24] Neverthelesscontroversy exists regarding the efficacy of this approachbecause the selected plants appear not to be effective in thetreatment of cancer [11] and mycoses caused by yeasts andAspergillus spp indeed for these types of fungi Svetaz et al[12] found no significant differences between the antimicro-bial activities of plants obtained by random selection versusethnopharmacological selection Moreover the fact that inthe present study only one species obtained by randomselection was highly active against the three Candida strainsindicates that the random approach should not be completelyabandoned but should instead be adapted to other selectionapproaches such as chemosystematic or ecological methods

The species that were obtained by direct ethnopharma-cological random and indirect ethnopharmacological selec-tions were active only against Gram-positive bacteria Theinactivity against Gram-negative bacteria might be observedbecause of the lipophilic outer layer among other reasonsas this layer most likely prevents the access of the extract tothe interior of the bacteria as observed by Nantitanon et al[25] in their assessment of the antimicrobial activity ofHyptissuaveolens Regarding antifungal activity it is noteworthythat most of the studies were conducted using C albicans[26] The exceptions were the studies by Cruz et al [9]who tested the use of plants from the Caatinga against Cguilliermondii and De Toledo et al [27] who tested the useof plants againstC parapsilosis strainsThe present study also

included C krusei and C tropicalis which are species that aremore resistant to commonly used drugs Among the speciesthat we investigated L octovalvis and species 1 inhibited thegrowth of the three Candida species with the most activeMICs ranging from 625 120583gmL to 3125120583gmL This findingof the present study is of great importance considering thehigh incidence of C albicans (70) and C tropicalis (20)in Latin America [28] the emergence of other Candidaspecies resistant to antifungal agents and mainly the limitednumber of drugs available to treat fungal infections [29]

Due to the cosmopolitan nature of the species analyzedherein studies on the antimicrobial activity involving some ofthese species have been conducted in various regions world-wide such as Ageratum conyzoides in Malaysia [30] which isused against cough and has displayed an MIC of 1600 120583gmLfor theMycobacterium strains However in the present studyall of the tested strains were resistant to the extract from Aconyzoides

Gachet et al [31] who tested plants traditionally usedagainst leishmaniasis in Ecuador found that the extract of Sdulcis is effective against axenic amastigotes of L donovaniIn the present study this species displayed moderate activityagainst S aureus and B subtilis Wiart et al [26] tested themethanol extract in an antimicrobial screening of plants fromMalaysia including among othersA conyzoides C hirtus Eprostrate andH suaveolens these species are effective againstvarious microorganisms such as B cereus P aeruginosaB subtilis S aureus and C albicans In the present studyhowever except forH suaveolenswhich inhibited the growthof S aureus S saprophyticus S epidermidis and B subtilis(resulting in inhibition halo between 15 and 18mm) the


other species displayed no activity against any of the analyzedmicroorganisms

Matsuse et al [32] studied plants from Panama withpotential antiviral activity and found that both the crudeextract and isolated compounds from E hyssopifolia wereeffective against HIV In the present study this species is oneof themost effective with anMIC of 1000120583gmL for S aureuswhich qualifies it as a potential candidate for studies aimingto develop drugs obtained from plants of the Caatinga thatare more effective against resistant strains In addition thisfinding supports the folk use of this species against microbialinfections in the semiarid region of Northeast Brazil [33]

In Brazil especially the Caatinga few studies exist onantimicrobial activity based on ethnobotanical data andinvestigations are almost nonexistent regarding species fromthe Caatinga whether native or spontaneous Cruz et al [9]studied the extract of Ziziphus joazeiro Mart Caesalpiniapyramidalis Tul Bumelia sartorum Mart and Hymenaeacourbaril L which are traditionally used to treat mycoses andfound that Z joazeiro and C pyramidalis display significantantifungal activities making them potential candidates forthe development of new strategies to treat fungal infections

Almeida et al [1] in a study comparing the antimicrobialefficiency of species selected in the Caatinga and AtlanticForest tested the crude extract of B diffusa which displayedmoderate activity against S aureus Streptococcus faecalis andMycobacterium smegmatis when collected in the CaatingaHowever the extract of the species from the Atlantic Forestexhibited no activity against any microorganism In thepresent study all of the tested strains were resistant to theextract of this plantThe authors concluded that the Caatingaregion appears to be a promising source in the search fornew compounds of plant origin due to the larger size ofthe inhibition halo generated when using extracts from thesespecies and due to their ability to inhibit a greater number ofmicroorganisms

Despite the fact that species 1 from the random selec-tion displays an MIC that classifies it as highly active(le625120583gmL) for the three Candida species tested thefindings in the present studies regarding species obtained bydirect ethnopharmacological selection (ieAcanthospermumhispidum Euphorbia hyssopifolia I suffruticosa Ludwigiaoctovalvis and Momordica charantia which exhibited MICsranging from 250 to 1000 120583gmL) indicate that this selectionapproach is an effective strategy for bioprospecting newdrugswith antimicrobial activity Furthermore additional in-depth studies should be conducted using compounds isolatedfrom the cited species The present study included onlyspontaneous herbaceous species of the Caatinga which is apractice that is still undervalued in bioprospecting but thatbased on our findings appears to be potentially useful inthe search for compoundswith antimicrobial activity indeedthe plant extracts used were able to inhibit yeasts that occurwith high incidence in Latin America and that exhibit highresistance to regular antibiotics Additionally these speciesare characterized by wide distribution high population num-bers and rapid growth which would facilitate their studyOur findings are supported by the investigations of Cruzet al [9] and Almeida et al [1] in considering the Caatinga

and ethnopharmacological selection in the search for newpharmaceutical products

3 Conclusions

It may be concluded that direct ethnopharmacological selec-tion is an important bioprospecting tool and that theCaatingais a type of vegetation that should be included in futurestudies on the bioprospection of new antimicrobial plantdrugs Additionally the above-mentioned species should beincluded in the studies investigating the production of newphytomedicines

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors are grateful to Lucilene Lima dos Santos forspecies identification and Edna Santos and Ingrid Suely Limafor technical assistance to FACEPE for scholarship to ACOSand toCNPq for financial support (Edital Universal 2012) andproductivity grant given to Ulysses Paulino Albuquerque

References

[1] C D F Castelo Branco Rangel De Almeida D L De Vascon-celos Cabral C C B Rangel De Almeida E L Cavalcanti DeAmorim J M De Arajo and U P De Albuquerque ldquoCom-parative study of the antimicrobial activity of native and exoticplants from the Caatinga and Atlantic Forest selected throughan ethnobotanical surveyrdquo Pharmaceutical Biology vol 50 no2 pp 201ndash207 2012

[2] D O Guimaraes L S Momesso andM T Pupo ldquoAntibioticosimportancia terapeutica e perspectivas para a descoberta edesenvolvimento de novos agentesrdquo Quımica Nova vol 33 no3 pp 667ndash679 2010

[3] G G F Nascimento J Locatelli P C Freitas and G L SilvaldquoAntibacterial activity of plant extracts and phytochemicals onantibiotic-resistant bacteriardquo Brazilian Journal of Microbiologyvol 31 no 4 pp 247ndash256 2000

[4] G P Silveira F Nome J C Gesser and M M Sa ldquoEstrategiasUtilizadas no Combate a Resistencia Bacterianardquo QuımicaNova vol 29 no 4 pp 844ndash855 2006

[5] H-F Ji X-J Li and H-Y Zhang ldquoNatural products and drugdiscovery can thousands of years of ancientmedical knowledgelead us to new and powerful drug combinations in the fightagainst cancer and dementiardquo EMBO Reports vol 10 no 3 pp194ndash200 2009

[6] D Chattopadhyay M Chawla-Sarkar T Chatterjee et al ldquoRe-cent advancements for the evaluation of anti-viral activities ofnatural productsrdquo New Biotechnology vol 25 no 5 pp 348ndash365 2009

[7] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[8] U P Albuquerque and N Hanazaki ldquoAs pesquisas etnodirigi-das na descoberta de novos farmacos de interesse medico efarmaceutico fragilidades e perspectivasrdquo Revista Brasileira DeFarmacognosia vol 16 pp 678ndash689 2006


[9] M C S Cruz P O Santos A M Barbosa Jr et al ldquoAntifungalactivity of Brazilian medicinal plants involved in populartreatment of mycosesrdquo Journal of Ethnopharmacology vol 111no 2 pp 409ndash412 2007

[10] S F van Vuuren andD Naidoo ldquoAn antimicrobial investigationof plants used traditionally in southern Africa to treat sexuallytransmitted infectionsrdquo Journal of Ethnopharmacology vol 130no 3 pp 552ndash558 2010

[11] J G Melo Estrategias de bioprospeccao e agentes anticancerıg-enos a partir da flora nordestina [PhD thesis] Rede Nordestede Biotecnologia Recife Brazil 2010

[12] L Svetaz F Zuljan M Derita et al ldquoValue of the ethnomedicalinformation for the discovery of plants with antifungal proper-ties A survey among seven Latin American countriesrdquo Journalof Ethnopharmacology vol 127 no 1 pp 137ndash158 2010

[13] Clinical and Laboratory Standards Institute (CLSI) Perfor-mance Standards for Antimicrobial Disk Susceptibility Tests M2-A8 Wayne Pa USA 8th edition 2003

[14] A M Saraiva R H A Castro R P Cordeiro et al ldquoIn vitroevaluation of antioxidant antimicrobial and toxicity propertiesof extracts of Schinopsis brasiliensi engl (Anacardiaceae)rdquoAfrican Journal of Pharmacy and Pharmacology vol 5 no 14pp 1724ndash1731 2011

[15] T M A Alves A F Silva M Brandao et al ldquoBiologicalscreening of brazilian medicinal plantsrdquo Memorias do InstitutoOswaldo Cruz vol 95 pp 367ndash373 2000

[16] Clinical and Laboratory Standards Institute (CLSI) ReferenceMethod for Broth Dilution Antifungal Susceptibility Testing ofYeasts Approved Standard M27-A3 NCCLS Wayne Pa USA2008

[17] Clinical and Laboratory Standards Institute (CLSI) Methodsfor Dilution Antimicrobial Susceptibility Tests for Bacteria thatGrow Aerobically Approved Standard M07-A8 CLSI WaynePa USA 8th edition 2009

[18] S Phongpaichit S Subhadhirasakul and C Wattanapirom-sakul ldquoAntifungal activities of extracts from Thai medicinalplants against opportunistic fungal pathogens associated withAIDS patientsrdquoMycoses vol 48 no 5 pp 333ndash338 2005

[19] W Fabry P O Okemo and R Ansorg ldquoAntibacterial activity ofEast African medicinal plantsrdquo Journal of Ethnopharmacologyvol 60 no 1 pp 79ndash84 1998

[20] S Gibbons ldquoAnti-staphylococcal plant natural productsrdquoNatu-ral Product Reports vol 21 no 2 pp 263ndash277 2004

[21] J O Adebayo and A U Krettli ldquoPotential antimalarials fromNigerian plants a reviewrdquo Journal of Ethnopharmacology vol133 no 2 pp 289ndash302 2011

[22] G M Cragg M R Boyd J H Cardellina et al ldquoThe searchfor new pharmaceutical crops drug discovery and developmentat the National Cancer Instituterdquo in New Crops J Janick and JE Simon Eds pp 161ndash167 John Wiley amp Sons New York NYUSA 1993

[23] P A Cox and M J Balick ldquoThe ethnobotanical approach todrug discoveryrdquo Scientific American vol 270 no 6 pp 82ndash871994

[24] E Douwes N R Crouch T J Edwards and D A Mulhol-land ldquoRegression analyses of southern African ethnomedicinalplants informing the targeted selection of bioprospecting andpharmacological screening subjectsrdquo Journal of Ethnopharma-cology vol 119 no 3 pp 356ndash364 2008

[25] WNantitanon S Chowwanapoonpohn and SOkonogi ldquoAnti-oxidant and antimicrobial activities of Hyptis suaveolens essen-tial oilrdquo Scientia Pharmaceutica vol 75 no 1 pp 35ndash46 2007

[26] CWiart S Mogana S Khalifah et al ldquoAntimicrobial screeningof plants used for traditional medicine in the state of PerakPeninsular Malaysiardquo Fitoterapia vol 75 no 1 pp 68ndash73 2004

[27] C EM De Toledo E A Britta L F Ceole et al ldquoAntimicrobialand cytotoxic activities of medicinal plants of the Braziliancerrado using Brazilian cachaca as extractor liquidrdquo Journal ofEthnopharmacology vol 133 no 2 pp 420ndash425 2011

[28] M A Pfaller and D J Diekema ldquoEpidemiology of invasivecandidiasis a persistent public health problemrdquo Clinical Micro-biology Reviews vol 20 no 1 pp 133ndash163 2007

[29] B P Mathew and M Nath ldquoRecent approaches to antifungaltherapy for invasive mycosesrdquo ChemMedChem vol 4 no 3 pp310ndash323 2009

[30] S Mohamad N M Zin H A Wahab et al ldquoAntituberculosispotential of some ethnobotanically selected Malaysian plantsrdquoJournal of Ethnopharmacology vol 133 no 3 pp 1021ndash10262011

[31] M S Gachet J S Lecaro M Kaiser et al ldquoAssessment of anti-protozoal activity of plants traditionally used in Ecuador in thetreatment of leishmaniasisrdquo Journal of Ethnopharmacology vol128 no 1 pp 184ndash197 2010

[32] I TMatsuse Y A LimMHattori M Correa andM P GuptaldquoA search for anti-viral properties in Panamanian medicinalplants The effects on HIV and its essential enzymesrdquo Journalof Ethnopharmacology vol 64 no 1 pp 15ndash22 1998

[33] U P de Albuquerque P M de Medeiros A L S de Almeida etal ldquoMedicinal plants of the caatinga (semi-arid) vegetation ofNE Brazil a quantitative approachrdquo Journal of Ethnopharmacol-ogy vol 114 no 3 pp 325ndash354 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


drugs may be produced either using crude plant extracts orby isolating and characterizing the isolated active compo-nents moreover the goal of these investigations is to under-stand the role of these components as the basis for the devel-opment of new drugs (both natural and synthetic) fromplants [5] It is estimated that 30 to 40 of the most recentantimicrobial drugs available on the market are derived fromnatural products However these resources have been poorlyexplored to produce additional antimicrobial drugs fromplants [6]

It is known that plants provide an unlimited range ofcompounds because of their high chemical diversity andbecause they have been used for centuries by several differentpeoples For exampleArctostaphylos uva-ursi (L) Spreng andVaccinium macrocarpon Aiton are used to treat urinary tractinfections and Melissa officinalis L Allium sativum L andMelaleuca alternifolia Cheel are well-known broad-spectrumantimicrobial agents [7]

Currently one of the major challenges is the selectionof plants for a bioprospecting study because this processis the first research step The various methods include theselection approach which is used to choose plants based oncertain criteria random selection which involves the arbi-trary collection of the species without consideration of forinstance taxonomic affinities and ethnobotanical informa-tion ethnodirected selection which includes ethnobotanicaland ethnopharmacological approaches and applies informa-tion about the traditional use of plants to treat specific dis-eases and the chemotaxonomic approach which is based onthe study of plants from the same family or genus of a speciesfromwhich active compounds have been already isolated [8]

Several studies have been focused on selecting plantsthrough the ethnodirected approach [1 9 10] corroboratingthe folk use of many species that are traditionally used Sev-eral authors also suggest that random selection should beavoided in favor of ethnodirected selection because the latterappears to be a more efficient method of selecting species forbioprospecting [7]

Despite the large body of published data (namely thestudies cited previously) suggesting that ethnodirected selec-tion is more efficient few studies have compared the differentselection approaches (eg see Melo [11] and Svetaz et al [12])to provide a more realistic scenario for bioprospecting stud-ies

The main aim of the present study was to assess wheth-er the probability of discovering plants with antimicro-bial potentials is greater when they already have indicationsof ethnopharmacological use for infectious and parasiticdiseases (direct ethnopharmacological selection) than whenthey are randomly selected (random selection) or do not havedirect indications of use for infectious and parasitic diseases(indirect ethnopharmacological selection) Furthermore weaimed to determine the selection group to which the plantsbelonged with the lowest minimum inhibitory concentration(MIC) that is to assesswhether plantswith higher antimicro-bial activities are among those with a history of folk use in thetreatment of infectious diseases Finally we aimed to definewhich species should be selected for future investigations

involving compound isolation and identification for the pro-duction of antimicrobial phytopharmaceuticals

11 Selection and Collection of Species For species selection adatabase was created from 10 floristic andor phytosociologi-cal studies conducted in the ldquoCaatingardquo (savanna-like vegeta-tion) in the semiarid region of Northeast Brazil Six hundredforty-five species belonging to 81 families and 319 generawere included Subsequently the folk use for each specieswas assessed in the literature totaling 147 species with indi-cations for use For the random selection all species withoutmedicinal uses were considered For the direct ethnopharma-cological selection plants with indications of use for infec-tious and parasitic diseases such as pleuritis helminthiasisgeneral inflammation urinary disorders prostate infectionand uterine inflammation were considered For the indirectethnopharmacological selection all of the species with anyindications of medicinal use were selected excluding thosespecies that were related to the direct ethnopharmacologicalapproach Thereafter the Biostat 50 software was used forspecies selection Initially 20 species were randomly selectedfor each type of approach However due to the unavailabilityof certain species during the collection period 19 speciesof herbs were selected for the random approach 20 for thedirect ethnopharmacological selection and 14 for the indirectethnopharmacological selection

In total 53 species of plants were collected from Aprilto July 2011 at the Experimental Station of the AgriculturalResearch Company of Pernambuco Agronomic Institute ofPernambuco (Instituto Agronomico de Pernambuco (IPA))(8∘141015840S and 35∘551015840W 537m altitude) in Caruaru Agresteregion of Pernambuco Northeast Brazil Voucher specimensare deposited in the Herbarium of Professor VasconcelosSobrinho of the Federal Rural University of Pernambuco(PEUFR) The area was selected because it is well studied interms of floristics and provides easy access for obtaining thevarious species

12 Extract Preparation Because the species were herba-ceous all of the extracts were prepared from the leavesTwenty grams of dried and powdered leaves wasmacerated inabsolute methanol for 24 hours The process was extensivelyrepeated The extract obtained was then evaporated underreduced pressure at 45∘Cuntil driedThe samples were placedin a desiccator for a one-week period The extracts werediluted in 20 dimethyl sulfoxide (DMSO) and used atconcentrations of 100mgmL and 50mgmL As there is nostandard regarding the maximum concentration of crudeextract of plants for screening the protocol of the Laboratoryof Microbiological Analyses from Federal University of Per-nambuco was adopted where the assays were performed

13 Antimicrobial Assay The antimicrobial activity was eval-uated in two trials First the antimicrobial activity wasdetermined by the agar diffusion method [13] Next for thespecies that displayed inhibition halo greater than 13mm theMIC [13] was determined

The crude extracts were tested against standard strains ofStaphylococcus aureus (ATCC 6538) S epidermidis (sperm)
















Random approach



2318

2013

2016



3025

2017

2214

2116












9 10 77




Sida urens L 10050

2020

2020



1010

86












Table 1 Continued



1412 mdash 12









1715

1817

1816

1313





1210

1010



2220

1915

1915





87

77


15 mdash



2016

1613

1815

1916





1816

1513

1816



3030

2222

2220 mdash 30





1311


151312

1312


1010

1411

87

87



2017

1515

1514

1815



Table 1 Continued






1212 mdash mdash 12




























3 Conclusions




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































Random approach



2318

2013

2016



3025

2017

2214

2116












9 10 77




Sida urens L 10050

2020

2020



1010

86












Table 1 Continued



1412 mdash 12









1715

1817

1816

1313





1210

1010



2220

1915

1915





87

77


15 mdash



2016

1613

1815

1916





1816

1513

1816



3030

2222

2220 mdash 30





1311


151312

1312


1010

1411

87

87



2017

1515

1514

1815



Table 1 Continued






1212 mdash mdash 12




























3 Conclusions




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Random approach



2318

2013

2016



3025

2017

2214

2116












9 10 77




Sida urens L 10050

2020

2020



1010

86












Table 1 Continued



1412 mdash 12









1715

1817

1816

1313





1210

1010



2220

1915

1915





87

77


15 mdash



2016

1613

1815

1916





1816

1513

1816



3030

2222

2220 mdash 30





1311


151312

1312


1010

1411

87

87



2017

1515

1514

1815



Table 1 Continued






1212 mdash mdash 12




























3 Conclusions




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued



1412 mdash 12









1715

1817

1816

1313





1210

1010



2220

1915

1915





87

77


15 mdash



2016

1613

1815

1916





1816

1513

1816



3030

2222

2220 mdash 30





1311


151312

1312


1010

1411

87

87



2017

1515

1514

1815



Table 1 Continued






1212 mdash mdash 12




























3 Conclusions




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued






1212 mdash mdash 12




























3 Conclusions




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































3 Conclusions




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Conclusions




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Approach to Identify Medicinal Plants A

Documents