Research Article Phytochemicals and Antioxidant Capacity

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 154606 9 pageshttpdxdoiorg1011552013154606

Research ArticlePhytochemicals and Antioxidant Capacity fromNypa fruticans Wurmb Fruit

Nagendra Prasad12 Bao Yang3 Kin Weng Kong4 Hock Eng Khoo2 Jian Sun5

Azrina Azlan26 Amin Ismail26 and Zulfiki Bin Romli7

1 Chemical Engineering Discipline School of Engineering Monash University Jalan Lagoon Selatan46150 Bandar Sunway Selangor Malaysia

2 Department of Nutrition and Dietetics Faculty of Medicine and Health Sciences Universiti Putra Malaysia43400 Serdang Selangor Malaysia

3 Key Laboratory of Plant Resources Conservation and Sustainable Utilization South China Botanical GardenChinese Academy of Sciences Guangzhou 510650 China

4Department of Molecular Medicine Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia5 Institute of Agro-food Science and Technology Guangxi Academy of Agricultural Sciences Nanning 530007 China6 Laboratory of Halal Science Research Halal Products Research Institute Universiti Putra Malaysia43400 Serdang Selangor Malaysia

7MUDA Agricultural Development Authority (MADA) Alor Setar 05990 Kedah Malaysia

Correspondence should be addressed to Nagendra Prasad nagendraprasadmonashedu andAmin Ismail aminmedicupmedumy

Received 9 October 2012 Revised 20 February 2013 Accepted 18 March 2013

Academic Editor Weena Jiratchariyakul

Copyright copy 2013 Nagendra Prasad et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Nypa fruticans Wurmb is one of the important underutilized fruit of Malaysia which lacks scientific attention Total phenolicsflavonoid content and antioxidant capacities from endosperm extracts of Nypa fruticans (unripe and ripe fruits) were evaluatedEndosperm extract of unripe fruits (EEU) exhibited the highest phenolics (1356 plusmn 45mg GAEg) flavonoid content (686 plusmn31REg) and antioxidant capacity Free radical scavenging capacity of EEU as assessed by 2-21015840-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) and 11-diphenyl-2-picryl hydrazyl (DPPH) radicals showed inhibitory activity of 78 plusmn 12 and 85 plusmn 26respectively Beta carotene bleaching coefficient of EEU was higher (2550 plusmn 123) when compared to endosperm extract of ripefruits (1729 plusmn 172) Additionally EEU exhibited high antioxidant capacity by phosphomolybdenum method and ferric reducingantioxidant power values Eight phenolic compounds from Nypa fruticans endosperm extracts were identified and quantified byultra-high-performance liquid chromatography Chlorogenic acid protocatechuic acid and kaempferol were the major phenoliccompounds Thus this fruit could be used as a potential source of natural antioxidant

1 Introduction

Antioxidants from plant sources have been an increasingconcern to consumers since synthetic antioxidants such asbutylated hydroxy toluene (BHT) have restricted usage infoods due to carcinogenic activity [1] Epidemiological stud-ies have shown that frequent consumption of fruits andvegetables high in natural antioxidants can lower the inci-dence of certain types of cancer cardiovascular diseases anddiabetes [2] These beneficial effects are related to bioactive

compounds like phenolic acids flavonoids anthocyaninsand carotenoids possessing antioxidant activity [3 4]

The years 2011ndash2020 are recognized as the ldquoDecadeon Biodiversityrdquo by the United Nations to promote theimportance and utilization of underutilized foods Recentlyresearch and development activities on antioxidants fromunderutilized fruits have become a great priority [5 6] Theyare notable by the fact that they are locally available butuniversally erratic and much related information is alsolimited Many of these fruits have a wide range of color

2 Evidence-Based Complementary and Alternative Medicine

(a) (b)

Figure 1 Nypa fruticans (a) and fruit bunch (b)

for skin and pulp with health-promoting benefits Howevermany of these fruits are still not familiar due to lack ofpublicity and promotional campaign [7]

Nypa fruticans Wurmb (NF) belongs to Araceae familyand is considered as ldquounderutilizedrdquo plant [8] Other syn-onym of this plant includes Cocos nypa Lour Nypa fruticansThunb and Nypa palm It is a monoecious palm foundgrowing in brackish water with upright stem (Figure 1(a))trunkless with fruits found commonly emerging from the soil(Figure 1(b)) This palm is typically found in India MalaysiaIndonesia Philippines and in some parts of QueenslandAustralia [9] Sap (obtained from the inflorescence stalk) isused as a drink by the indigenous peoples while young fruitsare eaten [10] The sap is a good source of sugar and usedfor making sweets vinegar beverage and alcohol production[8] The fruit is also rich in carbohydrates fibers mineralsand vitamin A [11] Traditionally leaves stem and rootsof NF are used to treat asthma leprosy tuberculosis sorethroat liver disease snake bite as a pain reliever and can alsobe used as sedative and carminative [12 13] Recently stemand leaf methanol extracts of NF have been shown to haveantidiabetic and analgesic effect [14] Till date no informationon antioxidant activity and identification of phytochemicalsfrom endosperm extract of ripe (EER) and unripe fruits(EEU) of NF has been documented This work is essentialsince it would determine the potency of the extract at differentmaturity levels Hence the objective of the present work is toevaluate the antioxidant capacity of EER and EEU of NF andalso to identify its phenolic compounds

2 Materials and Methods

21 Chemicals and Reagents 2-21015840-Azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) 2 4 6-tri (2-pyridyl)-l3 5-triazine (TPTZ) aluminium chloride tween-20 linoleicacid chlorogenic acid protocatechuic acid kaempferolrutin hydroxybenzoic acid beta carotene quercetin gal-lic acid BHT trichloroacetic acid thiobarbituric acidsodium azide tris-HCl buffer phosphate buffer and Hepeswere obtained from Sigma-Aldrich Co (MO USA) Folin-Ciocalteu reagent trifluoroacetic acid (HPLC Grade) ace-tonitrile (HPLC Grade) and hydrogen peroxide were

Table 1 Physical parameters of Nypa fruticans fruit

Unripe RipeWhole fruit bunch

Weight (kg) 72 plusmn 019b 161 plusmn 016a

Length (cm) 255 plusmn 25b 345 plusmn 27a

Perimeter (cm) 812 plusmn 45b 1089 plusmn 63a

Individual fruitWeight (g) 1381 plusmn 5b 1596 plusmn 77a


Breadth (cm) 78 plusmn 05 81 plusmn 03Endosperm

Weight (g) 36 plusmn 15b 196 plusmn 08a



For each treatment means in a row followed by different letters aresignificantly different at 119875 lt 005

obtained from Merck (Darmstadt Germany) All otherchemicals and solvents used were of analytical grade

22 Plant Material Whole bunch of unripe (Figure 2(a))and ripe fruits (Figure 2(b)) of Nypa fruticans at three andsix monthrsquos maturity respectively were collected on the 6thof February 2011 from Kedah Malaysia with the assistanceof Muda Agricultural Development Authority (MADA)Malaysia Voucher specimens (NFUR622011 andNFR622011)are preserved at MADA office The fruits were immediatelytransported to the laboratory of Universiti Putra MalaysiaUpon arrival the fruits were washed under running tap waterand air dried The individual fruits (Figures 2(c) and 2(d))were separated from the bunch andphysical parameters of thefruits weremeasured (Table 1) Later the fruits weremanuallyseparated to obtain the edible endosperm (Figures 2(e) and2(f)) The endosperm (150 g) was kept in an oven maintainedat 60∘C for drying until constant weight was obtained Thedried portion was allowed to cool and be powdered andsieved (particle size of 20 mesh) to get uniform particle sizeThe dry powder was used for extraction

Evidence-Based Complementary and Alternative Medicine 3

(a) (b)

(c) (d)

(e) (f)

Figure 2 Nypa fruticans unripe (a) and ripe (b) whole fruit bunch unripe (c) and ripe (d) individual fruit and unripe (e) and ripe (f)endosperm

23 Extraction Dry powder (10 g) from unripe and ripeendosperms of NF was extracted separately with 50 ethanol(100mL) using an orbital shaker (Unimax 1010 HeidolphGermany) The shaker was maintained at a speed of 400 rpmand the extraction was carried out at 30∘C for 1 h durationThe extracts were then filtered using Whatman filter paper(no 4) concentrated freeze dried using bench top freezedryer (Virtis NY USA) and stored at minus20∘C until furtheranalysis

24 Determination of Total Phenolics Content Total pheno-lics content (TPC) of the extracts was determined accordingto themethod developed by Singleton and Rossi [15] In brief100 120583L-aliquot of the extract was added to 2mL of 20 gL

Na2CO3solution After 2min 100 120583L of 50 Folin-Ciocalteu

reagent was added and the mixture was allowed to standfor 2 h at 25∘C The absorbance was measured at 750 nmusing a spectrophotometer (UV 1601 Shimadzu Co LtdKyoto Japan) The total phenolics content was determinedusing the standard gallic acid calibration curve and the resultswere expressed as milligram gallic acid equivalents per gramsample dry weight (mg GAEg DW)

25 Determination of Total Flavonoid Content Total fla-vonoid content (TFC) was measured using the aluminumchloride colorimetric assay described by Liu et al [16] Analiquot (01mL) of the extract was mixed with 02mL of 5sodiumnitrite After 5min 02mLof 10 aluminumchloride


and 2mL of 1M sodium hydroxide were added and mixedvigorously Absorbance was measured at 510 nm against ablank The total flavonoid content was determined using astandard curve of rutin and the results were expressed asmilligram rutin equivalent per gram sample dry weight (mgREg DW)

26 Analyses of Antioxidant Activities

261 ABTS Radical Scavenging Activity Radical scavengingactivity of the samples against ABTS was carried out accord-ing to the method described by Re et al [17] ABTS radicalcationwas produced by reactingABTS stock solution (7mM)with 245mM potassium persulfate and allowed the mixtureto stand in the dark at room temperature for 16 h The ABTSsolutionwas diluted to obtain an absorbance of 070 at 734 nmand equilibrated at 30∘C ABTS solution (1mL) was mixedwith the extracts (100 120583L) at different concentrations (10 50100 and 200120583gmL) and the decrease in absorbance after6min of incubationwasmonitored using spectrophotometerControl received only ABTS solution while distilled waterwas used as blankThe inhibition of ABTS radicals by the testsamples was calculated as scavenging activity () = (Controloptical density (OD) minus sample ODcontrol OD)) times 100

262 DPPHRadical Scavenging Activity DPPH radical scav-enging activities were determined based on a method devel-oped by Prasad et al [6] with some minor changes Analiquot of 01mL of the extract at different concentrations(10 50 100 and 200 120583gmL) mixed with 1mL of DPPH(200mM dissolved in methanol) The reaction mixture wasvortexed and incubated at 37∘C in dark light for 30minThe changes in absorbance were measured at 517 nm usinga spectrophotometer The inhibition of DPPHsdot radicals wascalculated as scavenging activity () = (Control ODminus sampleODcontrol OD) times 100 BHT was used for comparison

263 Ferric Reducing Antioxidant Power (FRAP) AssayFerric reducing antioxidant power assay of the extractswas performed according to the method of Re et al [17]Working FRAP reagent was prepared by mixing acetatebuffer (300mM pH 36) 10mM TPTZ solution in 40mMHCL 20mM ferric chloride solution in proportion of 10 1 1(vvv) An aliquot (50 120583L) of appropriately diluted extractwas mixed with 3mL of freshly prepared FRAP reagent andmixed thoroughly The reaction mixture was then incubatedat 37∘C for 30min Absorbance of the reaction mixture wasread at 593 nm against a blank The results were then cal-culated based on the calibration curve plotted using ferroussulphate and expressed as mmol Fe2+100 g dry weight

264 Beta Carotene Bleaching Assay Beta carotene bleach-ing assay was performed according to the method of Veliogluet al [18] with slight modifications One milliliter of 120573-carotene solution (2mgmL dissolved in chloroform) wasadded into brown color round-bottom flask containing002mL of linoleic acid and 02mL of Tween 20 Thechloroform in the mixture was evaporated under vacuum

and 100mL of deionized water was added The mixturewas shaken vigorously to form an emulsion The emulsion(1mL) and 100 120583L of the extract at different concentrations(10 50 100 and 200120583gmL) were pipetted in different testtubes and incubated at 45∘C for 2 h Control received onlythe emulsion without any sample while blank consists ofemulsion without 120573-carotene and the extract Absorbance ofthe solution was monitored at 470 nmThe rate of 120573-carotenebleaching was calculated as antioxidant activity coefficient(AAC) and calculated using the equation

AAC = [As (120) minusAc (120)

Ac (0)minus Ac (120)] times 1000 (1)

where As (120) is absorbance of the sample at time 120minAc (120) is absorbance of control at 120minAc (0) is absorbanceof control at 0min and Ac (120) is absorbance of controlat 120min The higher the AAC values the higher theantioxidant activity

265 Antioxidant Capacity by Phosphomolybdenum MethodAntioxidant capacity by phosphomolybdenum method wasdetermined by the method of Prieto et al [19] An aliquot(01mL) of the extracts at various concentrations (10 50 100and 200120583gmL) was mixed with 1mL of reagent solution(06M sulphuric acid 28mM sodium phosphate and 4mMammonium molybdate) The mixture was covered and incu-bated at 95∘C for 90min After the mixture was cooled itwas centrifuged and absorbance of the supernatant was mea-sured at 695 nm using a spectrophotometer The antioxidantcapacity was expressed as the absorbance value A higherabsorbance value indicates higher antioxidant capacity

27 Identification and Quantification of Phenolic Compou-nds Using Ultra-High-Performance Liquid Chromatography(UHPLC) Individual phenolic compounds in EEU and EERof NF were identified using validated UHPLC methoddescribed by Kong et al [20] on an Agilent 1290 Infinity LCsystem (Agilent Technologies Waldbronn Germany) Thesystemwas equipped with binary pump diode array detectorand an autosampler A C-18 Zorbax Eclipse column (50mmtimes 21mm ID 18120583m Agilent Darmstadt Germany) wasused for polyphenol separation and it was maintained at25∘C Five microliter of the sample was injected into thesystem and the elution program was set as follows Mobilephase A is comprised of 01 trifluoroacetic acid (TFA) whilemobile phase B contained acetonitrile and the flow ratewas set at 06mLmin The following linear gradient elutionwas carried out for separation of polyphenols 15 B for6min 25 B for 3min 60 B for 3min 80 B for 06100 B for 08min The total runtime was 14min UV-Visabsorption spectra were monitored by diode array detector(DAD) at 280 nmThe identification of phenolic compoundswas achieved by comparison with retention times and UV-Vis absorption spectra with standards availableThe phenoliccompounds were quantified on the bases of their peak areasand calibration curves of the corresponding standards andthen expressed as microgram per gram dry weight (120583ggDW)


28 Statistical Analysis Datawere expressed asmeansplusmn stan-dard deviations (SD) of three determinations and analyzedby SPSS V13 (SPSS Inc Chicago USA) One way analysisof variance (ANOVA) and Duncanrsquos multiple-range test wereused to determine the differences among the means P valuesof lt005 were considered to be significantly different

3 Results and Discussion

The fruit of NF was selected for the present investigationsince it is one of the important underutilized plants ofMalaysia Although the sap is often used as beverage [8] thefruits are discarded Many underutilized fruits in Malaysiahad been documented previously to be rich in antioxidantswith many health benefits [5ndash7] Weight (1596 plusmn 77 g) andlength (129 plusmn 07 cm) of ripe fruits were significantly higher(119875 lt 005) than unripe ones (Table 1) Unripe endospermis soft juicy and pale white in color (Figure 2(e)) while theripe endosperm is hard andmilkywhite in color (Figure 2(f))These data are important for the food processing industry

31 Total Phenolics and Flavonoid Contents Phenolics andflavonoids present in fruits and vegetables have receivedconsiderable attention due to their potential antioxidantactivities [21] Phenolic compounds undergo a complex redoxreaction with the phosphotungstic and phosphomolybdicacids present in Folin-Ciocalteu (FC) reagent However itshould be also noted that some chemical groups of proteinsorganic acids and sugars present in the extracts might alsoreact with FC reagent and therefore interfere with the result[22] EEU of NF had significantly (119875 lt 005) higher totalphenolics content (1356 plusmn 45mg GAEg) compared to EER(88 plusmn 20mg GAEg) In addition higher total flavonoidcontent (119875 lt 005) was also noticed in EEU (686 plusmn 31mgREg) compared to EER (36 plusmn 04mg REg)

32 Antioxidant Capacity Assay Different antioxidant com-pounds could react through differentmechanisms and hencea single method alone cannot fully evaluate the antioxidantcapacity of foods [23] For this reason different antioxidantcapacity tests with different approaches and mechanismswere carried out in the present study

321 ABTS Assay ABTS method is developed based on adecolorization technique Once added the antioxidant causesa reduction of ABTS which could be measured at 734 nmThis assay is performed to measure the ability of antioxidantsto inhibit radical cation induced by persulfate Strong antiox-idants have the ability to change the blue color of ABTS intolight blue color The change in absorbance is proportionalto the antioxidant concentration [22] Figure 3 illustrates theeffect of theNF extracts against ABTS radicals EEU exhibitedsignificantly (119875 lt 005) higher scavenging activity of 78plusmn12at a concentration of 200120583gmL higher than BHT standard(75 plusmn 26) EER exhibited moderate activity of 55 plusmn 43The IC

50(concentration of the extractsample to scavenge

50 ABTS radicals) values were also obtained The lowerthe IC

50value the higher the antioxidant activity The IC

50

0

25

50

75

100

0 10 50 100 200

UnripeRipeBHT

ABT

S ra

dica

lsc

aven

ging

activ

ity (

) lowast

Concentration (120583gmL)

Figure 3 ABTS radical scavenging activity of unripe and ripeendosperm extracts of Nypa fruticans lowastdenotes significant differ-ences at 119875 lt 005

values of EEU (52 plusmn 27 120583gmL) were parallel to BHT (51 plusmn17 120583gmL) but EER showed higher values (187 plusmn 47 120583gmL)Our results are supported by similar findings by Gordonet al [24] where unripe acacia fruits exhibited higher ABTSradical scavenging activity as compared to ripe fruits Unripecactus berry exhibited two times higher ABTS scavengingactivity compared to ripe fruits [25] The antioxidant activityof the extracts in the present investigation is probably due tothe action of hydroxyl groups of phenolic compounds whichmight act as hydrogen donors

322 DPPH Radical Scavenging Activity DPPH is a stablefree radical and accepts an electron or hydrogen radical tobecome a stable diamagnetic molecule which is widely usedto investigate radical scavenging activity In DPPH radicalscavenging assay antioxidants react with DPPH and con-vert it to yellow coloured 120572120572-diphenyl-120573-picryl hydrazineThe degree of discoloration indicates the radical-scavengingpotential of the antioxidant [26] DPPH radical scavengingactivity of NF extracts and BHT increased as concentrationincreased (Figure 4) EEU exhibited the highest scavengingactivity (85 plusmn 26) significantly (119875 lt 005) higher thanEER (32 plusmn 28) at a concentration of 200120583gmL and wascomparable to the scavenging activity of BHT (88 plusmn 12)IC50

value of EEU (36 plusmn 12 120583gmL) was more potent thanEER (312 plusmn 48 120583gmL) However BHT showed the highestantioxidant activity with IC

50value of 12 plusmn 07 120583gmL It

has been found that phenolics flavonoids and tocopherolsscavenge DPPH radicals by their hydrogen-donating ability[22]The results obtained in this investigation also reveal thatthe sample extracts act as free radical scavengers whichmightbe attributed to their electron-donating ability

323 Ferric Reducing Antioxidant Power (FRAP) Ferricreducing antioxidant power is widely used in evaluatingantioxidant activity of plant polyphenols Principally FRAPassay treats the antioxidants in the sample as reductantin a redox-linked colorimetric reaction [22] This assay is

6 Evidence-Based Complementary and Alternative MedicineD

PPH

scav

engi

ng ac

tivity

()

0 10 50 100 200Concentration (120583gmL)

0

25

50

75

100

UnripeRipeBHT

lowast lowast

Figure 4 DPPH radical scavenging activity of unripe and ripeendosperm extracts of Nypa fruticans lowastdenotes significant differ-ences at 119875 lt 005

0100200300400500600700800


UnripeRipeBHT

lowast

lowast

lowastlowast

(mM

Fe2+

100

g)

Figure 5 FRAP values of unripe and ripe endosperm extracts ofNypa fruticans lowastdenotes significant differences at 119875 lt 005

relatively simple and easy to conduct FRAP assay measuresthe reducing potential of antioxidant to react on ferrictripyridyltriazine (Fe3+-TPTZ) complex and produce bluecolor of ferrous form which can be detected at absorbance of593 nm [15] Antioxidant compounds which act as reducingagent exert their effect by donating hydrogen atom to ferriccomplex and thus break the radical chain reaction [27] Inthe present study EEU exhibited the highest reducing powerfollowed by BHT and EER (Figure 5) At 200120583gmL thereducing power of EEU BHT and EERwas 819plusmn43 250plusmn16and 147 plusmn 07mmol Fe2+100 g dry weight respectively Thereducing power of fruit fractions is probably due to the actionof hydroxyl group of the phenolic compounds which mightact as electron donors

324 BetaCarotene BleachingAssay Beta carotene bleachingmethod iswidely used tomeasure antioxidant activity of plant

0

5

10

15

20

25

30

Ant

ioxi

dant

activ

ity co

effici

ent

a

bb

a b

c

aa

b

a

b

c

10 50 100 200Concentration (120583gmL)

UnripeRipeBHT

times102

Figure 6 Antioxidant activity coefficient values determined by betacarotene bleaching method of unripe and ripe endosperm extractsof Nypa fruticans For each treatment means in a row followed bydifferent letters are significantly different at 119875 lt 005

extracts It is an in vitro assay that measures the inhibition ofcoupled autooxidation of linoleic acid and 120573-carotene Thismethod is based on lipid radicals as autooxidation productsof linoleic acid which attack the double bonds of 120573-carotenebut in presence of antioxidants they can inhibit the oxidationand retain the yellowish-orange color of beta carotene andthus reduce their bleaching activity [28] The bleachingactivities of NF extracts were concentration dependent andthe activities increased as the concentration increased EEUexhibited excellent antioxidant activity coefficient of 2550 plusmn28 at a concentration of 200120583gmL significantly higher(119875 lt 005) than EER (1729 plusmn 19) and BHT (1183 plusmn34) (Figure 6) The antioxidant activity of the extracts inthe present investigation is probably due to the transfer ofhydrogen atom from phenolic compounds to the free radicaltherefore inhibiting bleaching of beta carotene

325 Antioxidant Capacity by Phosphomolybdenum MethodAntioxidant capacities of NF extracts weremeasured spectro-photometrically using phosphomolybdenum method whichis based on the reduction ofMo (IV) toMo (V) by the sampleanalyte and subsequent formation of green phosphateMo(V) compounds with a maximum absorption at 695 nm [19]A high absorbance value of the sample indicates its strongantioxidant activity Figure 7 shows the total antioxidantcapacities of NF extracts and BHT All the extracts showeda concentration-dependent activity The total antioxidantactivity of EEU at 200120583gmL was 09 (Figure 3) significantlyhigher (119875 lt 005) than EEU (03) and BHT (07) Howeverthe total antioxidant activity of BHT at all other concen-trations tested was higher than other extracts PreviouslyJayaprakasha et al [29] indicated that total antioxidant activ-ity of citrus was due to the presence of bioactive compoundsin the form of phenolics and flavonoids Hence probably inthe present investigation the antioxidant capacity might beattributed to the reducing activity of phenolic compounds


Table 2 Phenolic compound composition of unripe and ripe endosperm extracts of Nypa fruticans (120583gg DW of plant material)

Peak number Compound Retention time (min) 120582max (nm) Unripe Ripe1 Gallic acid 21 272 047 plusmn 002 046 plusmn 001

2 Protocatechuic acid 29 294 552 plusmn 040a 388 plusmn 050b

3 Hydroxybenzoic acid 39 256 110 plusmn 007a 087 plusmn 006b

4 Chlorogenic acid 61 326 145 plusmn 130a 97 plusmn 110b

5 Rutin 95 354 27 plusmn 004a 22 plusmn 007b

6 Cinnamic acid 107 272 11 plusmn 001 09 plusmn 002

7 Quercetin 116 372 173 plusmn 002 174 plusmn 07

8 Kaempferol 119 364 32 plusmn 040 30 plusmn 010

For each treatment means in a row followed by different letters are significantly different at 119875 lt 005

0010203040506070809

1

a

bb

a

b c

a

bc

a

c

b

Abso

rban

ce at

695

nm


UnripeRipeBHT

Figure 7 Antioxidant capacity of unripe and ripe endospermextracts of Nypa fruticans determined by phosphomolybdenummethod For each treatment means in a row followed by differentletters are significantly different at 119875 lt 005

33 Identification and Quantification of Phenolic CompoundsHPLC chromatograms from EEU (Figure 8(a)) and EER(Figure 8(b)) of NF are provided Eight phenolic compoundswere identified where chlorogenic acid protocatechuic acidand kaempferol were the major compounds Among all thephenolic compounds identified chlorogenic acid was thehighest in EEU (145 plusmn 130 120583gg) and EER (97 plusmn 110 120583gg)while the lowest was gallic acid (Table 2) Protocatechuicacid occupied the second major compound as noted inEEU (55 plusmn 040 120583gg) and EER (38 plusmn 050 120583gg) followedby kaempferol In addition hydroxy benzoic acid rutincinnamic acid and quercetinwere detected inminor amountPhenolic compounds (chlorogenic acid protocatechuic acidrutin and hydroxybenzoic acid) of EEU were significantlyhigher (119875 lt 005) than EER

Our results are in good agreement with Monde et al[30] where chlorogenic acid was reported as the majorcompound in oil palm fruits In addition caffeic acid rutinand quercetin were also detected Da Silva Campelo Borgeset al [31] have identified gallic acid hydroxy benzoic acidferulic acid and quercetin from jussara palm plant Gordonet al [24] reported from acacia palm that protocatechuicacid chlorogenic acid hydroxy benzoic acid and gallic acid

decreased as fruit maturity increased In support to currentfindings Herrera-Hernandez et al [25] reported high gallicacid ellagic acid caffeic acid and quercetin in unripe fruitswhen compared to ripe fruits

Antioxidant activity and phenolic content of EEU deter-mined in the present study were higher compared to EERThis is in good agreement with other researchers wherethey had reported parallel results [25 32 33] Phenoliccompounds are synthesized rapidly during the early stages offruit maturity Once the fruit matures decrease in phenolicconcentration is noticed due to the dilution caused by cellgrowth [34] Reduction of primary metabolism in the ripefruit due to lack of substrates necessary for the biosynthesisof phenolic compounds also results in decrease of phenolicscompounds In addition polymerisation oxidation andconjugation of bound phenolics duringmaturation could alsoresult in decrease of phenolic composition [32]

The total phenolics content of NF is higher whencompared to acai palm (1231 GAEg) [24] and date palm(48GAEg) [35] Statistical correlations between total phe-nolics content and antioxidant activities were also deter-mined Total phenolics content exhibited the highest associa-tions with FRAP (119903 = 09815) total antioxidant capacity (119903 =09523) and DPPH (119903 = 08594) For total flavonoid contentits correlation with FRAP and antioxidant capacity was low(119903 = 0249 and 0315 resp) while negative correlation wasobserved for other antioxidant assays Thus the amount ofphenolic compounds could be used as an important indicatorof antioxidant capacity This also clearly indicated that totalphenolics in EEU and EER of NF are major contributors forantioxidant activities since they have a high correlation whileflavonoids are not the major contributors for antioxidantactivities In addition antioxidant activity could also becontributed by synergistic action of other phytochemicalswhich are not determined in the current study such ascarotenoids and polysaccharides Previously many authorshave reported a positive correlation between total phenolicscontent and antioxidant actity [16 34] Liu et al [16] reportednegative correlation between antioxidant activity and totalflavonoid content

4 Conclusion

Unripe endosperm extract of Nypa fruticans showed hightotal phenolics total flavonoid content and antioxidant


14

121

08060402

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

56

7

8

(a)

14

12

1

08

06

04

02

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

5 6

7

8

1

2

3

4

5 6

7

8

(b)

Figure 8 HPLC chromatogram of unripe (a) and ripe endosperm extract (b) ofNypa frutican The peak identifications are (1) gallic acid (2)protocatechuic acid (3) hydroxybenzoic acid (4) chlorogenic acid (5) rutin (6) cinnamic acid (7) quercetin and (8) kaempferol

capacities as compared to ripe endosperm extract Chloro-genic acid protocatechuic acid and kaempferol were iden-tified as major compounds in the extract Thus unripeendosperm extract of NF could be used as natural antioxi-dant Further investigations on the evaluation of nutritionalcomposition and other therapeutic uses of Nypa fruticansendosperm extracts are worth investigating

Conflict of Interests

The authors have no conflict of interest to declare

Acknowledgments

Thefinancial support of Science-Fund fromMinistry of Agri-culture Malaysia (02-02-10-SF0131 and 05-01-04-SF1137) isthankfully acknowledged The authors would like to thankthe assistance of laboratory staffs from the Department ofNutrition and Dietetic and the use of laboratories facilities ofUniversiti Putra Malaysia throughout the research project

References

[1] M Namiki ldquoAntioxidantsantimutagens in foodrdquo CriticalReviews in Food Science and Nutrition vol 29 no 4 pp 273ndash300 1990

[2] Z Laura C Caliceti F V D Sega et al ldquoDietary phenolicacids act as effective antioxidants in membrane models andin cultured cells exhibiting proapoptotic effects in leukaemiacellsrdquo Oxidative Medicine and Cellular Longevity vol 2012Article ID 839298 2012

[3] A Gordon A P G Cruz L M C Cabral et al ldquoChemicalcharacterization and evaluation of antioxidant properties ofAcai fruits (Euterpe oleraceae Mart) during ripeningrdquo FoodChemistry vol 133 pp 256ndash263 2012

[4] TNManojlovic P J Vasiljevic P ZMaskovicM Juskovic andG Bogdanovic-Dusanovic ldquoChemical composition antioxi-dant and antimicrobial activities of Lichen Umbilicaria cylin-drica (L) Delise (Umbilicariaceae)rdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2012 Article ID 4524318 pages 2012

[5] K N Prasad K W Kong N S Ramannan A Azrina andI Amin ldquoSelection of experimental domain using two levelfactorial design to determine extract yield antioxidant capacityphenolics and flavonoids from Mangifera pajang KostermrdquoSeparation Science and Technology vol 47 no 16 pp 2417ndash24232012

[6] K N Prasad L Y Chew H E Khoo B Yang A Azlan and AIsmail ldquoCarotenoids and antioxidant capacities fromCanariumodontophyllum Miq fruitrdquo Food Chemistry vol 124 no 4 pp1549ndash1555 2011

[7] E H K Ikram K H Eng A M M Jalil et al ldquoAntioxidantcapacity and total phenolic content of Malaysian underutilizedfruitsrdquo Journal of Food Composition and Analysis vol 22 no 5pp 388ndash393 2009

[8] L Joshi U Kanagaratnam and D Adhuri ldquoNypa fruticansmdashuseful but forgotten in mangrove reforestration programsrdquo2006 httpwwwworldagroforestrycentreorgSeaW-NewdatasAceh30Nov06720Nypa20fruticans-useful20butforgotten20in20mangrovepdf

[9] P Tamunaidu and S Saka ldquoChemical characterization ofvarious parts of nipa palm (Nypa fruticans)rdquo Industrial Cropsand Products vol 34 pp 1423ndash1428 2011

[10] S Y Tang S Hara L Melling K J Goh and Y HashidokoldquoBurkholderia vietnamiensis isolated from root tissues of nipapalm (Nypa fruticans) in Sarawak Malaysia proved to beits major endophytic nitrogen-fixing bacteriumrdquo BioscienceBiotechnology and Biochemistry vol 74 no 9 pp 1972ndash19752010

[11] V N Osabor G E Egbung and P C Okafor ldquoChemical profileof Nypa fruiticans from Cross River Estuary south easternNigeriardquo Pakistan Journal of Nutrition vol 7 no 1 pp 146ndash1502008

[12] M Rahmatullah S M I Sadeak S C Bachar et al ldquoBrineShrimp Toxicity Study of different Bangladeshi MedicinalPlantsrdquo Advances in Natural and Applied Sciences vol 4 pp163ndash173 2010

[13] WM Bandaranayake ldquoTraditional andmedicinal uses of man-grovesrdquo Mangroves and Salt Marshes vol 2 no 3 pp 133ndash1481998

[14] H Reza V M Haq A K Das S Rahman R Jahan and MRahmatullah ldquoAanti-hyperglycemic and antinociceptive activ-ity of methanol leaf and stem extract ofNypa fruticansWurmbrdquo


Pakistan Journal of Pharmaceutical Science vol 24 pp 485ndash4882011

[15] V A Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquo Jour-nal of Enology and Vitriculture vol 16 pp 144ndash158 1965

[16] H Liu N Qiu H Ding and R Yao ldquoPolyphenols contents andantioxidant capacity of 68 Chinese herbals suitable for medicalor food usesrdquoFoodResearch International vol 41 no 4 pp 363ndash370 2008

[17] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[18] Y S Velioglu GMazza L Gao and B D Oomah ldquoAntioxidantactivity and total phenolics in selected fruits vegetables andgrain productsrdquo Journal of Agricultural and Food Chemistry vol46 no 10 pp 4113ndash4117 1998

[19] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[20] KWKong SM JunitNAminudin I Amin andAAAzlinaldquoAntioxidant activities and polyphenolics from the shoots ofBarringtonia racemosa (L) Spreng in a polar to apolar mediumsystemrdquo Food Chemistry vol 134 pp 324ndash332 2012

[21] L Bravo ldquoPolyphenols chemistry dietary sources metabolismand nutritional significancerdquo Nutrition Reviews vol 56 no 11pp 317ndash333 1987

[22] D Huang O U Boxin and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005

[23] N Pellegrini M Serafini B Colombi et al ldquoTotal antioxidantcapacity of plant foods beverages and oils consumed in Italyassessed by three different in vitro assaysrdquo Journal of Nutritionvol 133 no 9 pp 2812ndash2819 2003


[25] MGHerrera-Hernandez F Guevara-Lara R Reynoso-Cama-cho and S H Guzman-Maldonado ldquoEffects of maturity stageand storage on cactus berry (Myrtillocactus geometrizans) phe-nolics vitamin C betalains and their antioxidant propertiesrdquoFood Chemistry vol 129 pp 1744ndash1750 2011

[26] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958

[27] N Singh and P S Rajini ldquoFree radical scavenging activity of anaqueous extract of potato peelrdquo Food Chemistry vol 85 no 4pp 611ndash616 2004

[28] N I Krinsky ldquoAntioxidant functions of carotenoidsrdquo FreeRadical Biology and Medicine vol 7 no 6 pp 617ndash635 1989

[29] G K Jayaprakasha B Girennavar and B S Patil ldquoRadicalscavenging activities of Rio Red grapefruits and Sour orangefruit extracts in different in vitro model systemsrdquo BioresourceTechnology vol 99 no 10 pp 4484ndash4494 2008

[30] A Monde M A Carbonneau F Michel et al ldquoPotential healthimplication of in vitro human low-density lipoprotein-vitaminE oxidation modulation by polyphenols derived from cotedrsquoIvoirersquos oil palm speciesrdquo Journal of Agriculture and FoodChemistry vol 59 pp 9166ndash9171 2011

[31] G Da Silva Campelo Borges F Gracieli Kunradi VieiraC Copetti et al ldquoChemical characterization bioactive com-pounds and antioxidant capacity of jussara (Euterpe edulis)fruit from the Atlantic Forest in southern Brazilrdquo Food ResearchInternational vol 44 no 7 pp 2128ndash2133 2011

[32] J Gruz F A Ayaz H Torun and M Strnad ldquoPhenolic acidcontent and radical scavenging activity of extracts frommedlar(Mespilus germanica L) fruit at different stages of ripeningrdquoFood Chemistry vol 124 no 1 pp 271ndash277 2011

[33] F Menichini M R Loizzo M Bonesi et al ldquoPhytochemi-cal profile antioxidant anti-inflammatory and hypoglycemicpotential of hydroalcoholic extracts from Citrus medica L cvDiamante flowers leaves and fruits at twomaturity stagesrdquo Foodand Chemical Toxicology vol 49 no 7 pp 1549ndash1555 2011

[34] A Ismail K N Prasad L Y Chew H E Khoo K W Kongand A Azlan ldquoAntioxidant capacities of peel pulp and seedfractions of canarium odontophyllum Miq fruitrdquo Journal ofBiomedicine and Biotechnology vol 2010 Article ID 871379 8pages 2010

[35] J Castillo O Benavente and J A Del Rıo ldquoNaringin and neo-hesperidin levels during development of leaves flower budsand fruits of citrus aurantiumrdquo Plant Physiology vol 99 no 1pp 67ndash73 1992

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


(a) (b)

Figure 1 Nypa fruticans (a) and fruit bunch (b)

for skin and pulp with health-promoting benefits Howevermany of these fruits are still not familiar due to lack ofpublicity and promotional campaign [7]

Nypa fruticans Wurmb (NF) belongs to Araceae familyand is considered as ldquounderutilizedrdquo plant [8] Other syn-onym of this plant includes Cocos nypa Lour Nypa fruticansThunb and Nypa palm It is a monoecious palm foundgrowing in brackish water with upright stem (Figure 1(a))trunkless with fruits found commonly emerging from the soil(Figure 1(b)) This palm is typically found in India MalaysiaIndonesia Philippines and in some parts of QueenslandAustralia [9] Sap (obtained from the inflorescence stalk) isused as a drink by the indigenous peoples while young fruitsare eaten [10] The sap is a good source of sugar and usedfor making sweets vinegar beverage and alcohol production[8] The fruit is also rich in carbohydrates fibers mineralsand vitamin A [11] Traditionally leaves stem and rootsof NF are used to treat asthma leprosy tuberculosis sorethroat liver disease snake bite as a pain reliever and can alsobe used as sedative and carminative [12 13] Recently stemand leaf methanol extracts of NF have been shown to haveantidiabetic and analgesic effect [14] Till date no informationon antioxidant activity and identification of phytochemicalsfrom endosperm extract of ripe (EER) and unripe fruits(EEU) of NF has been documented This work is essentialsince it would determine the potency of the extract at differentmaturity levels Hence the objective of the present work is toevaluate the antioxidant capacity of EER and EEU of NF andalso to identify its phenolic compounds

2 Materials and Methods

21 Chemicals and Reagents 2-21015840-Azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) 2 4 6-tri (2-pyridyl)-l3 5-triazine (TPTZ) aluminium chloride tween-20 linoleicacid chlorogenic acid protocatechuic acid kaempferolrutin hydroxybenzoic acid beta carotene quercetin gal-lic acid BHT trichloroacetic acid thiobarbituric acidsodium azide tris-HCl buffer phosphate buffer and Hepeswere obtained from Sigma-Aldrich Co (MO USA) Folin-Ciocalteu reagent trifluoroacetic acid (HPLC Grade) ace-tonitrile (HPLC Grade) and hydrogen peroxide were

Table 1 Physical parameters of Nypa fruticans fruit

Unripe RipeWhole fruit bunch

Weight (kg) 72 plusmn 019b 161 plusmn 016a



Individual fruitWeight (g) 1381 plusmn 5b 1596 plusmn 77a


Breadth (cm) 78 plusmn 05 81 plusmn 03Endosperm

Weight (g) 36 plusmn 15b 196 plusmn 08a



For each treatment means in a row followed by different letters aresignificantly different at 119875 lt 005

obtained from Merck (Darmstadt Germany) All otherchemicals and solvents used were of analytical grade

22 Plant Material Whole bunch of unripe (Figure 2(a))and ripe fruits (Figure 2(b)) of Nypa fruticans at three andsix monthrsquos maturity respectively were collected on the 6thof February 2011 from Kedah Malaysia with the assistanceof Muda Agricultural Development Authority (MADA)Malaysia Voucher specimens (NFUR622011 andNFR622011)are preserved at MADA office The fruits were immediatelytransported to the laboratory of Universiti Putra MalaysiaUpon arrival the fruits were washed under running tap waterand air dried The individual fruits (Figures 2(c) and 2(d))were separated from the bunch andphysical parameters of thefruits weremeasured (Table 1) Later the fruits weremanuallyseparated to obtain the edible endosperm (Figures 2(e) and2(f)) The endosperm (150 g) was kept in an oven maintainedat 60∘C for drying until constant weight was obtained Thedried portion was allowed to cool and be powdered andsieved (particle size of 20 mesh) to get uniform particle sizeThe dry powder was used for extraction


(a) (b)

(c) (d)

(e) (f)
















Ac (0)minus Ac (120)] times 1000 (1)














50

0

25

50

75

100

0 10 50 100 200

UnripeRipeBHT

ABT

S ra

dica

lsc

aven

ging

activ

ity (

) lowast










PPH

scav

engi

ng ac

tivity

()


0

25

50

75

100

UnripeRipeBHT

lowast lowast


0100200300400500600700800


UnripeRipeBHT

lowast

lowast

lowastlowast

(mM

Fe2+

100

g)




0

5

10

15

20

25

30

Ant

ioxi

dant

activ

ity co

effici

ent

a

bb

a b

c

aa

b

a

b

c


UnripeRipeBHT

times102















0010203040506070809

1

a

bb

a

b c

a

bc

a

c

b

Abso

rban

ce at

695

nm


UnripeRipeBHT







4 Conclusion



14

121

08060402

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

56

7

8

(a)

14

12

1

08

06

04

02

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

5 6

7

8

1

2

3

4

5 6

7

8

(b)





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e) (f)
















Ac (0)minus Ac (120)] times 1000 (1)














50

0

25

50

75

100

0 10 50 100 200

UnripeRipeBHT

ABT

S ra

dica

lsc

aven

ging

activ

ity (

) lowast










PPH

scav

engi

ng ac

tivity

()


0

25

50

75

100

UnripeRipeBHT

lowast lowast


0100200300400500600700800


UnripeRipeBHT

lowast

lowast

lowastlowast

(mM

Fe2+

100

g)




0

5

10

15

20

25

30

Ant

ioxi

dant

activ

ity co

effici

ent

a

bb

a b

c

aa

b

a

b

c


UnripeRipeBHT

times102















0010203040506070809

1

a

bb

a

b c

a

bc

a

c

b

Abso

rban

ce at

695

nm


UnripeRipeBHT







4 Conclusion



14

121

08060402

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

56

7

8

(a)

14

12

1

08

06

04

02

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

5 6

7

8

1

2

3

4

5 6

7

8

(b)





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Ac (0)minus Ac (120)] times 1000 (1)














50

0

25

50

75

100

0 10 50 100 200

UnripeRipeBHT

ABT

S ra

dica

lsc

aven

ging

activ

ity (

) lowast










PPH

scav

engi

ng ac

tivity

()


0

25

50

75

100

UnripeRipeBHT

lowast lowast


0100200300400500600700800


UnripeRipeBHT

lowast

lowast

lowastlowast

(mM

Fe2+

100

g)




0

5

10

15

20

25

30

Ant

ioxi

dant

activ

ity co

effici

ent

a

bb

a b

c

aa

b

a

b

c


UnripeRipeBHT

times102















0010203040506070809

1

a

bb

a

b c

a

bc

a

c

b

Abso

rban

ce at

695

nm


UnripeRipeBHT







4 Conclusion



14

121

08060402

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

56

7

8

(a)

14

12

1

08

06

04

02

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

5 6

7

8

1

2

3

4

5 6

7

8

(b)





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























50

0

25

50

75

100

0 10 50 100 200

UnripeRipeBHT

ABT

S ra

dica

lsc

aven

ging

activ

ity (

) lowast










PPH

scav

engi

ng ac

tivity

()


0

25

50

75

100

UnripeRipeBHT

lowast lowast


0100200300400500600700800


UnripeRipeBHT

lowast

lowast

lowastlowast

(mM

Fe2+

100

g)




0

5

10

15

20

25

30

Ant

ioxi

dant

activ

ity co

effici

ent

a

bb

a b

c

aa

b

a

b

c


UnripeRipeBHT

times102















0010203040506070809

1

a

bb

a

b c

a

bc

a

c

b

Abso

rban

ce at

695

nm


UnripeRipeBHT







4 Conclusion



14

121

08060402

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

56

7

8

(a)

14

12

1

08

06

04

02

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

5 6

7

8

1

2

3

4

5 6

7

8

(b)





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PPH

scav

engi

ng ac

tivity

()


0

25

50

75

100

UnripeRipeBHT

lowast lowast


0100200300400500600700800


UnripeRipeBHT

lowast

lowast

lowastlowast

(mM

Fe2+

100

g)




0

5

10

15

20

25

30

Ant

ioxi

dant

activ

ity co

effici

ent

a

bb

a b

c

aa

b

a

b

c


UnripeRipeBHT

times102















0010203040506070809

1

a

bb

a

b c

a

bc

a

c

b

Abso

rban

ce at

695

nm


UnripeRipeBHT







4 Conclusion



14

121

08060402

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

56

7

8

(a)

14

12

1

08

06

04

02

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

5 6

7

8

1

2

3

4

5 6

7

8

(b)





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























0010203040506070809

1

a

bb

a

b c

a

bc

a

c

b

Abso

rban

ce at

695

nm


UnripeRipeBHT







4 Conclusion



14

121

08060402

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

56

7

8

(a)

14

12

1

08

06

04

02

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

5 6

7

8

1

2

3

4

5 6

7

8

(b)





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















14

121

08060402

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

56

7

8

(a)

14

12

1

08

06

04

02

00 2 4 6 8 10 12 14

(min)

(mAU

)

1

2

3

4

5 6

7

8

1

2

3

4

5 6

7

8

(b)





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Phytochemicals and Antioxidant Capacity