Phytochemical and antimicrobial activity of Swietenia mahagoni crude methanolic seed extract
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274 Tropical Biomedicine 26(3): 274–279 (2009) Phytochemical and antimicrobial activity of Swietenia mahagoni crude methanolic seed extract Sahgal, G., 1 Ramanathan, S., 1 Sasidharan, S., 2, * Mordi, M.N., 1 Ismail, S. 1 and Mansor, S.M. 1 1 Centre for Drug Research, University Sains Malaysia, 11800 Penang, Malaysia. 2 Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. *Corresponding author E-mail: [email protected] Received 24 June 2009; received in revised form 26 August 2008; accepted 31 August 2009 Abstract. The present study was designed to evaluate the antibacterial activities of Swietenia mahagoni crude methanolic (SMCM) seed extract. The antimicrobial activity of the oily extract against Gram-positive, Gram-negative, yeast and fungus strains was evaluated based on the inhibition zone using disc diffusion assay, minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) values. The crude extract was subjected to various phytochemicals analysis. The demonstrated qualitative phytochemical tests exhibited the presences of common phytocompounds including alkaloids, terpenoids, antraquinones, cardiac glycosides, saponins, and volatile oils as major active constituents. The SMCM seed extract had inhibitory effects on the growth of Candida albicans, Staphylococcus aureus, Pseudomonas aeroginosa, Streptococcus faecalis and Proteus mirabillase and illustrated MIC and MBC values ranging from 25 mg/ml to 50 mg/ml. INTRODUCTION Swietenia mahagoni (Linn.) Jacq., (Meliaceae), is a large, deciduous, and economically important timber tree native to the west Indies. This timber tree is mainly cultivated at tropical zone, such as India, Malaysia, and Southern China (Mulholland et al., 2000). Swietenia mahagoni is a valuable timber tree closely related to the African genus Khaya and one of the most popular traditional medicines in Africa. The fruit is a brown, egg- to pear-shaped capsule about 6 to 10 cm long. When the fruit is fully ripe, the woody shell splits into five sections from the base upward and falls off to release the seeds. The decoction of the bark of these mahoganies is extensively used as febrifuge, which could be associated with its use as an antimalarial drug. Swietenia mahagoni seeds have been applied as a folk medicine for the treatment of hypertension, diabetes, and malaria (Nagalakshmi et al., 2001). The seeds have also been reported to have therapeutic value for treatment of cancer, amoebiasis, coughs, chest pains and intestinal parasitism. The biologically active ingredients, tetranortriterpenoids and fatty acids are considered to be responsible for these therapeutic effects (Bascal et al., 1997). Infectious diseases are the world’s leading cause of premature deaths, killing almost 50 000 people every day. In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world (Ahmad & Arina, 2001). In general, bacteria have the genetic ability to transmit and acquire resistance to drugs. To overcome the problem of antibiotic resistance, medicinal plants have been extensively studied as alternative treatments for diseases. According to World Health Organization (Santos et al., 1995) medicinal plants would be the best source to obtain a
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Phytochemical and antimicrobial activity of Swietenia mahagoni crude methanolic seed extract
Transcript of Phytochemical and antimicrobial activity of Swietenia mahagoni crude methanolic seed extract
274TropicalBiomedicine26(3):274279(2009)PhytochemicalandantimicrobialactivityofSwieteniamahagoni crude methanolic seed extractSahgal, G.,1 Ramanathan, S.,1 Sasidharan, S.,2,* Mordi, M.N.,1 Ismail, S.1 and Mansor, S.M.11 CentreforDrugResearch,UniversitySainsMalaysia,11800Penang,Malaysia.2 InstituteforResearchinMolecularMedicine(INFORMM),UniversitiSainsMalaysia,11800Minden,Penang,Malaysia.*CorrespondingauthorE-mail:[email protected];receivedinrevisedform26August2008;accepted31August2009Abstract.ThepresentstudywasdesignedtoevaluatetheantibacterialactivitiesofSwieteniamahagonicrudemethanolic(SMCM)seedextract.TheantimicrobialactivityoftheoilyextractagainstGram-positive,Gram-negative,yeastandfungusstrainswasevaluatedbasedontheinhibitionzoneusingdiscdiffusionassay,minimalinhibitionconcentration(MIC)andminimalbactericidalconcentration(MBC)values.Thecrudeextractwassubjectedtovariousphytochemicalsanalysis.Thedemonstratedqualitativephytochemicaltestsexhibitedthepresencesofcommonphytocompoundsincludingalkaloids,terpenoids,antraquinones,cardiacglycosides,saponins,andvolatileoilsasmajoractiveconstituents.TheSMCMseedextracthadinhibitoryeffectsonthegrowthofCandidaalbicans,Staphylococcusaureus,Pseudomonasaeroginosa, Streptococcus faecalis and Proteus mirabillase and illustrated MIC and MBC valuesranging from 25 mg/ml to 50 mg/ml.INTRODUCTIONSwieteniamahagoni(Linn.)Jacq.,(Meliaceae),isalarge,deciduous,andeconomicallyimportanttimbertreenativeto the west Indies. This timber tree is mainlycultivatedattropicalzone,suchasIndia,Malaysia,andSouthernChina(Mulhollandetal.,2000).SwieteniamahagoniisavaluabletimbertreecloselyrelatedtotheAfricangenusKhayaandoneofthemostpopular traditional medicines in Africa. Thefruit is a brown, egg- to pear-shaped capsuleabout 6 to 10 cm long. When the fruit is fullyripe, the woody shell splits into five sectionsfrom the base upward and falls off to releasethe seeds.Thedecoctionofthebarkofthesemahoganies is extensively used as febrifuge,which could be associated with its use as anantimalarialdrug.Swieteniamahagoniseeds have been applied as a folk medicinefor the treatment of hypertension, diabetes,and malaria (Nagalakshmi et al., 2001). Theseedshavealsobeenreportedtohavetherapeuticvaluefortreatmentofcancer,amoebiasis,coughs,chestpainsandintestinal parasitism. The biologically activeingredients,tetranortriterpenoidsandfattyacidsareconsideredtoberesponsibleforthesetherapeuticeffects(Bascaletal.,1997).Infectiousdiseasesaretheworldsleadingcauseofprematuredeaths,killingalmost50000peopleeveryday.Inrecentyears, drug resistance to human pathogenicbacteria has been commonly reported fromall over the world (Ahmad & Arina, 2001). Ingeneral, bacteria have the genetic ability totransmit and acquire resistance to drugs. Toovercometheproblemofantibioticresistance,medicinalplantshavebeenextensively studied as alternative treatmentsfordiseases.AccordingtoWorldHealthOrganization (Santos et al., 1995) medicinalplants would be the best source to obtain a275varietyofdrugs.Thesubstancesthatcaneither inhibit the growth of pathogens or killthemandhavenoorleasttoxicitytohostcellsareconsideredcandidatesfordevelopingnewantimicrobialdrugs.Thesystematicscreeningofmedicinalplantswiththepurposeofdiscoveringnewbioactive compounds is a routine activity inmanylaboratoriesdevotedtobiomedicalresearch.Therefore,S.mahagonicrudemethanolicseedextractisusedforthescreeningofvariousmicroorganismstodiscover new antimicrobial compounds.MATERIALS & METHODSMaterialsDragendorffreagent,sodiumhydroxide(NaOH)andseveralotherreagentsforphytochemical analysis were homemade andFolin-Ciacaleu reagent was purchased fromSigma-Aldrich,UnitedStates.Methanol(Analyticalgraded)andsulphuricacid(H2SO4)werepurchasedfromMerck,Germany.MuellerHintonagar,MuellerHintonbroth,nutrientagar,nutrientbroth,Saboarouddextroseagar,andSaboarouddextrosebrothwerepurchasedfromHimedia, India.Plant MaterialsThe S. mahagoni seeds were collected in thestate of Penang, Malaysia. An initial qualityevaluation of the plant material was carriedouttovalidateitsauthenticity.Theauthenticityworkwascarriedoutbybotanist from School of Biological Sciences,Universiti Sains Malaysia.Sample preparationTheseedswerewashedwithrunningtapwatertoremovedirtpriortothedryingprocess.Theseedswerecutintosmallpiecesanddriedat40Cforaweektoremovethemoisturecontent.Theseedswerepowderedusingablender(NewDeluhe, Suruchi, India). The powdered seedswere extracted with methanol by means ofmaceration method. The extract was filteredthroughfilterpaper(WhtamanNo.1).Thefiltrate was collected and concentrated in arotary evaporator (RII0 Buchi, Switzerland)at 40C. The concentrated extract was driedinanovenat40Cforthreedaystoobtainconsistent weight and freeze dried for 2 days.Thesamplewasstoredunderrefrigeration(-20C) condition for further analysis.PhytochemicalanalysisThecondensedS.mahagonicrudemethanolic(SMCM)seedextractwillbeusedforpreliminaryqualitativescreeningofphytochemicalssuchasalkaloids(Dragendorff test), flavonoids (NaOH test),glycosides(Keller-Kiliani,conc.H2SO4),lignins(Lignintest),phenols(phenoltest),saponins(Foamtest),sterolsandterpenes(Liberman-Burchard)andtannins(Sumitraet al., 2006).Antimicrobial assaySMCMseedextractwillbesubjectedtoantimicrobialassaysincludingagardiscdiffusion test, and broth dilution assay.Microbial strainsTwentypathogenicmicroorganisms(localisolates)culturewasusedfortheantimicrobialassays.Thebacteriastrainswere grown in 50 ml of nutrient broth at 37Cand maintained in nutrient agar slant at 4C.However the fungus strains were grown andmaintainedinSabourauddextroseagar(SDA). The Mueller Hinton agar (MHA) wasalso used for the determination of MIC andMBC.Agar disc diffusion assayThe disk diffusion (Kirby-Baurer) technique,which is of the recommended standards oftheNationalCommitteeforClinicalLaboratory Standards (NCCLS), was used forantimicrobial test. An overnight suspensioncultureoffifteenmicrobialstrainswasspreadonMHAmedia.Sterilediscswerepreparedandplacedontheculturespreadagarmedia.ThediscswereimpregnatedwiththeSMCMseedextractinrangeofvarious concentrations. Methanol was usedas negative control and standard antibioticsincludingchloramphenicolandamoxicillinaspositivereferencetodeterminethesensitivityofthestrains.Theinoculated276plateswereincubatedat37Cfor24hforbacterial strains and 48 h for fungal strains(Karamanetal.,2003).Theantimicrobialactivitywasevaluatedbymeasuringdiameteroftheinhibitionzonearoundthedisc (Table 2).Minimuminhibitionconcentration(MIC)TheMICvaluewasstudiedforthemicroorganisms,whicharedeterminedassensitivetotestsamples(SMCMseedextract) in disc diffusion assay. The inoculaof microorganisms were prepared from 18 hnutrient broth cultures. In this method, thebrothdilutiontechniquewasutilizedwheretheplantextractwaspreparedtothehighestconcentrationof100mg/ml(stockconcentration)inmethanolandseriallydiluted(two-fold)toaworkingconcentration ranging from 0.78 mg/ml to 50mg/mlusingnutrientbrothandlaterinoculated with 1 ml suspension of the testorganisms. The positive control was nutrientbrothwithstandardreferenceantibioticsandinoculumsandnegativecontrolwasthenutrientbrothandinoculums.After18hoursofincubationat37C,thetesttubeswereobservedforturbidity.TheleastconcentrationwherenoturbiditywasobservedandnotedastheMICvalue.Thetesttubeswerevortexgentlytomixthecontent and incubated at 37C for 24 h (Kueteetal.,2008).MICisdefinedasthelowestconcentrationwherenovisibleturbidityisobservedinthetesttube(bacteriostaticconcentration).Minimumbacterialconcentration(MBC)TheMBCisdefinedasthelowestconcentration where no bacterial growth isobserved (bactericidal concentration). ThiswasdeterminedfromthebrothdilutionresultingfromtheMICtubesbysub-culturingtoantimicrobialfreeagarasdescribedinReubenetal.(2008).Inthistechnique,thecontentsofthetesttubesresultingfromMICwasstreakedusingasterile wire loop on agar plate free of bacteriaand incubated at 37C for 24 h for bacteriaand48hforfungalstrains.Thelowestconcentration of the extract which showedno bacterial growth was noted and recordedas the MBC.RESULTS & DISCUSSIONSMCMseedsextractwassubjectedtovariousstudiesasmentionedabove.Preliminaryqualitativephytochemicalscreening reveals the presence of alkaloids,terpenoids,antraquinones,cardiacglycosides,saponins,andvolatileoilsinSMCM seeds extract while test for tannins,flavonoidsandsteroidsdemonstratednegative responses (Table 1). Initial work ontheSMCMseedextractforantimicrobialactivitywascarriedoutinourlaboratory.TheantimicrobialactivitiesofthisextractagainstGrampositiveandGram-negativebacteria,yeastandfunguswereexaminedintermsofdiskdiffusion,MICandMBCassays (Table 2 and Table 3).The diffusion test showed that the SMCMseedextractwasactiveagainst5Grampositive,9Gram-negativebacteria,andCandidaalbicans(fungus).HowevernosignificantactivityshowedagainstSaccharomycescerevisiae(yeast),Rhizopussp.,Penicilliumsp.,andAspergillusfumigatus.Wefoundthisextract more active compared to amoxicillinantibiotic(positivecontrol)whentestedagainstBacillusthuringiensisandPseudomonas aeroginosa at 1 mg/ml. ThoseGrampositiveandGram-negativebacteriaand C. albicans were further evaluated forMIC and MBC determination.The MIC wasdefined as the lowest value concentration oftheextract,whichisinhibitingthegrowthofmicroorganisms.TheMBCisthesignificanceconcentrationoftheextractwhichisabletokillthemicroorganisms.TheMICandMBCrangingfrom12.5to50mg/ml and 25 to 50 mg/ml for the SMCM seedextract respectively. The standard antibioticchloramphenicol and amoxicillin (bacteria)and miconazole nitrite (fungal) inhibited thegrowth of the test bacterial and fungal strainsatthesmallestconcentration.ThelowestMIC and MBC recorded were 12.5 mg/ml forStreptococcussp.,Staphylococcusaureus,277Table1.ThePhytochemicalsubstancesincrudemethanolicSwieteniamahagonicrudemethanolicseedsextract(SMCMseedextract)Phytochemicals Observation OutcomesAlkaloids Extractchangeintoyellow-orangecolour +Terpenoids Extractcolourchangedtopink +Antraquinones Formationoftomatoredcolour +Cardiacglycosides ExtractchangeintoGreen-bluecolour +Saponins Formationoffrothing +Volatileoils Whiteprecipitates +Tannins Nobluecolourindication Flavonoids Nopinkcolourindication Steroids Nogreencolourvisualized Notes:+Swieteniamahagonihasthephytochemicalsubstances,Swieteniamahagonidoesnothasthephytochemicalcompounds.Table2.Antibacterialactivityincludingdiskdiffusion,MICandMBCassaysofSwieteniamahagonicrudemethanolicseedextractsagainstvarioustypesofbacterialandfungalstrainsa The zone of inhibition is mean of three replicates for each microbial strain. Methanol does not give any affect against the microorganisms.NA- No activity against SMCM seed extract.Zone of inhibition (mm) of various concentration of extracts, Bacteria strainsmethanol and antibioticsa100 10 1 Chloram-Amoxicillin MethanolMIC MBCmg.ml-1mg.ml-1mg.ml-1phenicol(mg/ml)a(mg/ml)aGram positive strainsBacillus subtilis 11.2 2.4 09.6 1.3 09.0 0.7 24.0 1.4 25.0 2.8 NA NABacillus cereus 11.8 0.8 11.0 0.7 09.6 0.9 19.0 2.8 14.5 3.5 NA NABacillusthuringiensis 15.7 4.0 13.0 0.6 10.0 0.0 19.0 1.0 09.5 0.7 NA NAEnterococcusfaecalis 11.6 1.7 10.6 1.3 09.5 0.6 20.0 2.0 31.0 5.3 12.5 25Staphylococcus aureus 12.0 1.2 10.8 1.1 09.8 1.7 24.0 0.0 30.5 0.7 12.5 25Gram negative strainsEscherichia coli 12.0 0.0 11.7 0.6 11.7 0.6 16.0 1.0 30.7 1.2 NA NASalmonella typhi 10.6 0.9 09.6 0.9 10.0 1.0 20.7 3.0 11.0 2.0 NA NAShigella sonnei 15.5 2.1 11.8 1.5 11.0 3.0 20.7 0.6 14.0 4.0 NA NAKlebsiella pneumoniae. 10.2 0.8 09.4 0.5 08.3 1.0 19.7 0.6 18.3 0.8 NA NAPseudomonasaeroginosa 19.0 1.0 17.0 0.2 15.3 3.6 15.0 0.0 10.0 0.0 12.5 25Proteus mirabillase 15.3 2.1 11.8 1.5 11.0 3.0 20.7 0.6 14.0 4.0 25 50Enterobacteraeroginosa 12.0 1.2 10.5 0.5 09.3 0.4 23.0 0.0 16.0 0.0 NA NAAzatobacter brandi 14.0 4.2 14.5 4.9 09.5 0.7 24.5 2.3 29.5 1.1 NA NAAzospirillumlipoferum 12.2 1.1 10.8 1.6 10.0 1.2 21.0 1.7 15.3 0.6 NA NAHerbaspirillumsp. 14.0 1.4 12.0 1.6 10.3 1.3 16.0 2.6 39.0 1.7 NA NAFungal strains Myconazole 1 mg.ml-1MethanolCandida albicans 19.0 1.7 16.0 1.6 16.0 1.1 16.7 3.5 12.5 25Saccharomycescerevisiae NA NA NA 17.0 1.5 NA NARhizopus sp. 12.8 0.5 11.0 0.5 09.1 1.2 18.0 1.7 NA NA NAPenicilliumsp. NA NA NA 18.0 0.3 NA NA NAAspergillusfumigatus NA NA NA 19.0 1.1 NA NA NAP. aeroginosa, and C. albicans respectively.Similar study was also reported by Goun etal.(2003).TheyusedS.mahagoniseedextractextractedwithmethylenechlorideandmethanolbeforetestingtheirantimicrobialactivityagainst10microbialspecies, including four bacterial pathogens,Escherichiacoli,S.aureus,Xanthomonas278campestris, Bacillus subtilis; one yeasts, C.albicans; and five molds, Pythium ultimum,Rhizoctoniasolani,Sclerotiumrolfsii,A.fumigatusandPythiumparasiticausingthediskdiffusionassaytechnique.Themethylenechlorideextractwasfoundtobemosteffectiveandinhibited7microorganismstested.Incontrast,themethanolextractwasfoundnotactiveagainstallofthetestedmicroorganismsexcept for R. solani.Besides the seed, other parts of this plantsuch as leaf and bark have also been testedforantimicrobialactivitiesbyusingchloroform and ethyl acetate by Haque et al.(2009).Theextractsweretestedforantibacterialactivitiesagainst12(4GrampositivenamelyBacillusmegaterium,B.subtilis, S. aureus andStaphylococcus luteaand8GramnegativenamelySalmonellaparatyphi,Salmonellatyphi,Vibrioparahemolyticus, Vibrio mimicus, E. coli,Shigella dysenteriae, P. aeruginosa and S.boydii)humanpathogenicbacteria.Thechloroform and ethyl acetate extracts wereusedinconcentrationof500gdisc-1.Thechloroform extracts of leaf and bark showedactivity against most of the test bacteria. ButthechloroformextractofseedshowedactivityagainstonlyB.megaterium,S.paratyphi, S. dysenteriae, P. aeruginosa andS. boydii. The highest activity of chloroformextractwasrecorded16mmdisc-1incaseof seed against S. paratyphi. A good activitywas recorded by the ethyl acetate extract ofbarkagainstallthetestorganismsincomparisontoothers.AmongtheethylacetateextractsofS.mahagoni,barkshowedhighestactivityandwasfound18mmdisc-1againstB.megaterium,S.paratyphi and P. aeruginosaThis findings show that the extract mightbepotentialanticandidalandantibacterialagentsevenagainstsomeresistantstrainsuch as S. aureus. However, the SMCM seedextract was more active against C. albicansstraincomparedtotheotherscreenedbacterialandfungalstrains.Theinhibitionzoneforthisfungalyeastis16.01.1at1mg.ml-1.TheMICandMBCvaluesare12.5mg/ml and25mg/ml respectivelyforC.albicans.Thepresenceofbioactivecompoundsincludingthephytochemicalsubstancesintheextracthasbeenassociated to antibacterial activities and thushascurativepropertiesagainstpathogens.SeverallimonoidswerepreviouslyisolatedfromS.mahagoni(Govindacharietal.,1999).IsolationoftriterpenesalsowasreportedonS.mahagoni(Ekimotoetal.,1991).Theantibacterialactivityoftheisolated limonoids 1 and 2 has been reportedbyShahidurRahmanetal.(2009)againstpathogenic bacteria. They used the isolatedlimonoids 1 and 2 to test their antimicrobialactivity against eight multiple-drug-resistantbacterial strains (clinical isolates) includingfourGrampositive(GroupAbhaemolyticS.aureus,S.aureus,S.pneumoniaeandHaemophillusinfluenzae)andfourGram-negative (E. coli, Klebsiella pneumoniae, S.typhi,andS.paratyphi)strainsbytheconventionaldiscdiffusionmethod.Whileboth compounds were active against all testorganisms,compound2showedoverallmore potent activity than compound 1. Whilethemostpotentactivityoflimonoid1wasobserved against H. influenzae, S. typhi, andS.paratyphi,limonoid2wasmostactiveagainst Streptococcus pneumoniae, S. typhi,andS.paratyphi.ThelowestactivitywasobservedagainstK.pneumoniaeforbothcompounds.Moreover,theantimicrobialactivity of triterpenes isolated from Drypetesinaequalis was reported by Awanchiri et al.(2009)againstagainstpathogenicbacteriaincludingoneGrampositive(S.aureus)andfour Gram negative (E. coli, S. typhi, S.dysenteriae,K.pneumoniaeandP.aeruginosa)strains.Hence,itispossiblethatthesetwocompoundsweremainlyresponsiblefortheobservedantimicrobialeffects in this study.Therefore,SMCMseedextractcouldserveasaremedytosuchresistanceandinfectiousmicrobialstrains.Plant-basedantimicrobialshavevastmedicinalprospectiveastheycanservethepurposewithminorsideeffectsthatarehabituallyassociatedwithsyntheticantibiotics.Thisstudy also provides some validity for the useofthehigherplantpartsintraditionalmedicineandasasourceofchemotherapeuticagents.279Inconclusion,SMCMseedextractspossessabroadspectrumofactivityagainstapanelofbacteriaandfungusrespectively. These promising extracts openthepossibilityoffindingnewclinicallyeffectiveantibacterialcompounds.Furtherpurification of the active compounds and invivoevaluationofantimicrobialactivityalongwithtoxicitystudiesoftheextractsfromSMCMarethereforesuggestedforfurther studies.REFERENCESAhmad, I. & Arina, Z.B. (2001). Antimicrobialandphytochemicalstdieson45indianmedicinalplantsagainstmulti-drugresistanthumanpathogen.JournalofEthno-pharmacology74:113123.Bacsal,K.,Chavez,L.,Diaz,I.,Espina,S.,Javillo,J.,Manzanilla,H.,Motalban,J.,Panganiban, C., Rodriguez, A., Sumpaico,C., Talip, B. & Yap, S. (1997) The EffectofSwieteniaMahogani(Mahogany)Seed Extract On Indomethacin-InducedGastricUlcersInFemaleSprague-Dawley Rats. Acta Medica Philippina 3:127-139.Ekimoto,H.,Irie,Y.,Araki,Y.,Han,G.Q.,Kadota, S. & Kikuchi, T. (1991). 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