Efficiency of Pomegranate Peel Extract as Antimicrobial, Antioxidant and Protective Agents

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1 World Journl of Agriulturl Sienes 6 (4): , 2010 ISSN IDOSI Pulitions, 2010 Effiieny of Pomegrnte Peel Extrt s Antimiroil, Antioxidnt nd Protetive Agents M.I. Irhium Deprtment of Food Siene nd Tehnology, Fulty of Agriulture, AL-Azhr University, Ciro, Egypt Astrt: The im of this study ws to find plnt extrt with ntimiroil nd ntioxidnt properties tht ould e potentilly used s nturl preservtive in foods. Pomegrnte peel extrt (PPE) ws evluted their ntimiroil tivity (y the gr diffusion method ginst the growth of Stphyloous ureus, Esherihi oli, Aspergillus niger nd Shromyes erevisie) nd ntioxidnt effet (y determine the oxidtive stility of sunflower oil ontining PPE). Also, to lrify the heptoprotetive effet of PPE on levels of serum lipid frtions, liver enzymes nd kidney funtions, hyperholesterolemi rt groups were fed on sl diet ontining PPE (t levels 400 or 00 mg/kg rt ody weight) nd the remining groups were positive nd negtive ontrol. The results showed tht PPE hd high polyphenoli ontent (67 mg/g) nd ws effetive ginst the growth of tested miroorgnisms nd giving inhiition zones rnged from 9.6 to 25.7 mm. The ntioxidnt tivity of PPE t levels 400 nd 00 ppm were 41.5 nd 63.4%, respetively while BHT t level 200 ppm ws 43.9%. The iologil study exhiited tht feeding hyperholesterolemi rts on sl diet ontining PPE t different levels oviously redued (P<0.05) the levels of TC, TG nd TL in rts serum y rtes from % s ompred to (C-) group ( %). Also, results indited tht the lowest levels of ALT, AST, ALP, uri id, ure nd retinine were found in hyperholesterolemi rt group fed on sl diet ontining PPE t level 00 mg/kg etween tested rt groups. It ould e onluded tht, PPE exhiited strong ntimiroil effet nd high ntioxidnt tivity. In ddition it enhned liver nd kidney funtions. Consequently this extrt might e used s multifuntionl preservtive in foods. Key words:pomegrnte peel Polyphenols Antimiroil Antioxidnt Liver enzymes Kidney funtions INTRODUCTION other plnt mterils hs gretly inresed. The pomegrnte is one of the importnt dietry soures of Numerous food produts require protetion ginst ntioxidnt phenolis [4]. Pomegrnte peel is reognized miroil spoilge during their shelf life. The growing for its mny helth-promoting qulities nd pprent demnd of onsumers for sfe nd nturl produts hs wound-heling properties [5], ntimiroil tivity [6], resulted in thorough investigtions from food uthorities ntiner property [7], ntitherosleroti nd nd reserhers to ssess the fesiility of mild ntioxidtive pities []. This ntioxidnt pity hs preservtion tehniques nd to improve the miroil een minly ttriuted to the wter-solule polyphenols, qulity nd sfety of produts, with mintining their nthoynins nd hydrolysle tnnins. good nutritionl nd orgnolepti properties [1]. There hve een some investigtions of the The use of syntheti ntioxidnts in food hs een ntimiroil effets nd ntioxidnt tivities of severl deresed due to their suspeted tion s promoters of hers on vriety of foods. However, there is puity of rinogenesis, s well for the generl onsumer rejetion informtion regrding the inhiitory effets of of syntheti food dditives [2]. Severl studies indited pomegrnte peel extrt on food orne pthogens nd tht the use of syntheti ntioxidnts hs egun to e lipid oxidtion. Therefore, the ojetives of the present restrited euse of their helth risks nd toxiity [3]. study were to evlute the ility of pomegrnte peel Therefore, the importne of repling syntheti extrt to inhiit some miroorgnisms growing nd ntioxidnts with nturl ingredients from spies nd determine their ntioxidnt tivity. Also, investigting Corresponding Author: M.I. Irhium, Deprtment of Food Siene nd Tehnology, Fulty of Agriulture, AL-Azhr University, Ciro, Egypt 33

2 World J. Agri. Si., 6 (4): , 2010 the effet of this extrt on serum lipid frtions, liver nd sterile ssy gr ws dded to eh of two Petri dishes kidney funtions through rt feeding trils ws nother with slow shking. Wrmed gr ws inoulted with trget ml tive teril ulture (1x10 fu/ml) nd llowed to hrden in refrigertor t 4 C for 1 h. After wrds, four MATERIALS AND METHODS equidistnt holes were mde in the gr using sterile ork orers nd 100µl PPE (t onentrtions 300, 600 or Mterils: Ripened nd freshly hrvested pomegrnte 900 µg/ml) ws dded on the top of inoulted gr lyer fruits (seson Otoer 2009) were otined from lol then dried t 25 C for 30 min. Pltes were kept t 4 C for mrket. Freshly refined sunflower oil without syntheti 1 h then inuted t 37 C for 24-4 h [13]. At the end of ntioxidnt ws otined from Egypt oils nd sop this period, inhiition zones formed on medium were Compny, Zgzig, Shrki, Egypt. Butylted urtely mesured in mm. hydroxytoluene (BHT) nd Folin-Ciolteu regent were purhsed from Sigm Chemil Co. (St. Louis, Mo). Determintion of Antioxidnt Effiieny: Different Stphyloous ureus (Grm-positive teri), onentrtions of PPE (400 nd 00 ppm) or BHT Esherihi oli (Grm-negtive teri), Aspergillus (200 ppm) were individully dded to 50g sun niger s mold nd Shromyes erevisie s yest flower oil to study their ntioxidnt effiieny. were otined from Miroiology Deprtment, Sienes The designtion of indution period y rnimt Fulty, Al-Azhr University, Ciro, Egypt. Kits for totl 679 (Metrom Ltd. CH.9100 Herisu, Switzerlnd) ws holesterol, triglyerides, totl lipids, ALT, AST, ALP, tken s tool to ompre the effetiveness of uri id, ure nd retinine were otined from pomegrnte peel extrt nd syntheti ntioxidnt on Biodignosti Co., Egypt. oxidtive stility of oil ording to the method desried y Mendez et l. [14]. Methods Preprtion of Pomegrnte Peel Extrt (PPE): Biologil Investigtion: Twenty four dult mle white Pomegrnte fruits were wshed y distilled wter then lino rts (weighing g) were housed individully peeled nd their edile portions were refully seprted. in mesh-ottom stinless steel ges in ontrolled The peels were ir dried in ventilted oven t 40 C for 4 environment. All rts were fed on sl diet, prepred s h nd ground to fine powder nd pssed through 24- desried y Reeves et l. [15], for 7 dys. Diet nd mesh sieve. 100g powdered smple ws extrted with deionized wter were supplied d liitum throughout the 00ml ethnol t room temperture for 24 h in shking study. After wrds, rts were rndomly divided into four wter th s desried y Shn et l. [ 9]. The mixture groups s follows: filtered through Whtmn No. 2 filter pper for removl of peel prtiles. The extrt ws freeze-dried nd stored Group 1: Rts fed on hyperholesterolemi diet, prepred in refrigertor t 4 C until use. s desried y Gll [16], for 7 weeks (C+). Determintion of Polyphenoli Compounds: HPLC Group 2: Rts fed on hyperholesterolemi diet for 3 tehnique ws used for seprtion nd estimtion of weeks followed y sl diet for 4 weeks (C-). polyphenoli ompounds in PPE ording to the method desried y Crllo et l. [10]. While, totl polyphenols Group 3: Rts fed on hyperholesterolemi diet for 3 were determined olorimetrilly using Folin-Ciolteu weeks followed y sl diet ontining PPE t regent ording to the method desried y level 400 mg/kg rt ody weight for 4 weeks. Singleton et l. [11]. The sorne ws mesured y n utomted UV-VIS spetrophotometer t 750 nm. Group 4: Rts fed on hyperholesterolemi diet for 3 weeks followed y sl diet ontining PPE t Determintion of Antimiroil Ativity: The gr level 00 mg/kg rt ody weight for 4 weeks. diffusion method ws used to study the effet of PPE on growth of Stphyloous ureus, Esherihi oli, Biologil investigtion ws rried out t the niml Aspergillus niger nd Shromyes erevisie y house of Sienes Fulty, Al-Azhr University, Ciro, mesuring of the dimeter of the inhiition zone [12]. 10ml Egypt. 339

3 World J. Agri. Si., 6 (4): , 2010 Blood Smpling: Blood smples were otined from RESULTS oritl venous plexus (fter 0, 4 nd weeks) y mens of fine pillry glss tues. The smples were pled in dry Polyphenoli Composition of PPE: Tle 1 shows tht nd len entrifuge tues nd llowed to lot for 1-2 h t totl polyphenols ontent is reltively high in PPE 37 C. The serum ws seprted y entrifugtion t 3000 (67 mg/g). Also, the sme tle indits tht the r.p.m. for 10 min. The superntnt serum ws then pipette polyphenoli omposition of PPE is hrterized y into epindorff tues nd immeditely frozen t -70 C until highly proportion of punilgin (296 mg/g) in omprison use. At the end of experimentl period, rts were srified to the remining ompounds. y depittion fter n overnight fst. The seprted serum used for ssy of lood serum prmeters. Antimiroil Ativity of PPE: Pomegrnte peel extrt showed vrious degrees of inhiition ginst Determintion of Serum Lipid Frtions: Totl the growth of investigted miroorgnisms s shown holesterol, triglyerides nd totl lipids in lood serum in Tle 2. Dt showed tht there were signifint were determined ording to the methods desried y differenes (P<0.05) mong inhiition effets on the tested Allin et l. [17], Fossti nd Prenipe [1] nd Zollner miroorgnisms. Hene, the inhiitory effet of PPE nd Kirsh [19], respetively. inresed y inresing their onentrtions nd ws inhiition zones rnged from 9.6 to 25.7 mm. Also, Liver Funtion Tests: Serum lnine mino trnsferse from the sme tle, it is ler tht the highest inhiition (ALT), sprti mino trnsferse (AST) nd lkline ws otined for E. oli, while the lowest ws for phosphtse (ALP) were mesured y ultrviolet S. erevisie. spetrometry method ording to Oser [20]. Effet of PPE on Oxidtive Stility of Sunflower Oil: Kidney Funtion Tests: Uri id, ure nd retinine in Results presented in Tle 3 indited tht the indution lood serum were determined y enzymti olorimetri period of ontrol oil smple (without ntioxidnt) is.2 h. methods ording to Brhm nd Trinder [21], Fwett Inresing of PPE level from 400 to 00 ppm in sunflower nd Sott [22] nd Brtels nd Bohmer [23], respetively. oil oviously inresed the indution period from 11.6 to Sttistil Anlysis: The dt were sttistilly nlyzed 13.4 h with ntioxidnt tivity rehed to 63.4%. While, y nlysis of vrine (ANOVA) nd lest signifine ntioxidnt tivity of sunflower oil ontining 200 ppm differene (LSD) t signifine of proility 5% [24]. BHT ws 43.9%. Tle 1: Polyphenoli omposition nd TP of PPE Polyphenoli ompounds (mg/g) TP (mg/g) Punilgin 296 Delphinidin 34 Cinnmi id Punilin 15 Pelrgonidin 21 Coumri id 32 Ellgi id 1 Queretin 40 Feruli id 2 Glli id 71 Kempherol 62 Sinpi id 17 Cynidin 26 Luteolin 33 Cffei id 11 Tle 2: Effet of PPE on growth of tested miroorgnisms Dimeter of inhiition zone (mm) Smple Conentrtion (µg/ml) S. ureus E. oli A. niger S. erevisie PPE , nd mens in eh olumn with different supersripts re different signifintly (p 0.05) Tle 3: Effets of PPE nd BHT on oxidtive stility of sunflower oil PPE (ppm) Items Control BHT (200 PPM) Indution period (h) Antioxidnt tivity* (%) *AA% = Indution period of smple - Indution period of ontrol / Indution period of ontrol x

4 World J. Agri. Si., 6 (4): , 2010 Tle 4: Effet of PPE on levels of TC, TG nd TL of tested rts PPE (mg/kg) Prmeter (mg/d1) Period (week) C+ C TC TG TL , nd mens in eh rw with different supersripts re different signifintly (p 0.05) Tle 5: Effet of PPE on liver enzyme tivities of tested rts PPE (mg/kg) Prmeter (mg/d1) Period (week) C+ C ALT (GPT) AST (GOT) ALP , nd mens in eh rw with different supersripts re different signifintly (p 0.05) Tle 6: Effet of PPE on levels of uri id, ure nd retinine of tested rts PPE (mg/kg) Prmeter (mg/d1) Period (week) C+ C Uri id Ure Cretinine , nd mens in eh rw with different supersripts re different signifintly (p 0.05) Effet of PPE on Serum Lipid Frtions: The levels of Effet of PPE on Liver Enzyme Ativities: Results present totl holesterol, triglyerides nd totl lipids of rt in Tle 5 nd Fig. 2 show serum ALT, AST nd ALP groups fed on different diets re presented in Tle 4. levels initilly nd fter feeding rts on different diets. Dt indited tht feeding rts on hyperholesterolemi After feeding on hyperholesterolemi diet for 3 weeks, diet for 3 weeks signifintly inresed (P<0.05) levels of these levels signifintly inresed (P<0.05) from 12.3, TC, TG nd TL from 4.3, nd mg/d1 to nd mg/d1 to 1.79, nd mg/d1, , nd mg/d1, respetively. respetively. At the end of experimentl period, these From the sme tle, it ould e lso notied tht levels oviously deresed (P<0.05) to 12.96, 20.9 nd feeding the sme rts on sl diet ontining 400 or mg/dl, respetively fter feeding rts on sl diet 00 mg/kg PPE for nother 4 weeks oviously deresed ontining 00 mg/kg PPE, while it were 16.6, 26.7 nd (P<0.05) levels of TC, TG nd TL y different rtes s 0.59 mg/dl in (C-) group, whih fed on sl diet only, shown in Fig. 1. respetively. 341

5 World J. Agri. Si., 6 (4): , 2010 DISCUSSION Fig. 1: Redution % of TC, TG nd TL in tested rts serum Fig. 2: Redution % of ALT, AST nd ALP in tested rts serum Fig. 3: Redution % of uri id nd ure in tested rts serum Effet of PPE on Kidney Funtions: Results given in Tle 6 nd fig. 3 indited tht levels of uri id, ure nd retinine in the investigted rts t zero time were 3.72, nd 0.51 mg/d1, respetively. Feeding rts on hyperholesterolemi diet for 3 weeks signifintly inresed (P<0.05) these levels to 5.76, nd 0.9 mg/d1, respetively. Feeding these rts on sl diet ontining PPE t different levels exhiited extremely lower vlues of uri id, ure nd retinine s ompred with (C-) group. In the lst few yers, n inresed ttention hs een foused on the industril wstes, espeilly those ontining residul phenols from the plnt rw mteril used. Pomegrnte peel is one of the importnt dietry soures of ntioxidnt phenolis. Qulittive nlysis of polyphenoli omposition indited tht PPE hd high mount of totl polyphenols (67 mg/g). This result is in greement with Crllo et l. [10] who studied the physil nd hemil hrteristis of 19 pomegrnte ultivrs nd found tht the mount of TP vried etween 354 nd 73 mg/g. Pomegrnte peel extrt ws tive nd effetive ginst the growth of tested miroorgnisms. Wheres, the inhiition zones rnged from 9.6 to 25.7 mm depend on type of miroorgnism. These results provide evidene for the presene of ntimiroil phenoli ompounds in PPE. These ompounds n degrde the ell wll, disrupt the ytoplsmi memrne, dmge memrne proteins nd interfere with memrne-integrted enzymes, whih my eventully led to ell deth [25]. Regrding the ntioxidnt tivity, it ould e notied tht ddition of PPE t different levels to sunflower oil smples enhned their oxidtive stility. The gretest effet ws found t level 00 ppm peel extrt s ompred to syntheti ntioxidnt. Generlly, PPE exhiit good ntioxidnt pity nd is effetive svenger of severl retive oxygen speies, due to its high levels of polyphenoli ompounds [26]. Feeding rt trils exhiited tht PPE t level 00 mg/kg rt ody weight ws le to redue the levels of TC, TG nd TL in hyperholesterolemi rt's serum y rtes 52, 47 nd 4%, respetively s ompred with (C-) group (29, 30 nd 2%, respetively). The urrent study found mrked effet of PPE on rt liver funtions tht deresed ALT, AST nd ALP y 1 to 1.5-fold ompre to (C+) group. These results re in greement with Toklu et l. [27], who studied the effet of hroni dministrtion of PPE on liver firosis indued y ile dut legtion in rts nd found tht serum ALT nd AST were signifintly deresed y PPE tretment. These indite tht pomegrnte peel preserved the struturl integrity of the heptoellulr memrne nd liver ell rhiteture whih is onfirmed y histopthologil studies [2, 29]. Regrding the levels of uri id, ure nd retinine, it ould e notied tht lower levels were found in hyperholesterolemi rt group fed on sl diet ontining 00 mg/kg PPE thn other groups. 342

6 World J. Agri. Si., 6 (4): , 2010 It ould e onluded tht, PPE ontined high levels of phenoli ompounds, onsequently it exhiited effetive ntimiroil properties nd high ntioxidnt tivity. In ddition it enhned liver nd kidney funtions. Therefore this extrt might e used s multifuntionl preservtive in foods. REFERENCES 1. Burt, S., Essentil oils: Their ntiteril properties nd potentil pplitions in foods-a review. Int. J. Food Miroiol., 94: Nmiki, M., Crit. Rev. Food Si. nd Nutrition, Hirose, M., Y. Tkesd, H. Tnk, S. Tmno, T. Kto nd T. Shiri, 199. Crinogenesis, 19: Ozgen, M., C. Durg, S. Sere nd C. Ky, 200. Chemil nd ntioxidnt properties of pomegrnte ultivrs grown in Mediterrnen region of Turkey. Food Chem., 111: Chidmr, M.K., V.K. Reddy, J.M. Veigs nd U.D. Murthy, Study on wound heling tivity of Puni grntum peel. J. Med. Food, 7: Brg, L.C., A.A. Leite nd K.G. Xvier, Synergi intertion etween pomegrnte extrt nd ntiiotis ginst Stphyloous ureus. Cn. J. Miroiol., 51: Jeune, M.A., J. Kumi-Dik nd J. Brown, Antiner tivities of pomegrnte extrts nd genistein in humn rest ner ells. J. Med. Food, : Tzulker, R., I. Glzer, I. Br-Iln, D. Hollnd, M. Avirm nd R. Amir, Antioxidnt tivity, polyphenol ontent nd relted ompounds in different fruit juies nd homogentes prepred from 29 different pomegrnte essions. J. Agri. Food Chem., 55: Shn, B., Y.Z. Ci, J.D. Brooks nd H. Corke, Antiteril nd ntioxidnt effets of five spie nd her extrts s nturl preservtives of rw pork. J. Si. Food Agri., 9: Crllo, S., G. Rodriguez, C.G. Krueger, M. Dreher nd J.D. Reed, Pomegrnte (Puni grntum) supplements: Authentiity, ntioxidnt nd polyphenol omposition. J. Funtionl Foods, 35: Singleton, V.L., R. Orthofer nd R.M. Rventos, Anlysis of totl phenols nd other oxidtion sustrtes nd ntioxidnts y mens of Folin- Ciolteu regent. Methods Enzymol., 299: Ruh, J.P., S. Remes, M. Heinonen nd A. Hopi, Antimiroil effets of Finnish plnt extrts ontining flvonoids nd other phenoli ompounds. Int. J. Food Miroiol., 56: Collins, C.M, P. Lyne nd J. Grnge, 199. Antimiroil sensitivity nd ssy tests. Collins nd Lyne Miroiologil Methods. Butterworths, London, UK. 14. Mendez, E., J. Snhez, H. Speisky nd A. Vlenzuel, Comprison of rnimt evlution models to ssess oxidtive stility of fish oils. J. Am. Oil Chem. So., 74: Reeves, P.G., F.H. Nielsen nd F.C. Jr, Ain-93 purified diets for lortory rodents: finl report of the Amerin institute of nutrition d ho writing ommittee on the reformultion of the Ain-76 rodent diet. J. Nutr., 123: Gll, S.M., 195. Chemil nd iologil evlution of unsponifile mtter of vegetle oils. Ph. D. Thesis, F. Of Agri., Ciro Univ., Egypt, pp: Allin, C., L. Poon, C. Chn, W. Rihmnd nd P. Fu, Enzymti determintion of totl serum holesterol. Clin. Chem., 20: Fossti, P. nd L. Prenipe, 192. Determintion of triglyerides using enzymti methods. Clin. Chem., 2: Zollner, N. nd K. Kirsh, Determintion of totl lipid onentrtion in serum. Zentrlltt fur Gesmte experimentl, 135: Oser, B.L., Hwk's Phsiologil Chemistry Ltd, New Delhi, Indi, : Brhm, D. nd P. Trinder, Determintion of uri id using enzymti olorimetri method. Anlyst, 97: Fwett, A. nd J. Sott, Determintion of ure in serum using enzymti olorimetri method. J. Clin. Pth., 13: Brtels, H. nd M. Bohmer, Cretinine stndrd mesurement of serum retinine with piri id. Clin. Chem. At., 32: Steel, R.G. nd J.H. Torrie, 190. Priniples nd proedure of sttistis nlysis. Biometril pproh. 2 nd ed. MC Grw-Hill Book Co. INC New York, N.Y. 25. Shn, B., Y. Ci, J. Brooks nd H. Corke, The in vitro ntiteril tivity of dietry spie nd mediinl her extrts. Int. J. Food Miroiol., 117:

7 World J. Agri. Si., 6 (4): , Kulkrni, A. nd S. Ardhy, Chemil hnges 2. Friedmn, S.L., Moleulr regultion of hepti nd ntioxidnt tivity in pomegrnte rils during firosis n integrted ellulr response to tissue fruit development. Food Chem., 93: injury. J. Biol. Chem., 275: Toklu, H.Z., M.U. Dumlu, Ö. Sehirli nd G. Sener, 29. Chttopdhyy, R.R., Possile mehnism of Pomegrnte peel extrt prevents liver heptoprotetive tivity of Azdirht indi lef firosis in illiryostruted rts. J. Phrmy nd extrt: Prt II. J. Ethnophr. Mol., 9: Phrmology, 59: