Asian Journal of Pharmaceutical and Clinical Research

Vol. 4, Issue 3, 2011

ISSN - 0974-2441

ResearchArticle

TOXICITYSTUDIESOFEURYCOMALONGIFOLIA(JACK)BASEDREMEDIALPRODUCTS
MOHDFUATARAZAK*1,KOFIEAIDOO2
2

InstituteforMedicalResearch,JalanPahang,50588KualaLumpur,Malaysia DepartmentofBiomedicalSciences,GlasgowCaledonianUniversity,Scotland,UK Email:fuat@imr.gov.my

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ABSTRACT Eurycoma longifoliaJackisapopularherbthatiscommonlymixedwithotherreputedlynontoxicherbsinanontraditional(new)formulation.In this study, the effect of mixing herbs in a polyherbal product on the overall toxicity status of the polyherbal products was studied using three commercial polyherbal products containing mixture of E. longifolia with other herbs, a product which contains only E. longifolia and an authenticated E. longifolia. Eurycoma longifolia ortheherbalproductswereextractedwithmethanolchloroformandtheextractswerechallenged withhumancells,Hep2.EffectsoftheextractsonHep2cellsviabilitywasanalysedusing34,5dimethylthiazole2,5diphenyltetrazoliumbromide (MTT)assay.TheextractswerealsotestedformutagenicityusingAmestestemploying SalmonellaTA98and SalmonellaTA100.Cadmium,As,Pb, MnandCucontentoftheproductswereanalysedbyflameatomicabsorptionspectrometry.IC50ofcrudeextractofpure E.longifoliaandaproduct containingonly E.longifoliawas22.23gml1and50.00gml1,respectively.TwoofthepolyherbalproductshadIC50of15.20gml1and18.89g ml1,respectively.Allextracts,exceptaproductcontainingmixtureof E. longifoliaand Cistanche deserticola,wasnotmutagenic.Heavymetalswere detected in very low concentrations in all products. Under the condition of this study, it can be concluded that there is a risk of increased cytotoxicityandmutagenicityofextractof E. longifoliabasedremediescomparedtothetoxicityofremediescontainingsolely E. longifolia.Toxicity ofnewlyformulatedpolyherbalproductscannotbededucedfrom theinformationofthetoxicityofeachindividualcomponentofthepolyherbal products. Keywords:Cytotoxicity,Eurycomalongifolia,heavymetals,mutagenicity,polyherbalremedies. INTRODUCTION Eurycoma longifolia(Jack)isasmalltreethatgrowsalongthehilly slopes of the rainforests of Southeast Asia, including Indonesia, Malaysia,Thailand,Laos,CambodiaandVietnam 1.Ithasbeenused asamedicinalherbinSoutheastAsiamainlytoincreaselibidoand toalesserextenttoimprovegeneralhealth2.Othertraditionaluses includetreatmentofmalaria,bleeding,ulcersandhypertension 3. E. longifoliahasbeenmixedwithotherreputedlynontoxicherbsin many herbal products preparation for improving general health ratherthantoimprovestrengthandpower during sexualactivities alone. Many of the formulations are nontraditional 4. However, mixingof E. longifoliawithotherherbsmayincreasethechancesof E.longifolia basedherbal products being contaminated with toxic materials such as heavy metals and it may change the cytotoxicity statusduetocombinationofvariousplantsmetabolites. Although there have been a lot of E. longifolia preparations have beenmarketed,ineithersingleorcombinedpreparations,therehas notbeenmuchstudydoneontoxicityandsafetyof E.longifoliaorE. longifoliabased products in humans. Animal studies in mice have shownthatdoseof E.longifoliaof1500to2000mg/kgkilled50%of thetestanimals.Doseof600mg/kgdailywasassociatedwithsigns of toxicity such as increased weight of liver, kidneys, spleen, and testes, and toxicity increased to 100fold in intraperitoneal administrationascomparedtooral5. Theidentificationofproductswithchemicalsorcompoundscapable of inducing mutations is crucial in safety assessment since mutagenic compounds can potentially induce cancer 67. Gene mutations can bemeasuredin bacteria,wheretheycause achange in the growth requirements. The Ames test, which is conducted using Salmonella typhimurium, is a widely used bacterial assay for theidentificationofchemicalsthatcanproducegenemutations,and it shows a high predictive value with rodent carcinogenicity tests8. Pure E. longifolia extracts have been shown to be nonmutagenic at 250gml1 9. However, therewerelittleornoreport of mutagenic potential of extract of polyherbal products containing E. longifolia. Recentanalysisof100productsthatcontain E.longifoliainMalaysia showedthat36%and8%oftheseproductspossessed0.525.3and 10.6420.72 ppm of mercury4 and lead10 respectively and these values exceeded the limit set by the Malaysian government 11. Mercuryandleadarecytotoxicandmutagenic12. Inthisstudytheeffectofcombining E.longifoliawithotherherbson cytotoxicity was studied using tissue culture method. Mutagenic potentialoftheherbalproductswasdeterminedbymeasuringgene mutation in bacteria. Heavy metals of the products were also analysedtodeterminetheirpotentialastoxicantsintheproducts. MATERIALSANDMETHODS Herbalproducts Herbal products namely EG, PB, TA, and GE were purchased form retailshopsinKualaLumpur,Malaysia.ProductEGcontainssolelyE. longifolia while PB, TA and GE were mixed herbal products containing E. longifolia as major constituents. Table1 shows the constituents of the products and their intended usage. All herbal products were sold in bottles bearing the scientific name of the herbal plants used in the preparation of the products and production batch number. All products were fine powder (140 mesh) packed in capsules and were at least 6 months before the expirydate.Threebottlesofproductsfromdifferentbatchnumbers foreachherbalproductwereusedintheanalysis. SampleofE.longifolia EurycomalongifoliawascollectedbythestaffoftheForestResearch InstituteofMalaysia(FRIM)andauthenticatedbythebotanistofthe Institute. The specimen was deposited at FRIM and the root was driedandgroundtopowder. ExtractionofherbalproductsandE.longifolia Herbal samples (25 g) were extracted with 50 ml methanol/chloroform solution (1:1)13. The extract was filtered throughfilterpaper(Whatmanno.1)andthenevaporatedinrotary evaporatorat40oCtoapproximately3mlandthenstoredinadark glassvialat24oC.Thefiltratewasevaporatedtodrynessbeforethe cytotoxicityassayandmutagenicitytestwerecarriedout.Thedried filtratewasweighedanddissolvedinethanol. Cytotoxicitytest Humancelllines,Hep2,(purchasedfromTheEuropeanCollectionof CellCulture,Salisbury,UK)wereculturedasdescribedbyBetancur Garvisetal14.Thecellsweregrowninthe75cm2cellculturedishas amonolayerintheMinimumEssentialMediumwithEarlssaltsand glutamine (MEM, BioWhittaker BE12611F) and added with Non

Razaketal. AsianJPharmClinRes,Vol4,Issue3,2011,2327 essential amino acids (BioWhittaker Be1114E), Penicillin Streptomycin (100 IU/ml, Sigma P0906), Amphotericin B (0.25 g/ml, BioWhittaker BE17836E) and Foetal Bovine Serum (10%, BioWhittaker ). Culturedishwasincubatedat37oCin anincubator (5% CO2, 95% air) for 3 days or until the cells were 7080% confluence.Whenthecelllineswereapproximately80%confluence, the growth medium was removed by aspiration and the cells were rinsed with 5 ml phosphate buffer solution (PBS). The washing solution was discarded and 4 ml of trypsin (0.1%)/EDTA (0.04%) solutionwasadded. Thecellswereincubatedat37oCfor5minutesandthentheculture dishwastappedtodetachthecells.Phosphatebuffersolution(6ml) wasaddedandthecellsweretransferredintoacentrifugetube.The cellswerecentrifugedat1000rpmfor3min.Thesupernatantwas discardedand10mlMEMwasadded.Thecellswerecountedusing a Haemocytometer (Neubaur improved) and then diluted to approximately5x105cells/ml.Cellsuspension(195l)wasseeded onto a flat bottom 96well tissue microtitre culture plate and incubated at 37oC for 24 h. The crude extracts were diluted in the MEMinordertoachievetherequiredworkingstocksand5lofthe dilutedextractswereaddedtotheculturewellstogivefinalextracts concentrationof7.82,15.63,31.25,62.5and250gml1. Each concentration was done in quadruplicate and untreated cells servedasnegativecontrol.Allcultureplateswereincubatedin 5% CO2 at 37oC for 48 h15. Once the incubation was completed, the cytotoxicity of the herbal extracts was evaluated by using MTT 14. The supernatants were removed from all wells and 25 l of 34,5 dimethylthiazole2,5diphenyltetrazoliumbromide(MTT)inPBS(2 mgml1)wasaddedtoeachwellandtheplatewasincubatedin5% CO2 at 37oC for 2 hours. Then, 125 l Dimethylsulfoxide (DMSO) was added to dissolve any intracellular formazan crystals and agitatedfor15minonarotaryshaker.Absorbancewasmeasuredat 492nminanELISAplatereader. The absorbance reading of test materials and untreated cells (control)werecorrectedbysubtractingthebackgroundabsorbance from wells containing no cells (blank). The percentage of cytotoxicitywascalculatedas,[(AB)/A]x100,whereAisthemean opticaldensityofcontrolwellsandBistheopticaldensityofwells with plant extracts. The IC50 concentration, determined as an effective dose to reduce the growth to 50% of the control value (50%inhibitionofgrowth),wascalculatedbylinearinterpolation16. Inthiscalculation,two test points that bracket 50%inhibition was determined and the two percentages and the two concentrations wereinsertedintothefollowingformula: (High%Low%) IC50=[ x(Highconcentration (50%Low%)Lowconcentration)] +Low concentration The IC50 was used to rank the potential risk of acute toxicity of herbalproductsextracts. Mutagenicitytest
% of cell growth inhibition

inoculatedinnutrientbrothandincubatedat37oCfor1824h.The culture broth (5 l) was inoculated into the bottles and mixed thoroughly. The content of each bottle was transferred into a multichannelreagent boatand 200 laliquots of the mixture were dispensed into each well of a 96well microtitration plate using a multichannelpipette.Theplatewasplacedinanairtightplasticbag topreventevaporationandincubatedat37oCfor4days. Theblankplatewasobservedfirstandtherestoftheplateswere read only when all wells in the blank plate were coloured purple indicating the assay was not contaminated. The background, standard and test plates were scored visually and all yellow, partiallyyelloworturbidwellswerescoredaspositivewhilepurple wells were scored as negative. Numbers of all positive wells were recorded. The background plate (no herbal extract or standard mutagen added) showed the level of spontaneous or background mutationofthetestbacteria.Theextractwasconsideredtoxictothe teststrainifallwellsinthetestplateshowedpurplecoloration.For aherbalextracttobemutagenic,thenumberofpositivewellhadto bemorethantwicethenumberofpositivewellinthebackground plate(spontaneousmutation)18. Heavymetalanalysis Herbal powder was ashed at 525oC overnight. After cooling, 20% HClwasaddedtothesamples.Sampleswerefilteredandthefiltrate was diluted with deionised water. Metals in the filtrate were analysed using Flame Atomic Absorbtion Spectrometry (Perkin Elmer) with AirC2H2 flame. Cathode lamps wavelength and slit width used were as recommended by the manufacturer. The recovery efficiency of this method was carried out by spiking 1 g herbalpowderwiththecombinedstandards.Thespikedsamplewas placed in an oven at 80oC until dried followed by dry ashing and sampleswerepreparedasdescribedabove. RESULTS Figure 1shows concentrationresponse curvesofHep2 cellto72 h exposurewiththeherbalproductsextractsand E. longifolia.Table3 showsthatIC50of E longifolia(EL)andtheproductcontainingonly E. longifolia (EG) was 20.87 2.23 g/ml and 50.00 9.41 g/ml, respectively.ConcentrationsofIC50forextractsTA,PBandGEwere 15.20 2.43 g/ml, 55.77 3.69 g/ml and 18.89 7.18 g/ml respectively.Table4showsthatnoneoftheproductswasmutagenic whentestedon SalmonellastrainTA98whilemixtureof E.longifolia and C. deserticola (TA) was mutagenic when tested on Salmonella strainTA100.Cadmium,Pb,AsandCuwerenotdetectedinproduct EG,TAandGE(Table5).Cadmium,AsandCuwerefoundtobe0.2 ppm or less in PB and E. longifolia. All products contain Mn of not morethan12ppmandPbwasdetectedonlyinE.longifolia.

100 90 80 70 60 50 40 30 20 10 0 0 50 100 150 200 250 Concentration of extract (ug/ml)

A commercial test kit, the MutaChromplate, was purchased from EnvironmentalBiodetection ProductsIncorporation(EBPI,Ontario, Canada). This test kit was based on the validated Ames bacterial reversemutationtest17butwasperformedentirelyinliquidculture (fluctuation test). The following chemicals were purchased from EBPI:DavisMingiolisalt(5.5timesconcentrated),Dglucose(40%, w/v), bromocresol purple (2 mg/ml), Dbiotin (0.1 mg/ml), and L histidine(0.1mg/ml).Twosterilestandardmutagensweresodium azide (NaN3, 0.5 g/100 l) and 2nitrofluorene (2NF, 30 g/100 l). Reagent mixture comprising of DavisMingioli salt (21.62 ml), D glucose(4.75ml),bromocresolpurple(2.38ml),Dbiotin(1.19ml) and Lhistidine (0.06 ml) were mixed aseptically in a sterile bottle. Reagentmixture,herbalextract,steriledistilledwaterandstandard mutagen were mixed in several bottles at the amount indicated in Table 2. Two mutant strains, S. typhimurium TA98 and S. typhimurium TA100 were provided by EBPI. The bacteria were

TA GE PB EG EL

Fig.1:ConcentrationresponsecurvesofHep2cellto72h exposurewithchloroformmethanolextractsofE. longifolia(EL),aproductcontiningsolelyE.longifolia(EG) andthreepolyherbalproductscontainingE.longifolia(TA, GEandPB).Eachpointsofthecurverepresentthemean valueof3samplesandstandarddeviations.

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Razaketal. AsianJPharmClinRes,Vol4,Issue3,2011,2327 Table1:Herbalformulations,dosage,andintendedusageofsomeherbalproductscontainingE.longifoliapurchasedfrom retailshopsinKualaLumpur,Malaysia. Herbal Constituents Plantpartsuseda %inthe Dosage Intendeduse preparation formulation EG Eurycomalongifolia Radix 100 600mg,twicedaily To increase sexual staminaenergyinman PB Eurycomalongifolia Radix 40 1225mg, Increase passion in Curcumaesp Rhizoma 10 3timesdaily women Honey Whole 50 TA Eurycomalongifolia Radix 50 1200mg, To increase sexual Cistanchedeserticola Herba 50 twicedaily staminaenergyinman Eurycomalongifolia n.i 30.4 600mg,twicedaily For energy, increase Taccapalmate n.i 21.4 sexual stamina and Zingiberisaromaticae n.i 17.9 menshealth. 14.3 Zingiberisofficinale n.i 16 Helminthoctachyszeylanica. n.i a Plant parts used: n.i, not indicated; Radix, the root; Rhizoma, rhizome or a creeping horizontal stem generally bearing roots on its underside;Herba,theaerialpartsortheabovegroundpartsofplantswhichmayincludetheflower,leaf,andthestem; Table2:Setupofthefluctuationassay Treatment Blank Background Standardmutagen Testsample Table3:ConcentrationofextractsofherbalproductscontainingE.longifoliaandextractsofE.longifoliathatcaused50%decreasein Hep2cellviability(IC50)ascalculatedbylinearinterpolation.Sign#indicatessignificantlydifferentfromEG E.longifolia/Polyherbalproducts EG TA PB GE E.longifolia(EL) Table4:MutagenicactivityofextractofE.longifoliaandsomepolyherbalproductscontainingE.longifoliaintheAmesfluctuationtest. Allextractsweretestedat250gml1 TestonSalmonellaTA98 TestonSalmonellaTA100 Background 2Nitrofluorene(1.5g/ml) NaN3(0.025g/ml) EC PB TA GE E.longifolia Number of positive wells / 96wells# 8 93 7 4 4 4 8 Results (p0.05) Mutagenic Nonmutagenic Nonmutagenic Nonmutagenic Nonmutagenic Nonmutagenic Number of positive wells/96wells# 14 93 20 8 28 14 19 Results (p0.05) Mutagenic Nonmutagenic Nonmutagenic Mutagenic Nonmutagenic Nonmutagenic IC50(gml1) 50.009.41 15.202.43# 55.773.69 18.897.18# 20.872.23# MutagenStandard 0.1 Herbalextract 0.005 Volumeadded(ml) Reagentmixture DeionisedWater 2.5 2.5 2.5 2.5 17.5 17.5 17.4 17.5 Salmonellateststrain 0.005 0.005 0.005 GE

#Averagevalueof3samplesofdifferentbatchesofproducts Table5:Heavymetalscontent(ppm)*ofE.longifoliaandsomepolyherbalproductscontainingE.longifolia. Cd Pb As Herbalproducts EG ND ND ND PB 0.200.06 ND 0.010.00 TA ND ND ND GE ND ND ND E.longifolia 0.200.06 1.670.04 0.080.20 *Averageconcentrationstandarddeviation(n=3);ND=notdetected Cu ND 0.200.01 ND ND 0.200.05 Mn .500.01 11.000.01 3.000.01 6.670.02 4.000.07

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Razaketal. AsianJPharmClinRes,Vol4,Issue3,2011,2327 DISCUSSIONS Table3showsthatIC50fortheproductcontainingonly E. longifolia (EG)was50.009.41g/mlandthisisconsideredtobecytotoxic. Cytotoxicity of E. longifolia has been reported 9,19. However, E. longifoliahasbeenconsumedorallyforcenturieswithoutanyreport of adverse effect. Eurycoma longofolia extract was found to be 100 times less cytotoxic if administered orally than intraperitoneal in experimentalmice.Since E. longifoliaisnormallytaken orally from water decoction4, its cytotoxicity has been reduced possibly by elimination of cytotoxic substance in the liver or poorly absorbed intothebloodcirculation5.HigherIC50of E. longifoliainproductEG than pure E. longifolia powder suggest that E. longifolia used in commercial products were of different quality or were not standardisedandmayaffecttheeffectivenessoftheremedies. MixingCurcumaesp.withE.longifolia(1:4)asinPBdidnotresultin a product with high cytotoxocity property (IC50 = 55.77 3.69 g/ml).However,adding C. deserticolato E. longifolia(1:1)asinTA resultedinaproductwithhighcytotoxicstatus(IC50=15.202.43 g/ml).AcrudeextractwithIC50of<20g/mlisconsideredhighly cytotoxic20. High cytotoxicity of the latter mixture could not be explainedduetocytoxicityof C. deserticolaas C. diserticola hasnot beenreportedhighlycytotoxic. Combination of E. longifolia with of T. palmata, Z. aromaticae, Z. officinaleand H. zeylanica(reducingtheproportionof E.longifoliain the remedy to only 30%) as in GE resulted in a high cytotoxicity product (IC50 = 18.89 7.18 g/ml). None of T. palmata, Z. aromaticae, Z. officinaleand H. zeylanicahasbeenconsideredhighly cytotoxic. Thus,highercytotoxicityofTAandGEascomparedtoEGcould be duetointeractionbetweenthephytochemicalconstituentsoftwoor moreherbalextractsinTAandGEsynergisticallyresultinginhigher cytotoxic extract. Although IC50 of PB is comparable to EG, concentrationof E. longifoliainPBis50%lessindicatingthatthere mightbeapositiveinteractionof E. longifoliaand Curcumaespecies towardstheincreaseofIC50.Thesefindingsarenotinlinewiththe popular believed that mixture of two or more noncytotoxic herbs shouldnotbecytotoxic.Theseresultssupportthesuggestionbythe WorldHealthOrganizationthatacombinationofingredientsshould be considered as herbal medicine of uncertain safety and also regardsthisproductasanewsubstancewheresafetydataarethen required21. Inthisstudyproductcontaining E. longifoliaalonewasfoundtobe not mutagenic using Salmonella strain TA98 and TA100. Extract of E.longifolia was reported to be nonmutagenic9. However, E. longifolia was reported to contain mutagenic and genotoxic substance alkaloids carboline 2223. This study showed that some componentsof E. longifolia thatcouldbemutagenicareinverylow concentration. However, products containing E. longifolia and C. deserticola, TA, was found to be mutagenic. There were little or no reports of mutagenicity of C. deserticola. These results suggested that the mutagenicity of TA could be due to the effect of herbal combination and warrant further study on the combined effect of herbalplantsinmixedherbalpreparationonmutagenicity. Arsenic,CdandPbaretoxicforhumanbiosystemsevenatlowlevel of intake24. Lead may induce reduced cognitive development and may be associated with cardiovascular disease while Cd can affect kidney function, induce skeletal damage and reproductive deficiency25.Because of their toxicityandthe fact that theyare not requiredbythebody,levelofAs,CdandPbpermissibleinfoodand herbalproductshasbeenset. The World Health Organization prescribes limits for As, Cd and Pb for various medicinal plants of not more than 5, 0.3, and 10 ppm respectively26.NoneoftheherbalproductsexceptPBcontainedAs, CdorPb.InPB,PbwasnotdetectedwhileCdandAsconcentration werelessthanthepermissiblelimits.Manganeseismoreprevalent in the products but presence in low concentration. Despite of containing most of the metals detected in this study, PB was not mutagenic and less cytotoxic than the rest of the products. These results suggest that there is no toxicity risk of consuming these productsas faras heavymetalsisconcernedandheavymetals did not contribute to the cytotoxicity or mutagenicity of the products studied. CONCLUSIONS Under the conditions of this study, it can be concluded that some herbal products containing E. longifolia were highly cytotoxic. This cytotoxicity could be the result of interaction of phytochemicals in theherbalmixture.Cytotoxicitystatusofpolyherbalproducts could notbededucedfromthereportedcytotoxicitystatusofeachherbin the polyherbal products. Phytochemical interaction may have also contributed to mutagenicity of a product containing E. longifolia with a nonmutagenic herb. Heavy metals such as Lead, Cadmium and Arsenic were detected in some proucts and E. longifolia in concentrationthatdidnotexertmutageniceffect. ACKNOWLEDGEMENT The authors wish to thank the Director General of Health Malaysia andtheDirectorofInstituteformedicalResearchforthepermission to publish this paper. This study was financially supported by the Public Service Department of Malaysia through the Federal GovernmentTrainingScholarshipProgram. REFERENCES 1. KuoPC,ShiLH,DamuAG,SuCR,HuangCH,KeCH,WuJB,Lin AJ,BastowKF,LeeKH,WuTS.Cytotoxicandantimalarial carboline alkaloids from the roots of Eurycoma longifolia. J NatProd2003;66:13241327. 2. 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