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ISSN: 1511-3701 PertanikaJ. Trop. Agric. Sci. 28 (2): 111- 119 (2005) ©Universiti Putxa Malaysia Press Determination of Flavonoid Components from Motinda citrifolia (Mengkudu) and Their Antioxidant Activities •SAIDATUL HUSNI SAIDIN, ^UMI KALSOM YUSUF, 2 ASMAH RAHMAT & S ASPOLLAH MUHAMMAD SUKARI department of Biology, Faculty of Science , 2 Department of Nutrition and Community Health, Faculty of Medicine and Health, Sciences, ^Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia Keywords: Morinda citrifolia, flavanoid ABSTRAK Lima komponen flavonoid dapat diekstrak dan dikenal pasti daripada daun Morinda citrifolia: quercetin 3,7-0- dimethyl ether, quercetin 3-O-methyl ether, kaempferol 3,4'-O-dimethyl ether, kaempferol 5 y 7-O-diarabinoside dan apigenin Pengasingan komponen flavonoid dijalankan menggunakan teknik kertas kromatografi dengan empat sistem pelarut; BAW(n-butanol:asid asetik.air; 4:1:5), 15 % asid asetik (15 % HoAc), BEW(n-butanol:etanol:air; 4:1:2.2) dan air. Flavonoid dikenal pasti melalui warna di bawah cahaya ultra violet (UV) dengan atau tanpa wap NH 3 , nilai Rp hidrolisis asid kepada flavonoid aglikon dan gala, perbandingan ko-kromatografi dengan penanda piawai, analisis spektral dengan menggunakan beberapa bahan reagen. Kesemua komponen flavonoid pertama kali dilaporkan dalam genus Morinda. Aktiviti antioksidan bagi komponen flavonoid ditentukan dengan menggunakan kaedah pelunturan fi-karotena. Butylated hydroxyl tolmil (BHT) dan quercetin digunakan sebagai bahan penanda piawai positif Kesemua komponen flavonoid menunjukkan penurunan aktiviti antioksidan sepanjang tempoh uji kaji. Pada minit yang ke 40, quercetin 3-O-methyl ether menunjukkan aktiviti antioksidan yang tertinggi (77.67%); diikuti oleh quercetin 3,7-O-dimethyl ether (77.00%), kaempferol 3,4 f -O-dimethyl ether (66.47%), apigenin (58.10%) dan kaempferol 5,7- O-diarabinoside (56.14%). Ujian statistik ANOVA satu hala dari perisian SPSS versi 11.0 menunjukkan kesemua aktiviti antioksidan komponen flavonoid adalah berbeza secara signifikan daripada aktiviti antioksidan BHT dan quercetin (p < 0.05). ABSTRACT Five flavonoid components were isolated and identified from Morinda citrifolia's leaves; quercetin 3,7-O-dimethyl ether, quercetin 3-O-methyl ether, kaempferol 3,4'-O-dimethyl ether, kaempferol 5, 7-O-diarabinoside and apigenin. The isolation of flavonoid components was carried using paper chromatography methods with three different system solutions namely, BAW (n-butanol:acetic acid.water; 4:1:5), 15% acetic acid (15% HoAc), BEW(n-butanol:ethanol:water; 4:1:2.2) in addition to pare water. Flavonoid was identified through their colors under ultra violet (UV) with/without NHj, Rf values, acid hydrolysis to aglycone and sugar, co-chromatographic comparison with standard markers and spectral analysis by using a series of shift reagents. All of these flavonoid components were firstly reported in the genus Morinda. The antioxidant activities of flavonoid components were evaluated by using thefi-carotenebleaching method. Butylated hydroxyl toloul (BHT) and quercetin were used as positive markers. All isolated flavonoid components showed declining antioxidant activities through out the experiment. At the 4O h minute, quercetin 3-O-methyl ether showed the highest antioxidant activities (77.67%); followed by quercetin 3,7-O-dimethyl ether (77.00%), kaempferol 3,4 y -O- dimethyl ether (66.47%), apigenin (58.10%) and kaempferol 5,7-O-diarabinoside (56.14%). Statistical analysis was done using ANOVA with SPSS version 11.0 software. The results showed that all antioxidant activities belonging to the flavonoid components were significantly different from BHT and quercetins' antioxidant activites (p < 0.05). * Correspondence author

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ISSN: 1511-3701PertanikaJ. Trop. Agric. Sci. 28 (2): 111- 119 (2005) ©Universiti Putxa Malaysia Press

Determination of Flavonoid Components from Motinda citrifolia (Mengkudu)and Their Antioxidant Activities

•SAIDATUL HUSNI SAIDIN, ^UMI KALSOM YUSUF, 2ASMAH RAHMAT& SASPOLLAH MUHAMMAD SUKARI

department of Biology, Faculty of Science ,2 Department of Nutrition and Community Health,Faculty of Medicine and Health, Sciences, ^Department of Chemistry, Faculty of Science,

Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

Keywords: Morinda citrifolia, flavanoid

ABSTRAKLima komponen flavonoid dapat diekstrak dan dikenal pasti daripada daun Morinda citrifolia: quercetin 3,7-0-dimethyl ether, quercetin 3-O-methyl ether, kaempferol 3,4'-O-dimethyl ether, kaempferol 5y 7-O-diarabinoside dan apigenin.Pengasingan komponen flavonoid dijalankan menggunakan teknik kertas kromatografi dengan empat sistem pelarut;BAW(n-butanol:asid asetik.air; 4:1:5), 15 % asid asetik (15 % HoAc), BEW(n-butanol:etanol:air; 4:1:2.2) dan air.Flavonoid dikenal pasti melalui warna di bawah cahaya ultra violet (UV) dengan atau tanpa wap NH3, nilai Rphidrolisis asid kepada flavonoid aglikon dan gala, perbandingan ko-kromatografi dengan penanda piawai, analisisspektral dengan menggunakan beberapa bahan reagen. Kesemua komponen flavonoid pertama kali dilaporkan dalamgenus Morinda. Aktiviti antioksidan bagi komponen flavonoid ditentukan dengan menggunakan kaedah pelunturanfi-karotena. Butylated hydroxyl tolmil (BHT) dan quercetin digunakan sebagai bahan penanda piawai positif Kesemuakomponen flavonoid menunjukkan penurunan aktiviti antioksidan sepanjang tempoh uji kaji. Pada minit yang ke40, quercetin 3-O-methyl ether menunjukkan aktiviti antioksidan yang tertinggi (77.67%); diikuti oleh quercetin3,7-O-dimethyl ether (77.00%), kaempferol 3,4f-O-dimethyl ether (66.47%), apigenin (58.10%) dan kaempferol 5,7-O-diarabinoside (56.14%). Ujian statistik ANOVA satu hala dari perisian SPSS versi 11.0 menunjukkan kesemuaaktiviti antioksidan komponen flavonoid adalah berbeza secara signifikan daripada aktiviti antioksidan BHT danquercetin (p < 0.05).

ABSTRACTFive flavonoid components were isolated and identified from Morinda citrifolia's leaves; quercetin 3,7-O-dimethylether, quercetin 3-O-methyl ether, kaempferol 3,4'-O-dimethyl ether, kaempferol 5, 7-O-diarabinoside and apigenin. Theisolation of flavonoid components was carried using paper chromatography methods with three different system solutionsnamely, BAW (n-butanol:acetic acid.water; 4:1:5), 15% acetic acid (15% HoAc), BEW(n-butanol:ethanol:water;4:1:2.2) in addition to pare water. Flavonoid was identified through their colors under ultra violet (UV) with/withoutNHj, Rf values, acid hydrolysis to aglycone and sugar, co-chromatographic comparison with standard markers andspectral analysis by using a series of shift reagents. All of these flavonoid components were firstly reported in the genusMorinda. The antioxidant activities of flavonoid components were evaluated by using the fi-carotene bleaching method.Butylated hydroxyl toloul (BHT) and quercetin were used as positive markers. All isolated flavonoid components showeddeclining antioxidant activities through out the experiment. At the 4Oh minute, quercetin 3-O-methyl ether showed thehighest antioxidant activities (77.67%); followed by quercetin 3,7-O-dimethyl ether (77.00%), kaempferol 3,4y-O-dimethyl ether (66.47%), apigenin (58.10%) and kaempferol 5,7-O-diarabinoside (56.14%). Statistical analysiswas done using ANOVA with SPSS version 11.0 software. The results showed that all antioxidant activities belongingto the flavonoid components were significantly different from BHT and quercetins' antioxidant activites (p < 0.05).

* Correspondence author

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UMIKALSOMYUSUF ET AL.

INTRODUCTIONFlavonoid is a phenolic compound and wasdiscovered as early as the 1930s. At that time,it was known as vitamin P (Nijveldt etal 2001)because of its nutritional properties. Flavonoidis abundant in plants and can be found in allplant parts; bark, leaves, fruit and stem becauseflavonoid is a secondary metabolite in plants.There were five major groups of flavonoid;flavone, flavanone, isoflavone, flavonol, andanthocyanin (Wang and Halliwel 2001). Basedon in vitro experiments, flavonoid was foundto have anti-inflammatory, anti-allergy,antiviral and anti-carcinogenic properties(Middleton 1998). From a biological activityperspective, flavonoids have the ability toscavenge oxygen-derived free radicals and canact as antioxidant. According to Nijveldt et al(2001), flavones and catechins are the mostpowerful antioxidant against reactive oxygenspecies (ROS).

An antioxidant is a substance that canprevent the process of lipid peroxidation; aprocess whereby free radicals (FR) can damagemembrane cells. The damaged cells can leadto many degenerative diseases (Mohd Zin etal. 2002). In plants, antioxidants areproduced/developed to defend theirstructures against ROS attack duringphotosynthesis. By taking antioxidantsupplements such as vitamins or foods high inantioxidants such as vegetables and greenleaves can help counter detrimental effects offree radicals (Faridah et al 2003).

Morinda citrifolia or Mengkudu iscommonly used as a Malay traditional remedyand its young shoots are eaten as ulam or raw.Morinda citrifolia has been used a lot especiallyby post-natal women for healing menstrualproblems and curing diabetes (ZainatulSuhaida 2003). The plant belongs to theRubiaceae family and originated from tropicalAsia especially the coast of South East Asia(Dharma 1987). Apart from its utilization intraditional medicine, the pulp is also used ashair shampoo (Jaganath et al. 2000). Manyscientific and modern research studies havebeen conducted to investigate the active

compounds and special abilities of this plant.From previous research that has beenconducted, few pure active compounds havebeen revealed in the leaf of Morinda citrifolialike iridoids (Sang et al. 2003) andanthraquinone (Thomson 1987) while thebiological activities that has been discoveredfrom Morinda citrifolia are antioxidative (MohdZin et al 2002), and anti-inflammatory (Li etal 2003) activities.

MATERIALS AND METHODS

Plant SourcesThe leaves of Morinda citrifolia were collectedat Universiti Putra Malaysia's campus and driedin an oven at 50°C for four-five days.

Extraction of FlavonoidAir-dried leaves of Morinda citrifolia wereextracted using three solvents; 80% methanol,petroleum ether (60-80°C boiling point) andchloroform. 300-600 g dried leaves werepulverized and extracted using the solvents.After standing overnight for methanol andthree-four days for petroleum ether (60-80°Cboiling point) and chloroform, the extractswere concentrated using a rotary evaporator.

Isolation of FlavonoidThe concentrated extracts were separated bychromatography on 3 mm paper (46 x 57 cm)in BAW (4:1:5, top layer). Bands that appeareddark under UV light, changed to yellow-greenor unaffected by fuming with NH3 vapour wereeluted. The concentrated eluates wereseparated on 3 mm paper in 15% HOAc andthe same processes were repeated with BEW(?>-butanol: ethanol:water; 4:1:2.2) and water.Flavonoid components were identifiedthrough their colors under ultra violet (UV)with/without ammonia vapor, comparison Rfvalues with standard markers, acid hydrolysisand spectral data (Markham 1982). Theisolated compounds were also identified basedon the reported compounds in the previousstudies (Harborne 1967).

112 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 28 NO. 2 (2005)

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DETERMINATION OF FLAVONOID COMPONENTS FROM M. CITRIFOLIA

Quercetin 3y 7 O-dimethyl etherThe - max values for this component are 349,300 (shoulder) and 265; + NaOAc 393, 305(sh) 269; +NaOH 408, 325 (sh), 273; + A12C13

348, 300 (sh), 397 (iv), 273 nm. The absenceof a NaOAc shift indicates that the 7-positionis blocked. The component gave a dark colourunder UV light but changed to yellow-greenwhen fumed with ammonia vapor (NHS). Onpartial acid hydrolysis, it produced quercetin,quercetin 3-Omethyl ether and quercetin 7-0-methyl ether. Quercetin showed yellow toyellow reaction in UV + NH3 and showedidentical spectral and Rfproperties to thestandard marker. Quercetin 3-Omethyl ethershowed dark to yellow color reaction in UV +NH3 The location of the methyl group atposition 3 followed from the UV spectralanalysis: a

max in MeOH at 359, 266, 256 (sh),in MeOH + NaOH 385, 359 (sh), 270.Quercetin 7-O-methyl ether was identifiedbecause of the Rf value, colour reaction underUV + NH3 and Ccxhromatogram comparisionstandard marker

Quercetin 3-O-methyl EtherQuercetin 3-O-methyl ether was dark whenviewed under UV light on paper and yellowwith NH3. The - max values for this componentare 359, 299 (sh), 255, 268 (sh). The sodiumhydroxide UV spectral data established thepresence of a hydroxyl group on C3 waschanged (320 nm as band III). On acidhydrolysis no sugar was detected andtentatively this component was identified asQuercetin 3-Omethyl ether.

Kaempferol 3,4 '-O-dimethyl EtherThe color of the component was dark underUV light and unchanged after fuming. Spectralanalysis; in MeOH 350, 295sh; 266, in MeOH+ NaOH 406, 325 (hi), 274 nm (decompose),indicated that there was free C3 and C4 Afterhydrolysis with acid, three derivatives wereisolated: kaempferol that showed unchangedyellow under UV/UV+NH3, (366, 317sh; 266nm), kaempferol 3-O-methyl ether with darkto yellow reaction under UV/UV+NH3 and

kaempferol 4'-O-methyl ether also known askaempferide,'which showed unchanged darkreaction under UV/UV+NH3 as well as thedecompose reaction after NaOH was added toneutralise the sample for spectral dataindicates free C4. Identification kaempferol,kaempferol 3-O-methyl ether and kaempferide wasconfirmed from their spectral data, Rf values,color reaction under UV/UV+NH3> Co-chromatography comparison with standardmarker and bibliography data by Harborne(1967). This component was identified askaempferol3,4'-O-dimethyletherbec2iUse the sugartest produced negative results.

ApigeninThis component was isolated from petroleumether (60-80°C boiling point) extract andidentified using standard procedures. Itproduced a dark color under UV and changedto yellow after fuming with NH3 vapor,

Kaempferol 5y 7-O-diarabinosideKaempferol 5,7-Odiarabinoside was isolatedfrom chloroform and its color under UV lightwas dark and turned yellow after fuming withNH3. Its neutral spectral in MeOH was 360,283sh; 268, 257sh. No shift between neutralspectral and spectral after addition of NaOAcreagent on band II (268 nm) indicates thatthe hydroxyl group at C7was not free. Thehydroxyl group at C5 was not free because therewas no shift between the neutral spectral andthe spectral after addition of A12C13 reagent onband I (361 nm). After hydrolisis with acid,three derivatives were isolated. The firstderivative was identified as kaempferol becausethe yellow color under UV did not change afterNH3 fuming. Its Rf and spectral data wereidentical to the purchased kaempferol. Thesecond derivative was recognized askaempferol 7-Oarabinose based on thespectral characteristics, Rf value, and colorreaction under UV/UV+NH3. The sugar testdetected arabinose and tentatively thiscomponent is known as kaempferol 5,7-O-diarabinoside.

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 28 NO. 2 (2005) 113

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UMI KALSOMYUSUF ETAL

Antioxidant Assay (fi-carotene Bleaching Method)Fresh leaves were used to determine theantioxidant activities. The leaves were crushedby using mortar and extracted using 80%methanol for 2 hours by stirring with amagnetic stirrer. The antioxidant activity of thecrude extract and pure flavonoid compoundswere evaluated by the /carotene bleachingmethod (Veliglou 1998) with somemodification to suit the equipment in thelaboratory. A solution of/-carotene wasprepared by dissolving 1 mg of powder /carotene in 5 ml of chloroform. 60 ji\ of 95%linoleic acid, between 20 and 600 fi\ of thesample or control (80% methanol) or standardmarker were pipetted into a 250 ml roundbottom flask and covered with aluminium foil.Three ml of/carotene prepared previouslywas added into the same flask. Afterchloroform was removed by vacuum using arotary evaporator, 150 ml of distilled water wasadded to the flask and shaked vigorously for60 seconds. Aliquots (5 ml) of this emulsionwere transferred into test tubes to maketriplicates. As soon as the emulsion was addedto each tube, the zero time absorbance wasmeasured at 470 nm using aspectrophotometer. The tubes were placed at50°C in dri-block. Absorbance measurementswere recorded every 20 minutes for 2 hours.Butylated hydroxyl toloul (BHT) andquercetin were used as positive standardmarkers for comparative purposes. Theantioxidant activities of the methanolic extractand flavonoid compounds were determinedby using the following formula. Theantioxidant activity was calculated in terms ofpercent inhibition relative to the control after40 minutes incubation.

Antioxidant activity (%) formula:

(In a/b)1. d r -

b = absorbance value at observationtime (0,20,40,60,80,100,120minute)

t = observation time (0, 20, 40, 60, 80,100, 120 minute)

2. Antioxidant activity (%)

dr control - dr samplex 100

dr control

In = natural logdr = degradation ratea = absorbance value at 0 minute

The principle o f / C B determines theoxidative degradation of/carotene in anemulsion containing linoleic acid. To measurethese products, spectrophotometry has beenused (Cruz et al 1999). /carotene in themodel system undergoes rapid discolorationin the absence of an antioxidant. This isbecause of the coupled oxidation of/caroteneand linoleic acid, which generates free radicals.The linoleic acid free radical formed upon theabstraction of a hydrogen atom from one ofits methylene groups attacks the highlyunsaturated /ca ro tene molecules. As / -carotene molecules loose their double bonds,the system loses its characteristic orangecolour, which can be monitored usingspectrophotometry. The presence of aflavonoid as an antioxidant can hinder theextent of /-carotene destruction byneutralizing the linoleate free radical andother free radicals formed in the system.

RESULT AND DISCUSSION

Flavonoid IdentificationIn this study, five flavonoid components wereisolated and identified from the leaves ofMorinda citrifoliaxiz; quercetin 3,7-O-dimethylether, quercetin 3-O-methyl ether, kaempferol3,4'-O-dimethyl ether, kaempferol 5,7-0-diarabinoside and apigenin. Quercetin 3,7-O-dimethyl ether, quercetin 3-O-methyl ether andkaempferol 3,4'-Odimethyl ether were isolatedfrom methanolic extract. Kaempferol 5,7-0-diarabinose and apigenin were isolated fromchloroform and petroleum ether extractsrespectively. The color reactions under UV/

114 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 28 NO. 2 (2005)

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DETERMINATION OF FLAVONOID COMPONENTS FROM Af. OTRIFOUA

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UMI KALSOM YUSUF ETAL

UV+ NH3 vapor, Rf values and spectralproperties of the identified flavonoidcomponents are shown in Table 1.

Flavonoids that have been revealed inRubiaceae family in previous study were rutinand kaempferol 3-O^utinoside from the leavesoi Randiaformosa (Sahpaz etal 2000). Cimangaet al (1995), identified quercetin, quercetin7,4'-Odimethyl ether, luteolin 7-O-glucoside,apigenin 7-O-glucoside, quercetin 3-0-rhamnoside, kaempferol 3-O-rhamnoside,quercetin 3-O-rutinoside, kaempferol 3-0-rutinoside and chrysoeriol neohesperidosidefrom the leaves of Morinda morindoides. Froma similar study, Siti Juhaida (2000) foundquercetin 3-O-rhamnoside, kaempferol 3-O-rutinoside, myricetin 3-O-galactoside from theleaves of Morinda citrifolia. The differencebetween the results of previous studies and thisstudy is the flavonoid's rate of syhthesis andacumulation. There were many factors thatcontribute to both processes such as light, pHand soil's salinity (Onyilagha et al 2003). Thecondition of the plants played a major role too,as plants infected with any kind of disease willtend to synthesize flavonoids in higherconcentration as a mechanism of protection.(Manthey et al 2000). According to Manach etal (1996) the concentration of flavonoidsvaries for different plant organs.

Quercetin 3,7-O-dimethyl ether has beenfound before in the leaves and trichome ofNicotiana attenuata (wild tabacco) (Roda et al2003). Quercetin 3-O-methyl ether has beenfound in the fruits of Rhamnus disperma(Marzouk et al 1999), stem and fruit of Optuniaficus-indica van saboten (Go et al 2003). Tothe bestfo our knowledge, all the five flavonoidcomponents isolated from the leaves of M.citrifolia in this study are pioneer findings forthe genus Morinda,

Structure of Isolated Flavonoid from Morindacitrifolia

MeO

OMe

Qu 3,7-Odimethyl ether

Arabinoside

OH

Km 5,7-O-diarabinoside

Apigenin

HO,

OMe

Qu 3-O-methyl ether

116 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 28 NO. 2 (2005)

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DETERMINATION OF FLAVONOID COMPONENTS FROM M. CITRIFOUA

OMe

OH O

Km 3,4'-O-dimethyl ether

owe emulsion conta in ing linoleic acid andflavonoid methylated antioxidant propertieswith t ime. The corre la t ions indicateddecreasing antioxidant activity during thereaction for all flavonoid components isolatedfrom Morinda citrifolia leaves (Fig. 7). For thepurpose of comparison, the 40th minute wasselected and all antioxidant activities of thesamples and standards for that time werecompared (Fig. 2). Unfortunately, antioxidant

60 80

Time (minute)

-Qu 3,7-0- dimethyl ether

Km 3,4'-O-dimethyl ether

-Km 5,7-O-diarabinoside

Quercetin

Qu 3-O- methyl etherApigenin

BHT

Fig. 1: Flavonoid components' antioxidant activities during 120 minutes

Quercetin

BKT

Kaempferol 5,7-O-diarabinoside

Apigenin

Kaempferol 3,4'-O-dimethyl ether

Quercetin 3-0-methyl ether

Quercetin 3,7-0- dimethyl ether

(

1— 1 — • • • • • • I MM) 937

• i m ^ t H 56.14

ZH 58.1

H 66.4/

2J 77

) 20 40 60 80 100 120

Antioxidant activity (%)

Fig. 2: Comparison of flavonoid antioxidant activities at 40 minutes

Antioxidant Activities

Based on the antioxidant activities formula,there were inverse correlations between theoxidative degradation of j#-carotene in an

properties of all the samples were lower thanthe standards; quercetin (93.7%) and BHT(92.55%). Based on the statistical testcomparing all the mean values, all antioxidant

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 28 NO. 2 (2005) 117

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UMI KALSOM YUSUF ETAL.

TABLE 2Differences of antioxidant properties values

between samples and standards

Samples Antioxidantpropertiesvalues

Quercetin 3,7-Odimethyl ether 77.00 % ± L4a

Quercetin 3-O-methyl ether 77.67 % + 3.4a

Kaempferol 3,4'-Odimethyl ether 66.47 % + 4.2a

Kaempferol 5,7-Odiarabinoside 58.10 % ± 2.5a

Apigenin 56.14 % + 4.6a

BHT 92.55 % ± 4.0Quercetin 93.70 % + 0.7

a: p<0.05

activities belonging to the flavonoidcomponents were significandy different fromBHT and quercetins' antioxidant activites(p<0.05) (Table 2).

The main characteristics of flavonoids tobe an antioxidant are presence of the hydroxylgroup at C3, a double bond between C2 andC*, a carbonyl group on C4 andpolyhydroxylation on aromatic rings A and B(Cook and Samman 1996). A study by Go etal. (2003) showed that quercetin 3-methylether can act as an antioxidant because it hasthe ability to scavenge free radicals.

ACKNOWLEDGEMENTThe authors wish to thank the Ministry ofScience, Technology and Environmental forfunding provided under the IntensifiedResearch in Priority Area Proposal Grant(IRPA No: 54075 )

REFFERENCESBATE-SMITH, E. C and J. B. HARBORNE. 1971.

Differences in flavonoid content between freshand herbarium leaf tissue in Dillenia.Phytochemistry 10: 1055-1058.

CIMANGA, K., T. DE BRUYNE, A. LASURE, Q. LI,L. PIETERS, M. CLAEYS, D.V. BERGHE,K. KAMBU, L. TONA and A. VLIETINCK. 1995.Flavonoid O-glycosides from the leaves ofMorinda morindoides. Phytochemistry 38:1301-1303.

COOK, N. C. and S. SAMMAN. 1996. Flavonoid -chemistry, metabolisme, cardioprotectiveeffects and dietary sources. The Journal ofNutrition Biochemistry 7: 66-76.

DHARMA, A. P. 1987. Indonesia Medicinal Plant,Jakarta: Balai Pustaka.

FARIDAH, A., S. KHOZIRAH, N. H. LAJIS, D. A. ISRAF andY. UMI KALSOM. 2003. Antioxidative and radicalscavenging properties of the constituentsisolated from Cosmos caudatus Kunth. NaturalProduct Sciences 9: 245-248

Go. H. D., K. H. LEE, H. J. KIM, E. H. LEE, Y. S. SONG,Y.H. LEE, C.JIN, Y. S. LEE and J. CHO. 2003.Neuroprotective effects of antioxidantflavonoids, quercetin, (+) - dihydroquercetinand quercetin-3-methyl ather isolated fromOptuniaficus - indica van saboten. Brain Research965: 130-136.

JAGANATH, I. B., S. MOHD NASIR, R. A. RAHMAN andC. MUTHUVELU. 2000. Herba Berpotensi diMalaysia. Kuala Lumpur: Institut Penyelidikandan Kemajuan Pertanian Malaysia.

HARBORNE, J. B. 1967. Comparative Biochemistry of theFlavonoids. London and New York: AcademicPress.

U R- W., S. P. MYERS, D. N. LEACH, G. D. LIN andG. LEACH. 2003. A cross-cultural study: anti-inflammatory activity of Australian andChinese plants./owma/ ofEthnopharmacology 85:25-32.

MARKHAM, K.R. 1982. Techniques of FlavonoidIdentification. New York: Academic Press.

MARZOUK, S. M., S. A. A. EL-TOUMY, I. MERFORT andM. A. M. NAWWAR. 1999. Polyphenolicmetabolites ofRhamnus disperma. Phytochemistry52: 943-946.

MIDDUETON, E. Jr. 1998. Effect of plant flavonoidson immune and inflammatory cell function.Adv. Exp. Med. Biol 439: 175-182.

MOHD ZIN, Z., A. ABDUL-HAMID and A. OSMAN. 2001.Antioxidative activity of extracts fromMengkudu (Morinda dtrifoliaL.) root, fruit andleaf. Food Chemistry 78: 227-231.

118 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 28 NO. 2 (2005)

Page 9: Determination of Flavonoid Components from Motinda ... · hidrolisis asid kepada flavonoid aglikon dan gala, perbandingan ko-kromatografi dengan penanda piawai, analisis spektral

DETERMINATION OF FLAVONOID COMPONENTS FROM M. CITRIFOLIA

NlJVELDT R. J., E. VAN NOOD, D. F. VAN HoORN, P. G.BOELENS, K. VAN NORREN and P. A. M VANLEEUWEN. 2001. Flavonoids: a review ofprobable mechanisms of action and potentialapplications The American Journal of ClinicalNutrition 74e 418-425.

RODA, A. L., N. J. OLDHAM, A. SVATOS and I, T.BALDWIN. 2003. Allometric analysis of theinduced flavonols on the leaf surface of wildtabacco (Nicotiana attenuate). Phytochemistry 62:527-536.

SANG, S., G. LIU, K. HE, N. ZHU, Z. DONG, Q. ZHENG,R. T. ROSEN and C. T. Ho. 2003. New unusualiridoids from the leaves of noni (Morindacitrifolia L.) show inhibitory effect onultraviolet B-induced transcriptional activatorprotein-1 (AP-1) activity. Bioorganic &MedicinalChemistry 11: 2499-2502.

SITI NORJUHAIDA, S. A. H. 2000. Ekstraksi sebatianflavonoid daripada daun dan buah Morindacitrifolia L. Bac. Sc. Tesis, Universiti PutraMalaysia, Selangor.

THOMSON, R. H. 1987. Naturally Occurring QuinonesIII. Recent Advances. London: Chapman & Hall.

VEUGLOU, Y S., G. MAZZA., L. GAO and B. D. OOMAH.1998. Antioxidant activity and total phenolicsin selected fruits, vegetable and grain products.Journal Agricultural Food Chemistry 46: 4113-4117.

WANG, H. and K. HELLIWELL. 2001. Determinationof flavonols in green and black tea leaves andgreen tea infusions by high-performance liquidchromatography Food Research International 34:223-227.

ZAINATUL SHUHAIDA, A. R. 2003. Herba dan UbatanTradisi Zaman-berzaman. Jilid 1: 64-65. KualaLumpur: Zebra Editions Sdn. Bhd.

(Received: 25 February 2006)(Accepted: 13 October 2006)

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