Influence of Different Extraction Media on PhenolicContents and Antioxidant Capacity of Defatted Dabai(Canarium odontophyllum) Fruit

Hock Eng Khoo & Azrina Azlan & Amin Ismail &Faridah Abas

Received: 4 April 2011 /Accepted: 6 May 2011 /Published online: 17 May 2011# Springer Science+Business Media, LLC 2011

Abstract Dabai (Canarium odontophyllum) is a potential“functional fruit”. Future commercialization of dabai fat ashealthy oil may result in the accumulation of defatted dabai asa by-product. This study was carried out to determine the totalphenolics and antioxidant capacity of defatted dabai parts as anew source of functional food and nutraceutical ingredient. Inthis study, defatted dabai parts were extracted using differentextraction media (methanol, ethanol, ethyl acetate, acetone,and water) and analyzed for total phenolics, total flavonoids,total anthocyanins, and antioxidant capacity (ABTS+ radicalscavenging and ferric-reducing antioxidant power assays)using spectrophotometric and high-performance liquid chro-matography methods. Major phenolics in defatted dabai peelextracted using methanol were catechin and epigallocatechinwhile in water extract, major phenolic acid was ellagic acid.Defatted dabai peel also had higher anthocyanidin contentthan its pulp. The peel of a defatted dabai fruit extractedusing methanol contained a high total phenolics and Troloxequivalent antioxidant capacity (TEAC). Ethyl acetateextract of defatted dabai parts had the least phenolicscompared in ethanol and acetone extracts. Higher totalphenolics and TEAC values were observed in water extractof a defatted dabai peel than ethanol, acetone, and ethylacetate extracts. Hence, methanol extract of a defatted dabaipeel could probably be used as a natural antioxidant.

Keywords Canarium odontophyllum . Total phenoliccontent . Total flavonoid content . Total anthocyanincontent . ABTS+ radical scavenging . Ferric-reducingantioxidant power

Introduction

Dabai (Canarium odontophyllum Miq.) is a species ofCanarium from the family of Burseraceae. In Latin,C. odontophyllum means “toothed leaf.” Dabai trees exhibitbiennial fruit-bearing season (Lau 2009). The tree can befound in Sumatra, Borneo (Sarawak, Sabah, and EastKalimantan), and the Philippines. Nowadays, dabai isplanted in Queensland, Australia. In the Borneo region,local communities commonly soak the whole fruit in warmwater for 5–10 min and eat with soy sauce or sugar (TopTropicals 2009). The fruit is nutritious and has a greatpotential to be exploited as nutraceutical food due to itshigh nutrient and antioxidant properties (Jackson 2008; Lau2009; Azlan et al. 2010a).

Dabai skin is dark purple in color and has yellow-brownish fleshy drupe. The edible portion is the unctuousmesocarp of about 6–8 mm thick that surrounds the hardthree-angular seed (Slik 2006; Lau 2009). The fruit is about3.7–4.1 cm in length, 2.4–2.8 cm in width, and weighs anaverage of 15.5 g each (Azlan et al. 2009). The photo of thedabai fruit was published by Chew et al. (2011a). Good-quality fruit is large in size, about 18 g or more, with thickaril, nutty flavor, and fine texture (Lau 2009). Dabai isconsidered as one of the hundred high-value fruit species inSarawak (Jamadon et al. 2007). The skin and flesh areedible after soaking in warm water (60°C).

In East Malaysia, specifically Sarawak, dabai is locallyknown as “Sibu olive” and has been reported to have

H. E. Khoo :A. Azlan (*) :A. IsmailDepartment of Nutrition and Dietetics, Faculty of Medicineand Health Sciences, Universiti Putra Malaysia,43400 UPM Serdang, Selangor, Malaysiae-mail: azrina@medic.upm.edu.my

F. AbasDepartment of Food Science, Faculty of Food Scienceand Technology, Universiti Putra Malaysia,43400 UPM Serdang, Selangor, Malaysia

Food Anal. Methods (2012) 5:339–350DOI 10.1007/s12161-011-9250-0

similar characteristics to olive. Previous study has reportedthat olive fruit contained numerous substances of phenols(Tripoli et al. 2005). Other than olive, studies have alsoshown that dabai fruit contained high amount of oil andphenolic contents (Azlan et al. 2010a,b), which had beenfound in olive since the past few decades (Lesage-Meessenet al. 2001; Marsilio et al. 2001). The fatty acid compositionof dabai oil is similar to palm oil (Azlan et al. 2010b). Azlanet al. (2010a) have reported high total phenolic of dabai withthe peel having the highest total phenolics (387.5±33.23 mggallic acid equivalent (GAE)/100 g), followed by pulp andkernel, which had 267.0±4.24 and 51.0±0.01 mg GAE/100 g, respectively. Chew et al. (2011b) have reported thatdabai fruit contained five flavonoids (catechin, epicatechin,epicatechin gallate, epigallocatechin gallate, and apigenin)and three anthocyanidins (cyanidin, pelargonidin, anddelphinidin). Surprisingly, findings of our study (data notshown) showed that triglyceride and total cholesterol ofhypercholesterolemic-induced rabbits treated with defatteddabai parts were significantly decreased (50 g−1 kg−1 day−1)similar to statin-treated animals.

Nowadays, scientists are keen to explore the antioxidantproperties of by-products arising from the fruit processingindustry. These by-products can potentially be used as newvalue-added product rather than be disposed or producedinto fertilizers and cattle feeds. As dabai fat is considered tobe a potential functional oil and an ingredient forcommercialization (Azlan and Ismail 2008, 2010), theby-products then arise from oil processing, especially thedefatted pulp and peel, should be fully explored as a newsource of antioxidant. To date, specific phenolic com-pounds which occurred in the defatted dabai pulp and peelremains unknown. Besides, the phenolic contents andantioxidant capacity of the defatted dabai pulp and peelextracted using various extraction media have not beenstudied. Therefore, this study is aimed to investigate theinfluence of different extraction media on phenolics contentand the antioxidant capacity of defatted dabai fruit parts.

Materials and Methods

Chemicals and Reagents

Methanol (AR grade), methanol (high-performance liquidchromatography (HPLC) grade), absolute ethanol (AR grade),acetonitrile (HPLC grade), ethyl acetate (AR grade), acetone(AR grade), and n-hexane (AR grade) were purchased fromFisher Scientific (Pennsylvania, USA). Sodium bicarbonate,sodium acetate, aluminum chloride, potassium acetate,potassium persulphate, iron chloride, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sul-fonate) (ABTS), Trolox, Folin–Ciocalteu reagent, gallic acid,

and quercetin were purchased from Sigma Chemical(Missouri, USA). Acetic acid, trifluoroacetic acid, andhydrochloric acid (37% fuming) were obtained from J.T.Baker (New Jersey, USA).

Sample Preparation and Extraction

Fresh dabai fruits were obtained from a few differentlocations in Sarawak, Malaysia. These fruits were regis-tered at Sarawak Herbarium of the Forestry Department andhave been recorded under voucher no. S73759 (Tebedu),S40073 (Niah), and S64872 (Kapit). The fruits weretransported to laboratory and stored at −20 °C beforesample preparation. The kernel of dabai fruit was separatedfrom its pulp. The prepared dabai samples consist of pulp,peel, and pulp together with peel (pulp and peel) weresubjected to freeze drying using a Virtis’s bench-top freezedryer (New York, USA). The lyophilized sample powderswere defatted using hexane before being subjected todifferent extraction media. The defatting procedure wasadapted from a method described by Khetarpaul et al.(2004), where the lyophilized dabai samples were soakedovernight in hexane at a ratio of 1:10 and re-extracted threetimes using the same ratio of hexane.

The defatted dabai powders (2.0 g each) were addedwith 10 mL of five different extraction media (ethanol,methanol, ethyl acetate, acetone, and distilled water), whichwas based on a method described by Earp et al. (1981) withsome modifications. These extraction media were chosenbecause methanol, ethanol, ethyl acetate, acetone, and waterare the typical extraction media used for phenolic com-pounds. The mixture was incubated at room temperature for10 min after being vortexed for 1 min and filtered using afunnel. The funnel was rinsed with 5-mL aliquot ofextraction solvents prior to filtration. Triplicate extractionswere performed for each defatted dabai sample. All extractswere stored at −20 °C before further analysis.

Estimation of Total Phenolics in Defatted Dabai ExtractsUsing Spectrophotometric Methods

Determination of Total Phenolic Content

Determination of total phenolic content was performedbased on a method by Hajimahmoodi et al. (2009), whichwas originated from the Folin–Ciocalteu reagent assay byVelioglu et al. (1998). The defatted dabai extracts (200 μL)were mixed with 1.5 mL of Folin–Ciocalteu reagent(previously diluted 10-fold with distilled water), andallowed to stand at room temperature for 5 min. Then,1.5 mL of sodium bicarbonate solution (60 g/L) was addedinto each mixture. The absorbance was measured at 750 nmusing a Secomam’s RS232 ultraviolet–visible (UV–vis)

340 Food Anal. Methods (2012) 5:339–350

spectrophotometer (Cedex, France) after a 90-min incuba-tion at room temperature. The total phenolic content wascalculated using regression equations from standard curveof gallic acid (1–200 μg/mL in methanol). The results wereexpressed as milligrams of GAE per 100 g of dry weight.Triplicate determinations were performed for total phenoliccontent assay.

Determination of Total Flavonoid Content

Total flavonoid content was determined using aluminumchloride colorimetric method based on a procedurereported by Chang et al. (2002). Quercetin (1–100 μg/mLin methanol) was used for standard calibration curve. Thedefatted dabai extracts and quercetin (standard) weredissolved in methanol or the studied extraction media. Tostart the reaction, the diluted defatted dabai extract asdescribed previously or standard (0.5 mL) was then addedwith 1.5 mL of 95% ethanol, 0.1 mL of 10% aluminumchloride, 0.1 mL of 1 M potassium acetate and 2.8 mL ofdistilled water. After 30 min incubation at room temperature,the absorbance of the reaction mixture was measured at415 nm with a Secomam’s RS232 spectrophotometer. Forblank, 10% aluminum chloride was substituted by the sameamount of distilled water. All tests were carried out intriplicates. Total flavonoid content of the defatted dabaiextracts were expressed as milligrams of quercetin equivalentper 100 g of dry weight.

Determination of Total Anthocyanin Content

Total anthocyanin content (TAC) of defatted dabai extractswas determined based on a method described by Xu et al.(2007). The extraction solution was diluted with theextraction media studied at pH 3.0. Then the absorptionvalue was measured at 535 nm. The calculation formula isas follows:

TAC ¼ A535nm � V � N

98:2� m

where TAC is the total anthocyanin content in the defatteddabai extracts (mg/100 g), A535 nm is the absorption value at535 nm, V is the total volume (mL), N is the dilutionmultiple, 98.2 is the extinction coefficient in 535 nm, and mis the mass of the sample.

HPLC Determination of Phenolic and AnthocyanidinCompounds in Defatted Dabai Extracts

Individual phenolic compounds in defatted dabai extractswere identified and determined using an Agilent 1100HPLC system equipped with an Agilent 1100 Series

DAD (Agilent Technologies, Germany) on a LichrospherC-18 column (250×4 mm i.d., 5 μm) (Merck KGaA,Darmstadt, Germany). Chromatographic conditions usedfor HPLC analysis were based on a method described byChew et al. (2011b) (procedure modified slightly from Heand Xia 2007). Defatted dabai extracts were acidified with1% HCl (Naczk and Shahidi 2004) and incubated at 90 °Cfor 5 min to remove phenolic esters from bound phenoliccompounds, especially anthocyanidin. The mobilephases used were 0.5% acetic acid (v/v) (A) andmethanol (B). Gradient elution used was as follows:0–20 min, linear gradient from 0–90% B; 20–25 min,90% B isocratic; 25–30 min, linear gradient from 90–0% B,and finally, washing and reconditioning of the column. Theflow rate and column temperature were set at 0.6 mL/min and30 °C, respectively, with 50 μL of injection. UV absorptionspectra were recorded from 210–400 nm during HPLCanalysis.

A total of 15 phenolics standard were used for theidentification of phenolic compounds in defatted dabaiextracts. However, nine phenolic compounds such ascatechin, epicatechin, epicatechin gallate, epigallocatechin,methyl gallate, ellagic acid, vanillic acid, protocatechuicacid, and apigenin were identified in the samples studied.Using the nine phenolics standards, calibration curves wereplotted for phenolics determination, with a range of R2 from0.969 to 0.999. Determination of GAE for HPLC methodwas calculated based on the equation for gallic acidstandard calibration curve (y=60.889x+660.06; R2=0.992); where x is the concentration of GAE, and y is theabsorbance of total peaks area of the defatted dabai extractsobtained from HPLC result.

Anthocyanidin identification and determination werecarried out based on a method described by Chew et al.(2011b), which was adapted from Abad-García et al.(2009). Mobile phases 0.5% trifluoroacetic acid (v/v) (A)and acetonitrile (B) were applied for this analysis. Thegradient elution condition was pre-set at 0–15 min, lineargradient from 12–15% B; 15–25 min, 15% B isocratic;25–40 min, linear gradient from 15–30% B; 40–45 min,30% B isocratic; 45–50 min, linear gradient from 30–80%B; 50–60 min, linear gradient from 80–100% B, andfinally, washing and reconditioning of the column. Theflow rate was set at 0.8 mL/min and the column wasoperated at 30 °C, with 50 μL of injection. Anthocyani-dins were detected at 530 nm while UV–vis absorptionspectra were recorded in the range of 250–600 nm. A totalof four anthocyanidins standard (delphinidin, cyanidin,malvidin, and pelargonidin) were used for the identifica-tion of anthocyanidins in the defatted dabai extracts. Thedetermination of anthocyanidins content in the defatteddabai extracts were based on the standard calibrationcurves plotted (R2=0.995–0.997).

Food Anal. Methods (2012) 5:339–350 341

Trolox Equivalent Antioxidant Capacity Assays

ABTS+ Scavenging Assay

The ABTS+ radical scavenging assay was performedbased on a method described by Hurtado et al. (2009).Briefly, 7 mM ABTS and 2.45 mM potassium persul-phate were added into an amber bottle. Then, the mixturewas incubated in the dark at room temperature for 16 hto produce ABTS+ radical cation. Before the assay wascarried out, ABTS+ solution was diluted with ethanol toan absorbance of 0.70±0.1 at 734 nm and was measuredusing a Secomam’s RS232 spectrophotometer. Defatteddabai extracts (20 μL) were diluted with the respectiveextraction media to give 20–80% inhibition of the blankabsorbance. The assay was performed by adding 20 μLof defatted dabai extracts or standard (Trolox) to 1.0 mLABTS+ solution. Before the experiment, Trolox standardat a final concentration of 0.004–0.24 mM was preparedin methanol and assayed under the same conditions. TheTrolox equivalent antioxidant capacity (TEAC) of thedefatted dabai extracts was calculated based on theinhibition exerted by the standard Trolox solution at6 min.

% of scavenging capacity ¼ ð1� Absorbancesample

AbsorbancecontrolÞ � 100

Ferric-Reducing Antioxidant Power Assay

Ferric-reducing antioxidant power (FRAP) assay wascarried out using a method described by Benzie andStrain (1996) with some modifications. The FRAP reagentwas prepared by mixing 0.3 M acetate buffer (pH 3.6),0.01 M of TPTZ, and 0.02 M of FeCl3.6H2O with a ratioof 10:1:1 at 37 °C. An aliquot of 0.2 mL of defatted dabaiextract was mixed with 3 mL of FRAP reagent and wasincubated at 37 °C. Each extract was run in threedeterminations. The changes in absorbance was measuredat 593 nm after initial mixing and at 15 min. Troloxconcentrations of 0.004–0.2 mM were used for calibrationof the FRAP assay, and antioxidant power was expressedas millimolars of TEAC per 100 g dry weight.

Statistical Analysis

Total phenolics and antioxidant capacity of the defatteddabai pulp and peel extracts were expressed as means±standard deviations. Data were subjected to two-samplet test and analysis of variance (ANOVA) using a statisticalsoftware Minitab version 15. The significant value was setat p<0.05.

Results and Discussion

Total Phenolics in Defatted Dabai Extracts

The total phenolic contents of defatted dabai pulp and peelextracts were estimated and determined using three differentspectrophotometric methods. The total phenolic content(TPC) was determined by measuring the absorbance ofreaction of phenolic compounds in the defatted dabai extractsand Folin–Ciocalteu reagent against gallic acid standardcalibrations (Hajimahmoodi et al. 2009). The total flavonoidcontent (TFC) was measured based on color change due tothe reaction between flavonoid compounds in the defatteddabai extracts and aluminum chloride (Chang et al. 2002)while the TAC of the defatted dabai extracts was estimatedbased on color intensity of the extracts and calculated basedon extinction coefficient of anthocyanin (Xu et al. 2007).The defatted dabai pulp was assumed to contain trace or noanthocyanins; therefore, the determination of TAC was notperformed.

The values of TPC, TFC, and TAC in defatted dabaiextracts are shown in Fig. 1. The result showed thatdefatted dabai peel extracts contained the highest TPC andTAC as compared with defatted dabai pulp extracts.Additionally, it was found that methanol extracts of defatteddabai peel contained the highest total phenolics (TPC, TFC,and TAC) when compared with other defatted dabaiextracts. Apart from that, in terms of the extraction mediaused, it was found that defatted dabai extracted using ethylacetate and acetone had a lower TPC as compared withmethanol, ethanol, and water. However, when comparisonwas made between ethyl acetate and acetone extracts, it wasfound that acetone extract had a higher TPC. On the otherhand, it was also found that ethanol extract of defatteddabai pulp contained the lowest TPC and TFC comparedwith methanol and water. For defatted dabai sampleextracted using ethyl acetate, no significant difference wasfound for total phenolics between pulp and peel of defatteddabai. Statistical result showed no significant difference wasfound for TFC between peel and pulp and peel of defatteddabai sample extracted using acetone. Similarly, no significantdifference was found for TFC between pulp and pulp and peelof defatted dabai sample extracted using water. The TFC ofacetone and water extracts of defatted dabai pulp weresignificantly higher than its peel, respectively.

This study showed that methanol extract of defatteddabai peel contained the highest antioxidant properties,which was characterized by the highest total phenolics incomparison with other defatted dabai extracts. This obser-vation was supported by a study of Azlan et al. (2010a) andShakirin et al. (2010) who found that methanolic extract ofnondefatted dabai peel had higher TPC than its pulp.Acetone extracts of defatted dabai samples had low TPC.

342 Food Anal. Methods (2012) 5:339–350

However, Musa et al. (2011) found that acetone extract ofpink guava has the highest TPC (using Folin–Ciocalteuindex), followed by methanol, ethanol, and water extracts.For water extract, defatted dabai peel was also shown tocontain the highest total phenolics, except for TFC, whichwas found to be significantly higher in defatted dabai pulp.

Prasad et al. (2011) have reported that water fractions ofdabai pulp and peel had TPC three times lower than ethylacetate fractions and TFC about 18 times lower than ethylacetate fractions. From their study, the ethyl acetatefractions of nondefatted dabai pulp contained phenolic-rich oil, which is also high in phenolics content (Azlan etal. 2010a). Fuleki and Francis (1968) have reported that

ethyl acetate was commonly used to remove lipids andunwanted phenolics. Removal of unwanted phenolic com-pounds using ethyl acetate will contribute to a minor lost ofantioxidant properties. No previous study has been done onthe determination of TPC in dabai fruit extracted usingvarious organic solvents and distilled water. Besides, mostof the flavonoids from defatted dabai pulp and phenolicacids from defatted dabai peel are highly soluble in waterwhile anthocyanins from dabai peel are both highly solublein methanol and water.

The solubility of phenolics in organic solvent is notwidely studied. Pellegrini et al. (2007) reported thatantioxidant properties in food samples were stronglyinfluenced by different extraction solvents. As there is nouniform or universal procedure for extraction of phenoliccompounds, the solubility of phenolic compounds is notonly dependent on the type of solvent used but also on thedegree of polymerization and interaction of phenolics withother phytochemicals, vitamins, and minerals (Naczk andShahidi 2004). HPLC result showed that ethyl acetateextract contained lesser amount of phenolic compoundscompared with methanol and water extracts. Therefore, thestudy of different extraction media is vital for phenolicsanalysis.

HPLC Identification and Quantification of Phenolicand Anthocyanidin Compounds

Phenolic compounds in the defatted dabai extracts wereidentified based on specific retention time and peak spectraof the phenolic standards. The identified phenolic peakswere from 0% to 83% of total peak areas while <10% foranthocyanidin. Two to three major peaks from differentextraction media were not possible to be identified based onspiking test using the available phenolic standards anddetermined based on the peak spectra. All unidentifiedpeaks were categorized as unknown. The trend for phenolicand anthocyanidin peaks identified in the studied HPLCchromatogram is similar as reported by Chew et al.(2011b), except for gallic acid which was not found in thedefatted dabai extracts.

The quantification of phenolics and anthocyanidin in thedefatted dabai extracts was based on the linear curveobtained using phenolics and anthocyanidins standards,which have a linearity of 0.90 to 0.99. The recovery ofphenolic compounds in the defatted dabai pulp and peelwas carried using methanol and water as extractionmediums while anthocyanidins were recovered in defatteddabai peel using methanol. Catechin, epicatechin, epicate-chin gallate, epigallocatechin, methyl gallate, and ethylgallate were recovered in defatted dabai pulp and peel usingmethanol while ellagic acid, vanillic acid, and protocate-chuic acid were recovered in defatted dabai pulp and peel

Total Phenolic Contents

0200400600800

100012001400

Methanol Ethanol EthylAcetate

Acetone Water

Extraction medium

Methanol Ethanol EthylAcetate

Acetone Water

Extraction medium

Methanol Ethanol EthylAcetate

Acetone Water

Extraction medium

GA

E (

mg

/100

g)

Pulp

Peel

Pulp & Peel

a a a b b

Total Flavonoid Contents

020406080

100120140160

Qu

erce

tin

Eq

uiv

alen

t(m

g/1

00 g

) Pulp

Peel

Pulp & Peel

b ba a a

Total Anthocyanin Contents

0

10

20

30

40

TA

C (

mg

/100

g)

Pulp

Peel

Pulp & Peel

a a

A

B

C

Fig. 1 a Total phenolic content, b total flavonoid content and c totalanthocyanin content of defatted dabai extracts. Dabai pulp, peel, andpulp and peel were extracted using five different extraction media(methanol, ethanol, ethyl acetate, acetone, and distilled water). Similarlower case letters in the bar chart show no significant differences intotal phenolics for each extract among the defatted dabai samples(p≥0.05, one-way ANOVA)

Food Anal. Methods (2012) 5:339–350 343

using distilled water as extraction medium. A moderatelyhigh recovery was found for methyl gallate (81.2%),catechin (81.6%), epicatechin gallate (81.7%), epigalloca-techin (87.2%), and ethyl gallate (88.4%) while a highrecovery was found for ellagic acid (93.2%), vanillic acid(96.7%), protocatechuic acid (98.6%), and epicatechin(108.9%). For anthocyanidins, a high recovery was foundfor pelargonidin (96.2%), followed by delphinidin (86.3%)and cyanidin (81.4%).

HPLC results showed that the defatted dabai extractscontained a total of more than 15 peaks. In this study, ninepeaks of phenolic compounds were identified as ellagic acid(13.6±0.5 min), epigallocatechin (14.5±0.5 min), protocate-chuic acid (14.6±0.5 min), catechin (15.1±0.5 min), epica-techin (16.3±0.5 min), methyl gallate (16.7±0.5 min),epicatechin gallate (17.6±0.5 min), vanillic acid (17.9±0.5 min), and apigenin (25.5±0.5 min) (Table 1) while threepeaks of anthocyanidins were delphinidin (34.1±0.3 min),cyanidin (39.5±0.3 min), and pelargonidin (43.5±0.3 min)(Table 2). Identification of the phenolics and anthocyanidinspeak was based on spiking tests and the peak spectrum foreach compound. The recoveries of the phenolic andanthocyanin compounds were in between 80% to 105%.The retention time for each phenolic compound was similaras reported by Chew et al. (2011b) with a slight shift of±0.5 min in retention time for all peaks.

The values for phenolics content found in defatted dabaiextracts determined using HPLC method are shown inTable 1. Results showed that the methanol extracts ofdefatted dabai samples contained epigallocatechin, cate-chin, epicatechin, methyl gallate, epicatechin gallate,vanillic acid, and apigenin. Epigallocatechin, catechin,epicatechin, and apigenin were the phenolics found in theethanol extracts of defatted dabai samples; catechin andapigenin for ethyl acetate extracts while apigenin foracetone extracts of defatted dabai samples. Water extractshad the more phenolic acids identified compared withflavonoids where the identified phenolic compounds wereellagic acid, epigallocatechin, protocatechuic acid, methylgallate, epicatechin gallate, and vanillic acid. Catechin,apigenin, and protocatechuic acid were the phenolics notdetected in defatted dabai pulp extracts but can be identifiedin the ethanol extract of defatted dabai pulp. The resultsalso showed a high concentration of ellagic acid (~200 mg/100 g) was found in water extracts of defatted dabai pulpand peel; catechin (~280 mg/100 g) and epigallocatechin(650–680 mg/100 g) in methanol extracts of defatted dabaipeel; and apigenin (23–209 mg/100 g) in methanol andacetone extracts of defatted dabai peel.

Methanol and water were the stronger extractionmedia which are able to dissolve most of the phenoliccompound from defatted dabai samples. Methanol is ableto extract semi-polar phenolics while water is more

favored to polar phenolic acid. The defatted dabai peelcontained delphinidin (5.8–7.6 mg/100 g), cyanidin (3.7–5.0 mg/100 g), and pelargonidin (1.8–3.0 mg/100 g), whichwere found in methanol and ethanol extracts. No anthocya-nidin peaks were detected in ethyl acetate extracts of defatteddabai peel and pulp and peel. Cyanidin and delphinidin werefound in acetone and water extracts of defatted dabai peel,respectively. The concentration of anthocyanidins determinedin defatted dabai extracts were <10% of total anthocyanins,which another 90% to 95% of anthocyanins were notdetermined.

In this study, defatting of dabai pulp and peel causeddegradation or slight loss of phenolic compounds (Bau andDerby 1980), where some of the unknown degradationproducts were remained in the defatted dabai extracts.Defatting process of dabai sample may also cause a majorloss of flavonoids, especially quercetin and rutin whichwere not detected in the defatted dabai parts. This is inagreement with Fernández-Caldas et al. (2001) who foundthat large amount of flavonoids have been removed duringdefatting process. For the defatted dabai pulp and peel, amixture of anthocyanidin glucosides was identified inmethanol, ethanol and water extracts based on UVabsorption spectra. The used of different extraction mediaaffected the results obtained. The water extracts of defatteddabai peel contained protocatechuic acid, which is knownto be the degradation product of quercetin or rutin (Makrisand Rossiter 2000). Besides, gallic acid was not found inthe defatted dabai extracts due to degradation. The fact issupported by Nardini and Ghiselli (2004) that commonlyused experimental condition might cause a total loss ofphenolic acids, especially gallic acid.

In general, the defatted dabai peel has the highest totalphenolics and total anthocyanidins determined usingHPLC. However, many phenolic compounds found in thewater extracts of defatted dabai sample were not identifiedand determined. Malvidin, a typical anthocyanidin found inberries (Montoro et al. 2006) was not detected in defatteddabai parts. Further studies are needed to characterize theunknown phenolics and flavonoids in defatted dabaisample.

Comparison of GAE Between HPLCand Spectrophotometric Methods

Comparisons of the concentration of total phenolicsapplying GAE (mg/100 g) for defatted dabai extracts weredone using UV–vis spectrophotometric and HPLC methods(Fig. 2). The results of two-sample t test showed that totalphenolics (GAE) were significantly different between thetwo methods used (p<0.001), except for acetone extract ofdefatted dabai pulp and peel. Besides, total phenolics(GAE) in water and methanol extracts of defatted dabai

344 Food Anal. Methods (2012) 5:339–350

Tab

le1

Selectedph

enolic

compo

unds

determ

ined

inthedefatteddabaiextracts

Sam

ple

Pheno

liccompo

unds

Ellagicacid

Epigallo

catechin

Protocatechuicacid

Catechin

Epicatechin

Methy

lgallate

Epicatechin

gallate

Vanillic

acid

Apigenin

Methano

l

Pulp

–19

.1(1.65)

a–

–1.19

(0.11)

a30

.26(0.09)

a24

.47(0.13)

a3.53

(0.22)

a–

Peel

–68

1.71

(29.03

)b

–28

0.04

(5.78)

a1.67

(0.16)

b30

.15(0.21)

a24

.81(0.11)

b5.69

(0.27)

b26

.05(3.32)

a

Pulpandpeel

–65

1.78

(60.95

)b

–27

3.28

(12.97

)a

1.2(0.15)

a30

.13(0.19)

a23

.15(0.43)

a6.48

(0.13)

c23

.07(3.06)

Ethanol

Pulp

–3.47

(0.98)

a–

3.76

(0.21)

a7.8(0.07)

––

––

Peel

–12

.1(2.04)

b–

16.97(0.72)

b–

––

–2.01

(0.3)a

Pulpandpeel

–59

.39(3.41)

a–

31.77(2.23)

c–

––

–2.77

(0.23)

b

Ethyl

acetate

Pulp

––

––

––

––

–

Peel

––

–3.54

(0.19)

a–

––

–7.21

(0.51)

a

Pulpandpeel

––

–3.78

(0.19)

a–

––

–7.87

(0.36)

b

Acetone

Pulp

––

––

––

––

–

Peel

––

––

––

––

23.61(4.22)

a

Pulpandpeel

––

––

––

––

209.29

(25.86

)b

Water

Pulp

227.65

(0.81)

a42

.72(1.84)

––

–27

.51(0.5)a

19.4

(0.97)

a4.59

(0.34)

a–

Peel

223.62

(3.93)

b–

11.49(0.3)a

––

27.32(0.51)

a18

.07(0.91)

a,b

6.18

(0.17)

b–

Pulpandpeel

198.07

(22.17

)a

–8.85

(0.5)b

––

27.12(0.12)

a17

.45(0.62)

b4.54

(0.16)

a–

Dataareexpressedas

mean(stand

arddeviation)

inmg/10

0g.

Different

lower

case

lettersshow

sign

ificantd

ifferences

inph

enolicsconcentrationforeach

extractamon

gthedefatteddabaisam

ples,

inwhich

sign

ificantvalueissetat

p<0.05

,on

e-way

ANOVA

Food Anal. Methods (2012) 5:339–350 345

pulp and peel measured using HPLC method weresignificantly higher than the total phenolics determined byspectrophotometric method (p<0.05).

The difference in GAE determined for these twomethods used could be due to several reasons. TPC assaymeasured the total phenolics using Folin–Ciocalteureagents, where the reagent could be reacted with aminoacids. Folin–Ciocalteu reagent is widely used in Lowry’smethod for plasma protein determination (Lowry et al.1951). HPLC is used to identify and measure the UV–visabsorbance of individual phenolic compound at specificabsorption wavelength. Spectrophotometric method, how-ever, measures the absorbance of the end-product ofphenolic compounds reacted with Folin–Ciocalteu reagent.

GAE was found higher in water extract of defatted dabaipulp determined by HPLC method compared with spectro-photometric method. This may be due to the defatted dabaipulp containing more polar phenolic compounds which arenot strong reducing agents. There was a small different inGAE between methanol extracts of defatted dabai pulp andpulp and peel determined using HPLC methods. However,for the GAE of both defatted dabai extracts determinedusing spectrophotometric method, the defatted dabai peelusing methanol extract contained total phenolics (in GAE)more than four times higher than the defatted dabai pulpand peel methanol extract. As compared with methanol andwater, ethyl acetate and acetone are weak extraction media

(Tian et al. 2009). These solvents have yielded low totalphenolics and low antioxidant capacity, which is in agreementwith Miliauskas et al. (2004) that ethyl acetate extractshave low antioxidant properties. The higher GAE value ofdefatted dabai peel water extract determined using spec-trophotometric method compared with HPLC method ismainly due to high amount of anthocyanin found in thedefatted dabai peel. However, no evidence can be obtainedto show the strong reducing ability of anthocyanin inFolin–Ciocalteu reagent. In addition, anthocyanin is astrong antioxidant and it possesses high antioxidantcapacity (Gould et al. 2002).

0

30

60

90

120

150

180

210

GA

E (

mg

/100

g) HPLC

Spectrophotometer

A

0

200

400

600

800

1000

1200

1400

GA

E (

mg

/100

g)

GA

E (

mg

/100

g)

HPLC

Spectrophotometer

B

0

100

200

300

400

500

600

700

800

HPLC

Spectrophotometer

C

a a

Methanol Ethanol EthylAcetate

Acetone Water

Extraction medium

Methanol Ethanol EthylAcetate

Acetone Water

Extraction medium

Methanol Ethanol EthylAcetate

Acetone Water

Extraction medium

Fig. 2 Comparison of gallic acid equivalent (GAE) between HPLCand UV–vis spectrophotometric methods for defatted dabai a pulp, bpeel, c pulp and peel extracts. Similar lower case letters in the barchart show no significant different in GAE values for each extractbetween the two methods used (p≥0.05, two-sample t test)

Table 2 Selected anthocyanidin compounds determined in thedefatted dabai extracts

Sample Anthocyanidin

Delphinidin Cyanidin Pelargonidin

Methanol

Peel 7.61 (0.2) a 5.0 (0.04) a 2.97 (0.04)

Pulp and peel 6.85 (0.04) b 4.19 (0.07) b –

Ethanol

Peel 6.03 (0.06) a 3.69 (0.03) 1.84 (0.01)

Pulp and peel 5.85 (0.01) a – –

Ethyl acetate

Peel – – –

Pulp and peel – – –

Acetone

Peel – 3.72 (0.04) –

Pulp and peel – – –

Water

Peel 5.87 (0.01) – –

Pulp and peel – – –

Data are expressed as mean (standard deviation) in mg/100 g.Different lower case letters show significant difference in anthocya-nidins concentration for each extract between the defatted dabaisamples in which significant value is set at p<0.05, two-sample t test

346 Food Anal. Methods (2012) 5:339–350

As shown in Tables 2 and 3, methanol extract ofdefatted dabai peel contained higher amount of catechin,epigallocatechin, and anthocyanidins compared with meth-anol extract of pulp. These phenolic compounds are strongreducing agents, and have more lone pair of electrons onthe terminal O atom and phenol O–H group, whichsignificantly involve in electron transfer reaction (DiLabioand Johnson 2007). In Folin–Ciocalteu reagent assay, these –OH containing phenol or phenoxyl have strong ability toreduce the molybdenum and tungsten cations in the 6+ statein the reagent (Ikawa et al. 2003). When Folin–Ciocalteureagent reacts with phenols and other reducing agents, colorchanges can also be observed. Various phenolic compoundswith reducing end hydroxyl group (−OH) can be acted asreducing agents, hydrogen donors, and singlet oxygenquenchers (Rice-Evans et al. 1996; Middleton et al. 2000).Rice-Evans et al. (1996) revealed that removing of 3-OHgroup in the C-ring in the polyphenol had decreasedantioxidant activity. As anthocyanin contains more of thesegroups, it can act as strong reducing agent.

Trolox Equivalent Antioxidant Capacity Assays

Antioxidant capacity of defatted dabai pulp and peel wasdetermined using two different antioxidant capacity assays

(ABTS+ radical scavenging and FRAP), where results ofboth assays were expressed as Trolox equivalents (mM/100 g). These two assays involved scavenging of free radicalthrough electron transfer reaction (Huang et al. 2005).

The values for TEAC of the defatted dabai extracts areshown in Fig. 3. As observed in both antioxidant capacitymethods used, the defatted dabai peel had the highestantioxidant capacity (TEAC), followed by defatted dabaipulp and defatted dabai pulp and peel. Methanol extracts ofthe defatted dabai sample have contributed to the highestTEAC values, followed by water extract, ethanol extractand acetone extract; while ethyl acetate extract of defatteddabai sample had the lowest TEAC values. Conversely,Musa et al. (2011) have reported that acetone had yieldedthe highest antioxidant activity (DPPH and FRAP assays)compared with methanol, ethanol, or water in pink guavasamples. The reason is unknown. They also revealed thatmethanol and acetone have similar polarity. Possibly, pinkguava contained higher amount of phytochemicals thatstrongly solubilized in acetone as compared with methanol.However, the studied methanol extracts contained highertotal phenolics compared with acetone extract. Besides,ethanol extract of defatted dabai peel had TEAC value(~2.0 mM/100 g) comparable to Dacryodes rostrata fruitextract (70% ethanol) (Kong et al. 2010).

Table 3 Relative standard deviation (RSD%) and slope (M) for total phenolics and antioxidant capacity estimation in the defatted dabai extracts

Sample Extraction media Total phenoliccontent

Total flavonoidcontent

Total anthocyanincontent (TAC)

ABTS FRAP

RSD M RSD M RSD M RSD M RSD M

Pulp Methanol 1.63 0.0021 1.21 0.0009 – – 4.50 0.0032 2.45 0.0049

Ethanol 5.03 0.0004 1.11 0.0009 – – 8.49 0.0033 3.20 0.0007

Ethyl acetate 5.61 0.0005 4.81 0.0003 – – 10.79 – 7.37 –

Acetone 2.86 0.0002 1.66 0.0009 – – 6.83 – 8.13 –

Water 1.31 0.0035 2.48 0.0035 – – 4.12 0.0018 2.58 0.0034

Peel Methanol 0.92 0.0032 2.83 0.0038 2.84 0.0016 2.67 0.1204 1.69 0.1418

Ethanol 1.11 0.0038 2.89 0.0023 2.71 0.0038 9.59 0.0247 3.77 0.0119

Ethyl acetate 7.32 0.0005 6.01 0.0005 17.59 0.0007 4.42 – 7.88 –

Acetone 3.08 0.0013 2.09 0.0009 19.34 0.0022 0.69 – 1.86 –

Water 1.97 0.0073 2.33 0.0006 9.04 0.0031 3.39 0.0622 2.67 0.0343

Pulp and peel Methanol 1.55 0.0084 3.62 0.0019 3.16 0.0015 2.94 0.0407 2.00 0.0407

Ethanol 1.99 0.0009 6.09 0.0023 1.76 0.0019 3.11 0.0097 2.06 0.0097

Ethyl acetate 6.36 0.0006 16.05 0.0004 15.56 0.0007 8.09 – 5.65 –

Acetone 2.87 0.0003 10.47 0.0004 7.85 0.0012 8.40 – 5.89 –

Water 1.51 0.0028 2.59 0.0041 2.06 0.0015 5.79 0.0114 1.61 0.0114

Results show high linearity (R2 =0.968–0.999). M represents the power of antioxidant concentrations in each extracts; the studied samples withlarger M will have higher total phenolics/stronger antioxidant capacity with even a slight increase in the extract concentration applied. TAC is notdetermined using defatted dabai pulp extract due to the defatted dabai pulp not containing anthocyanin compounds, while TEAC assays aredetermined for methanol, ethanol, and water extracts of defatted dabai samples as ethyl acetate and acetone extracts have low antioxidantcompounds

Food Anal. Methods (2012) 5:339–350 347

Using appropriate regression analysis, the regressionequation predicted that the TEAC values of both ABTS+

radical scavenging and FRAP assays will significantlyincrease if there is an increase in TFC and TPC concen-trations of defatted dabai samples extracted using methanoland ethanol, respectively. However, the TEAC value ofABTS+ radical scavenging and FRAP will increase signif-icantly if there is an increase in the concentrations of TPCand TAC of the defatted dabai sample using water extracts.Similarly, an increase in the concentration of TPC of thedefatted dabai pulp and peel extracts will significantlycontribute to an increase in TEAC values for both ABTS+

radical scavenging and FRAP assays.The regression equation also predicted that there was a

significant correlation between the TEAC values and thetotal polyphenols concentration for defatted dabai peelextracts, but no significant correlation was found betweenthe TEAC values of FRAP assay and the concentration ofTFC in defatted dabai peel extracts. Besides, an increase inthe concentration of TAC of the defatted dabai pulp andpeel extracts will significant contribute to an increase inTEAC value for ABTS+ radical scavenging assay.

Quality Control

This study has also taken into consideration the precisionand linearity of the experimental methods. The precisionvalues for this study were determined based on thepercentage of relative standard deviation (RSD%) of threeextraction replications (Table 3). For TPC, TFC, and TACassays, most of the RSD values for the defatted dabaiextracts were ranging from 0.9–9.0%, except for foursamples with 10.5–17.6%. These values however werelower than 20%, which is acceptable for food science andnutrition research. These RSD values are also agreed byLee et al. (2009). Similarly, the RSD values of defatteddabai extracts for TEAC assays were lesser than 10.0%,except in ethyl acetate extract of defatted dabai pulp.

Higher RSD values were mainly found for the ethylacetate extract of defatted dabai sample, which could bedue to the low concentrations of total phenolics and TEACvalues that added to higher deviation from mean. Besides,there is a decrease in reliability when a low concentration ofanalyte is measured using spectrophotometric method, asreported by Naylor et al. (1992) who had observed analyteswere unstable at trace concentrations. In this study, most ofthe samples had low RSD values; therefore, we canconclude that the experimental methods are precise.Although a few studied samples had higher RSD values,the high RSD% are probably due to the shortcoming ofstatistical deviation from the mean values.

Linearity of the studied defatted dabai extracts wasabsolutely high. The calculated R2 values for most of thedefatted dabai extracts were higher than 0.99 (Table 3).Only a few of the defatted dabai extracts had R2 valuesranging from 0.97 to 0.99. The decreased in R2 for linearityfor the studied samples is mainly due to dilution of extractconcentration that was not closely matched with the linearrange of calibration (Zhang et al. 2007). Overall, thelinearity of this study can be considered good. Consequently,we conclude that whether a high or a low concentration of theextracts used for different samples will not affect the totalphenolics and TEAC values.

Based on the regression equations obtained, the coeffi-cient of determination (R2) values predicted that more than99% of the proportions of the variability in the TEACvalues were accounted by the total phenolics concentra-tions, except for two regression equations which had about60% of the proportions of the variability in the TEACvalues were accounted by the total phenolics concentra-tions. The values of the slope (M) represent the strength ofextracts concentration.

The studied samples with larger M will have higher totalphenolics or stronger antioxidant capacity even when slightincrease in the extract concentration applied. For all studiedassays, methanol and water extracts of the defatted dabai

ABTS Radical Scavenging Assay

0

2

4

6

8

10

12

14

TE

AC

(m

M/1

00 g

)T

EA

C (

mM

/100

g)

Pulp

Peel

Pulp & Peel

a a

Ferric-Reducing Antioxidant Power

0

2

4

6

8

10

12

Pulp

Peel

Pulp & Peel

a a b b

B

A

Methanol Ethanol EthylAcetate

Acetone Water

Extraction medium

Methanol Ethanol EthylAcetate

Acetone Water

Extraction medium

Fig. 3 Trolox equivalent antioxidant capacity of defatted dabaiextracts accessed using a ABTS+ radical scavenging and b ferric-reducing antioxidant power assays. Dabai pulp, peel, and pulp andpeel were extracted using five different extraction media (methanol,ethanol, ethyl acetate, acetone, and distilled water). Similar lower caseletters in the bar chart show no significant differences in TEACvalues for each extract among the defatted dabai samples (p≥0.05,one-way ANOVA)

348 Food Anal. Methods (2012) 5:339–350

samples had the highest slope values. However, the resultsfrom TAC assay revealed that ethanol extracts of the defatteddabai samples had the highest slope values, while ethanolextracts of the defatted dabai pulp for the ABTS+ radicalscavenging assay had the highest slope values. The exactreason is unknown. However, the crude extracts of defatteddabai samples may contain a mixture of antioxidantcompounds, which each antioxidant compound has theirown pathway of free radical scavenging and antioxidanteffect (Huang et al. 2005).

Conclusions

Dabai (C. odontophyllum) is an antioxidant-rich fruit.Although high phenolics content was found in dabai oil,the defatted dabai pulp and peel also contained high totalphenolics and antioxidant capacity. Defatted dabai pulpcontained higher amount of total flavonoids than defatteddabai peel. We have found that the defatted dabai peel isa potential source of phenolics and anthocyanins. Ellagicacid, epigallocatechin, protocatechuic acid, catechin,epicatechin, methyl gallate, epicatechin gallate, vanillicacid, and apigenin were found in defatted dabai extracts.Catechin and epigallocatechin were two major phenolicsfound in methanol extract of defatted dabai peel whileellagic acid was found abundantly in water extract ofdefatted dabai samples.

Delphinidin, cyanidin, and pelargonidin were the poten-tial anthocyanidin identified in methanol extract of defatteddabai parts. The highest TPC and TAC were determined indefatted dabai peel, especially in methanol and waterextracts. High water soluble phenolic compounds werefound in defatted dabai pulp compared with its peel.Methanol extracts of defatted dabai peel had the highestTEAC values while ethyl acetate extracts of defatted dabaipulp and peel had the lowest TEAC values. A mixture ofdefatted dabai pulp and peel yielded no synergetic effect forantioxidant capacity, with a minor increment in totalphenolics as compared with defatted dabai peel. Morestudies are needed to determine the mechanism of reactionor pathway on antioxidant effect for specific phenoliccompound.

Acknowledgment This work was funded by the Research UniversityGrant Scheme (RUGS) (04-02-10-0921RU, 9192600), ResearchManagement Center, Universiti Putra Malaysia.

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