Food Chemistry 123 (2010) 423–429

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Food Chemistry

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Effect of microwave treatment on phenolic content and antioxidant activityof citrus mandarin pomace

Khizar Hayat, Xiaoming Zhang *, Umar Farooq, Shabbar Abbas, Shuqin Xia, Chengsheng Jia,Fang Zhong, Jing ZhangState Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, Jiangsu 214122, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 August 2009Received in revised form 5 March 2010Accepted 26 April 2010

Keywords:Microwave treatmentCitrus mandarin pomacePhenolic contentAntioxidant activityKinnowFeutrell’s Early

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.04.060

* Corresponding author. Tel.: +86 510 85919106; faE-mail addresses: khizaraura@hotmail.com (K.

edu.cn (X. Zhang).

The effects of microwave treatment on the phenolic compounds and antioxidant capacity of citrus man-darin pomace were evaluated. After treatment, methanol extracts of citrus pomace were prepared andthe contents of phenolic acids (free and bound) and flavanol, flavanone and flavonol compounds (FCs)were determined by HPLC. Antioxidant capacity of pomace extracts was measured using DPPH radical-scavenging assay, hydroxyl radical-scavenging assay and reducing power. After microwave treatment,the free fraction of phenolic acids increased, whereas the bound fractions decreased and antioxidantactivity was increased. The content of total FCs increased with power but, at longer irradiation time, itdeclined which meant that some FCs might be degraded. The results indicated that appropriate micro-wave treatment could be an efficient process to liberate and activate the bound phenolic compoundsand to enhance the antioxidant activity of citrus mandarin pomace.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Antioxidants are both natural and synthetic compounds, able toscavenge free radicals and to inhibit oxidation processes. Currently,the growing interest to find cheap, renewable and abundantsources of antioxidant compounds has fostered research on vegeta-ble sources of residual origin from agricultural industries (Moureet al., 2001). The antioxidants from natural sources, with potentialnutritional and therapeutic value, can be used for increasing thestability of foods by preventing lipid peroxidation (Zia-ur-Rehman,2006).

Pomace is the residue remaining when fruits are processed forjuice, wine, or other products. Many studies have reported thatfruit pomaces are rich sources of phenolic compounds and thesebyproducts, obtained from the juice and wine industry, might beuseful raw materials for creating new value-added products (Zhouet al., 2009). Citrus is an important crop, mainly used in food indus-tries for fresh juice production, and peel and pomace are the mainbyproducts during its processing (Li, Smith, & Hossain, 2006a,2006b). Citrus pomace has been used as a source for molasses,pectin, and limonene. Citrus pomace has also been widely studied,because it contains numerous biologically active compounds,

ll rights reserved.

x: +86 510 85884496.Hayat), xmzhang@jiangnan.

including natural antioxidants such as phenolic acids and flavo-noids (Kim, Lee, Lee, Nam, & Lee, 2008b).

Phenolic acids have attracted increasing attention for their anti-oxidant behaviour and beneficial health-promoting effects andthey account for about one-third of the phenolic compounds inplant foods. It is assumed that many antioxidative phenolic com-pounds in plants are usually presented in a covalently-bound form(Xu, Ye, Chen, & Liu, 2007). Therefore, reliable and practical meth-ods for liberation of natural antioxidants from plant materials areof considerable interest. Microwave energy can potentiate the bio-availability of free pharmacologically active natural compounds bypreventing the binding of polyphenols to the plant matrix (Gulati,Rawat, Singh, & Ravindranath, 2003). The internal temperature dis-tribution of a material subjected to conventional-heating dependson its thermal conductivity, whereas microwave-heating results inthe heating of all the individual elements of a material instanta-neously. Consequently, heating time, using microwaves can be sig-nificantly reduced as compared to conventional-heating methods(Robinson et al., 2009). Several methods, such as heat treatment,far-infrared radiation, electron-beam irradiation, fermentationand protease treatment have been studied to liberate and activatelow-molecular weight natural antioxidants (Jeong, Kim, & Kim,2004; Kim, Bae, Kim, & Lee, 2008a; Kim et al., 2008b; Niwa, Kanoh,Kasama, & Neigishi, 1988; Xu et al., 2007). However, to the best ofour knowledge, there has been no detailed report on the use ofmicrowaves to liberate phenolic compounds in plant materialsand particularly in citrus pomace.

424 K. Hayat et al. / Food Chemistry 123 (2010) 423–429

This research evaluates the effect of microwave treatment onphenolic compounds and antioxidant activity of citrus mandarinpomace. For this study, Kinnow (Citrus reticulata Blanco cv Kinnow)and Feutrell’s Early (C. reticulata Blanco cv Feutrell’s Early) fruitpomaces were chosen because they are the two main citrus man-darin cultivars in Pakistan, besides being common in the wholesouth Asian region. The aim of this work was to propose a suitablemethod for the liberation, and separation of phenolic compoundsfrom citrus mandarin pomace and to evaluate antioxidant activitybefore and after microwave treatment.

2. Materials and methods

2.1. Material

Kinnow and Feutrell’s Early fruits were purchased from a localmarket in Jhang, Pakistan. Fruits were hand-peeled and the citruspomaces were obtained after juice extraction by a stainless screwsqueezer. Pomaces were sun-dried for about 15 days. The dried cit-rus pomaces were ground into fine powder in a mill (DFY-500 sam-ples mill, Da De Zhong Yao Ji Xie Company, Wenling, China) andthe material that passed through a 60-mesh sieve was stored inairtight plastic bags at room temperature.

2.2. Chemicals

Standards of gallic acid, q-hydroxybenzoic acid, vanillic acid, q-coumaric acid, ferulic acid, catechin, naringin, naringenin, hesper-idin, rutin, kaempferol and isorhamnetin were purchased from Sig-ma (St. Louis, MO, USA). All other chemicals were purchased fromSinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.3. Microwave treatment

A household microwave oven (700 W, 2450 MHz, Foshan MideaMicrowave Oven Manufacture Co., Fo Shan, Guang Dong, China)was used for heating citrus pomace powder. Microwave outputpower was measured according to the method of Zhang, Zhang,Shan, and Fang (2007). Kinnow and Feutrell’s Early pomace pow-ders had water contents of 8.4% and 9.7%, respectively. Powder ofcitrus pomace (4 g) was put into a 500 ml glass beaker and heatedin a microwave oven at 250 W for 5, 10 and 15 min and at 125 and500 W for 5 min. Non-heated (untreated) powder was used as acontrol.

2.4. Extraction of phenolic compounds from citrus pomace

The extraction was performed according to a modified methodof Xu, Ye, Liu, Ma, and Chen (2008). Citrus pomace powder (4 g)was extracted with 80 ml of 80% methanol in an ultrasonic device(200 W, 59 kHz, Shanghai Kudos Sonication Machine CompanyLtd., China) for 60 min at room temperature. The mixture was thenfiltered through Whatman no. 4 filter paper and the filtrate wasevaporated under reduced pressure at 30 �C until its volume wasabout 40 ml. The final volume of the extract was made to 50 mlwith the extraction solvent and then it was taken out for the anal-ysis of FCs, free phenolic acids and evaluation of antioxidantcapacity.

2.5. Isolation of phenolic acids

Phenolic acids were isolated from the extracts according to themethod described by Xu et al. (2008), with modifications. A 20 mlaliquot of the extract was used for sequential hydrolysis with base,followed by an acid for the determination of ester-bound and gly-

coside-bound phenolic acid contents, respectively. Twenty millili-tres of 4 M NaOH were added to the extract and the mixture wasstirred for 1 h at ambient temperature. The reaction mixture wasadjusted to pH 2 using 6 M HCl and extracted three times with25, 20 and 20 ml of diethyl ether–ethyl acetate (DE/EA, 1:1) atroom temperature. The DE/EA extracts were combined and evapo-rated to dryness under vacuum at 30 �C. The dry residue was dis-solved in 5 ml of methanol and analysed for ester-boundphenolic acid content. Afterwards, 10 ml of 6 M HCl were addedto the aqueous phase and the mixture was hydrolysed at 85 �Cfor 30 min. Then it was cooled to room temperature and the glyco-side-bound phenolic acids were extracted with DE/EA as describedabove. After evaporation under vacuum, the residue was re-dis-solved in 5 ml methanol and analysed for glycoside-bound pheno-lic acids. Each sample was done in duplicate.

2.6. HPLC analysis

The analysis of phenolic acids and FCs was carried out accordingto the method of Hayat et al. (2009). The chromatographic systemconsisted of a Waters 1525 binary pump and a Waters 2996 pho-todiode array (PDA) detector (Waters, Milford, MA, USA). C18 col-umn (SunFire™ 5 lm, 4.6 mm � 150 mm, Waters, USA) was usedfor separation. The column thermostat was set at 30 �C. Solvent Aconsisted of 0.1% formic acid, and solvent B consisted of 100%methanol. Sample injection volume was 15 ll and the flow ratewas 1 ml/min. The solvent gradient in volume ratios was as fol-lows: 0–13 min, 15–33% B; 13–21 min, 33–39% B; 21–23 min,39–45% B; 23–25 min, 45% B; 25–27 min, 45–15% B; 27–33 min,15% B. The PDA detector was operated between 220 and 350 nm,with a resolution of 1.2 nm. Phenolic acids were identified by theretention time and the UV–vis spectra of standards. Phenolic acidswere quantified from peak area at 270 nm.

The contents of FCs were determined with a high-performanceliquid chromatography system module 1100 (Agilent) using aDiamonsil C18 column (4.6 mm � 250 mm). The column tempera-ture was set at 30 �C and the injection volume for all samples was5 ll. Solvent A consisted of 0.05% phosphoric acid and solvent Bwas methanol and the flow rate was maintained at 0.8 ml/min.The elution gradient was carried out as follows: 0 min, 80% B;5 min, 60% B; 20 min, 30% B; 23 min, 20% B; 26 min, 20% B;27 min, 80% B; 32 min, 80% B. The eluent was monitored at280 nm and the identification of the FCs was accomplished bycomparing the retention times of peaks in extract samples to thoseof FCs standards.

All of the identified phenolic compounds were quantified by anexternal standard method, using calibration curves, and their con-centrations were expressed as microgrammes per gramme of dryweight (lg/g DW). The standard response curve for each phenolicacid was a linear regression fitted to values obtained at each con-centration within the range 1–16 lg/ml, with correlation coeffi-cients (R2) > 0.999 and within the range of 1–30 lg/ml for FCs(R2 > 0.998).

2.7. DPPH radical-scavenging activity

The effect of extracts on 1,1-diphenyl-2-picrylhydrazyl (DPPH)free radical was estimated according to the procedure describedby Yi, Yu, Liang, and Zeng (2008), with some modifications. An ali-quot of 1.5 ml of sample solution (1 mg/ml) was mixed with 1.5 mlof methanolic solution of DPPH� (0.2 mM). The reaction mixturewas incubated for 30 min in the dark at room temperature. Theabsorbance of the resulting solution was measured at 517 nm witha spectrophotometer. For the control, the assay was conducted inthe same manner but methanol was used instead of samplesolution. DPPH�-scavenging capacity of the tested samples was

K. Hayat et al. / Food Chemistry 123 (2010) 423–429 425

measured as a decrease in the absorbance and was calculated byusing the following equation:

Scavenging activity ð%Þ ¼ Ac � AsAc

� 100

where Ac and As are the absorbances at 517 nm of the control andsample, respectively.

2.8. Hydroxyl radical (OH)-scavenging activity

Hydroxyl radical-scavenging activity of extracts was deter-mined according to the modified method of Halliwell, Gutteridge,and Aruoma (1987). The reaction mixture, containing 0.1 ml of ex-tract (2 mg/ml) dissolved in phosphate buffer (50 mM, pH 7.4),0.1 ml of 60 mM 2-deoxy-D-ribose in phosphate buffer, 0.2 ml ofa premixed 100 mM FeCl3 and 1.04 mM EDTA (1:1, v/v) solution,0.1 ml of 10 mM H2O2 and 0.1 ml of 2 mM ascorbic acid, was incu-bated at 37 �C for 1 h. After incubation, 1 ml of 20% trichloroaceticacid (TCA) and 1 ml of 0.8% thiobarbituric acid (TBA) were added.The reaction mixture was heated in a boiling water bath for20 min. The absorbance of the pink colour developed was mea-sured at 532 nm, using a spectrophotometer. Distilled water, inplace of extracts, was used as blank and the sample solution with-out adding deoxyribose as sample blank. The percentage of hydro-xyl radical-scavenging activity was calculated as:

Scavenging activity ð%Þ ¼ Ab� ðAs� AsbÞAb

� 100

where Ab, As and Asb are the absorbances at 532 nm of the blank,extract, and sample blank, respectively.

2.9. Determination of reducing power (RP)

The reducing power of the extracts was measured according tothe method of Oyaizu (1986). One millilitre of sample (10 mg/ml)was mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and2.5 ml of 1% potassium ferricyanide. The mixture was incubated

Table 1Effects of microwave treatment on phenolic acid content (lg/g DW) of Kinnow pomace.

Forms MW treatmenth power/time Gallic q-Hydroxy

Free Untreated 114 ± 0.3e 89.4 ± 0.5f

125 W, 5 min 119 ± 2.3d 96.1 ± 0.8e

250 W, 5 min 130 ± 1.9c 115 ± 1.4d

250 W, 10 min 146 ± 0.1b 129 ± 0.3b

250 W, 15 min 163 ± 0.8a 140 ± 1.8a

500 W, 5 min 166 ± 1.1a 120 ± 0.1c

Ester-bound Untreated 106 ± 0.8b 120 ± 2.0a

125 W, 5 min 111 ± 2.1a 113 ± 1.1b

250 W, 5 min 89.8 ± 0.1c 102 ± 0.5c

250 W, 10 min 76.2 ± 1.0d 94.3 ± 0.1d

250 W, 15 min 62.5 ± 0.1e 80.1 ± 0.2f

500 W, 5 min 51.4 ± 0.4f 85.8 ± 0.1e

Glycoside-bound Untreated 32.2 ± 0.9a 80.4 ± 1.1a

125 W, 5 min 33.4 ± 0.1a 76.7 ± 0.6b

250 W, 5 min 30.0 ± 0.1b 73.5 ± 0.1c

250 W, 10 min 25.7 ± 0.2c 68.6 ± 0.4d

250 W, 15 min 20.2 ± 0.9d 64.3 ± 0.1e

500 W, 5 min 25.1 ± 0.3c 76.9 ± 1.8d

Total Untreated 252 ± 2.0b 290 ± 3.6ab

125 W, 5 min 263 ± 4.5a 285 ± 2.5ab

250 W, 5 min 250 ± 2.1bc 290 ± 2.0ab

250 W, 10 min 248 ± 1.3bc 292 ± 0.7a

250 W, 15 min 246 ± 1.8bc 285 ± 2.1ab

500 W, 5 min 242 ± 1.8c 283 ± 2.0b

Means with different letters in the column for each fraction are significantly different (Pg SPC: sum of individual phenolic acids content.h MW treatment: microwave treatment.

for 20 min at 50 �C. After incubation, 2.5 ml of 10% trichloroaceticacid were added to the mixture, followed by centrifugation at3000g for 10 min at room temperature. Aliquots of 2.5 ml of thesupernatant were mixed with 2.5 ml of distilled water and 0.5 mlof 0.1% ferric chloride. The absorbance was measured at 700 nmagainst the blank that contained all the reagents except the sampleextract.

2.10. Statistical analysis

The data were subjected to analysis of variance (ANOVA) andthe significance of the difference between means was determinedby Duncan’s multiple range test (P < 0.05), using SAS (Version8.1, 2000; SAS Inst., Cary, NC, USA).

3. Results and discussion

3.1. Effect of microwave treatment on the phenolic acids of citrusmandarin pomace

Most of the phenolic acids in the citrus fruits are present inbound forms. Phenolic acids, such as caffeic, q-coumaric, ferulic,and sinapic acids, have been detected in citrus fruits and theirco-products in the form of amides, esters and glycosides (Peleg,Naim, Rouseff, & Zehavi, 1991).

For this study, a range of process parameters was selected,based on the physical condition and the antioxidant activity ofthe pomace powder during preliminary trials. For example, duringpreliminary trials, when pomace powder was treated for 10 min at125 W or for 5 min at 250 W, it showed almost the same antioxi-dant activities (data not shown). When treated for 10 min at500 W, the pomace power turned dark in colour and showed lowerantioxidant activity than did powder treated at 250 W for 15 min(data not shown). So, a medium power level, i.e. 250 W, was cho-sen to see the effect of different irradiation times. The treatmenttime was fixed at 5 min to determine the effects of different levels

benzoic Vanillic q-Coumaric Ferulic SPCg

292 ± 1.1e 234 ± 1.0f 399 ± 1.1f 1128 ± 4.0f

297 ± 1.0e 242 ± 0.9e 409 ± 1.2e 1163 ± 6.2e

329 ± 0.1d 260 ± 1.6d 424 ± 0.1c 1258 ± 5.1d

354 ± 0.9b 276 ± 0.7b 450 ± 0.2b 1355 ± 2.3b

368 ± 4.2a 297 ± 0.6a 455 ± 0.9a 1424 ± 8.3a

342 ± 1.0c 268 ± 1.1c 420.1.0d 1317 ± 4.3c

325 ± 1.7a 570 ± 1.0a 1092 ± 3.2a 2214 ± 8.7a

315 ± 0.7b 559 ± 1.4b 1064 ± 2.5b 2161 ± 7.8b

284 ± 1.3c 533 ± 3.6c 1047 ± 5.2c 2055 ± 10.7c

263 ± 1.4d 511 ± 1.2d 1009 ± 0.4d 1954 ± 4.1d

251 ± 1.1e 491 ± 0.6f 987 ± 1.2e 1872 ± 3.2e

265 ± 2.2d 503 ± 1.5e 959 ± 0.6f 1864 ± 4.8e

46.5 ± 1.5a 18.4 ± 0.7b 65.0 ± 2.2b 243 ± 6.4a

48.7 ± 0.1a 16.1 ± 0.1c 69.4 ± 0.4a 244 ± 1.3a

42.3 ± 0.5b 16.6 ± 0.1c 64.5 ± 1.7b 227 ± 2.5b

34.3 ± 0.1c 12.8 ± 0.1d 43.3 ± 0.4c 185 ± 1.2d

28.0 ± 0.4e 10.0 ± 0.8e 38.8 ± 0.1d 161 ± 2.3e

31.4 ± 0.1d 21.5 ± 0.3a 44.3 ± 0.5c 199 ± 3.0c

664 ± 4.3a 823 ± 2.7a 1556 ± 6.5a 3585 ± 19.1a

660 ± 1.8a 817 ± 2.4ab 1543 ± 4.1ab 3569 ± 15.3a

655 ± 1.9ab 809 ± 5.3bc 1536 ± 7.0b 3540 ± 18.3ab

652 ± 2.4ab 800 ± 2.0cd 1502 ± 1.0c 3493±.7.6bc

647 ± 5.7bc 798 ± 2.0cd 1481 ± 2.2d 3457 ± 13.8c

639 ± 3.3c 792 ± 2.9d 1423 ± 2.1e 3379 ± 12.1d

< 0.05).

426 K. Hayat et al. / Food Chemistry 123 (2010) 423–429

of microwave power on the phenolic content and antioxidantactivity of citrus pomace.

Five phenolic acids, namely, gallic acid, q-hydroxybenzoic acid,vanillic acid, q-coumaric acid and ferulic acid, were identified andquantified from pomaces of two citrus mandarin cultivars. Pheno-lic acids in the citrus pomace extracts were divided into three frac-tions: free, ester-bound and glycoside-bound forms. The contentsof the individual phenolic acids from Kinnow and Feutrell’s Earlypomaces treated at different microwave powers and times aresummarised in Tables 1 and 2, respectively.

Results indicated that, in the free phenolic acid fraction, thesum of the individual phenolic acid contents (SPC) increased signif-icantly (P < 0.05) with the microwave power. For example, byincreasing microwave power from 125 to 500 W, the SPC increasedfrom 1163 to 1317 lg/g DW and 664 to 854 lg/g DW, in Kinnowand Feutrell’s Early pomace extracts, respectively. Except for feru-lic acid in Kinnow pomace, the content of individual phenolic acids

Table 3aEffects of microwave treatment on FCs content (lg/g DW) of Kinnow pomace.

MW treatmenth power/time Catechi Narinj Nargnink

Untreated 15.6 ± 0.16c ndo nd125 W, 5 min 15.5 ± 1.04c nd nd250 W, 5 min 18.9 ± 0.23b nd nd250 W, 10 min 22.3 ± 0.51a nd nd250 W, 15 min 19.2 ± 0.65b nd nd500 W, 5 min 20.9 ± 0.09ab nd nd

Means with different letters in the column are significantly different (P < 0.05).g FCs: flavanol, flavanone and flavonol compounds.h MW treatment: microwave treatment.i Catech: catechin.j Narin: naringin.k Nargnin: naringenin.l Hesp: hesperidin.

m Kaemp: kaempferol.n Isorham: isorhamnetin.o nd: not detected.

Table 2Effects of microwave treatment on phenolic acid content (lg/g DW) of Feutrell’s Early po

Forms MW treatmenth power/time Gallic q-Hydrox

Free Untreated 65.7 ± 1.8d 105 ± 1.2f

125 W, 5 min 68.4 ± 0.4d 114 ± 2.5e

250 W, 5 min 74.5 ± 0.1c 129 ± 0.8d

250 W, 10 min 88.0 ± 1.6b 141 ± 0.1c

250 W, 15 min 103 ± 0.1a 161 ± 0.9a

500 W, 5 min 103 ± 1.4a 152 ± 1.6b

Ester-bound Untreated 70.6 ± 2.7a 131 ± 1.4a

125 W, 5 min 65.4 ± 0.3b 123 ± 2.1b

250 W, 5 min 54.8 ± 1.1c 109 ± 0.5c

250 W, 10 min 41.7 ± 0.9d 95.3 ± 1.0d

250 W, 15 min 36.6 ± 0.1e 70.7 ± 1.0e

500 W, 5 min 35.6 ± 0.3e 71.2 ± 1.3e

Glycoside-bound Untreated 45.0 ± 1.2a 65.1 ± 1.4a

125 W, 5 min 44.4 ± 0.1a 66.8 ± 0.2a

250 W, 5 min 41.4 ± 0.4b 60.5 ± 0.6b

250 W, 10 min 36.5 ± 0.8c 52.7 ± 0.1d

250 W, 15 min 34.6 ± 0.1c 53.0 ± 0.1d

500 W, 5 min 30.6 ± 0.2d 57.6 ± 0.7c

Total Untreated 181 ± 5.7a 301 ± 4.0a

125 W, 5 min 178 ± 0.8ab 303 ± 4.8a

250 W, 5 min 171 ± 1.6bc 299 ± 1.9a

250 W, 10 min 166 ± 3.3c 289 ± 1.2b

250 W, 15 min 174 ± 0.3abc 285 ± 2.0c

500 W, 5 min 169 ± 1.9bc 281 ± 3.6c

Means with different letters in the column for each fraction are significantly different (Pg SPC: sum of individual phenolic acids content.h MW treatment: microwave treatment.

also significantly increased with microwave power. For instance,on treating for 5 min at 125, 250 and 500 W, the content of vanillicacid in Kinnow pomace extracts were 297, 329 and 342 lg/g DW,respectively. A similar trend was observed in the Feutrell’s Earlypomace but ferulic acid showed a different behaviour in Kinnowpomace, where it gave the highest content at 250 W, followed by500 and 125 W, respectively. This divergence can be attributed tothe varietal difference of citrus pomace. Water content, being themost common absorbing phase for microwave energy, plays akey role during microwave treatment of a material. Another reasonfor the above mentioned divergence could be the difference inwater contents of Kinnow and Feutrell’s Early pomaces. Some otherstudies have also reported that the phenolic content was increasedafter heat or radiation treatment of the plant materials (Gulatiet al., 2003; Jeong et al., 2004; Lee et al., 2003, 2006; Xu et al.,2007). In ester and glycoside-bound fractions, the content of phe-nolic acids was decreased with increase in microwave power. For

Hespl Rutin Kaempm Isorhamn Total FCsg

4541 ± 4.88f nd nd nd 4556 ± 5.04f

4823 ± 9.13e nd nd nd 4838 ± 10.2e

5665 ± 4.37c nd nd nd 5684 ± 4.60c

6166 ± 12.0a nd nd nd 6188 ± 12.46a

5228 ± 7.69d nd nd nd 5247 ± 8.34d

5780 ± 2.71b nd nd nd 5801 ± 2.80b

mace.

ybenzoic Vanillic q-Coumaric Ferulic SPCg

134 ± 0.7e 85.9 ± 0.6f 229 ± 2.1e 620 ± 6.3f

147 ± 2.0d 98.1 ± 1.2e 238 ± 3.4d 664 ± 9.5e

167 ± 0.1c 107 ± 0.2d 254 ± 1.9c 732 ± 3.1d

189 ± 1.8b 129 ± 0.3c 277 ± 0.6b 823 ± 4.4c

203 ± 0.4a 144 ± 0.8a 306 ± 0.2a 916 ± 2.4a

187 ± 1.0b 132 ± 1.3b 281 ± 2.7b 854 ± 8.0b

163 ± 2.9a 188 ± 1.1a 567 ± 6.0a 1119 ± 14.1a

150 ± 0.9b 181 ± 2.6b 548 ± 1.9b 1068 ± 7.8b

131 ± 0.6c 167 ± 0.1c 537 ± 2.2c 998 ± 4.5c

113 ± 0.1d 142 ± 0.8d 511 ± 0.1d 903 ± 2.9d

96.3 ± 1.1e 125 ± 0.1e 473 ± 1.2e 802 ± 3.5e

102 ± 1.7e 123 ± 2.4e 475 ± 1.1e 806 ± 6.8e

89.6 ± 0.7a 77.6 ± 1.8a 52.1 ± 2.0a 329 ± 7.1a

80.2 ± 0.1b 80.1 ± 0.5a 44.9 ± 0.1b 316 ± 1.0b

76.4 ± 1.0c 69.4 ± 1.2b 43.5 ± 0.1b 291 ± 3.3c

70.8 ± 0.2d 66.0 ± 0.3c 37.5 ± 0.1c 264 ± 1.5d

64.0 ± 0.1e 59.6 ± 0.2d 27.3 ± 0.2d 239 ± 0.7e

61.1 ± 0.8f 58.0 ± 0.1d 28.6 ± 1.1d 236 ± 2.9e

386 ± 4.3a 351 ± 3.5ab 849 ± 10.1a 2069 ± 27.5a

377 ± 3.0ab 360 ± 4.3a 831 ± 5.4ab 2049 ± 18.3ab

b 375 ± 1.7b 344 ± 1.5bc 834 ± 4.2ab 2022 ± 10.9ab

c 373 ± 2.1bc 336 ± 1.4cd 826 ± 0.8b 1990 ± 8.8bc

363 ± 1.6c 328 ± 1.1d 806 ± 1.6c 1956 ± 6.6c

350 ± 3.5d 312 ± 3.8e 785 ± 4.9d 1896 ± 17.7d

< 0.05).

a

d cb

acbc b

a

c cb

a

0

10

20

30

40

50

60

untreated 125 W 250 W 500 WMicrowave power

Scav

engi

ng (%

)

0

0.2

0.4

0.6

0.8

1

1.2

Abs

orba

nce

(700

nm

)

DPPH (scavenging)OH (scavenging)RP (absorbance)

b

abb

c

a

bcc

ab

cd

10

15

20

25

30

35

40

Scav

engi

ng (%

)

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orba

nce

(700

nm

)DPPH (scavenging)OH (scavenging)RP (absorbance)

Table 3bEffects of microwave treatment on FCs content (lg/g DW) of Feutrell’s Early pomace.

MW treatmenth power/time Catechi Narinj Nargnink Hespl Rutin Kaempm Isorhamn Total FCsg

Untreated 12.7 ± 1.14b ndo nd 3593 ± 5.94f nd nd nd 3605 ± 7.08f

125 W, 5 min 13.9 ± 0.87ab nd nd 4051 ± 2.29e nd nd nd 4065 ± 3.16e

250 W, 5 min 14.2 ± 0.06ab nd nd 4160 ± 5.51c nd nd nd 4175 ± 5.57c

250 W, 10 min 16.4 ± 0.53a nd nd 4566 ± 8.96a nd nd nd 4582 ± 9.49a

250 W, 15 min 13.5 ± 1.08b nd nd 4128 ± 6.11d nd nd nd 4142 ± 7.19d

500 W, 5 min 14.8 ± 0.22ab nd nd 4213 ± 4.74b nd nd nd 4228 ± 4.96b

Means with different letters in the column are significantly different (P < 0.05).g FCs: flavanol, flavanone and flavonol compounds.h MW treatment: microwave treatment.i Catech: catechin.j Narin: naringin.k Nargnin: naringenin.l Hesp: hesperidin.

m Kaemp: kaempferol.n Isorham: isorhamnetin.o nd: not detected.

K. Hayat et al. / Food Chemistry 123 (2010) 423–429 427

instance, after treating the Kinnow pomace powder at 125 and500 W for 5 min, the SPC in the ester- and glycoside-bound fractiondecreased from 2161 to 1864 and 244 to 199 lg/g DW, respec-tively, and a similar trend was found in Feutrell’s Early pomace.On increasing microwave power, the contents of individual pheno-lic acids were also significantly decreased in bound fractions of cit-rus pomace extracts, except for q-coumaric acid which wasincreased in the glycoside-bound fraction of Kinnow pomaceextract.

During microwave treatment, the phenolic acid content of cit-rus pomace was significantly affected by irradiation time. WhenKinnow and Feutrell’s Early pomace powders were treated at250 W for 5, 10 and 15 min, the SPC in the free fraction was signif-icantly increased with increasing treatment time. For example, theSPC values in the free phenolic acid fraction of Kinnow pomace ex-tract were 1258, 1355 and 1424 lg/g DW, when irradiated for 5, 10and 15 min, respectively. As can be seen in Tables 1 and 2, for bothKinnow and Feutrell’s Early pomace extracts, the individual pheno-lic acid contents in the free fraction were also significantly in-creased with increasing irradiation time. With the exception ofgallic acid and q-hydroxybenzoic acid in the glycoside-bound frac-tion of Feutrell’s Early pomace extract, the contents of all individ-ual phenolic acids and SPC in the bound fractions weresignificantly decreased by increasing irradiation time (Tables 1and 2). The decrease in the bound fractions with microwave powerand irradiation time indicated that the esterified and glycoside-bound phenolic acids could be cleaved by microwave treatmentand this might be attributed to the heating effect caused by theelectromagnetic radiations during the microwave process. Theseresults are in agreement with the findings of other researchers(Jeong et al., 2004; Xu et al., 2007), who found that heat treatmentcan liberate the bound phenolic compounds in citrus peels.

The total phenolic acid content, which was equal to the sum offree and bound (ester- and glycoside-bound) phenolic acid con-tents, was decreased with increasing microwave power and treat-ment time (Tables 1 and 2). This indicated that some phenolic acidsprobably were degraded by microwave treatment. Xu et al. (2007)reported similar results on heating citrus peels in an electric oven.

0

5

untreated 125 W 250 W 500 WMicrowave power

0

Fig. 1. Effects of microwave power on antioxidant activity of citrus mandarinpomace. (a) Kinnow; (b) Feutrell’s Early. Bars with different letters for eachantioxidant assay at different power levels are significantly different. DPPH, 1,1-diphenyl-2-picrylhydrazyl; OH, hydroxyl radical; RP, reducing power.

3.2. Effect of microwave treatment on the FCs of citrus mandarinpomace

In the byproducts of citrus fruit after juice production, there arephenolic compounds rich in flavones, glycosylated flavones, phe-nolic acids and flavonols (Bocco, Cuvelier, Richard, & Berset,1998). The effect of microwave treatment on the contents of the se-

lected FCs from Kinnow and Feutrell’s Early pomace is given in Ta-bles 3a and 3b, respectively. The content of FCs, for both Kinnowand Feutrell’s Early pomace, was increased with increasing power.For instance, when Kinnow pomace powder was treated for 5 minat 125, 250 and 500 W, the total FCs contents were 4838, 5684 and5801 lg/g DW, respectively. An increase in the FCs content was ob-served with increasing irradiation time; however, when pomaceswere treated at 250 W for 15 min, there was a sudden decreasein the content, which indicated that a longer irradiation time washarmful to the FCs. The highest content of total FCs was obtainedwhen pomace powder was treated at 250 W for 10 min. Gulati

a

R2 = 0.9589 R2 = 0.9554

R2 = 0.9059

05

101520253035404550

1000 1100 1200 1300 1400 1500

SPC in free fraction ( µg/g DW)

Scav

engi

ng (%

)

00.10.20.30.40.50.60.70.80.91

Abs

orba

nce

(700

nm

)

DPPHOHRP

b

R2 = 0.9819

R2 = 0.9147

R2 = 0.9861

0

5

10

15

20

25

30

35

500 600 700 800 900 1000

Scav

engi

ng (%

)

-0.05

0.05

0.15

0.25

0.35

0.45

0.55

Abs

orba

nce

(700

nm

)

DPPHOHRP

428 K. Hayat et al. / Food Chemistry 123 (2010) 423–429

et al. (2003) found that the total phenols and catechins of green teawere increased by microwave treatment during the manufacturingprocess, and suggested that the application of microwave energyprevented the binding of polyphenol and catechin to the leaf ma-trix, which could increase catechins in green tea. Park et al.(2009) also found similar results for the use of FIR on green tealeaves; however, our results differed from the findings of Xuet al. (2007).

3.3. Effect of microwave treatment on the antioxidant capacity ofcitrus mandarin pomace extracts

The antioxidant capacities of Kinnow and Feutrell’s Early pom-ace extracts were evaluated by DPPH radical assay, hydroxyl radi-cal assay and reducing power assay. As shown in Fig. 1a and b, theantioxidant capacity of Kinnow and Feutrell’s Early pomace ex-tracts increased with increasing microwave power. For instance,after being heated at 500 W, the DPPH�-scavenging activity of Kin-now and Feutrell’s Early pomace was increased from 18.4% to29.1% and 8.5% to 16.2%, respectively. The treatment time also sig-nificantly increased the antioxidant activity of citrus pomace(Fig. 2a and b). Reducing power of Kinnow pomace was increasedfrom 0.448 to 0.764 when heated for 15 min at 250 W. Feutrell’sEarly showed a similar trend of antioxidant capacity and the high-est antioxidant capacities (DPPH�, OH and RP) were found after amicrowave treatment of 250 W for 15 min. These results are simi-lar to the findings of Kim et al. (2008b), who reported that the anti-oxidant activity of citrus pomace was increased after electron-beam irradiation.

SPC in free fraction ( µg/g DW)

Fig. 3. Correlations among SPC in free fraction: DPPH�-scavenging assay, OHradical-scavenging assay and RP. (a) Kinnow pomace; (b) Feutrell’s Early pomace.SPC, sum of individual phenolic acids content; DPPH, 1,1-diphenyl-2-pic-rylhydrazyl; OH, hydroxyl radical; RP, reducing power.

a

ab

cd

abc

d

ab

cc

0

10

20

30

40

50

60

untreated 5 min 10 min 15 minTreatment time

Scav

engi

ng (%

)

0

0.2

0.4

0.6

0.8

1

1.2

Abs

orba

nce

(700

nm

)

DPPH (scavenging)OH (scavenging)RP (absorbance)

b

dc

b

a

ab

c

d

d

cb

a

0

5

10

15

20

25

30

35

40

untreated 5 min 10 min 15 minTreatment time

Scav

engi

ng (%

)

0

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orba

nce

(700

nm

)DPPH (scavenging)OH (scavenging)RP (absorbance)

Fig. 2. Effect of treatment time on antioxidant activity of citrus mandarin pomace.(a) Kinnow; (b) Feutrell’s Early. Bars with different letters for each antioxidant assayat different treatment times are significantly different. DPPH, 1,1-diphenyl-2-picrylhydrazyl; OH, hydroxyl radical; RP, reducing power.

3.4. Correlation of DPPH radical, OH radical and RP assays with SPC inthe free phenolic acid fraction

The correlation coefficients (among DPPH, OH, RP and SPC) inthe free fraction for Kinnow and Feutrell’s Early pomace are shownin Fig. 3a and b, respectively. It can be seen that correlation coeffi-cients, in each case, were above 0.9059, which meant that the in-crease in the antioxidant capacities of citrus pomaces was due atleast in part to the increase of SPC in the free fraction. Free phenoliccompounds have been shown to have greater antioxidant effectsthan bound forms (Niwa et al., 1988). In addition, the increase inthe free fraction could be due to the liberation of low-molecularantioxidant compounds from the repeating subunits of high molec-ular weight polymers.

4. Conclusions

Results of this study indicated that, in general, the microwavetreatment of citrus pomace cleaved and liberated phenolic com-pounds, hence resulting in the increase of free phenolic com-pounds and enhancement of antioxidant capacity of the extracts.Covalently-bound phenolic compounds in citrus peels could be re-leased by electric oven heat treatment (Xu et al., 2007). However,microwave treatment, due to its simplicity and rapidity, could bemore effective in releasing antioxidant compounds from agricul-tural byproducts, such as citrus pomace, on an industrial scale.Notably, higher power and longer treatment time resulted in thedegradation of some FCs. Therefore, we suggest that a proper and

K. Hayat et al. / Food Chemistry 123 (2010) 423–429 429

reasonable microwave treatment should be carefully considered toenhance the antioxidant capacity of citrus mandarin pomace.

Acknowledgements

The authors gratefully acknowledge the financial support by theNational Key Technology R&D Program of China (2006BAD12B05-07) and PCSIRT0627.

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