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Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 434057 13 pageshttpdxdoiorg1011552013434057

Research ArticleAntioxidative and Cardioprotective Properties ofAnthocyanins from Defatted Dabai Extracts

Hock Eng Khoo1 Azrina Azlan12 M Halid Nurulhuda1 Amin Ismail12

Faridah Abas3 Muhajir Hamid4 and Suri Roowi5

1 Department of Nutrition and Dietetics Faculty of Medicine and Health Sciences Universiti Putra Malaysia (UPM)43400 Serdang Selangor Malaysia

2 Laboratory of Halal Science Research Halal Products Research Institute Universiti Putra Malaysia 43400 SerdangSelangor Malaysia

3 Laboratory of Natural Products Institute of Bioscience Universiti Putra Malaysia (UPM) 43400 Serdang Selangor Malaysia4Department of Microbiology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (UPM)43400 Serdang Selangor Malaysia

5 Food Technology Research Centre MARDI Headquarters Serdang PO Box 12301 50774 Kuala Lumpur Malaysia

Correspondence should be addressed to Azrina Azlan azrinaazupmedumy

Received 5 February 2013 Accepted 6 October 2013

Academic Editor Ki-Wan Oh

Copyright copy 2013 Hock Eng Khoo et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study aimed to determine anthocyanins and their antioxidative and cardioprotective properties in defatted dabai partsAnthocyanins in crude extracts and extract fractions of defatted dabai peel and pericarp were quantified using UHPLC whiletheir antioxidant capacity and oxidative stress inhibition ability were evaluated by using DPPH and CUPRAC assays as well aslinoleic acid oxidation system hemoglobin oxidation and PARP-1 inhibition ELISA Cardioprotective effect of the defatted dabaipeel extract was evaluated using hypercholesterolemic-induced New Zealand white rabbits Six anthocyanins were detected in thedefatted dabai peel with the highest antioxidant capacities and oxidative stress inhibition effect compared to the other part Thedefatted dabai peel extract has also inhibited lipid peroxidation (plasmaMDA) and elevated cellular antioxidant enzymes (SOD andGPx) in the tested animal model Major anthocyanin (cyanidin-3-glucoside) and other anthocyanins (pelargonidin-3-glucosidemalvidin-3-glucoside cyanidin-3-galactoside cyanidin-3-arabinoside and peonidin-3-glucoside) detected in the defatted dabaipeel are potential future nutraceuticals with promising medicinal properties

1 Introduction

Anthocyanins are pigments contributed to red purple andblue colors of many fruits and vegetables Anthocyanins haveimportant roles in plant metabolism such as phytoprotectiveagents and pollination attractants [1] As a member of pheno-lic compounds anthocyanin is potential antioxidant Due totheir chemical structures anthocyanins are considered strongantioxidants Anthocyanin has been reported to activelyscavenge free radicals [2] and acts as a potent cardioprotectiveagent [3]

Dabai (Canarium odontophyllum) is a type of olive-likepurple-colored fruit that is rich in anthocyanin Dabai treescan be found in Sumatra Borneo (Sarawak Sabah and East

Kalimantan) and Philippines Chew et al [4] reported thatdabai fruit contained cyanidin-3-glucoside as the majoranthocyaninThe anthocyanin is predominantly found in thepeel of dabai fruit Besides dabai berries such as bilberryblack berry chokeberry mulberry and dogwood berry havehigh anthocyanin contents Most of these berries have beenfound to contain high amounts of cyanidin-3-glucoside[5ndash7]

Extraction of fat fromdabai fruit results in purplish defat-ted dabai powder that is rich in anthocyanins During thisprocess small amount of anthocyanin could be lost in theextracted oil butmost anthocyanins are retained in the defat-ted dabai powder especially its peel Anthocyanins in thedefatted dabai powder have great potential in reducing

2 Evidence-Based Complementary and Alternative Medicine

in vivo oxidative stress and further prevent cardiovasculardiseases Antiatherosclerotic effects of defatted and nondefat-ted dabai pericarp and peel have been determined previouslyin hypercholesterolemic rabbits [8] Results from several invitro antioxidant assays have also shown protective effect ofdabai pericarp [9 10] Several biological oxidation activityassays such as linoleic acid oxidation hemoglobin oxidationand PARP-1 inhibition activity as well as lipid peroxidationmarker (plasma MDA) and antioxidant enzymes (SOD andGPx) in blood are good predictors for inhibition of oxidativestress and cardioprotective effect Therefore these markersare useful in provision of information and confirmation ofthe cardioprotective effect of anthocyanins extracted fromdefatted dabai powder especially from its peel

Due to the potential health benefits offered by antho-cyanins in defatted dabai it is of great interest to elucidatethe specific anthocyanins in the defatted dabai especially inits peel Solid phase extraction (SPE) is a separation techniquethat had been widely used for purification of polyphenols [1112] and pesticides [13] However no previous study has beendone to fractionate potential anthocyanins in defatted dabaiextract using this separation technique Therefore this studyaimed to determine the anthocyanins content in the extractsand extract fractions of defatted dabai peel and pericarptheir antioxidant capacity and related-health benefits usingin vitro assays (DPPHassay copper (II) reduction antioxidantcapacity (CUPRAC) assay linoleic acid oxidation systemhemoglobin oxidation and PARP-1 inhibition ELISA) Invivo study to evaluate cardioprotective effect of the defat-ted dabai peel extract on antioxidant enzymes (SOD andGPx) and lipid peroxidation parameter (plasma MDA) ofhypercholesterolemic-induced New Zealand white rabbitswas also carried out to support the beneficial effect of theextract

2 Materials and Methods

21 Sample Preparation Fresh dabai fruits were obtainedfrom a few locations from fruit plantations in Kapit SarawakMalaysia A homogenized sample was selected randomlyfrom a few different trees in each location Kernel of dabaifruit was discarded while dabai peel and a mixture of itspericarp were collected Both of the samples were freeze-dried using a freeze dryer (Virtis New York USA) and thelyophilized samples were ground into smaller particles usinga household grinder To prepare anthocyanin-rich defattedsamples the freeze-dried peel and pericarp were initiallydefatted by soaking in hexane for 24 h The defatted sampleswere redried and further ground into powder using thegrinder

22 Sample Extraction and Fractionation Anthocyaninsfrom the defatted dabai powders were extracted based onthe optimized conditions that were previously establishedBriefly anthocyanins in the defatted dabai peel and pericarppowders were extracted based on the optimized extractionparameters (53 methanol as solvent and sonicated for1min) [14] The extraction parameters have been optimizedpreviously based on response surfacemethodologywhere the

optimized extraction parameters had yielded optimal levelsof total phenolics and antioxidant capacity The mixture wasfiltered and anthocyanin-rich crude extract was collectedThe sample residue was reextracted twice using equal amountof 53 methanol Methanol was removed using a rotaryevaporator (Buchi Switzerland) at 39∘C The leftover watercomponent and methanol residue of the extracts were fullyremoved by freeze-drying and stored at minus40∘C until furtheranalyses

To obtain a purified anthocyanins fraction 50mg ofthe lyophilized anthocyanin-rich crude extract was dissolvedwith 80methanol (2mL vv) and fractionated by SPE usingactivated Sep-PakCNandC18 cartridges fromMerck (Darm-stadt Germany) Visiprep SPE vacuum manifold (12 ports)(Supelco Pennsylvania USA) was connected to a vacuumpump and the extracts were fractionated using the cartridgescontaining 100mg adsorbent by passing methanol and dis-tilled water (2mL each) through the cartridges Methanolfractions were collected but water fractions were discardedThe collected methanolic fractions together with the crudeextracts of defatted dabai peel and pericarp were determinedfor anthocyanins contents and antioxidant capacity togetherwith their oxidative stress inhibition ability

The flow of samples fractionation is shown in Figure 1Crude extracts of defatted dabai peel and pericarp (2mLinjection volume 5mgmL extract concentration) wereinjected into the activated Sep-Pak CN and C18 cartridgesrespectively using 5mL syringes [15] The adsorbed antho-cyanins in both cartridges were eluted by passing 20mL ofmethanol through theCNcartridge (Figure 1(a)) (for defatteddabai peel) and C18 cartridge (Figure 1(b)) (for defatted dabaipericarp) and collected as samples 1 and 2 respectively

23 HPLC Analysis Anthocyanins in the defatted dabaisamples were identified and quantified based on a modifiedmethod described by Chew et al [4] In this study UHPLCmethod using an Agilent 1290 Infinity LC system (AgilentTechnologies Waldbronn Germany) equipped with a binarypump diode array detector and an autosampler was appliedin the quantification of anthocyanins Separation of antho-cyanins was achieved on a Lichrospher C-18 column (250times 4mm id 5120583m) (Merck KGaA Darmstadt Germany) at30∘C The mobile phases consisted of A 05 trifluoroaceticacid and B 100 acetonitrile The gradient was achievedusing the following system 90ndash85 B 0ndash5min 85 B 5ndash10min 85ndash70 B 10ndash15min 70 B 15ndash20min 70ndash20B 20ndash25min 20ndash0 25ndash30min The column was washedand reconditioned after every run The flow rate was set at06mLmin and the injection volume was 20120583L

Anthocyanins were detected at a wavelength of 530 nmwhile UV-Vis absorption spectra were recorded in therange of 250ndash600 nm A total of six anthocyanin standardscyanidin-3-glucoside (C3G) cyanidin-3-galactoside (C3L)cyanidin-3-arabinoside (C3A) pelargonidin-3-glucoside(L3G) malvidin-3-glucoside (M3G) and peonidin-3-glu-coside (P3G) were used for identification of anthocyaninsin the defatted dabai extracts The anthocyanins content ofthe defatted dabai extracts was determined and calculatedusing cyanidin-3-rutinoside (C3R) as internal standard This

Evidence-Based Complementary and Alternative Medicine 3

CNcartridge

CNcartridge

Tovacuum

Waste Sample 1(peel fraction)

2mL ofdefatted dabaipeel extracts(5mgmL) in80 methanol

100

methanol

(a)

cartridge

Tovacuum

Waste

C18cartridge

C18

Sample 2(PP fraction)

2mL ofdefatted dabai

PP extracts(5mgmL) in80 methanol

100

methanol

(b)

Figure 1 Flow diagrams of sample fractionation [15]

is because C3R was not found in all samples The calculationof anthocyanin content was based on the following equationas suggested by Willis [17]

Anthocyanin content (mgg extract)

= (weight of standard times area of anthocyanin times 119865119877

times100 times dilution factor)

times (weight of sample times area of standard)minus1(1)

where

119865

119877=

absorbanceweight for anthocyanin standardabsorbanceweight for anthocyanin sample

(2)

24 DPPH Radical Scavenging Assay The antioxidant capac-ities of anthocyanin-rich crude extracts of defatted dabaipeelpericarp and extract fractions were evaluated by DPPHradical scavenging assay based on the method of Lai et al[18] Briefly aliquots of the anthocyanin-rich crude extractsor extract fractions (04mgmL) or reference standards (C3Gor P3G 10 120583gmL) were mixed with 01M Tris-HCl buffer(08mL pH 74) and then added to 10mL of 05mM DPPHin methanol The mixture was vortexed for 10 s and left tostand at room temperature in the dark After 20min theabsorbance was measured spectrophotometrically at 517 nmThe antioxidant capacities of the samples were calculatedbased on the following equation

Antioxidant capacity ()

=[1minus

absorbance of sample at 517 nmabsorbance of control at 517 nm

]times100

(3)

25 Copper (II) Reduction Antioxidant Capacity (CUPRAC)Theantioxidant capacities of anthocyanin-rich crude extractsof defatted dabai peelpericarp and extract fractions weremeasured using copper (II) reduction assay based on a

method described by Celik et al [19] with some modifica-tions The anthocyanin-rich crude extracts or extract frac-tions (04mgmL) or reference standards (C3G or P3G10 120583gmL) (01mL) were mixed with 10mL of 7 methyl-120573-cyclodextrin solution to completely solubilize the mixturefollowed by the addition of 10mL of CuCl

2 10mL of copper

(II)-neocuproine reagent solution and 10mL of ammoniumacetate solutionThe absorbance of final solution (41mL totalvolume)wasmeasured at 450 nmagainst a reagent blank after30min incubation at room temperature Calibration curves(absorbance versus concentration graph) of Trolox (01ndash4mM) were plotted Trolox equivalent antioxidant capacity(TEAC) was calculated and expressed as mM per gramsample

26 Inhibition of Linoleic Acid Oxidation The inhibition oflinoleic acid oxidation was carried out based on a methoddescribed by Sultana et al [20] with some modificationsThediluted anthocyanin-rich crude extracts or extract fractions(04mgmL) or reference standards (C3G or P3G 10 120583gmL)(02mL) were added to 0026mL of linoleic acid solution20mL of methanol (80 vv) and 10mL of 02M sodiumphosphate buffer (pH 7) Then the mixture was made upto 40mL by adding methanol (80 vv) and incubated at40∘C for 24 h The inhibition of oxidation was measured asperoxide value applying thiocyanate method [21]

For thiocyanate method a reacting mixture (01mL) wasadded with 10mL of methanol (80 vv) 01mL of aqueoussolution of ammonium thiocyanate (30 wv) and 01mLof FeCl

2solution (20mM in 35 HCl vv) After 3min of

stirring the absorption was measured at 500 nm Referencestandards (C3G or P3G) were used as positive control Theprotective capacities () of the samples were calculated byapplying the following equation

Protective capacity ()

= [1 minus

Absorbance increase of sample at 24 hrAbsorbance increase of control at 24 hr

] times 100

(4)


27 Hemoglobin Oxidation The hemoglobin oxidation wascarried out based on a method described by Rodrıguez et al[22] with some modifications The approval of experimentalprotocol involving blood withdrawal from ratsrsquo tails wasgranted from the Animal Care andUse Committee of Facultyof Medicine and Health Sciences Universiti Putra Malaysia(UPM) Selangor Malaysia (Approval no UPMFPSKPADSBR-UUH00384) Fresh blood was drawn from agroup of ten healthy male Sprague-Dawley rats in EDTA-containing tubes Plasma platelets and buffy coat wereremoved by centrifugation for 10min at 1853timesg at 4∘C Redblood cells (RBCs) were washed with PBS two times

After second wash the packed RBCs were resuspendedgently with PBS to obtain a 5 haematocrit The RBCs werepreincubated at 37∘C for 10min in the presence of 1mMNaN3(to inhibit catalase activity) Various aliquots of RBCs

(16mL) were added to test tubes for each sample treatmentAll the test tubes except the controls tubes were added withor without the anthocyanin-rich crude extracts or extractfractions (04mgmL) or reference standards (C3G or P3G10 120583gmL) After 24 h of incubation at 37∘C the reactingmixtures were placed in an ice bath for 60 s and centrifuged at1853timesg for 10min at 4∘CThe supernatantswere collected anddetermined for TBARS values resulting from autooxidation

To measure the TBARS 10mL of the peroxidationproduct was placed in glass test tube and mixed with 20mLof TCA-TBA-HCl mixture (15 TCA 0375 TBA 025 N ofHCl) for 10 s using a vortex mixer The mixture was placed inboiling water for 15min and left to cool at room temperaturefor 10minThemixture was centrifuged at 1000timesg for 10minThe absorbance of the supernatant was read at 535 nm againstblank The protective capacities () of the samples werecalculated based on the following equation


= [1 minus


] times 100

(5)

The TBARS values of the samples were calculated using amolar extinction of 156 times 105Mminus1 cmminus1 The results wereexpressed as TBARS value and calculated as follows [23]

TBARS value (mM)

=

AbsorbanceMolar extinction

times length of cuvette (1 cm) (6)

28 PARP-1 Inhibition ELISA Theability of the anthocyanin-rich samples to inhibit PARP-1 activity was determined usingan inhibition assay as described by Geraets et al [24] withslight modifications Briefly human rPARP-1 (400 ngmL)was incubated with a reaction mixture containing 50120583M120573-NAD+ (biotinylated 120573-NAD+) 1mM 14-dithiothreitol(DTT) 125 120583gmL nickedDNA and anthocyanin-rich crudeextracts or extract fractions (04mgmL) or reference stan-dards (C3G or P3G 10120583gmL) at 4∘C Nicked DNA wasprepared by incubating calf thymus DNA with 10 ngmL

DNase I at 37∘C for 40minThe plate was seededwith rPARP-1 overnight (8ndash12 h) at 4∘C before addition of the reactionmixture

After incubations with the reaction mixture the plateswere washed and the formation of poly(ADP-ribose)-pol-ymers was detected after 1 h incubation at room tempera-ture with peroxidase-labeled streptavidin (2120583gmL 1 500dilution) followed by a 15min incubation with 331015840551015840-tetramethylbenzidine (TMB) (01mgmL) in the presenceof hydrogen peroxide (03) at 37∘C The reaction wasterminated by addition of 075M HCl and the absorbancewas measured at 450 nm All measurements were carried outin triplicate

29 In Vivo Study A total of 21 male New Zealand whiterabbits at age of 8ndash10 weeks were obtained The initial bodyweights (15ndash17 kg) of the rabbits were recorded and rabbitswere caged individually After twoweeks of acclimatization inthe ambient temperature of 28∘C the rabbits were randomlydistributed into three groups (119899 = 7 per group) normal dietgroup (NDG) hypercholesterolemic treatment group (HTG)and hypercholesterolemic control group (HCG) also knownas positive control group In NDG the rabbits were just givennormal basal diet The rabbits from both HTG and HCGreceived 20 g of hypercholesterolemic diet (normal basal dietcontaining 05 cholesterol) [25] but HTG was addition-ally supplemented with 2000mgday of defatted dabai peelextract throughout the 8-week experimental period Thedosage of 2000mgday had been shown to exhibit protectiveeffect [26]

High cholesterol diets were prepared based on 05 oftotal cholesterol in a daily diet where 06 g of cholesterol wasdissolved in 2mL chloroform and the cholesterol solutionwas sprayed on the pellets of the rabbits The pellets weredried in an oven (Memmert GmbH Schwabach Germany) at40∘C overnight to allow evaporation of the chloroform [27]

At baseline (week 0) and week 8 of the study 10mLof blood was drawn from marginal ear of the fasting rab-bits (12 h of fasting) The blood was collected into EDTA-containing tubes for malondialdehyde (MDA) determina-tion while it was collected in tubes containing lithium hep-arin for the determination of superoxide dismutase (SOD)and glutathione peroxidase (GPx) The blood samples werecentrifuged at 1000timesg for 10min at 4∘C using a centrifuge(Universal 32 Hettich Zentrifugen Germany) to separateerythrocytes from the bloodrsquos plasma

MDA levels of the EDTA containing plasmas fromHTG and HCG rabbits were evaluated using the TBARSmethod [23] The SOD activity of blood samples collectedfrom the rabbits was determined by RANSOD kit (RandoxLaboratories Crumlin UK) using aVitalab Selectra Analyzer(Merck Darmstadt Germany) [28] Briefly the erythrocytescollected were washed four times with 3mL of 09 NaClsolution by centrifugation at 1000timesg for 10min The packederythrocytes were made up to 2mL by adding cold distilledwater vortexed for 10 s and left to stand at 4∘C for 15minThe lysate was diluted with 001molL phosphate buffer atpH 7 and mixed thoroughly The absorbance of the mixturewas read at 505 nm The GPx activity was determined based


200

175

150

125

100

75

50

25

0

0 5 10 15 20 25

(mAU

)

(min)

(Internalstandard)

C3R

C3GC3G

C3LC3L

Unknown L3GM3G

C3AP3G

Figure 2 UHPLC chromatogram for the defatted dabai peel extractSee text for the full names of anthocyanins

on the oxidation of glutathione The oxidized glutathione isimmediately converted to the reduced form with the oxida-tion of NADPH A blood sample was prepared by diluting005mL of the whole blood with 2mL of the diluting agentRANSEL kit (Randox Laboratories Crumlin UK) was usedto determine the GPx activity of the blood samples [28] Thedecrease in absorbance was measured at 340 nm using theVitalab Selectra Analyzer

210 Statistical Analysis Anthocyanin contents and antiox-idant capacities of the defatted dabai peel and pericarpcrude extracts and their SPE fractions were expressed asmean plusmn standard deviation Data were subjected to analysisof variance (ANOVA) using a statistical software Minitabversion 15 Data for the in vivo study were analyzed using(Statistical Package Social Sciences SPSS) version 200 (SPSSCorporation Chicago IL) for Windows The differencesin group means were determined using one-way ANOVATukey post hoc test was used for multiple group comparisonThe level of significance was set at 119875 lt 005

3 Results and Discussion

31 Anthocyanins in Defatted Dabai Samples Six antho-cyanins were identified in the defatted dabai peel They wereL3G M3G C3L C3G C3A and P3G These anthocyaninswere eluted from 140min to 191min (Table 1) The RSD() of the samples analyzed was lower than 20 Theconcentration of each anthocyanin identified in the defatteddabai peel extract was linear (1198772 = 0953ndash0999) The valuesfor LODandLOQof the defatted dabai peel extract are shownin Table 1 The UHPLC chromatogram for the defatted dabaipeel is shown in Figure 2

The peel of purple fruit such as dabai has high antho-cyanin content Anthocyanin especially C3G is the majorantioxidant compound in many purple fruits The resultshowed that defatted dabai peel had the highest amount ofC3G (5512 plusmn 082mgg) compared to other anthocyanins

identified The amount of other anthocyanins in the defatteddabai peel includes C3L (442 plusmn 008mgg) C3A (417 plusmn027mgg) M3G (561 plusmn 022mgg) and L3G (442 plusmn008mgg) Trace amount (below LOQ) of P3G was detectedin the defatted dabai peel In the defatted dabai pericarp L3GM3G and P3G were not detected (below LOD) while traceamount of C3Awas found C3G andC3L in the defatted dabaipericarp were at 607 plusmn 002 and 584 plusmn 013mgg extractrespectively

SPE had been used to concentrate and purify variouscrude extracts [29 30] However SPE has not been used inpurifying anthocyanin from dabai In this study methanolicfractions of the defatted dabai peel and pericarp were col-lected These fractions were CN-methanolic fraction of thedefatted dabai peel (sample 1) and C18-methanolic fraction ofthe defatted dabai pericarp (sample 2) The results obtainedfrom our previous study revealed that samples 1 and 2 hadthe highest total phenolic contents and total anthocyanincontents [15] Therefore samples 1 and 2 were considered asthe best anthocyanin-rich fractions of the defatted dabai partsif compared to the other extract fractions

The results obtained from UHPLC analysis revealed thatthe SPE fractions of the defatted dabai peel had higheramount of C3L compared to the crude extract of the defatteddabai peel This indicates that SPE method could be ableto concentrate anthocyanin compounds such as C3L TheC3G content determined in the SPE fraction (sample 1) ofthe defatted dabai peel was sim4 times lower compared to thecrude extract of defatted dabai peel while the amount ofC3L found in sample 1 was about four times higher than inthe defatted dabai peel crude extract Trace amounts of L3GM3G and P3G were detected in sample 1 The SPE fractionof the defatted dabai pericarp (sample 2) had lower amountsof C3G (432 plusmn 017mgg) and C3L (472 plusmn 007mgg) ascompared to the crude extract of defatted dabai pericarpTrace amounts of M3G and P3G were detected in sample 2while L3G was not detected in the sample A higher amountof C3A (047plusmn001mgg) was found in sample 2 as comparedto the level in defatted dabai pericarp crude extract

Major anthocyanins found in the defatted dabai sampleswere C3G C3L and C3A These anthocyanins were alsofound in many other purple-colored fruits [7 31] Althoughanthocyanins are highly available in blackcurrant and mostberries the anthocyanin especiallyC3G in the defatted dabaipeel is a valuable source of potent antioxidant Moreover thedefatted dabai peel is a by-product of dabai oil extraction thatpotentially uses as new source of nutraceutical

Generally the synthesis of anthocyanins in plant is genet-ically controlled [32] and regulated initially by phenylalanineammonia lyase Phenylalanine as the precursor for antho-cyanin synthesis has been reported to be regulated by severalenzymes until synthesis of various stable anthocyanidinglycosides [33] Cyanidin glycoside is the major bioactivecompound found in the defatted dabai peel where narin-genin chalcone is known as the main precursor of cyanidinsynthesis Previously Chew et al [4] and Khoo et al [34]reported that naringenin was not detected in dabai fruitThisshows that during fruit ripening most of the naringenin hadbeen used up for synthesis of cyanidin


Table1Antho

cyaninsc

ontent

ofthed

efatteddabaiextractsa

ndextractsfractio

nsandotherU

HPL

Cparameters

Sample

Antho

cyanins(mggextract)

Pelargon

idin-3-glucosid

eMalvidin-3-glucoside

Cyanidin-3-galactosid

eCy

anidin-3-glucosid

eCy

anidin-3-arabino

side

Peon

idin-3-glucosid

eDefatteddabaip

eelextract

442plusmn008

561plusmn022

442plusmn008

5512plusmn082

417plusmn027

Trace

Defatteddabaip

ericarpextract

ND

ND

584plusmn013

607plusmn002

Trace

ND

1Trace

Trace

1745plusmn04

1346plusmn028

108plusmn003

Trace

2ND

Trace

472plusmn007

432plusmn017

047plusmn001

Trace

Other

parameters

Retentiontim

e140plusmn02

153plusmn03

173plusmn04

178plusmn02

184plusmn05

191plusmn03

RSD(

)for

allsam

ples

177ndash322

127ndash463

156ndash

998

014ndash396

025ndash6

78

042ndash1549

RSD(

)for

defatte

ddabaip

eel

177

40

177

148

658

901

Linearity

(1198772

)for

defatte

ddabaip

eel

0953

0978

0953

0999

0989

0963

LODford

efatteddabaip

eel(mgmL)

027

115

028

031

001

082

LOQford

efatteddabaip

eel(mgmL)

123

515

468

138

003

368

lowast

NDnot

detectedLODlim

itof

detection

LOQlim

itof

quantitation

Seetextfor

then

ames

ofsamples

1and

2


Naringenin Naringenin

chalcone

N3HOH

OHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

CHI

Dihydrokaempferol

Dihydroquercetin

DF4R

AS

UF3GT

CyanidinCyanidin-3-glucoside Leucocyanidin

O+O+

HOHO

HO

HOHOHO

HOHO

HOOO

OO

O

O

O

O

O

OF3998400H

Figure 3 Biosynthetic pathway of cyanidin-3-glucoside fromnaringenin chalcone in defatted dabai peel [16] See text for the full names of thevarious enzymes

In the C3G synthesis specific gene regulates naringenin-chalcone synthase to produce naringenin chalcone Severalenzymes have been known to be involved in the synthesisof C3G such as chalcone isomerise (CHI) and naringenin3-hydrolase (N3H) The biosynthesis pathway of C3G isshown in Figure 3 The flavanone naringenin is hydrolyzedby N3H into dihydrokaempferol and further hydroxylatedto dihydroquercetin by flavonoid 31015840-hydroxylase (F31015840H) [1632] The dihydrokaempferol is then converted to leucocyani-din by dihydroflavonol 4-reductase (DF4R) During fruitripening leucocyanidin is readily converted to cyanidin byanthocyanidin synthase (AS) Due to the unstable form ofanthocyanidins uridine diphosphate glucose flavonoid 3-glucosyltransferase (UF3GT) is readily catalyzed the unstableform of anthocyanidin to a stable anthocyanin [35]

The crude extracts and extract fractions of the defatteddabai parts also contained a trace to undetectable amountof anthocyanidins where the HPLC peaks of anthocyanidinswere below quantitation and detection limits [32] Duringextraction degradation of some anthocyanins anthocyani-dins and other phenolic compounds might occur Exposureto light high temperature and storage duration are thepossible factors for the degradation of anthocyanidins [36]Besides degradation of anthocyanins may be due to activityof enzyme dihydroflavonol 4-reductase [37]

32 Antioxidant Capacity and Inhibition of Oxidative StressAnthocyanins in the defatted dabai parts (peel and pericarp)are potential source of nutraceutical In vitro antioxidantassays were used to determine the free radical scavengingeffects of the anthocyanin-rich crude extracts of defatteddabai parts The crude extracts of the defatted dabai peelshowed high antioxidant capacities which were also thehighest among the studied samples (Figures 4 and 5) Besidesthe crude extracts of defatted dabai parts contained antho-cyanins as the major bioactive compounds For antioxidant

80

70

60

50

40

30

20

10

0Scav

engi

ng ca

paci

ty (

)

Peel PP 1 2 C3G P3G

b

d e

f

a

c

Figure 4 Percentage of scavenging capacities of the anthocyanin-rich extracts extract fractions and anthocyanin standards based onDPPH assay Peel is referring to the defatted dabai peel extract PP isreferring to the pericarp extract of defatted dabai See text for thefull names of anthocyanins

Trol

ox eq

uiva

lent

(mM

g)

0

5

10

15

20

25

30

35

0

05

1

15

2

Peel PP 1 2

Peel PP 1 2

C3G P3G

b

d e f

ac

Figure 5 Trolox equivalent antioxidant capacities of the antho-cyanin-rich extracts extract fractions and anthocyanin standardsbased on CUPRAC assay

assays 04mgmL of crude extract or extract fractionsand 001mgmL of anthocyanin standards (C3G and P3G)were tested for their antioxidant capacities and inhibitionsof oxidative stress Our initial screening has shown that04mgmL of the crude extracts of defatted dabai peel gavethe optimum antioxidant activity The standards were usedfor comparison purpose


Theantioxidant capacities of the defatted dabai parts (peeland pericarp) based on chemical assays are shown in Figures4 and 5 The results showed that the crude extract of defatteddabai peel had significantly highest (119875 lt 005) antioxidantcapacity based on DPPH and CUPRAC assays as comparedto other samples Other samples such as the defatted dabaipericarp extract and their extract fractions (samples 1 and 2)had lower antioxidant capacities

As assessed using DPPH assay samples 1 and 2 had theleast percentages of scavenging capacities (Figure 4) com-pared to C3G and P3GThese samples had scavenging capac-ity less than 50 except for the crude extract of defatted dabaipeel which had 60 of scavenging capacity The scavengingcapacities for both C3G and P3G were 69 and 41 respec-tively The results obtained from CUPRAC assay revealedsimilar trend of antioxidant capacity as found forDPPHassayTE values of the anthocyanin standards were the highest(3042mM for C3G and 761mM for P3G) followed by thecrude extract of defatted dabai peel (175mM) (Figure 5)Based on these results the crude extract of defatted dabaipeel had antioxidant capacity of 4ndash17 times lower than theanthocyanin standards This observation suggests that thecrude extract of defatted dabai peel is a potentially strongantioxidant where it mainly consists of anthocyanins (C3Gcomprised of 75 of the total anthocyanins) (Table 1) Ascompared with the defatted dabai extracts lower antioxidantcapacities observed for the SPE fractions are possibly due tothe presence of other polyphenolics and saponins [15]

The oxidative stress inhibition effect of the anthocyanin-rich crude extracts and extract fractions of defatted dabaiwas also studied The effects were determined based on threeassays mimicking human vascular system (linoleic acid oxi-dation system assay hemoglobin oxidation assay and PARP-1 inhibition ELISA) In these assays the same concentrationof the extract was applied as in DPPH and CUPRAC assaysOverall the results showed that the crude extract of defatteddabai peel is a potential nutraceutical for inhibition of oxida-tive stress Among the studied samples the crude extractof defatted dabai peel showed the highest percentages ofprotective capacity and inhibition activity On the other handother phytochemicals such as flavonoids and saponins havealso been detected in the peel and pericarp of dabai fruit[15] where saponin derivative is being one of the majorcompounds in the defatted dabai pulpThese phytochemicalscould have contributed to the protective effects

The results from linoleic acid oxidation system assayrevealed that samples 1 and 2 had low protective capacitywhich were at sim20 (Figure 6) The crude extract of defatteddabai peel showed 32 of the protective capacity which wassignificantly higher than the rest of the studied samples Ascompared to defatted dabai peel the pericarp crude extracthad low percentage of protective capacity A possible reasonfor the low protective capacity is that the defatted dabai peelhas high anthocyanins while the defatted dabai pericarpconsists more of the nonanthocyanin compounds especiallyfrom the defatted portion of dabai pulp Using hemoglobinoxidation assay concentration of the samples used waslow (Figure 7) and was not able to sufficiently result in

0

5

10

15

20

25

30

35

40

Peel PP 1 2 C3G P3G

bb

d d

a

c

Control

Prot

ectiv

e cap

acity

()

Figure 6 Percentage of scavenging capacity of the anthocyanin-rich extracts extract fractions and anthocyanin standards based onlinoleic acid oxidation system

0

5

10

15

20

25

Peel PP 1 2 C3G P3G

b

a a a

c

Control

Prot

ectiv

e cap

acity

()

b c

Figure 7 Percentage of protective capacity of the anthocyanin-rich extracts extract fractions and anthocyanin standards based onmeasurement of conjugated diene

stronger protective effect in the in vitro human vascularmodel tested

Based on the linear curve of the crude extract of defatteddabai peel (Table 1) it can be postulated that sim100 timeshigher extract concentration is needed to give a 50 ofprotective capacity to the RBC solutionTherefore we suggestthat 1mgmL of anthocyanin standards (C3G or P3G) shouldbe able to result in 50 protective capacity in the hemoglobinoxidation system Besides a lot of water soluble anthocyanins(L3G and M3G) was also found based on UHPLC analysisas some of the hydrophilic compounds from the extracthad been removed by SPE (Table 1) Therefore the crudeextract of the defatted dabai peel could be the best source ofnutraceutical for inhibition of oxidative stress

Generally the percentages of protective capacity of thecrude extracts and extract fractions of the defatted dabaipeel and pericarp determined using linoleic acid (Figure 6)and hemoglobin oxidation (Figure 7) assays were less than50 as compared to the DPPH (Figure 4) and CUPRAC(Figure 5) assays Comparing the similar extract concen-tration of defatted dabai peel for both DPPH and linoleicacid oxidation assays there was about 30 difference in thepercentage of inhibition activity One possible explanation forthe difference is that DPPH assay was involved in electron-transfer (ET) reaction pathway while lipid oxidation assayswere involved in hydrogen atom transfer (HAT) reactionpathway Huang et al [38] reported that in ET-based assayantioxidant acts as a reducing agent but HAT-based assayinvolves a radical chain-breaking reaction

On the other hand the anthocyanin-rich fraction of thedefatted dabai peel showed a low TBARS value based onthe hemoglobin oxidation assay (Figure 8) Similarly the


0

1

2

3

4

5

6

7

8

Peel PP 1 2 C3G P3G

b

d

a

c

Control

c d

a b

c d

TBA

RS v

alue

(mM

)

Figure 8 TBARS value of the anthocyanin-rich extracts extractfractions and anthocyanin standards based on hemoglobin oxida-tion assay

Inhi

bitio

n ac

tivity

()

0

20

40

60

80

Peel PP 1 2 C3G P3G

b

dd

ac

Control

c d

Figure 9 PARP-1 inhibition activity of the anthocyanin-richextracts extract fractions and anthocyanin standards based onPARP-1 inhibition ELISA

methanolic fractions of defatted dabai peel (sample 1) andpericarp (sample 2) obtained from the SPE also had lowTBARS values Besides all samples had significantly lowerTBARS values than the control (119875 lt 005) except for thedefatted dabai pericarp extract The standard C3G hadTBARS value lower than P3G as assessed by hemoglobin oxi-dation assay while the protective capacity () and inhibitionactivity () of the standard C3G were higher than the P3Gand the protective capacities of the C3G and P3G were notsignificantly different (119875 ge 005) Similar observation hasbeen reported by Kahkonen and Heinonen [39] where lowconcentration of C3G had higher antioxidant activity thanP3G based on LDL oxidation assay They also found thatat higher concentration P3G showed a stronger antioxidantactivity compared to C3G

For the PARP-1 inhibition ELISA all samples showedsimilar inhibition trends as found for the hemoglobin oxida-tion assay (Figure 9) Among the samples crude extract ofdefatted dabai peel had the highest percentage of inhibitionactivity (65) while samples 1 and 2 had the least percent-ages of inhibition activity (lt50) A comparable inhibitionactivity was found for both of the crude extract of defatteddabai peel and anthocyanin standardsThehigh percentage ofinhibition activity for the defatted dabai crude extract (65)and C3G (70) suggested that C3G in the peel extract wasable to inhibit production of PARP-1 compared to P3G (50)and the other samples which is in agreement with the resultsobtained based on the linoleic acid oxidation and hemoglobinoxidation assaysTherefore C3G as themajor anthocyanin indefatted dabai peel can be one of the best nutraceuticals forinhibition of oxidative stress

Previous literatures have consistently shown that antho-cyanins possess strong antioxidant activities [40 41] Saint-Cricq de Gaulejac et al [42] postulated that the negativelycharged free radicals are prone to react with positivelycharged flavylium ions and thus prevent oxidation of cellularlipids As reported by Rice-Evans et al [43] phenolic com-pounds with o-dihydroxy structure in the B ring have thehighest antioxidant activities while flavonoid with additionalhydroxyl group in the B ring has increasing antioxidant activ-ity Similar to catechin cyanidin has o-dihydroxy structure inthe B ring Therefore it has high antioxidant activity Amonganthocyanidins delphinidin has additional hydroxyl groupin the B ring Kahkonen and Heinonen [39] revealed thatdelphinidin has significantly higherDPPHradical scavengingactivity (42) compared to cyanidin (33) Chew et al [4]also reported that delphinidin was detected in dabai fruitobtained from different locations in which the concentrationof delphinidin ranged from not detected to 011mg100 gdry weight Although delphinidin-3-glucoside was not deter-mined in the defatted dabai extract the occurrence ofdelphinidin in the extract could have contributed to highantioxidant activity Previous study has also reported thatmost of the anthocyanin pigments are unstable under neutralor alkaline pH [44] In this study the DPPH assay was per-formed at neutral pH As observed anthocyanins such asC3G in neutral pH have shown stronger antioxidant capacitycompared to P3G The possible explanation is that theflavylium cation of C3G formed at low pH may not be acrucial factor for the expression of antioxidative activity andC3G has been shown as a strong antioxidant at neutral pH[45] Besides Kahkonen and Heinonen [39] also showeda significant difference in antioxidant activity for C3G andC3L

In general the moderately high antioxidant capacity andprotective capacity found for the defatted dabai peel extractwere mainly because C3G was the major compound How-ever the actual antioxidative mechanism of C3G still remainsunknown Previous studies have reported that exposure ofC3G to epithelial cells upregulated nitric oxide (NO) synthaseactivity by phosphorylation of the enzyme [46 47] This isbecause NO functions as vasodilator [48] and upregulatesintracellular cGMP [49] that prevents transportation of cal-cium into endothelial cells and reduces intracellular calciumconcentrations Increased level of cellular NO has beenproven to possess protective effect against vascular damage[50] Bell and Gochenaur [51] also reported anthocyaninas potent ROS scavenger that could enhance NO-mediatedvasorelaxationHence C3Ghas protective effect againstDNAdamage by inhibition of xanthine oxidase activity [52]

On the other hand Hoe and Siong [53] reported thatdabai fruit contained 7mg copper and 13mg iron per 100 gedible portion These small amounts of trace mineral mightaffect the antioxidant capacity of dabai extract in CUPRACand hemoglobin oxidation assays In the CUPRAC assayCu2+ from the CUPRAC reagent receives an electron fromantioxidant and the Cu (II) is reduced to Cu (I) [54]Trace amount of copper ions from dabai extract might alsoreceive electrons from the antioxidant in the extract and thecopper ion from the extract possibly acted as chromogen


Table 2 MDA levels SOD and GPx activities of the blood samples from in vivo study

Groups Plasma MDA (molL) SOD (UmL) GPx (UmL)Baseline Week 8 Baseline Week 8 Baseline Week 8

NDG 239 plusmn 008 367 plusmn 005 480 plusmn 013 294 plusmn 009 076 plusmn 005 071 plusmn 009HTG 270 plusmn 007 289 plusmn 009 473 plusmn 009 324 plusmn 012 078 plusmn 008 056 plusmn 012HCG 283 plusmn 012 427 plusmn 014 477 plusmn 009 503 plusmn 011 081 plusmn 006 059 plusmn 009NDG normal diet normal HTG hypercholesterolemic treatment group and HCG hypercholesterolemic control group

in the CUPRAC assay Thus these copper ions could havecontributed to some inconsistency in the results obtainedSimilarly trace iron ions found in dabai extract could affectthe results obtained from hemoglobin oxidation assay Innonstress condition heme ion in hemoglobin normallyremains unaffected Under oxidative stress condition hemeion from the hemoglobin is released and coupled with theions from the extract which has high possibility to furtherinduce oxidative stress in the hemoglobin solution Additionof antioxidant in the reacting mixture might yield inaccuracyand inconsistency in the results obtained These metal ionsand heme compounds have also been found to decomposelipid peroxidase [55] where decrease in peroxidase activ-ity enhances oxidative stress Therefore further study onhemoglobin oxidation assay needs to consider the degree ofoxidative stress induced to hemoglobin

33 Cardioprotective Effect of Defatted Dabai Peel To furtherstrengthen the beneficial effect observed using in vitro sys-tems an in vivo study using the defatted dabai peel extractwas performed As compared to baseline values significantincrease of the plasma MDA levels was detected for bothHCG and HTG at week 8 of high cholesterol diet In HCGthe plasma MDA increased about 144molL (sim50) after8 weeks of the study As compared to NDG 8 weeks ofnormal chow diet has resulted in elevation of plasma MDAlevels by about 16 (128molL) which indicate the normalphysiological oxidation of the in vivo system However asignificantly low increment (019molL) of plasma MDA wasfound for the HTG after 8 weeks of supplementation with theanthocyanin-rich extract of defatted dabai peel (Table 2) ascompared to the HCG

From baseline to 8 weeks of the study the HCG had asignificant increase in SOD activity The result also showedthat 8-week supplementation of the defatted dabai peelextract (HTG) resulted in significant reduction of SODactivity (356) compared to the HCG while the GPx levelsfor bothHTG andHCGwere significantly reduced comparedto baseline after 8 weeks of the study High cholesterol dietgiven to the rabbit for 8 weeks has been demonstrated tosignificantly increase the SOD activity (42) and inhibitthe GPx activity (18) as compared to the NDG (Table 2)Excessive dietary cholesterol has enhanced oxidative stress inthe hypercholesterolemic rabbits thus increased the cellularSOD activity Similarly oxidative stress is known to accelerateproduction of MDA in cellular system of the rabbits Highcholesterol diet in the experimental rabbits has also exacer-bated the MDA production in both HTG and HCG

MDA is a secondary lipid peroxidation product whileSOD is an enzyme that catalyzes twomolecules of superoxideradicals to hydrogen peroxide and oxygen in order to reducethe oxidative stress in cellular level Increased cellular MDAand SOD indicate acceleration of oxidative stress level initi-ated by free radicals High levels of MDA and SOD are goodindicators of oxidative stress as the increasing level of MDAindicates the acceleration of lipid peroxidation while highSOD activity shows the high level of superoxide radicals inthe cellular system In this study reduction ofMDA and SODis a good indicator of cardioprotection

On the other hand the increase in GPx activity may notshow cardioprotective effect It is due to the involvementof GPx enzymes in breaking down of hydrogen peroxidethat has been produced by SOD in high oxidative stresscondition The result of this study demonstrated that the nosignificant change of GPx activity between HTG and HCGdoes not imply any cardioprotective effect of the defatteddabai peel extract as the activity of GPx is an adaptation tothe abundant activity of SOD in the system GPx enzyme isone of the indicators formonitoring oxidative stress instead ofindicating the reduction of oxidative stress Besides hydrogenperoxide in the cellular level should have been catalyzedor converted to water and oxygen by potential catalase andantioxidants [56]

Previous study has shown that 5 supplementation ofdefatted dabai powder was able to improve plasma lipidprofile of hypercholesterolemic rabbits [25] The cholesterollowering effect of the defatted dabai powder has provenits protective effect in hypercholesterolemic condition [825] In this study the use of defatted dabai peel extract at2000mgday has successfully reduced oxidative stress condi-tion in the hypercholesterolemic rabbits We have also con-firmed that the defatted dabai peel extract used containsgt70 of anthocyanin Based on this in vivo study we are ableto show that about 150mg of the total anthocyanins in thedefatted dabai powder would protect an in vivo system fromexcessive damage due to the oxidative stress condition

Several reported benefits of anthocyanins have furtherstrengthened the protective effect of defatted dabai peelextract Anthocyanin-rich potato flake has significantlyimproved the antioxidant status in the serum of hyperc-holesterolemic rats treated with the flake (300 gkg diet)[57] Besides anthocyanin-rich juice extracts of berries haveshown to be antihyperlipidemic in the hypercholesterolemicanimals [58 59] Conversely anthocyanin-rich extract andjuice from blackcurrant had no improvement in plasma lipidprofile in hyperlipidemic rabbits [60] thus suggesting that


anthocyanins are somehow prooxidant However the resultsfrom our study demonstrated the ability of anthocyaninsfrom defatted dabai peel for cardioprotection Besides MDAand SOD as indicators for CVD cellular oxidized LDL is alsoone of the risk factors for DNA damage

As oxidative stress plays an important role in DNA dam-age DNA damages induced by oxidative stress coupled withLDL oxidation were closely related to atherosclerosis [61]Kong et al [62] also suggested possible defence mechanismfor protection of anthocyanin against oxidativeDNAdamagewhere copigmentation of cyanidin-DNAhas helped to reduceDNA damage Besides NO synthase heme oxygenase-1 couldbe one of the possible protective mechanisms for C3G inendothelial dysfunction [63] Cellular NAD+ and PARP-1 areother parameters that are closely related to oxidative stressDuring oxidative stress activation of PARP-1 is closely linkedto reduction in NAD+ [64] El-Mahdy et al [65] reportedthat naringenin was found to inhibit activation of PARP-1 cleavage Similar to naringenin the catechol structure ofanthocyanin is also able to show protective effect againstoxidative stress through inhibition of PARP-1 activationThusanthocyanin could help in reduction of rapid depletion ofcellular NAD+ during high stress condition

4 Conclusions

Anthocyanins in the defatted dabai peel are potential antiox-idants In the crude extract of defatted dabai peel C3G wasthemajor anthocyanin with stronger antioxidative propertiescompared to P3G C3G also acted as reducing agent andpowerful free radical scavenger as determined by DPPH andCUPRAC assays C3G also possessed higher oxidative stressinhibition effects through inhibitions of linoleic acid oxida-tion hemoglobin oxidation and PARP-1 activity comparedto the other anthocyanins Higher amount of C3G was alsoobtained from the extract fraction of defatted dabai peel ascompared to the extract fraction of the pericarp The resultsof in vitro assays had also proven that the C3G-rich extract ofdefatted dabai peel is a potential cardioprotective agent Theprotective effect was further supported by in vivo study wherethe C3G-rich extract of the defatted dabai peel has inhibitedMDA production in the hypercholesterolemic rabbits andable to maintain the physiological regulation between theoxidant and the antioxidant in the in vivo system

Ethical Approval

Ethical approval was obtained from the Animal Care andUse Committee of the Faculty of Medicine and Health Sci-ences Universiti Putra Malaysia (Approval no UPMFPSKPADSBR-UUH00385)

Conflict of Interests

This study was performed using the funding from UniversitiPutra Malaysia and Ministry of Agriculture amp Agro-BasedIndustry Malaysia as acknowledged Neither company norindustry is directly or indirectly involved in this study

Acknowledgments

This study was funded by the Research University GrantScheme (04-02-10-0921RU 9192600) from the ResearchManagement Centre Universiti Putra Malaysia and theScience Fund from theMinistry of Agriculture ampAgro-BasedIndustry Malaysia (05-01-04-SF1137 5450555)

References

[1] A D Quartarolo and N Russo ldquoA computational study(TDDFT and RICC2) of the electronic spectra of pyranoan-thocyanins in the gas phase and solutionrdquo Journal of ChemicalTheory and Computation vol 7 no 4 pp 1073ndash1081 2011

[2] K S Gould JMcKelvie andK RMarkham ldquoDo anthocyaninsfunction as antioxidants in leaves Imaging of H

2

O2

in red andgreen leaves after mechanical injuryrdquo Plant Cell and Environ-ment vol 25 no 10 pp 1261ndash1269 2002

[3] S Zafra-Stone T YasminM Bagchi AChatterjee J AVinsonand D Bagchi ldquoBerry anthocyanins as novel antioxidants inhuman health and disease preventionrdquo Molecular Nutrition ampFood Research vol 51 no 6 pp 675ndash683 2007

[4] L Y ChewH E Khoo I Amin A Azrina andC Y Lau ldquoAnal-ysis of phenolic compounds of dabai (Canarium odontophyllumMiq) fruits by high-performance liquid chromatographyrdquo FoodAnalytical Methods vol 5 no 1 pp 126ndash137 2012

[5] J M Cooney D J Jensen and T K McGhie ldquoLC-MS identifi-cation of anthocyanins in boysenberry extract and anthocyaninmetabolites in human urine following dosingrdquo Journal of theScience of Food andAgriculture vol 84 no 3 pp 237ndash245 2004

[6] T Ichiyanagi Y Hatano S Matsugo and T Konishi ldquoStructuraldependence of HPLC separation pattern of anthocyanins frombilberry (Vaccinium myrtillus L)rdquo Chemical and Pharmaceuti-cal Bulletin vol 52 no 5 pp 628ndash630 2004

[7] S E Kulling andHM Rawel ldquoChokeberry (Aronia melanocar-pa)mdasha review on the characteristic components and potentialhealth effectsrdquo Planta Medica vol 74 no 13 pp 1625ndash16342008

[8] F H Shakirin A Azlan A Ismail Z Amom and C Y LauldquoAntiatherosclerotic effect of Canarium odontophyllum Miqfruit parts in rabbits fed high cholesterol dietrdquo Evidence-BasedComplementary and Alternative Medicine vol 2012 Article ID838604 10 pages 2012

[9] F H Shakirin K N Prasad A Ismail L C Yuon and A AzlanldquoAntioxidant capacity of underutilized Malaysian Canariumodontophyllum (dabai) Miq fruitrdquo Journal of Food Compositionand Analysis vol 23 no 8 pp 777ndash781 2010

[10] L Y Chew K N Prasad I Amin A Azrina and C Y LauldquoNutritional composition and antioxidant properties of Cana-rium odontophyllum Miq (dabai) fruitsrdquo Journal of Food Com-position and Analysis vol 24 no 4-5 pp 670ndash677 2011

[11] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[12] B Sun C Leandro J M Ricardo da Silva and I SprangerldquoSeparation of grape and wine proanthocyanidins according totheir degree of polymerizationrdquo Journal of Agricultural and FoodChemistry vol 46 no 4 pp 1390ndash1396 1998

[13] M V Russo G Goretti and T Nevigato ldquoSequential solid-phase extraction with cyanopropyl bonded-phase cartridges fortrace enrichment of PCBs and chlorinated pesticides fromwatersamplesrdquo Chromatographia vol 50 no 7-8 pp 446ndash452 1999


[14] H E Khoo A Azlan A Ismail and F Abas ldquoResponse surfacemethodology optimization for extraction of phenolics and anti-oxidant capacity in defatted dabai partsrdquo Sains Malaysiana vol42 no 7 pp 949ndash954 2013

[15] H E Khoo A Azlan A Ismail and F Abas ldquoAntioxidativeproperties of defatted dabai pulp and peel prepared by solidphase extractionrdquoMolecules vol 17 no 8 pp 9754ndash9773 2012

[16] T A Holton and E C Cornish ldquoGenetics and biochemistry ofanthocyanin biosynthesisrdquo Plant Cell vol 7 no 7 pp 1071ndash10831995

[17] D E Willis ldquoInternal standard method calculationsrdquo Chromat-ographia vol 5 no 1 pp 42ndash43 1972

[18] L-S Lai S-T Chou and W-W Chao ldquoStudies on the antiox-idative activities of Hsian-tsao (Mesona procumbensHemsl) leafgumrdquo Journal of Agricultural and Food Chemistry vol 49 no 2pp 963ndash968 2001

[19] S E Celik M Ozyurek K Guclu and R Apak ldquoCUPRACtotal antioxidant capacity assay of lipophilic antioxidants incombination with hydrophilic antioxidants using the macro-cyclic oligosaccharide methyl 120573-cyclodextrin as the solubilityenhancerrdquo Reactive and Functional Polymers vol 67 no 12 pp1548ndash1560 2007

[20] B Sultana F Anwar and R Przybylski ldquoAntioxidant activityof phenolic components present in barks of Azadirachta indicaTerminalia arjunaAcacia nilotica andEugenia jambolana Lamtreesrdquo Food Chemistry vol 104 no 3 pp 1106ndash1114 2007

[21] G-C Yen P-D Duh and D-Y Chuang ldquoAntioxidant activityof anthraquinones and anthronerdquo Food Chemistry vol 70 no4 pp 437ndash441 2000

[22] J Rodrıguez D di Pierro M Gioia et al ldquoEffects of a naturalextract from Mangifera indica L and its active compoundmangiferin on energy state and lipid peroxidation of red bloodcellsrdquo Biochimica et Biophysica Acta vol 1760 no 9 pp 1333ndash1342 2006

[23] J A Buege and S D Aust ldquoMicrosomal lipid peroxidationrdquo inMethods in Enzymology F Sidney and P Lester Eds vol 52 pp302ndash310 1978

[24] L Geraets H J J Moonen E F M Wouters A Bast and GJ Hageman ldquoCaffeine metabolites are inhibitors of the nuclearenzyme poly(ADP-ribose)polymerase-1 at physiological con-centrationsrdquo Biochemical Pharmacology vol 72 no 7 pp 902ndash910 2006

[25] M H Nurulhuda A Azlan A Ismail Z Amom and F HShakirin ldquoCholesterol-lowering and artherosclerosis inhibitoryeffect of sibu olive in cholesterol fed-rabbitrdquo Asian Journal ofBiochemistry vol 7 no 2 pp 80ndash89 2012

[26] R L Wayne and Y A Mahinda ldquoCardioprotective actions ofgrape polyphenolsrdquo Nutrition Research vol 28 no 11 pp 729ndash737 2008

[27] T Shimizu K Nakai Y Morimoto et al ldquoSimple rabbit mod-el of vulnerable atherosclerotic plaquerdquo Neurologia Medico-Chirurgica vol 49 no 8 pp 327ndash332 2009

[28] I Jasprica A Mornar Z Debeljak et al ldquoIn vivo study ofpropolis supplementation effects on antioxidative status and redblood cellsrdquo Journal of Ethnopharmacology vol 110 no 3 pp548ndash554 2007

[29] S M Cardoso S Guyot N Marnet J A Lopes-da-Silva C MGC Renard andMACoimbra ldquoCharacterisation of phenolicextracts from olive pulp and olive pomace by electrospray massspectrometryrdquo Journal of the Science of Food and Agriculturevol 85 no 1 pp 21ndash32 2005

[30] A Ataide da Silva E S Pereira do Nascimento D R Cardosoand DW Franco ldquoCoumarins and phenolic fingerprints of oakand Brazilian woods extracted by sugarcane spiritrdquo Journal ofSeparation Science vol 32 no 21 pp 3681ndash3691 2009

[31] F C Stintzing A S Stintzing R Carle B Frei and R EWrolstad ldquoColor and antioxidant properties of cyanidin-basedanthocyanin pigmentsrdquo Journal of Agricultural and Food Chem-istry vol 50 no 21 pp 6172ndash6181 2002

[32] J Z Ju L C Liu and Y Y Yuan ldquoActivities of chalcone synthaseand UDPGal flavonoid-3-o-glycosyltransferase in relation toanthocyanin synthesis in applerdquo Scientia Horticulturae vol 63no 3-4 pp 175ndash185 1995

[33] J E Lancaster ldquoRegulation of skin color in applesrdquo CriticalReviews in Plant Sciences vol 10 no 6 pp 487ndash502 1992

[34] H E Khoo A Azlan A Ismail and F Abas ldquoInfluence of dif-ferent extraction media on phenolic contents and antioxidantcapacity of defatted dabai (Canarium odontophyllum) fruitrdquoFood Analytical Methods vol 5 no 3 pp 339ndash350 2012

[35] H K Dooner T P Robbins and R A Jorgensen ldquoGenetic anddevelopmental control of anthocyanin biosynthesisrdquo AnnualReview of Genetics vol 25 pp 173ndash199 1991

[36] TWicklundH J Rosenfeld B KMartinsen et al ldquoAntioxidantcapacity and colour of strawberry jam as influenced by cultivarand storage conditionsrdquo LWTmdashFood Science and Technologyvol 38 no 4 pp 387ndash391 2005

[37] J-I Nakajima Y Tanaka M Yamazaki and K Saito ldquoReac-tion mechanism from leucoanthocyanidin to anthocyanidin 3-glucoside a key reaction for coloring in anthocyanin biosynthe-sisrdquo Journal of Biological Chemistry vol 276 no 28 pp 25797ndash25803 2001

[38] 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

[39] M P Kahkonen and M Heinonen ldquoAntioxidant activity ofanthocyanins and their aglyconsrdquo Journal of Agricultural andFood Chemistry vol 51 no 3 pp 628ndash633 2003

[40] H Wang G Cao and R L Prior ldquoOxygen radical absorbingcapacity of anthocyaninsrdquo Journal of Agricultural and FoodChemistry vol 45 no 2 pp 304ndash309 1997

[41] 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

[42] N Saint-Cricq de Gaulejac Y Glories and N Vivas ldquoFreeradical scavenging effect of anthocyanins in red winesrdquo FoodResearch International vol 32 no 5 pp 327ndash333 1999

[43] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[44] R Brouillard ldquoFlavonoids and flower colorrdquo inThe FlavonoidsJ B Harborne Ed Chapman amp Hall London UK 1988

[45] T Tsuda K Ohshima S Kawakishi and T Osawa ldquoOxidationproducts of cyanidin 3-O-120573-D-glucoside with a free radicalinitiatorrdquo Lipids vol 31 no 12 pp 1259ndash1263 1996

[46] J-W Xu K Ikeda and Y Yamori ldquoCyanidin-3-glucoside reg-ulates phosphorylation of endothelial nitric oxide synthaserdquoFEBS Letters vol 574 no 1ndash3 pp 176ndash180 2004

[47] J-W Xu K Ikeda and Y Yamori ldquoUpregulation of endothelialnitric oxide synthase by cyanidin-3-glucoside a typical antho-cyanin pigmentrdquoHypertension vol 44 no 2 pp 217ndash222 2004


[48] L V drsquoUscio S Milstien D Richardson L Smith and Z SKatusic ldquoLong-term vitamin C treatment increases vasculartetrahydrobiopterin levels and nitric oxide synthase activityrdquoCirculation Research vol 92 no 1 pp 88ndash95 2003

[49] S D Rybalkin C Yan K E Bornfeldt and J A Beavo ldquoCyclicGMP phosphodiesterases and regulation of smooth musclefunctionrdquoCirculation Research vol 93 no 4 pp 280ndash291 2003

[50] D E Burger X Lu M Lei et al ldquoNeuronal nitric oxide syn-thase protects against myocardial infarction-induced ventricu-lar arrhythmia and mortality in micerdquo Circulation vol 120 no14 pp 1345ndash1354 2009

[51] D R Bell andKGochenaur ldquoDirect vasoactive and vasoprotec-tive properties of anthocyanin-rich extractsrdquo Journal of AppliedPhysiology vol 100 no 4 pp 1164ndash1170 2006

[52] R Acquaviva A Russo F Galvano et al ldquoCyanidin and cya-nidin 3-O-120573-D-glucoside as DNA cleavage protectors andantioxidantsrdquoCell Biology and Toxicology vol 19 no 4 pp 243ndash252 2003

[53] V B Hoe and K H Siong ldquoThe nutritional value of indigenousfruits and vegetables in SarawakrdquoAsia Pacific Journal of ClinicalNutrition vol 8 no 1 pp 24ndash31 1999

[54] R Apak K Guclu M Ozyurek and S E Celik ldquoMechanismof antioxidant capacity assays and the CUPRAC (cupric ionreducing antioxidant capacity) assayrdquo Microchimica Acta vol160 no 4 pp 413ndash419 2008

[55] P J OrsquoBrien ldquoIntracellular mechanisms for the decompositionof a lipid peroxide I Decomposition of a lipid peroxide bymetalions heme compounds and nucleophilesrdquo Canadian Journal ofBiochemistry vol 47 no 5 pp 485ndash492 1969

[56] A Kamsler and M Segal ldquoHydrogen peroxide as a diffusiblesignal molecule in synaptic plasticityrdquo Molecular Neurobiologyvol 29 no 2 pp 167ndash178 2004

[57] K-H Han A Matsumoto K-I Shimada M Sekikawa andMFukushima ldquoEffects of anthocyanin-rich purple potato flakeson antioxidant status in F344 rats fed a cholesterol-rich dietrdquoBritish Journal of Nutrition vol 98 no 5 pp 914ndash921 2007

[58] C-C Chen L-K Liu J-D Hsu H-P Huang M-Y Yangand C-J Wang ldquoMulberry extract inhibits the development ofatherosclerosis in cholesterol-fed rabbitsrdquo Food Chemistry vol91 no 4 pp 601ndash607 2005

[59] S Valcheva-Kuzmanova K Kuzmanov V Mihova I Kras-naliev P Borisova and A Belcheva ldquoAntihyperlipidemic effectof Aronia melanocarpa fruit juice in rats fed a high-cholesteroldietrdquo Plant Foods for Human Nutrition vol 62 no 1 pp 19ndash242007

[60] I L F Nielsen S E Rasmussen A Mortensen et al ldquoAntho-cyanins increase low-density lipoprotein andplasma cholesteroland do not reduce atherosclerosis in watanabe heritable hyper-lipidemic rabbitsrdquoMolecular Nutrition amp Food Research vol 49no 4 pp 301ndash308 2005

[61] W Martinet M W M Knaapen G R Y de Meyer A G Her-man and M M Kockx ldquoElevated levels of oxidative DNAdamage and DNA repair enzymes in human atheroscleroticplaquesrdquo Circulation vol 106 no 8 pp 927ndash932 2002

[62] J-M Kong L-S Chia N-K Goh T-F Chia and R BrouillardldquoAnalysis and biological activities of anthocyaninsrdquo Phytochem-istry vol 64 no 5 pp 923ndash933 2003

[63] V Sorrenti FMazza A Campisi et al ldquoHeme oxygenase induc-tion by cyanidin-3-O-120573-glucoside in cultured human endothe-lial cellsrdquoMolecular Nutritionamp Food Research vol 51 no 5 pp580ndash586 2007

[64] J B Pillai A Isbatan S-I Imai and M P Gupta ldquoPoly(ADP-ribose) polymerase-1-dependent cardiac myocyte cell deathduring heart failure is mediated by NAD+ depletion andreduced Sir2120572 deacetylase activityrdquo Journal of Biological Chem-istry vol 280 no 52 pp 43121ndash43130 2005

[65] MA El-MahdyQ ZhuQ-EWang et al ldquoNaringenin protectsHaCaT human keratinocytes against UVB-induced apoptosisand enhances the removal of cyclobutane pyrimidine dimersfrom the genomerdquo Photochemistry and Photobiology vol 84 no2 pp 307ndash316 2008


in vivo oxidative stress and further prevent cardiovasculardiseases Antiatherosclerotic effects of defatted and nondefat-ted dabai pericarp and peel have been determined previouslyin hypercholesterolemic rabbits [8] Results from several invitro antioxidant assays have also shown protective effect ofdabai pericarp [9 10] Several biological oxidation activityassays such as linoleic acid oxidation hemoglobin oxidationand PARP-1 inhibition activity as well as lipid peroxidationmarker (plasma MDA) and antioxidant enzymes (SOD andGPx) in blood are good predictors for inhibition of oxidativestress and cardioprotective effect Therefore these markersare useful in provision of information and confirmation ofthe cardioprotective effect of anthocyanins extracted fromdefatted dabai powder especially from its peel

Due to the potential health benefits offered by antho-cyanins in defatted dabai it is of great interest to elucidatethe specific anthocyanins in the defatted dabai especially inits peel Solid phase extraction (SPE) is a separation techniquethat had been widely used for purification of polyphenols [1112] and pesticides [13] However no previous study has beendone to fractionate potential anthocyanins in defatted dabaiextract using this separation technique Therefore this studyaimed to determine the anthocyanins content in the extractsand extract fractions of defatted dabai peel and pericarptheir antioxidant capacity and related-health benefits usingin vitro assays (DPPHassay copper (II) reduction antioxidantcapacity (CUPRAC) assay linoleic acid oxidation systemhemoglobin oxidation and PARP-1 inhibition ELISA) Invivo study to evaluate cardioprotective effect of the defat-ted dabai peel extract on antioxidant enzymes (SOD andGPx) and lipid peroxidation parameter (plasma MDA) ofhypercholesterolemic-induced New Zealand white rabbitswas also carried out to support the beneficial effect of theextract

2 Materials and Methods

21 Sample Preparation Fresh dabai fruits were obtainedfrom a few locations from fruit plantations in Kapit SarawakMalaysia A homogenized sample was selected randomlyfrom a few different trees in each location Kernel of dabaifruit was discarded while dabai peel and a mixture of itspericarp were collected Both of the samples were freeze-dried using a freeze dryer (Virtis New York USA) and thelyophilized samples were ground into smaller particles usinga household grinder To prepare anthocyanin-rich defattedsamples the freeze-dried peel and pericarp were initiallydefatted by soaking in hexane for 24 h The defatted sampleswere redried and further ground into powder using thegrinder

22 Sample Extraction and Fractionation Anthocyaninsfrom the defatted dabai powders were extracted based onthe optimized conditions that were previously establishedBriefly anthocyanins in the defatted dabai peel and pericarppowders were extracted based on the optimized extractionparameters (53 methanol as solvent and sonicated for1min) [14] The extraction parameters have been optimizedpreviously based on response surfacemethodologywhere the

optimized extraction parameters had yielded optimal levelsof total phenolics and antioxidant capacity The mixture wasfiltered and anthocyanin-rich crude extract was collectedThe sample residue was reextracted twice using equal amountof 53 methanol Methanol was removed using a rotaryevaporator (Buchi Switzerland) at 39∘C The leftover watercomponent and methanol residue of the extracts were fullyremoved by freeze-drying and stored at minus40∘C until furtheranalyses

To obtain a purified anthocyanins fraction 50mg ofthe lyophilized anthocyanin-rich crude extract was dissolvedwith 80methanol (2mL vv) and fractionated by SPE usingactivated Sep-PakCNandC18 cartridges fromMerck (Darm-stadt Germany) Visiprep SPE vacuum manifold (12 ports)(Supelco Pennsylvania USA) was connected to a vacuumpump and the extracts were fractionated using the cartridgescontaining 100mg adsorbent by passing methanol and dis-tilled water (2mL each) through the cartridges Methanolfractions were collected but water fractions were discardedThe collected methanolic fractions together with the crudeextracts of defatted dabai peel and pericarp were determinedfor anthocyanins contents and antioxidant capacity togetherwith their oxidative stress inhibition ability

The flow of samples fractionation is shown in Figure 1Crude extracts of defatted dabai peel and pericarp (2mLinjection volume 5mgmL extract concentration) wereinjected into the activated Sep-Pak CN and C18 cartridgesrespectively using 5mL syringes [15] The adsorbed antho-cyanins in both cartridges were eluted by passing 20mL ofmethanol through theCNcartridge (Figure 1(a)) (for defatteddabai peel) and C18 cartridge (Figure 1(b)) (for defatted dabaipericarp) and collected as samples 1 and 2 respectively

23 HPLC Analysis Anthocyanins in the defatted dabaisamples were identified and quantified based on a modifiedmethod described by Chew et al [4] In this study UHPLCmethod using an Agilent 1290 Infinity LC system (AgilentTechnologies Waldbronn Germany) equipped with a binarypump diode array detector and an autosampler was appliedin the quantification of anthocyanins Separation of antho-cyanins was achieved on a Lichrospher C-18 column (250times 4mm id 5120583m) (Merck KGaA Darmstadt Germany) at30∘C The mobile phases consisted of A 05 trifluoroaceticacid and B 100 acetonitrile The gradient was achievedusing the following system 90ndash85 B 0ndash5min 85 B 5ndash10min 85ndash70 B 10ndash15min 70 B 15ndash20min 70ndash20B 20ndash25min 20ndash0 25ndash30min The column was washedand reconditioned after every run The flow rate was set at06mLmin and the injection volume was 20120583L

Anthocyanins were detected at a wavelength of 530 nmwhile UV-Vis absorption spectra were recorded in therange of 250ndash600 nm A total of six anthocyanin standardscyanidin-3-glucoside (C3G) cyanidin-3-galactoside (C3L)cyanidin-3-arabinoside (C3A) pelargonidin-3-glucoside(L3G) malvidin-3-glucoside (M3G) and peonidin-3-glu-coside (P3G) were used for identification of anthocyaninsin the defatted dabai extracts The anthocyanins content ofthe defatted dabai extracts was determined and calculatedusing cyanidin-3-rutinoside (C3R) as internal standard This


CNcartridge

CNcartridge

Tovacuum



100

methanol

(a)

cartridge

Tovacuum

Waste

C18cartridge

C18




100

methanol

(b)







where

119865

119877=


(2)



=[1minus


]times100

(3)



2 10mL of copper







= [1 minus


] times 100

(4)







= [1 minus


] times 100

(5)


TBARS value (mM)

=











200

175

150

125

100

75

50

25

0

0 5 10 15 20 25

(mAU

)

(min)

(Internalstandard)

C3R

C3GC3G

C3LC3L

Unknown L3GM3G

C3AP3G













Table1Antho

cyaninsc

ontent

ofthed


ndextractsfractio

nsandotherU

HPL

Cparameters

Sample

Antho

cyanins(mggextract)

Pelargon

idin-3-glucosid



eCy

anidin-3-glucosid

eCy

anidin-3-arabino

side

Peon

idin-3-glucosid

eDefatteddabaip

eelextract

442plusmn008

561plusmn022

442plusmn008

5512plusmn082

417plusmn027

Trace

Defatteddabaip

ericarpextract

ND

ND

584plusmn013

607plusmn002

Trace

ND

1Trace

Trace

1745plusmn04

1346plusmn028

108plusmn003

Trace

2ND

Trace

472plusmn007

432plusmn017

047plusmn001

Trace

Other

parameters

Retentiontim

e140plusmn02

153plusmn03

173plusmn04

178plusmn02

184plusmn05

191plusmn03

RSD(

)for

allsam

ples

177ndash322

127ndash463

156ndash

998

014ndash396

025ndash6

78

042ndash1549

RSD(

)for

defatte

ddabaip

eel

177

40

177

148

658

901

Linearity

(1198772

)for

defatte

ddabaip

eel

0953

0978

0953

0999

0989

0963

LODford

efatteddabaip

eel(mgmL)

027

115

028

031

001

082

LOQford

efatteddabaip

eel(mgmL)

123

515

468

138

003

368

lowast

NDnot

detectedLODlim

itof

detection

LOQlim

itof

quantitation

Seetextfor

then

ames

ofsamples

1and

2



chalcone

N3HOH

OHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

CHI

Dihydrokaempferol

Dihydroquercetin

DF4R

AS

UF3GT


O+O+

HOHO

HO

HOHOHO

HOHO

HOOO

OO

O

O

O

O

O

OF3998400H





80

70

60

50

40

30

20

10

0Scav

engi

ng ca

paci

ty (

)

Peel PP 1 2 C3G P3G

b

d e

f

a

c


Trol

ox eq

uiva

lent

(mM

g)

0

5

10

15

20

25

30

35

0

05

1

15

2

Peel PP 1 2

Peel PP 1 2

C3G P3G

b

d e f

ac








0

5

10

15

20

25

30

35

40

Peel PP 1 2 C3G P3G

bb

d d

a

c

Control

Prot

ectiv

e cap

acity

()


0

5

10

15

20

25

Peel PP 1 2 C3G P3G

b

a a a

c

Control

Prot

ectiv

e cap

acity

()

b c







0

1

2

3

4

5

6

7

8

Peel PP 1 2 C3G P3G

b

d

a

c

Control

c d

a b

c d

TBA

RS v

alue

(mM

)


Inhi

bitio

n ac

tivity

()

0

20

40

60

80

Peel PP 1 2 C3G P3G

b

dd

ac

Control

c d





















4 Conclusions


Ethical Approval




Acknowledgments


References



2

O2




































































CNcartridge

CNcartridge

Tovacuum



100

methanol

(a)

cartridge

Tovacuum

Waste

C18cartridge

C18




100

methanol

(b)







where

119865

119877=


(2)



=[1minus


]times100

(3)



2 10mL of copper







= [1 minus


] times 100

(4)







= [1 minus


] times 100

(5)


TBARS value (mM)

=











200

175

150

125

100

75

50

25

0

0 5 10 15 20 25

(mAU

)

(min)

(Internalstandard)

C3R

C3GC3G

C3LC3L

Unknown L3GM3G

C3AP3G













Table1Antho

cyaninsc

ontent

ofthed


ndextractsfractio

nsandotherU

HPL

Cparameters

Sample

Antho

cyanins(mggextract)

Pelargon

idin-3-glucosid



eCy

anidin-3-glucosid

eCy

anidin-3-arabino

side

Peon

idin-3-glucosid

eDefatteddabaip

eelextract

442plusmn008

561plusmn022

442plusmn008

5512plusmn082

417plusmn027

Trace

Defatteddabaip

ericarpextract

ND

ND

584plusmn013

607plusmn002

Trace

ND

1Trace

Trace

1745plusmn04

1346plusmn028

108plusmn003

Trace

2ND

Trace

472plusmn007

432plusmn017

047plusmn001

Trace

Other

parameters

Retentiontim

e140plusmn02

153plusmn03

173plusmn04

178plusmn02

184plusmn05

191plusmn03

RSD(

)for

allsam

ples

177ndash322

127ndash463

156ndash

998

014ndash396

025ndash6

78

042ndash1549

RSD(

)for

defatte

ddabaip

eel

177

40

177

148

658

901

Linearity

(1198772

)for

defatte

ddabaip

eel

0953

0978

0953

0999

0989

0963

LODford

efatteddabaip

eel(mgmL)

027

115

028

031

001

082

LOQford

efatteddabaip

eel(mgmL)

123

515

468

138

003

368

lowast

NDnot

detectedLODlim

itof

detection

LOQlim

itof

quantitation

Seetextfor

then

ames

ofsamples

1and

2



chalcone

N3HOH

OHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

CHI

Dihydrokaempferol

Dihydroquercetin

DF4R

AS

UF3GT


O+O+

HOHO

HO

HOHOHO

HOHO

HOOO

OO

O

O

O

O

O

OF3998400H





80

70

60

50

40

30

20

10

0Scav

engi

ng ca

paci

ty (

)

Peel PP 1 2 C3G P3G

b

d e

f

a

c


Trol

ox eq

uiva

lent

(mM

g)

0

5

10

15

20

25

30

35

0

05

1

15

2

Peel PP 1 2

Peel PP 1 2

C3G P3G

b

d e f

ac








0

5

10

15

20

25

30

35

40

Peel PP 1 2 C3G P3G

bb

d d

a

c

Control

Prot

ectiv

e cap

acity

()


0

5

10

15

20

25

Peel PP 1 2 C3G P3G

b

a a a

c

Control

Prot

ectiv

e cap

acity

()

b c







0

1

2

3

4

5

6

7

8

Peel PP 1 2 C3G P3G

b

d

a

c

Control

c d

a b

c d

TBA

RS v

alue

(mM

)


Inhi

bitio

n ac

tivity

()

0

20

40

60

80

Peel PP 1 2 C3G P3G

b

dd

ac

Control

c d





















4 Conclusions


Ethical Approval




Acknowledgments


References



2

O2









































































= [1 minus


] times 100

(5)


TBARS value (mM)

=











200

175

150

125

100

75

50

25

0

0 5 10 15 20 25

(mAU

)

(min)

(Internalstandard)

C3R

C3GC3G

C3LC3L

Unknown L3GM3G

C3AP3G













Table1Antho

cyaninsc

ontent

ofthed


ndextractsfractio

nsandotherU

HPL

Cparameters

Sample

Antho

cyanins(mggextract)

Pelargon

idin-3-glucosid



eCy

anidin-3-glucosid

eCy

anidin-3-arabino

side

Peon

idin-3-glucosid

eDefatteddabaip

eelextract

442plusmn008

561plusmn022

442plusmn008

5512plusmn082

417plusmn027

Trace

Defatteddabaip

ericarpextract

ND

ND

584plusmn013

607plusmn002

Trace

ND

1Trace

Trace

1745plusmn04

1346plusmn028

108plusmn003

Trace

2ND

Trace

472plusmn007

432plusmn017

047plusmn001

Trace

Other

parameters

Retentiontim

e140plusmn02

153plusmn03

173plusmn04

178plusmn02

184plusmn05

191plusmn03

RSD(

)for

allsam

ples

177ndash322

127ndash463

156ndash

998

014ndash396

025ndash6

78

042ndash1549

RSD(

)for

defatte

ddabaip

eel

177

40

177

148

658

901

Linearity

(1198772

)for

defatte

ddabaip

eel

0953

0978

0953

0999

0989

0963

LODford

efatteddabaip

eel(mgmL)

027

115

028

031

001

082

LOQford

efatteddabaip

eel(mgmL)

123

515

468

138

003

368

lowast

NDnot

detectedLODlim

itof

detection

LOQlim

itof

quantitation

Seetextfor

then

ames

ofsamples

1and

2



chalcone

N3HOH

OHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

CHI

Dihydrokaempferol

Dihydroquercetin

DF4R

AS

UF3GT


O+O+

HOHO

HO

HOHOHO

HOHO

HOOO

OO

O

O

O

O

O

OF3998400H





80

70

60

50

40

30

20

10

0Scav

engi

ng ca

paci

ty (

)

Peel PP 1 2 C3G P3G

b

d e

f

a

c


Trol

ox eq

uiva

lent

(mM

g)

0

5

10

15

20

25

30

35

0

05

1

15

2

Peel PP 1 2

Peel PP 1 2

C3G P3G

b

d e f

ac








0

5

10

15

20

25

30

35

40

Peel PP 1 2 C3G P3G

bb

d d

a

c

Control

Prot

ectiv

e cap

acity

()


0

5

10

15

20

25

Peel PP 1 2 C3G P3G

b

a a a

c

Control

Prot

ectiv

e cap

acity

()

b c







0

1

2

3

4

5

6

7

8

Peel PP 1 2 C3G P3G

b

d

a

c

Control

c d

a b

c d

TBA

RS v

alue

(mM

)


Inhi

bitio

n ac

tivity

()

0

20

40

60

80

Peel PP 1 2 C3G P3G

b

dd

ac

Control

c d





















4 Conclusions


Ethical Approval




Acknowledgments


References



2

O2




































































200

175

150

125

100

75

50

25

0

0 5 10 15 20 25

(mAU

)

(min)

(Internalstandard)

C3R

C3GC3G

C3LC3L

Unknown L3GM3G

C3AP3G













Table1Antho

cyaninsc

ontent

ofthed


ndextractsfractio

nsandotherU

HPL

Cparameters

Sample

Antho

cyanins(mggextract)

Pelargon

idin-3-glucosid



eCy

anidin-3-glucosid

eCy

anidin-3-arabino

side

Peon

idin-3-glucosid

eDefatteddabaip

eelextract

442plusmn008

561plusmn022

442plusmn008

5512plusmn082

417plusmn027

Trace

Defatteddabaip

ericarpextract

ND

ND

584plusmn013

607plusmn002

Trace

ND

1Trace

Trace

1745plusmn04

1346plusmn028

108plusmn003

Trace

2ND

Trace

472plusmn007

432plusmn017

047plusmn001

Trace

Other

parameters

Retentiontim

e140plusmn02

153plusmn03

173plusmn04

178plusmn02

184plusmn05

191plusmn03

RSD(

)for

allsam

ples

177ndash322

127ndash463

156ndash

998

014ndash396

025ndash6

78

042ndash1549

RSD(

)for

defatte

ddabaip

eel

177

40

177

148

658

901

Linearity

(1198772

)for

defatte

ddabaip

eel

0953

0978

0953

0999

0989

0963

LODford

efatteddabaip

eel(mgmL)

027

115

028

031

001

082

LOQford

efatteddabaip

eel(mgmL)

123

515

468

138

003

368

lowast

NDnot

detectedLODlim

itof

detection

LOQlim

itof

quantitation

Seetextfor

then

ames

ofsamples

1and

2



chalcone

N3HOH

OHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

CHI

Dihydrokaempferol

Dihydroquercetin

DF4R

AS

UF3GT


O+O+

HOHO

HO

HOHOHO

HOHO

HOOO

OO

O

O

O

O

O

OF3998400H





80

70

60

50

40

30

20

10

0Scav

engi

ng ca

paci

ty (

)

Peel PP 1 2 C3G P3G

b

d e

f

a

c


Trol

ox eq

uiva

lent

(mM

g)

0

5

10

15

20

25

30

35

0

05

1

15

2

Peel PP 1 2

Peel PP 1 2

C3G P3G

b

d e f

ac








0

5

10

15

20

25

30

35

40

Peel PP 1 2 C3G P3G

bb

d d

a

c

Control

Prot

ectiv

e cap

acity

()


0

5

10

15

20

25

Peel PP 1 2 C3G P3G

b

a a a

c

Control

Prot

ectiv

e cap

acity

()

b c







0

1

2

3

4

5

6

7

8

Peel PP 1 2 C3G P3G

b

d

a

c

Control

c d

a b

c d

TBA

RS v

alue

(mM

)


Inhi

bitio

n ac

tivity

()

0

20

40

60

80

Peel PP 1 2 C3G P3G

b

dd

ac

Control

c d





















4 Conclusions


Ethical Approval




Acknowledgments


References



2

O2




































































Table1Antho

cyaninsc

ontent

ofthed


ndextractsfractio

nsandotherU

HPL

Cparameters

Sample

Antho

cyanins(mggextract)

Pelargon

idin-3-glucosid



eCy

anidin-3-glucosid

eCy

anidin-3-arabino

side

Peon

idin-3-glucosid

eDefatteddabaip

eelextract

442plusmn008

561plusmn022

442plusmn008

5512plusmn082

417plusmn027

Trace

Defatteddabaip

ericarpextract

ND

ND

584plusmn013

607plusmn002

Trace

ND

1Trace

Trace

1745plusmn04

1346plusmn028

108plusmn003

Trace

2ND

Trace

472plusmn007

432plusmn017

047plusmn001

Trace

Other

parameters

Retentiontim

e140plusmn02

153plusmn03

173plusmn04

178plusmn02

184plusmn05

191plusmn03

RSD(

)for

allsam

ples

177ndash322

127ndash463

156ndash

998

014ndash396

025ndash6

78

042ndash1549

RSD(

)for

defatte

ddabaip

eel

177

40

177

148

658

901

Linearity

(1198772

)for

defatte

ddabaip

eel

0953

0978

0953

0999

0989

0963

LODford

efatteddabaip

eel(mgmL)

027

115

028

031

001

082

LOQford

efatteddabaip

eel(mgmL)

123

515

468

138

003

368

lowast

NDnot

detectedLODlim

itof

detection

LOQlim

itof

quantitation

Seetextfor

then

ames

ofsamples

1and

2



chalcone

N3HOH

OHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

CHI

Dihydrokaempferol

Dihydroquercetin

DF4R

AS

UF3GT


O+O+

HOHO

HO

HOHOHO

HOHO

HOOO

OO

O

O

O

O

O

OF3998400H





80

70

60

50

40

30

20

10

0Scav

engi

ng ca

paci

ty (

)

Peel PP 1 2 C3G P3G

b

d e

f

a

c


Trol

ox eq

uiva

lent

(mM

g)

0

5

10

15

20

25

30

35

0

05

1

15

2

Peel PP 1 2

Peel PP 1 2

C3G P3G

b

d e f

ac








0

5

10

15

20

25

30

35

40

Peel PP 1 2 C3G P3G

bb

d d

a

c

Control

Prot

ectiv

e cap

acity

()


0

5

10

15

20

25

Peel PP 1 2 C3G P3G

b

a a a

c

Control

Prot

ectiv

e cap

acity

()

b c







0

1

2

3

4

5

6

7

8

Peel PP 1 2 C3G P3G

b

d

a

c

Control

c d

a b

c d

TBA

RS v

alue

(mM

)


Inhi

bitio

n ac

tivity

()

0

20

40

60

80

Peel PP 1 2 C3G P3G

b

dd

ac

Control

c d





















4 Conclusions


Ethical Approval




Acknowledgments


References



2

O2





































































chalcone

N3HOH

OHOH

OHOH

OH

OHOH

OHOHOH

OHOH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

CHI

Dihydrokaempferol

Dihydroquercetin

DF4R

AS

UF3GT


O+O+

HOHO

HO

HOHOHO

HOHO

HOOO

OO

O

O

O

O

O

OF3998400H





80

70

60

50

40

30

20

10

0Scav

engi

ng ca

paci

ty (

)

Peel PP 1 2 C3G P3G

b

d e

f

a

c


Trol

ox eq

uiva

lent

(mM

g)

0

5

10

15

20

25

30

35

0

05

1

15

2

Peel PP 1 2

Peel PP 1 2

C3G P3G

b

d e f

ac








0

5

10

15

20

25

30

35

40

Peel PP 1 2 C3G P3G

bb

d d

a

c

Control

Prot

ectiv

e cap

acity

()


0

5

10

15

20

25

Peel PP 1 2 C3G P3G

b

a a a

c

Control

Prot

ectiv

e cap

acity

()

b c







0

1

2

3

4

5

6

7

8

Peel PP 1 2 C3G P3G

b

d

a

c

Control

c d

a b

c d

TBA

RS v

alue

(mM

)


Inhi

bitio

n ac

tivity

()

0

20

40

60

80

Peel PP 1 2 C3G P3G

b

dd

ac

Control

c d





















4 Conclusions


Ethical Approval




Acknowledgments


References



2

O2








































































0

5

10

15

20

25

30

35

40

Peel PP 1 2 C3G P3G

bb

d d

a

c

Control

Prot

ectiv

e cap

acity

()


0

5

10

15

20

25

Peel PP 1 2 C3G P3G

b

a a a

c

Control

Prot

ectiv

e cap

acity

()

b c







0

1

2

3

4

5

6

7

8

Peel PP 1 2 C3G P3G

b

d

a

c

Control

c d

a b

c d

TBA

RS v

alue

(mM

)


Inhi

bitio

n ac

tivity

()

0

20

40

60

80

Peel PP 1 2 C3G P3G

b

dd

ac

Control

c d





















4 Conclusions


Ethical Approval




Acknowledgments


References



2

O2




































































0

1

2

3

4

5

6

7

8

Peel PP 1 2 C3G P3G

b

d

a

c

Control

c d

a b

c d

TBA

RS v

alue

(mM

)


Inhi

bitio

n ac

tivity

()

0

20

40

60

80

Peel PP 1 2 C3G P3G

b

dd

ac

Control

c d





















4 Conclusions


Ethical Approval




Acknowledgments


References



2

O2

















































































4 Conclusions


Ethical Approval




Acknowledgments


References



2

O2



































































antioxidative and cardioprotective properties of ......dards (c3g or p3g, 10𝜇g/ml) at 4∘c....

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