Antioxidant Activity of Ethanol Extract of Pomegranate Seed

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)中国煤化工 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中国煤化工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]. 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