Agricultural Science Technology, 2014, 15(2): 304-306Copyright C) 2014, Information Institute of HAAS. All rights reservedStorage and processingAntioxidant Activity of Ethanol Extract ofPomegranate SeedYuzhong SHI, Yuan LU, Benguo LIU, Sasa ZUoSchool of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, ChinaAbstract In this study, the ethanol extract of pomegranate seed was prepared andDPPH radical scavenging assayts antioxidant activities were investigated. It was found the total phenolic content in was done according to a publishedthe extract was as high as 41 791 mg GAE/g. And the extract showed high antioxidant activity measured as scavenging of DPPH radicals, hydroxyl radicals. It alsood 141. Briefly, 2 ml of DPPHexhibited strong antioxidant activity in reducing power and Rancimat test. These re-ethanol solution (0.2 mmol/L, inults demonstrated Pomegranate seeds could serve as a new source of natural an- ethanol)was incubated with differentoxidantconcentrations of FE and BHT(ButyKey words Pomegranate seed; Antioxidant; Free radicalslated hydroxytoluene). The reactionmixture was shaken and incubated inthe dark for 30 min, at room temperaomegranates (Punica grana- Plant Beijing Co, Ltd, China), about ture. And the absorbance was read attum) have been used exten- 1. 844 g of ethanol extract(EE)was 517 nm against ethanol Controls con-ively in the folk medicine of obtainedtaining ethanol instead of the antioximany cultures. Pomegranate seed is Determination of total phenolic con- dant solution, and blanks containingthe main waste fraction of pomeg. tent in EEethanol instead of dPPh solution wereranate fruits 1-2. In order to provide the100 mg of EE was dissolved in also made. the inhibition of the DPPHcorresponding development strategy ethanol and diluted to 100 ml with radical by the samples was calculatedthe ethanol extract (EE)of pomeg- ethanoldetermine total phenolic according to the following formularanate seed was prepared and itscontentDPPH scavenging activittioxidant activities were investigated inTPC was determined according to [Abs of control-(Abs of sample-Absthe present study.the Folin-Ciocalteu method 3. Briefly, of blank)/Abs of control%0. 2 ml of the extract was added to a 25 Reducing power assayMaterials and Methodsml volumetric flask. and additionalThe reducing power of EE andMaterials and chemicalsdistilled water was added to make a fi- BHT was determined according to theThe defatted pomegranate seeds nal volume of 10 ml. A reagent blank method of A. Strivastava et a/5. 1 ml ofprovided by Jisanduo Oil &Fatrepared using distilled water. the extract in ethanol was mixed withTechnology Co, Ltd( Beijing, China). Folin-Ciocalteu phenol reagent (2 ml) phosphate buffer (2.5 ml, 0.2 mol/L,Folin-Ciocalteu's phenol reagent and was added to the mixture and shaken pH 6.6)and potassium ferricyanide1,1-diphenyl-2-picrylhydrazyl(DPPH) vigorously. After 5 min, 5 ml of 5%(2.5 ml, 1%); the mixture was incubatwas purchased from Sigma. Gallic Na CO3 solution was added with mix- ed at 50C for 20 min. A portion(2.5acid was the product of BBL. Other ing. The solution was immediately di- ml)of trichloroacetic acid (10%)washemicals were of analytical gradeluted to 25 ml with distilled water and added to the mixture, which was thenPreparation of the ethanol extractixed thoroughly and then allowed to centrifuged at 3 000 r/min for 10 minstand for 90 min. After that. the ab- The upper layer of solution (2.5 ml)Powdered pomegranate seed(50 sorbance was measured at 750 nm was mixed with distilled water (2.5 ml)g)was soaked in 500 ml of 80% versus the prepared blank. The total and FeCl(0.5 ml, 0.1%), and the ab-ethanol, sonicated for 30 min and then phenolic content of the sample was sorbance was measured at 700 nmfiltered. The filtrate was concentrated expressed as gallic acid equivalents Increased absorbance of reaction mixunder vacuum at 45C and freeze- (mg GAE/ml)ture indicated increased reducingdried( Four-Ring Science Instrument DPPH radical scavenging assaypoweSupported by Foundation for Science and Technology Research Program of Henan say oxyl radicals scavenging as-Hydrprovince (132102110007: 102102210194): Natural Science Foundation of EducationThe hydroxyl radical scavengingDepartment in Henan province(2011A550006): Program for Innovative Research Team activity of the extract was determined(in Science and Technology) in University of Henan Province(13IRTSTHNO06)中國(guó)煤化工 of Jin et a/ACorrespondingauthorE-mail:syz6511@163.comReceived: January 2, 2014 Accepted: February 2, 2014anthroline (5CNMHGh phosphateAgricultural Science Technologybuffer (0.6 ml, 0. 1 mol/L, pH 7. 4), absorbance maximum at 517 nm, to cause DNA strand breakage, whichFeSO4(0.6 ml, 5 mmol/ L), EDTA(15 which decreases with the scavenging contributes to carcinogenesis, mutammol/L, 0.6 ml). 0.6 ml of distilled wa- of the proton radical. Hydrogen-donat- genesis and cytotoxicity. In additionter(Adarece, Aortro )or sample(Asarrple), 0.8 ing ability of the antioxidant molecule this radical species is considered asml of 0. 1%H2O2(Damege, Asarrple)or dis- contributes to its free radical scaveng- one of the quick initiators of the LPOtilled water(Aoorntrod)were added into the ing nature a high DPPH radical scav- process, abstracting hydrogen atomsmixture. The mixture was incubated at enging activity of EE and BHT was ob- from unsaturated fatty acids. In this as37C for 1 h And the absorbanceserved in a concentration manner say, the activity of the extract fromread at 536 nm. The hydroxyl radicals (Fig. 1), and the extract was more ac- pomegranate seeds showed high acscavenging activity was calculated us- tive than BHT, which should attribute tivity and concentration-dependenting the following equationto the high content of phenolic com-(Fig 3)Hydroxyl radicals scavenging ac- pounds in itAntioxidant activity in rancimat testtivity (%)=[(Asample-AdarregeM(Aontror-A derrege)]x Reducing powerIn the rancimat method the sam-is believed that antioxidant ac- ple is exposed to a stream of air atRancimat testtivity and reducing power are related. temperatures from 50-220C.TheThe antioxidant activities of BHT In this assay, though the extract from volatile oxidation products(chieflyand EE were measured on a 743 pomegranate seeds showed high ac- formic acid)are transferred to theRancimat analyzer (Switzerland)ac- tivity, but its activity was lower than measuring vessel by the air streamcording to the method of Proestos et that of BHT(Fig. 2) And the equation and absorbed there in the measurita/m Samples of lard oil (3 g) containing of reducing power and amount of solution (distilled water). When the0.02% of dihydromyricetin-lecithin FE(x) was y=0. 323 8X+0.031 4(=0.996 conductivity of this measuring solutioncomplex, lecithin and BHTsub- 2), indicating that reducing ability cor-recorded continuously, an oxidationjected to oxidation at 110 Clow related well with amount of fecurve is obtained whose point of infec-20 W/h). Induction periods, IP (h), were Reducing hydroxyl radical scav- tion is known as the induction timerecorded automatically. The protection enging activitythis provides a good characteristic val-factors(PF)were calculated accordingThe hydroxyl radical is an ex- ue for the oxidation stability. As shownto the following formulatremely reactive free radical formed in in Fig 4 the induction times of controlbiological systems and has been im- EE and BHT were 7.12, 9.23 and 16.5Statistical analysisplicated as a highly damaging species h, respectively. The performance ofThe data obtained in this study in free radical pathology capable of EE with PF of 1. 30 was inferior to thatwere expressed as the mean of threeng biomolecules of the living of BHT withreplicate determinations and standardHydroxyl radical has the capacitydeviation(SD). Statistical comparisorwere carried out using student ttest. Pvalues of <0.05 were considered to besignificantResults and AnalysisTotal phenolic content in EEcompounds havetracted more and more attentiontheir antioxidant behavior and benefi45L。L5202510cial health-promoting effects in chronic02 04 5 08 L0and degenerative diseases la). In thisroentrutie取Croentrutiniwnlstudy, it was found that the total phe- Fig-1 DPPH radical scavenging activities of Fig 3 Hydroxyl radicals scavenging activitynolic content in EE was as high asEE and BHT1.791 mg GAE/g. The extract frompomegranate seeds maybe becomeInfustion tinnew source of natural antioxidant andantrolhealth food with great commercial inE L2terest in the food and phyto-pharmaceutical marketAnti-proliferation of EE on Hep G2cells04Proton-radical scavengingis an important attribute of ant255D1.1515。125dants, which is measured by DPPH5L。L:2●2510中國(guó)煤化工dical scavenging assay. DPPH, aEE and BHT inprotonated radical, has characteristic Fig. 2 Reducing power of EE and BHTCNMHG306I Agricultural Science & Technology2014Conclusions[2 SINGH RP, MURTHY KNC, JAYAPR- 2006, 39: 1059-1065In this study, it was found that theKASHA GK. Studies on the antioxi- [6]JIN M, CAI X, LI J, et al. 1, 10-Phenan-ethanol extract from pomegranatedant activity of pomegranate peel and throline-Fez*oxidative assay of hydroxseed extracts using in vitro models[J]. J radical produced by H2OaFe[J]. Progseeds showed strong antioxidant activ-Agr Food Chem, 2002, 50: 81Biochem Biophys, 1996, 23: 553-555ity in DPPH and hydroxyl radicals [3] CHUN OK, KIM DO, SMITH N, et al. [7] PROESTOS C, SERELI D, KOMAITISscavenging assays, reducing power Daily consumption of phenolics and to-M. Analysis of flavonoids and phenolicand Rancimat tests. Pomegranatetal antioxidant capacity from fruit andacids in Greek aromatic plants: Investiseeds could serve as a source of anvegetables in the American diet[J]. J Sci gation of their antioxidant capacity andFood Agric,2005,85:1715-1724oxidants or nutraceuticals with poten-[4] SaNCHEZ-MORENO C Methods usedantimicrobial activity [J]. Food Chem,006,95:664-671tial applicationsto evaluate the free radical scavenging [8]KIM DO, LEE CY Comprehensive studyvitamin C equivalent antioxidant caReferences). Food Sci Technol Int, 2002, 8: 121pacity (VCEAC)of various polypheno-1LIY, GUO C, YANG J, et aL. Evaluation137lics in scavenging a free radical and itsof antioxidant propertieshe- [5] STRIVASTAVA A, HARISH SR, SHIstructural relationship[J]. Crit Rev Foodgranate peel extract in comparison with ANANDAPPA T. Antioxidant activity ofSci nutr,2004,44:253-273pomegranate pulp extract [J]. Foodthe roots of Decalepis hamiltonii(WightChem,2006,96:254-2Arn. )[J. LWT-Food Sci TechnolResponsible editor: Qingqing YINResponsible proofreader: Xiaoyan WU(Continued from page 214)cological effects of heavy metal pollu-[31 WANG X(王新), ZHOU QX(周啟星)Journal of Ecotoxicology(生態(tài)毒理學(xué)tion in soils on earthworms(土壤重金he ecological process, effect and re-報(bào)),2009,4(1):93-100屬污染對(duì)蚯蚓的急性毒性效應(yīng)研究)Jmediation of heavy metals contami-21]JAXY(賈秀英), CHEN ZW(陳志偉Chinese Journal of Applied Ecology (Enated soil(土壤重金屬污染生態(tài)過程The subacute toxicity of cadmium on用生態(tài)學(xué)報(bào),2002,13(2):187-190應(yīng)及修復(fù))|J] Ecological Science(生Carassias auratus(鎘對(duì)鯽魚酶活性毒[26]QUJP(邱江平), Earthworm and envi態(tài)科學(xué)),2004,23(3):278-281性的研究)J], Acta Agriculturae Zheronment protection(蚯蚓與環(huán)境保護(hù))〖32]GAOY(高巖), LUO YM(駱永明Lagensis(浙江農(nóng)業(yè)學(xué)報(bào)),2002,14(3)J] Guizhou Science(貴州科學(xué)),2008earthworms as bioindicators of soil155-15818(1):16-13ollution and their potential for remedi2]LNsQ(林少琴), LAN RF(蘭瑞芳).氏②2刁] XIE WH(謝衛(wèi)華), YAO JF(姚菊芳)ation of contaminated soils(蚯蚓對(duì)土fect of metal lons on activities of Cat,YUAN QS(袁勤生),eta. Improvement壤污染的指示作用及其強(qiáng)化修復(fù)的潛GSH-PX and sod from earthwormsof biphenyl three phenolic autoxidation力)小]. Acta Pedologica Sinica(土壤學(xué)Eisenia Foelide(金屬離子對(duì)蚯蚓CAT、method for determination of activity of報(bào))2005,42(1):140-145GSH-Px及SOD酶活性的影響)[]peroxide dismutase(聯(lián)苯三酚自氧(33] ZHANG V(張薇), SONG YF(宋玉芳)Strait Pharmaceutical journal(海峽藥化法測(cè)定超氧化物歧化酶活性的改進(jìn))SUN TH(孫鐵珩),eta. Effects of loy學(xué)),2001,13(2):23-25小] Medical Industry(醫(yī)藥工業(yè)),1988dosage pyrene pollution on biochemi-23] LIANG HY(梁海燕),LYS(李銀生),19(5):217-219cal characters of earthworm(EiseniaSUN J(HP), et a/ Effect of aluminum [28 ]LAVELLE P. Earthworm activity andfetida)inso(土壤低劑量芘污染對(duì)蚯蚓onon activities of important antioxithe soil system[J]. Bio Fert Soils, 1988若干生化指標(biāo)的影響)]. Chinesedant enzymes from earthworms, Eise6:237-25Journal of Applied Ecology(應(yīng)用生態(tài)nia andrei(鋁離子污染脅迫對(duì)蚯蚓重要[29 SPURGEON DJ, STURZENBAUM2007,18(9):2091-2103抗氧化酶活性的影響)[J. Journal ofSR, SVENDSEN C, et al. Toxicologi- [34] WILLEMS JGM, MAIRINISSEN JCYShanghai Jiaotong University: Aglular and gene expression inBLAIR J. Effects of earthworm on ni-cultural Science(上海交通大學(xué)學(xué)報(bào):農(nóng)earthworm exposed to copper andtrogen mineralization [J]. Biol Fertile業(yè)科學(xué)版),2007,25(6):551-556cadmium [J]. Comparative Biochemsoil,1996.23:57-63[24]LUDH(劉德鴻),LUDH(劉德輝),stry and Physiology, Part C,2004,35] YUAN YF(袁宇飛),凵GD(李光德,LNCHENG JM(成杰民), Acute toxicity of138:11-21L(林立),etal. Change of availablecu, Cd to two species of earthworms3 O] YAN ZG(顏增光,HEoL(何巧力),Lstate of pb. Cd in soil and enrichmentinso(土壤Cucd污染對(duì)兩種蚯蚓種FS(李發(fā)生). The use of earthwormand transfer abilities of brassica cl的急性毒性)[J]. Chinese Journal ofecotoxicological test in risk assess-nensis to Pb, Cd as affected by earthApplied& Environmental Biology(應(yīng)用ment of soil contamination(蚯蚓生態(tài)毒worm(在蚯蚓影響下土壤PbCd有效與環(huán)境生物學(xué)報(bào)),2005,11(6):706理試驗(yàn)在土壤污染風(fēng)險(xiǎn)評(píng)價(jià)中的應(yīng)用)的變化及小白菜對(duì)PbCd的富集轉(zhuǎn)710[]. Research of Environmental Sci-移作用川J] Journal of plant resources25] SONG YF(宋玉芳), ZHOU QX(周啟ences(環(huán)境科學(xué)研究),2007,20(1):and Environment(植物資源與環(huán)境學(xué)星),XUHX(許華夏), et al acute toxi134142報(bào)),2010,19(2):79-85Responsible editor: Qingqing YINResponsible proofreader: Xiaoyan WU中國(guó)煤化工CNMHG