Proceeding of The 5th Annual Basic Science International Conference

Received, , Accepted, , Published online, .

IsolationofSecondaryMetabolitesCompoundandAntioxidant
ofNeemRoot(Azadiracthaindica)FromPoteranMaduraIsland
*PrimaAgustiLukis1TaslimErsam2
1,2
Department of Chemistry, Faculty of Mathematic and Science, Institut
TeknologiSepuluhNopember,Surabaya,Indonesia
*Correspondingauthors:[paktichem@gmail.com]
Abstract Numerousstudiesonmedicinalplantsreportedthatitcontainlarge
amount of antioxidants. Antioxidant effects mainly due to the secondary
metabolites compounds. In this study, carried out the isolation of secondary
metabolites compound of the neem roots (Azadirachta indica) from Poteran
MaduraIslandandquantitativelyantioxidanttestusingDPPH(2,2diphenyl1
picrylhydrazyl)method.Isolationmethodusedwasmaceratedwithmethanoland
thenfractionatedbyvacuumliquidchromatographyusingtheeluentnhexane,
dichloromethane,ethylacetateandmethanolbasedonincreasingpolarity.The
resultingfractionisthenpurifiedbyrecrystallizationusingthesolventnhexane.
Pure compound were obtained to test the melting point, solubility test, and
antioxidanttestwithDPPHmethod.Compoundsobtainedwhiteneedleshaped
crystals withamelting pointof133134C.BasesonGCMSanalysisofthis
compoundobtainedmolecularformulaisC29H48Oandthatsupportedthedata1H
NMR and 13CNMR. Antioxidant test using DPPH method showed that this
compoundhavingIC50ofmorethan600ppm.Thisindicatesthatthesecompound
inactiveantioxidant.
1.INTRODUCTION
Antioxidants are compounds electron donor or reductant. This compound has a small
molecularweight,butisabletoinactivatethedevelopmentoftheoxidationreactionwith
radicalbinding.Relatedoxidationreactionsinthebody,antioxidantstatusisanimportant
parametertomonitorthehealthofaperson.Antioxidanthasgainedimportanceinthecurrent
scenarioasithasanabilitytotrapfreeradicalswhichareproducedduringthedegenerative
diseases.Thehumanbodyhasasystemofantioxidantstocounteractthereactivityoffree
radicals,whicharecontinuouslyformedbythebody[1].
Several new compounds have been isolated from medicinal plants such as alkaloids,
terpenoids,flavonoidsandxanthoneswhichhavepotentialantioxidantactivity.Oneislandin
Indonesia, Poteran island has some endemic plants are considered potential sources of
phenoliccompounds asantioxidants.Severalpotentiallyproductiveplants asmedicine are
Moringa (Moringa oleifera), Neem (Azadirachta indica), Saga (Adenanthera pavonima),
Breadfruit(Artocarpusaltilis),Chilliherbs(Piperretrofactum,Vahl),Kesambi(Schleichera
aleosa),Pulai(Alstoniascholaris),andPigeonpea(Cajanuscajan).Inthisresearch,selecteda
plant that has potential as an antioxidant that Neem (A. indica). Its have been used
medicinallytotreatvariousdiseasessuchasfever,abdominalpain,itching,andareductionin
bloodsugarlevels.Aerialpartsofneemhasbeenisolatedmorethan140chemicalcompounds.
Thecompoundscontainedintheneemplantthroughresearchbioactivityinvitroandinvivo
studieshavedemonstratedactivityasanantiinflammatory,antihistamine,antipyretic,and
antifungal,butitisstillraretofindcompoundsfromneempotentialasantioxidants [2].

Researchonneemroothasnotbeendone,sointhisstudywillbeconductedtodeterminethe
activityofcompoundstheneemrootsfromPoteranMaduraIsland.Thesecondarymetabolites
ofneempotentiallycontainingcompoundsareexpectedtobepotentiallyasanantioxidant
compound.
2.METHODS
2.1Materials.
Plant Materials. Roots of A. indica were collected from Poteran IslandMadura,
IndonesiainMarch,2014.
InstrumensandChemicals.Meltingpointsweredetermined onFisherJohnmelting
point Apparatus. IR spectra were measured with FTIR PRESTIGE 21 (SHIMADZU)
spectrophotometers,respectively.GCMSspectraweremeasuredwithGC/MSAutosampler
AgilentTechnologies,5973inert,MassSelectiveDetector. 1Hand 13CNMRspectrawere
recordedwithJEOLNuclearMagneticResonance(JNMECS400),operatingat400.0MHz
usingstandardsfromresidualanddeuteratedsolventpeaks,respectively.VLCwascarriedout
usingMerckSigel60GF254andTLCanalysisonprecoatedSigelplates(MerckKieselgel60
F254,0.25mm).
2.2Procedures
ExtractionandIsolation
Thedriedpowderedofneemroots(5kg)wasmaceratedinMeOHandtheMeOHextract(30
g)wassequentiallyfractionatedbyVLCSigelusing nhexane,dichloromethane(CH2Cl2),
ethyl acetate (EtOAc),andMeOHbasedonincreasingpolarity togive7fractions (IO).
Thereare2fractions(LandM)whichhasrelativelythesameR fvalue,sothatthecombined
(P=4.5680g)andfurtherfractionatedbyVLCmethodusing nhexane:CH2Cl2 basedon
increasingpolarity.Resultsfromthesecondfractionationcombinedfractionsobtained4(P 1
P4) are monitored by TLC. At P3 fraction of precipitation, then filtered using vacuum
filtration,purifiedbyrecrystallizationusingnhexaneandP3cascompound1(0.1303g)were
obtained.
DPPHfreeradicalscavengingactivity
The DPPH method was used to evaluated the antioxidant activity of the phenolic
compounds[7],[8].Theantioxidantactivityofthecompound1wasdeterminedintermsof
hydrogendonatingorradicalscavengingability,usingthestableradicalDPPH.Samplesat
variousconcentrations(600;400;200;100;50;10g/ml)wereaddedto1mlofDPPHand2
mlmethanolandallowedtostandfor30minat37 C.Theabsorbanceofthesamplewas
measuredat517nm.Allexperimentswererepeatedthreetimes.Radicalscavengingactivity
wasexpressedastheinhibitionpercentageoffreeradicalbythesampleandwascalculated
usingtheformula[9].
..(1)
Compound1:whitesolid,meltingpoint133134C,1HNMR(CDCl3,400MHz)and13C
NMR(CDCl3,400MHz)seeTable1;GCMS(m/z):412[M+],394,369,351,314,300,
271,255,213,159,133,83,55;IRmaxKBr(cm1):3429,2958,1654,1463,1379,1054.
3.RESULTSANDDISCUSSION

Compound1wasisolatedintheformofawhitesolidwithameltingpointof133134
C.MSdataindicatethatthecompoundhasamolecularformulaC 29H48O,sothatthevalueof
DBE=6.Then,itwassupportedbythedataofIR, 1HNMRand13CNMR,andcompared
withdatafromprevious studies.TheIRsignalabsorptionbandobservedat3429cm1 is
characteristic of OH stretching. Absorption at 2958 cm 1 is typical of cyclic olefinic
HC=CHstretchingwhiletheabsorptionatfrequenciessuchas1654cm1 isasaresultof
C=Cabsorption.However,thissignalisweak,theabsorptionat1463cm 1wasassignableto
cyclic(CH2)nbendingabsorptionfrequency.Whiletheoneat1379cm 1isattributabletoOH
deformingabsorption.Theabsorptionat1054cm1istypicalofcycloalkanemoieties.These
absorptionfrequencieswereinverycloseagreementwiththeonesobservedforStigmasterol
[3].1HNMRspectraofcompound1(Table1)showedthepresenceoftwomethylsingletsat
H0.78ppm(s,3H)and1.01ppm(s,3H);threemethyldoubletthatappearedatH0.80ppm
(d,3H,J=6.8Hz),0.82ppm(d,3H,J=6.8Hz),and0.91ppm(d,3H,J=6,4Hz);andamethyl
tripletatH0.84ppm(t,3H,J=6.8Hz).Compound1alsoshowedprotonsatH4.99,5.14,
and 5.34 ppm suggesting the presence of three protons corresponding to that of a
trisubstitutedandadisubstitutedolefinicbond.Theprotonmultipletsignalat H 3.52ppm
showedahydroxyproton.ThesignalatH1,01ppm(s,3H)correspondstoC28andC29
protonsrespectivelyofStigmasterol[3].
Table1.DataComparationof1HNMRand13CNMRCompound1withStigmasterol[4]
inCDCl3
Position

Stigmasterol
H (ppm)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

3.51 (tdd, 1H, J = 4.5,

4.2, 3.8 Hz)
5.31 (t, 1H, J = 6.1 Hz)
0.91 (d, 3H, J = 6.2 Hz)
4.98 (m, 1H)
5.14 (m, 1H)
0.83 (t, 3H, J = 7.1 Hz)
0.82 (d, 3H, J = 6.6 Hz)
0.80 (d, 3H, J = 6.6 Hz)
0.71 (s, 3H)
1.03 (s, 3H)

Compound 1
C
(ppm)
37.6
32.1
72.1

H (ppm)
3.52 m

C
(ppm)
37.3
31.8
71.8

42.4
141.1
121.8
31.8
31.8
50.2
36.6
21.5
39.9
42.4
56.8
24.4
29.3
56.2
40.6
21.7
138.7
129.6
46.1
25.4
12.1
29.6
20.2
19.8
18.9
12.2

5.34 (t, 1H, J = 4.8 Hz)

0.91 (d, 3H, J = 6.4 Hz)
4.99 (m, 1H)
5.14 (m, 1H)
0.84 (t, 3H, J = 6.8 Hz)
0.82 (d, 3H, J = 6.8 Hz)
0.80 (d, 3H, J = 6.8 Hz)
0.68 (s, 3H)
1.01 (s, 3H)

42.3
140.8
121.8
31.9
31.9
51.3
36.6
21.2
39.8
42.4
56.8
24.4
28.3
56.1
40.6
21.3
138.4
129.3
45.9
25.5
11.9
31.9
21.3
19.5
18.9
12.1

Then,thedataisreinforcedbythedata13CNMRthatthecompound1hasaminimumof
29carbonatoms.ThisisconsistentwiththedataMSthattheamountofcarbonasmuchas
29.The 13CNMR(Table1)showssomerecognizablesignalsat C 140.8and121.8ppm
whichisassignabletothedoublebondatC5andC6[3],signalatC138.4and129.3ppmis
alsoassignabletothedoublebondatC20andC21[4].ThesignalatC71.8ppmisdueto
C3hydroxylgroup[3].AgainthesignalsobservedatC18.9and12.1ppmcorrespondto
angularcarbonatomsatC28andC29respectively(Table1).Ithadbeenreportedthatthe
lowervalueforC29isattributabletogaucheinteractionthatincreasesthescreeningofC
29therebycausing lowerchemicalshift[3].LikewisethelossofprotoninC6resultsin
decreaseinscreeningofC28leadingtoanincreasein13Cchemicalshifttohigherfrequency
[3].ThisexplanationisalsoacceptedC18.9and12.1ppm(forC28andC29respectively).
CarbonsignalsatC28moredeshieldingthanC29duetotheprotonatC6isexperiencing
dehydrogenationolefinicprotons,sothatitismoredeshielding,whileC29onlyhaveC12.1
ppmwhichisamethylcarbon.Then,13CNMRdataofcompound1werecomparedwithdata
fromtheliterature(Table1)thatithasastructuralsimilaritywithstigmasterol[4].
19
29
12
28

11

21

17

22

26
25

16

14
10

3
HO

18

13

24

23

20

15

27

7
6

Compound1:Stigmasterol
Thus,thestructureofcompound1wasassignedastheknowncompoundstigmasterol.
Thephysicalandspectraldataareconsistenttothereportedliteraturevaluesofstigmasterol
[3],[4].AntioxidanttestusingDPPHmethodshowedthatthiscompoundhavingIC 50ofmore
than600ppm.Thisindicatesthatthesecompoundinactiveantioxidant,asacompoundhaving
IC50oflessthan200ppmshowedithaspotentantioxidantactivity[5].Whencomparedwith
theantioxidantactivityofvitaminCthathaveIC50values6.316ppm,theantioxidantactivity
ofthesecompoundfarexceedsthestandards.
4.CONCLUSIONS
PhytochemicalstudyofmethanolextractofNeem(A.indica),amedicinalplantfromPoteran
IslandMaduraresultedtotheisolationofknowncompoundstigmasterol(1).Thiscompound
wasevaluatedforitsinvitroantioxidantactivityusing2,2diphenyl1picrylhydrazyl(DPPH)
radicalscavenginghave IC50 ofmorethan 600ppm.This indicated that these compound
inactiveantioxidant.
5.REFERENCES
1.
2.
3.

Winarsi, H., 2007, Natural Antioxidants and Free Radicals (Potential and Its
ApplicationinHealth),Kanisius,Yogyakarta.
Soegihardjo,C.J.,2007,Neem(AzadirachtaindicaA.Juss,TribeMeliaceae),MultiCrop
BenefitsIssuestoTackleRakyatIndonesia,SIGMA,Yogjakarta,10,83102.
Isah,Y.,Ndukwe,I.,andAmupitan,J.O.,2012,IsolationOfStigmasterolFromAerial
PlantPartOfSpillanthesAcmellaMurr,WorldJLifeSci.andMedicalResearch,2(2):
77.

4.

5.

Chaturvedula,V.S.P,andPrakash,I.,2012, IsolationOfStigmasterolAnd Sitosterol

From The Dichloromethane Extract Of Rubus Suavissimus, International Current
PharmaceuticalJournal,1(9):239242.
Blois,MS.,1958,Antioxidantdeterminationbytheuseofastablefreeradical,Nature
181,11991200.