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버진 시어버터의 항산화, 항염, 천연 방부력 효과에 관한 연구

버진 시어버터의 항산화, 항염, 천연 방부력 효과에 관한 연구

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NaturalProductScience
15(2):76-82(2009
7
NMRAssignmentandAntimicrobial/AntioxidantActivitiesof
β 
-HydroxyeuscaphicacidfromtheSeedsof
Butyrospermumparki
LaurentineBouquetTankeuNyaa
,LéonAzefackTapondjou
,LucianoBarboni
,JeanDeDieuTamokou 
,JulesRogerKuiaté 
,PierreTan
,Hee-JuhnPark
DepartmentofChemistry,FacultyofScience,UniversityofDschang,Box183,Dschang,Cameroo
DipartimentodiScienzeChimiche,UniversitàdiCamerino,ViaS.Agostino1,62032Camerino(MC),Ital
DepartmentofBiochemistry,FacultyofScience,UniversityofDschang,Box67,Dschang,Cameroo
DepartmentofPharmaceuticalEngineering,SangjiUniversity,Wonju220-702,RepublicofKorea 
Abstract 
− 
PhytochemicalinvestigationoftheEtOAcand
-BuOHfractionsobtainedfromthecrudemethanol extractoftheseed
of
Butyrospermumparki
ledtotheisolationofsevencompoundsincluding1 
β 
,
α 
,
α 
trihydroxyurs-12-en-28-oicacid(
β 
-hydroxyeuscaphicacid)whichfullNMRassignmentishereinreportedfor thefirsttime.Theaboveextract,fractionsandsomeoftheisolatedcompoundswerescreenedforantimicrobial andantioxidantactivities;
β 
-hydroxyeuscaphicacidwasthemorepotent
Keywords 
− 
Butyrospermumparki
,Sapotaceae,Triterpenoids,Antimicrobialactivity,Antioxidantactivit
Introduction 
Butyrospermunparki
Kotschy( 
Butyrospermunpara- doxum 
(Don)Hepper)isasmalltreethatgrowsupt14mhigh,itbelongstothefamilySapotaceaeinwhich triterpenoidsandsaponinsarefairlyencountered(Wandj
eta
.,2003;Lavaud
etal
,1996).Theseedsofthisplancontainanediblefat(Kapseu
etal 
.,2001)whichisused intraditionalmedicinetotreatscabies,ulcers,woundandnasalstiffness(Ogunwande
eta
.,2001).Previouworksonthesheabutter
B.parki
)fromthenorthernpart ofCameroonrevealedthepresenceoffattyacids(mainloleicacidandstearicacid)andfourteentriglycerides detectedbygaschromatography(Kapseu
eta
.,2001)Recently,Ogunwande
etal
(2001)evaluatedtheantibacterialandantifungalpropertiesofextractsfromthleaves,stembark,rootbark,fruitandseedkernelsof
Bparki
.Inourcontinuoussearchfornewand/orknowbioactivecompoundsfromCameroonianmedicinalplant(Tapondjou
eta
.,2005a;Teponno
etal
,2006;Teponno
eal
,2007,Ponou
etal 
.,2008),wehavestudiedthe methanolextractfromtheseedkernelsof
B.parki
growinginthewesternhighlandsofCameroon 
Thiarticledealswiththeisolationandstructuralelucidatioofsevencompoundsincluding
β 
-hydroxyeuscaphicaci
)whichfullNMRassignmentishereinreportedforthfirsttime.Theantimicrobialandantioxidantactivitiesothecrudeextract,fractionsandisolatedcompoundswere evaluatedandtheresultsobtainedarepresented
Experimental 
General 
− 
IRspectraweremeasuredasafilmona KBrpelletusingaFTIR-8400SShimadzuspectrometerESImassspectrawerecarriedoutonaHewlettPackard HP-1100seriesLC-MSDsystem.
Hand
1
CNMR, DEPT,COSY,HSQC,HMBCandROESYexperimentwereperformedindeuteratedMeOHonaVariaMercuryPlusSpectrometer(400MHzfor
Hand100 MHzfor
1
C).Allchemicalshifts( 
δ 
)aregiveninppunitswithreferencetotetramethylsilane(TMS)asthe internalstandardandthecouplingconstants
)areinHz. Columnchromatographywasperformedusingsilicagel 60Merck(63-200 
µ 
mand32-63 
µ 
m).TLCwascarried outonprecoatedKieselgel60
25
(Merck)plates developedwithC
C
-MeOH(96-4;92-8)andEtOAc- MeOH-
O(95-5-2).TLCplateswerevisualisedby sprayingwith50%
S
andheatingfor10minat11
C
Plantmaterial 
− 
Theseedsof
B.parki
(Sapotaceae) werecollectedinBangwavillage,nearthecityof Bangangte(WesternRegionofCameroon)inAugust 2005.Specimensdocumentingthecollectionweredeposited 
*AuthorforcorrespondencTel:+82-33-730-0564;E-mail:hjpark@sangji.ac.kr 
 
Vol.15,No.2,20097
intheCameroonNationalHerbarium,Yaoundé(Ref:
58927/HNC)
Extractionandisolation 
− 
Driedandpowderedseed kernelsof
B.parki
(2.5kg)wereextractedthreetimes withMeOHatroomtemperature.Thefiltrateobtained wasevaporatedunderreducedpressuretogiveadarresidue(258g),whichwassuspendedinwaterand successivelypartitionedwithEtOAcand
-BuOH.The EtOAcfraction(10g)wassubmittedtosilicagel(7020
µ 
m)columnchromatographyelutedwiththemixture Hexane-EtOAcinincreasingorderofpolaritytogivefoumainsub-fractions(A-D).Sub-fractionC(5g)Hexane/ EtOAc(6:4to4:6)wasrepeatedlychromatographedon silicagelcolumnelutedwithC
C
-MeOH(96:4)tgive
β 
-hydroxyeuscaphicacid
,30mg),euscaphicaci
,10mg),myrianthicacid
,25mg)and( 
)
-(4hydroxyphenethyl)-4-hydroxycinnamamide
20mg)The
-butanolfraction(52g)wasdissolvedindistilled water,pouredintoaDiaionHP-20column.Initiallywater wasusedforelutiontoremovesugarsandionisubstancesandthen1.5LMeOHwasusedandthe resultedeluatewasevaporated.ApartoftheMeOresidue(23g)wassubjectedtoasilicagelcolumchromatographyelutedwithEtOAc-MeOH(100:0t80:20),yieldingfourmainsub-fractions.Thesub- fractionelutedwithEtOAc(2g)waschromatographedon silicagelcolumnusingthemixtureEtOAc-MeOH-
(100:10:5)forelutiontogive9,10,13-trihydroxyoctadec- 11-enoicacid
,11mg).Filtrationofthesub-fractioelutedwithEtOAc-MeOH(90:10)gave(
)
-2'hydroxytetracosanoyl-1- 
-D-glucopyranosyl-4-hydroxy- 8-sphingogenine
,14mg).Thelastsub-fraction(1g) wasrechromatographedonsilicagelcolumnelutedwitC
C
-MeOH(92:8)togivegallocatechin( 
10mg)
β 
,
α 
,
α 
-trihydroxyurs-12-en-28-oicacid(
β 
-hydro- xyeuscaphicacid)( 
),whitepowderC
C
-MeOH,I(KBr)cm 
− 
:3373,2912,1720,1461,1118;
Hand
1
NMRdata:seeTable1;ESI-MS(pos.):
m/
527[M+Na] 
(
3
4
)
Microorganism
− 
Themicroorganismsusedinthistudyconsistedofthreebacteria( 
Staphylococcusaureus 
ATCC25922,
Klebsiellapneumoniae 
ATCC13883,
Salmonell
typhi
ATCC6539)andtwo
Candida 
species 
Candida 
albican
ATCC9002and
Candida 
tropicali
ATCC750);allofwhicharereferencestrainsobtained fromtheAmericanTypeCultureCollection.Also, includedwasonestrainof 
Cryptococcusneoformans 
IP95026obtainedfromthePasteurInstitute(IP,Paris- France).Thebacterialandyeaststrainsweregrownat3
Candmaintainedonnutrientagar(NA,Conda,Madrid, Spain)andSabouraudDextroseAgar(SDA,Conda) slantsrespectively. 
Determinationofminimuminhibitoryconcentration (MIC)andminimummicrobicidalconcentration (MMC) 
− 
MICsweredeterminedbybrothmicrodilution methodasdescribedbyKuete
etal 
withslight modifications.Thetestsampleswerefirstofalldissolved indimethylsulfoxide(DMSO).Thesolutionobtainedwas thenaddedtoMuellerHintonBroth(MHB)forbacteriorSabouraudDextroseBroth(SDB)foryeaststogivea finalconcentrationof4000µg/ml.Thiswasserialldilutedtwofoldtoobtainconcentrationrangesof4000t0.48µg/ml.Onehundredmicrolitersofeachconcentration wasaddedineachwell(96-wellsmicroplate)containin95µlofMHBorSDBand5µlofinoculumforfinal concentrationsvaryingfrom2000to0.24µg/ml.The inoculumwasstandardizedat1.
× 
1
CFU/mlbadjustingtheopticaldensityto0.1at600nmJENWA6105UV/Visspectrophotometer.Thefinalconcentration ofDMSOineachwellwaslessthan1%(preliminaranalyseswith1%(v/v)DMSOdonotinhibitthegrowtofthetestorganisms).Thenegativecontrolwellconsisted of195µlofMHBorSDBand5µlofthestandarinoculum.Theplateswerecoveredwiththesterilelidthenagitatedtomixthecontentsofthewellsusingaplate shakerandincubatedat35 
Cfor24hours(forbacteria) orfor48hours(foryeasts).Theassaywasrepeated thrice.TheMICsofsamplesweredeterminedbyadding 50µlofa0.2mg/ml
-iodonitrotetrazoliumchloridsolutionfollowedbyincubationat35 
Cfor30min. Viablemicro-organismsreducedtheyellowdyetoapincolour.MICsweredefinedasthelowestsampleconcentrationsthatpreventedthischangeincolourindicatinga completeinhibitionofmicrobialgrowth.ForthedeterminationofMMCs,aportionofliquid(µl)fromeachwellthatshowednogrowthof microorganismwasplatedonMuellerHintonAgaror SabouraudDextroseAgarandincubatedat35 
Cfor24 hours(forbacteria)or3
Cfor48hours(foryeasts).Thlowestconcentrationsthatyieldednogrowthafterthisub-culturingweretakenastheMMCs(Kuete
etal
2008).Gentamicin(Sigma-Aldrich,Steinheim,GermanyandNystatin(Merck,Darmstadt,Germany)forbacteriandyeastsrespectivelywereusedaspositivecontrols. 
Antioxidantassay:DPPHassaymethod 
− 
Thefree radicalscavengingactivityofthecrudeextractaswellas theirisolatedcompoundswasevaluatedasdescribedbMensor
eta
.(2001)withslightmodifications.Thetest samples,priordissolvedinDMSO(SIGMA)weremixed witha20mg/l2,2-diphenyl-1-picryl-hydrazyl-hydrat
 
7
NaturalProductScience
(DPPH)methanolsolution,togivefinalconcentrationso10,50,100,500and1000µg/ml.After30minatrootemperature,theabsorbancevaluesweremeasuredat51nmandconvertedintopercentageofantioxidantactivitasfollows:%inhibition=[(Absorbanceofcontrol 
− 
Absorbance oftestsample)/Absorbanceofcontrol]×100.Ascorbiacidwasusedasastandardcontrol.Theinhibitionratiwasconvertedinprobits.Theprobitvalueswereplotted againstthelogarithmicvaluesofconcentrationsofthetest samplesandalinearregressioncurvewasestablishediordertodeterminedtheI
5
(µg/ml);beingtheamount ofsamplenecessarytodecreaseby50%theabsorbance ofDPPH.Alltheanalyseswerecarriedoutintriplicatandtheresultswereexpressedasthemean 
± 
standard deviation(SD)andcomparedusingWaller-DuncantestAvalueofp<0.05wasconsideredstatisticallysignificant
ResultsandDiscussio
Compound
wasisolatedasawhitepowderinMeOHItsESI-MS(positive-ionmode)exhibitedapseudomolecularionpeakatm/z527[M+Na] 
consistentwitthemolecularformulaof
3
4
.ItshowedIabsorptionsbandsat3373(OH),2912(CH)and1720 (C=O)cm 
− 
.The
HNMRspectrumrevealedthepresence ofsevenmethylgroups(sixsingletsandonedoublet)anavinylicprotonsignalat
δ 
5.32(brs,H-12)suggestingan ursanetriterpenoidderivativewithoneoxidizedmethy(Wandji
eta
.,2003;Lavaud
eta
.,1996).Thisspectru
Table1.
NMRdataforcompound1,C
OD,
(Hz)inbracketPositio
1
C
δ 
H
δ 
)HMB179.8(d)3.43(d,11.4)C-25,C-5,C-270.4(d)3.63(dd,4.4,11.4)C-379.2(d)3.40(d,4.4)C-5,C-2,C-437.6(s
− 
548.1(d)2.01(nd)C-6,C-9,C-10,C-3,C-7,C-2618.1(t)1.62(m732.7(t)n840.4(s
− 
923.8(d)2.43(m1041.1(s
− 
1148.4(t)3.31C-18;C-112129.3(d)5.32(brs)C-10;C-9;C-113137.4(s
− 
1441.6(s
− 
1527.7(t)2.01(nd)C-11622.6(t)1.38(nd1748.2(s
− 
1853.5(d)2.48(s)C-14;C-17;C-19;C-12;C-13;C-21972.2(s
− 
2042.9(d)1.38(m2128.2(t)0.90(m)C-22;C-22237.5(t)2.57(m)C-21;C-12316.3(q)0.78(s)C-2421.1(q)0.83(s)C-5;C-2511.6(q)1.02(s)C-10;C-2627.8(q)n2725.9(q)1.20(s)C-128180.9(s
− 
2925.2(q)1.38(s3015.3(q)0.85(d,7.0)C-29;C-19;C-20
n
:notdetermined 

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