Phytochemicals in Pomegranate Seeds and Their Effect as Hypolipidemic Agent in Hypercholesterolemic Rats

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1 Worl Journl of Diry & Foo Sienes 7 (1): 85-92, 2012 ISSN X IDOSI Pulitions, 2012 DOI: /iosi.wjfs Phytohemils in Pomegrnte Sees n Their Effet s Hypolipiemi Agent in Hyperholesterolemi Rts 1 2 M.A. Elny n I.S. Ashoush 1 Foo Siene n Tehnology Dept., F. of Environ. Agri. Si., Suez Cnl Univ., El Arish, North Sini, Egypt 2 Foo Siene Dept., F. of Agri., Ain Shms Univ., Ciro, Egypt Astrt: In the present stuy, pomegrnte see oils (PSO) were extrte n nlyze for their ftty i profiles n totl toopherols. The eftte pomegrnte see resiue (PSR) ws evlute for its totl phenoli n svenging pities ginst DPPH. Dietry effet of pomegrnte see oil, eftte see resiue n their mixture (PSOR) on hyperholesterolemi rts ws investigte. The results revele tht, pomegrnte see oil ws rih in polyunsturte ftty i (88.4%) n totl toopherols (905.5 µg/100g). Menwhile, the pomegrnte see resiue ontine mount of totl phenolis (14.8 mg/g) n exhiite high svenging tivity (98.2%). After 28 ys of feeing perio, therogeni rts group (AC) revele n inresing in level of totl holesterol, triglyerie, low ensity lipoprotein holesterol (LDL-C), risk rtio, therogeni inex n lipi peroxition ompre with norml ontrol rts group (NC). Supplemente iet with 5% pomegrnte see oil, 10% pomegrnte see resiue n 15% of their mixture t the rtio of 2:1 respetively improve lipi profile of plsm s ompre to AC group. Furthermore, the supplementtion iets inue signifint inrese in levels of reue glutthione n reution in mlonilehye level (MDA) when ompre to AC group. In ition, the histopthologil exmintion of the hert n ort in rts group fe on hyperholesterolemi iet showe severe histologil hnges s ompre to supplemente PSO, PSR n PSOR groups, these results reflet the protetive effet of pomegrnte see frtions (oil n resiue) ginst theroslerosis n it hve more potentil s helth supplement rih in nturl phytohemils. Key wors: Pomegrnte see oil Deftte resiue DPPH Hypolipiemi Lipi profile Oxitive stress Atherogeni rts INTRODUCTION juie inustry re out 20% (w/w) of the whole fruit. Sine, they n e useful for foo pplitions (espeilly The high prevlene of oesity, theroslerosis n in juie n everge inustries) s funtionl gent riovsulr isese (CVD) is lrgely ttriutle to the Tehrnifr et l. [3] n Mohgheghi et l. [4]. Reent ontemporry lifestyle tht is often seentry n stuies foun tht pomegrnte see my hve the inlues iet high in sturte fts n sugrs n potentil to e goo soure of nutrients n low ingestion polyunsturte ftty is (PUFAs), ntioxints. It hs een suggeste tht ietry fruit, vegetles n fier [1]. Pomegrnte fruit supplementtion with pomegrnte sees my reue (Puni grntum L.) is use from nient times n weight gin n type 2 ietes risk [5], reue the risk reports of its therpeuti qulities hs ehoe throughout of ner n llevite menopusl symptoms [6]. the ges. Both in vitro n in vivo stuies hve The enefiil effets of pomegrnte sees my e emonstrte how this fruit ts s ntioxint, relte to the presene of vriety of iologilly tive ntiieti n hypolipiemi s well s improves ompouns, prtiulrly polyphenols, whih hve een riovsulr helth. The helth enefits of pomegrnte stuie for their ntioxint effets [7]. Signifint levels hve een ttriute to its wie rnge of phytohemils of phenoli ontent were etete in pomegrnte sees [2]. Pomegrnte sees re y-prouts of pomegrnte reommene y Pne n Akoh [8], Elflleh et l. [9]. Corresponing Author: Ih S. Ashoush, Deprtment of Foo Siene, Fulty of Agriulture, Ain Shms Univ. Ciro, Egypt. 85

2 Worl J. Diry & Foo Si., 7 (1): 85-92, 2012 Pomegrnte see oil (PSO) is mjor soure of Totl Phenoli Content: The totl phenoli ontent in the polyunsturte ftty is (PUFAs) inluing methnoli extrt of pomegrnte see resiue (PSR) ws onjugte linoleni i (CLA) isomers s well s low etermine oring to Singleton et l. [13]. The retion sturte ftty i whih is importnt for therpeuti mixture ontine 0.5 ml of Folin-Ciolteu regent, 0.5 ml uses in humn helth [10]. of 7.5% N2CO 3n 0.5 ml of extrt. The sorne ws The present stuy ws esigne to investigte mesure t 765 nm. Results were expresse s glli i the phytohemil omposition of pomegrnte see equivlents (GAE) per grm pomegrnte see resiue oil n its resiue. Also, ssess the hypolipiemi power (mg GAE/g power) using glli i stnr effets of pomegrnte see oil n its resiue on urve. the lipi profile, oxitive stress prmeter n histopthologil ltertion in rts fe DPPH Ril Svenge Ativity%: The ility of the hyperholesterolemi iet. methnoli extrt of pomegrnte see resiue (PSR) to svenge 1, 1-iphenyl-2-pirylhyrzyl (DPPH) free MATERIALS AND METHODS rils ws etermine using the metho esrie y Singh et l. [14]. Mterils Plnt Mteril: Full ripene pomegrnte fruits Biologil Experiment: The rts were house in (Puni grntum L.) Wonerful vriety ws otine sreen-ottome luminum ges in room mintine t from the lol mrket. Pomegrnte sees from the rest of 25±1 C with lternting yles of light n rk of 12h the fruit were seprte from the juie n wshe urtion. The nimls were fe on the ontrol iet for refully to remove sugrs n other hering mterils. three onseutive ys. Rts were then rnomly ivie Seprte sees were rie t C in n oven for 12 h. into five groups, eh of whih onsists of 6, the first The rie, len sees were then finely groun n oil group (the ontrol) ws fe on the norml ontrol (NC) ws extrte y steeping in hexne s solvent iet, the seon ws therogeni ontrol (AC) group fe overnight in rk ple with gentle shking t 20 C. on hyperholesterolemi iet, the thir ws fe on The resultnt oil n eftte see power were store hyperholesterolemi iet supplemente with t -18 C until use. pomegrnte oil t 5% (PSO), whih ws equl to the no oservle verse effet level (NOAEL) s reporte y Chemil: Soium ronte, hexne n methnol Meerts et l. [15], the fourth ws fe on were otine from El-Gomhorey Co., Ciro, Egypt. 1, hyperholesterolemi iet supplemente with 1-iphenyl-2-pirylhyrzyl ril (DPPH), Folin- pomegrnte see resiue t 10% (PSR) n the fifth ws Ciolteus phenol regent n Glli i were purhse fe on hyperholesterolemi iet supplemente with from Sigm-Alrih In. (St Louis, MO, USA). Commeril mixture of 5% pomegrnte see oil n 10% its resiue kits use for etermining gluose, totl holesterol, (PSOR) for 28 ys. The omposition of the experimentl triglyeries (TG), high ensity lipoprotein (HDL), iets is shown in Tle 1 oring to AIN-93 guielines mlonlehye (MDA) n reue glutthione (GSH) [16]. The hnges in oy weight were reore weekly. were purhse from Bioignosti Co. Dokki, Egypt. Animls: Thirty mle Wistr rts with n verge weight of 140 g were otine from the Orgniztion of Biologil Prouts n Vines (Helwn Frm, Ciro, Egypt). Methos of Anlysis Ftty Ai Composition n Vitmin E Content: Ftty is were etermine using gs hromtogrphy (GC Hewlett Pkr 6890) oring to the metho esrie in AOAC [11]. Vitmin E in pomegrnte see oil ws etermine using HPLC system (Hewlett Pkr series 1100) mesure t 292 nm oring to Pyk n Sliwiok [12]. Tle 1: Compositions of the experimentl iets (%) Component% NC AC PSO PSR PSOR Corn strh Csein Corn oil Cellulose Slt mixture Vitmin mixture PSO PSR Choli i Cholesterol

3 Worl J. Diry & Foo Si., 7 (1): 85-92, 2012 Bloo smples were lso otine from the retro-oritl RESULTS AND DISCUSSION plexus of the eyes from ll nimls of eh group t the en of the experiment; the orgn (hert) ws exise Vitmin E n Ftty i Chemil Composition of immeitely fter leeing for weight. Plsm ws Pomegrnte See Oil: Results in Tle 2 show tht the otine from loo smples y entrifugtion t 1500 see oil of pomegrnte ontine high mounts of rpm for 15 min t mient temperture. toopherols (vitmin E) whih reently hve ttrte sientifi ttention for its ntiging, ntiner n Gluose n Lipi Profiles: Enzymti etermintion of ntitheroslerosis effet. Beuse of its rihness in gluose in plsm ws rrie out olorimetrilly t 510 vitmin E, the see oil of pomegrnte woul e nm oring to Triner [17]. Fully enzymti potentil nturl soure for nutritionl n meiinl uses etermintion of totl holesterol (TC) ws rrie out s mentione y Elflleh et l. [9] n Jing et l. [26]. oring to Allin [18], totl triglyeries (TG) in plsm Ftty is omposition of pomegrnte see oil (PSO) ws mesure olorimetrilly t 546 nm, oring to presente in Tle 2 revele tht the oil is rih in Fossti n Prinipe [19], low ensity lipoprotein polyunsturte ftty is (PUFAs), the most holesterol (LDL-C) ws etermine y enzymti preominnt PUFA ws linoleni i C18:3. Conerning methos of Wieln n Seiel [20] n the high ensity the sturte ftty is/unsturte ftty is rtio lipoprotein holesterol (HDL-C) ws etermine oring (SEA/USFA) were very low. Therefore, onsumption of to the metho of Lopez-Virell et l. [21]. pomegrnte see oil, rih in highly poly unsturte ftty is (PUFAs), protets ginst riovsulr Oxitive Stress: The extent of lipi peroxition in isese s reporte y Jing et l. [26] n Prshr et l. plsm ws etermine y mesurement of [27]. mlonilehye (MDA) formtion t 534 nm using the thiorituri i retive sustnes (TBARS) metho Totl Phenolis Content n Antioxint Ativity of s esrie y Ohkw et l. [22]. The reue Pomegrnte See Resiue (PSR): From the t glutthione (GSH) in the plsm ws estimte y its presente in Tle 3, it oul e notie tht the retion with ithio-is-2-nitroenzoi i (DTNB) tht pomegrnte see resiue (the eftte pomegrnte gve yellow olore omplex with mximum sorption see) is goo soure of totl phenolis ontent n h t 412 nm, oring to the metho esrie y Beutler gret free ril svenging tivity. This fining et l. [23]. showe tht PSR oul e utilize s nutreutil resoure [28]. Histopthology: Autopsy smples were tken from the hert n ort of the ifferent groups of rts n fixe in 10% formol sline for twenty four hours. Wshing ws one with tp wter then seril ilutions of lohol (methyl, ethyl n solute ethyl) for ehyrtion. Speimens were lere in xylene emee in prffin t 56 egree in hot ir oven for twenty four hours. Prffin ees wx tissue loks were prepre for setioning t 4 mirons thikness y slige mirotome. The otine tissue setions were ollete on glss slies, eprffinize n stine y hemtoxylin n eosin stins for histopthologil exmintions through the light mirosope [24]. Sttistil Anlysis: All t were expresse s men vlues±sd for 6 rts in eh group. Sttistil nlysis ws performe using one wy nlysis of vrine (ANOVA) followe y Dunn s Multiple Rnge Test with P < 0.05 eing onsiere sttistilly signifint. Sttistil nlysis ws onute with SAS progrm [25]. Tle 2: Vitmin E n ftty i omposition of pomegrnte see oil: Prmeters Vitmin E (µg/100g) Ftty i nlysis (%) C16: C16: C17: C17: C18: C18: C18: C18: C20: C20: SFA MUFA PUFA SEA/USFA Tle 3: Totl phenolis ontent n ntioxint tivity of pomegrnte see resiue Components Totl phenolis (mg/g) Antioxint tivity%

4 Worl J. Diry & Foo Si., 7 (1): 85-92, 2012 Tle 4: Effets of experimentl iets on oy weight gin n reltive herts weight of rts fe on hyperholesterolemi iets. Prmeters NC AC PSO PSR PSOR Boy weight (g) Initil 141.6± ± ± ± ±1.2 Finl 203.6± ± ± ± ±1.8 Gin 61.8± ± ± ± ±1.7 Reltive hert weight (g/100 g BW) 0.90± ± ± ± ±0.19 Mens hving ifferent letters in the sme row re signifintly ifferent (P * 0.05). Tle 5: Effet of experimentl iets on plsm lipis profiles in rts fe on hyperholesterolemi iets Prmeters NC AC PSO PSR PSOR Triglyeries (mg/l) 45.92± ±2.93 e 36.32± ± ±1.02 Totl holesterol (mg/l) 65.93± ± ± ± ±3.67 HDL-holesterol (mg/l) 45.41±0.86 e 24.04± ± ± ±1.48 LDL-holesterol (mg/l) 11.84± ± ± ± ± Risk rtio 1.450± ± ± ± ± Atherogeni inex 0.45± ± ± ± ± Risk rtio= Totl holesterol / HDL-holesterol 2 Atherogeni inex = [(Totl holesterol - HDL-holesterol)/HDL-holesterol] Mens hving ifferent letters in the sme rw re signifintly ifferent (P * 0.05). Effet of Experimentl Diets on Boy Weight Gin n signifintly reue plsm TC onentrtion y 58.9, 44 Reltive Herts Weight of Rts: The initil oy weights n 52.8% n LDL-C y 82.2, 80.3 n %, of ll rt groups were not signifintly ifferent, however, respetively (Tle 5). These finings re in fter 28 ys of feeing; the oy weight gin were greement with those otine y Meerts et l. [15]. signifintly reue in therogeni ontrol (AC) group The onentrtion of TG ws foun in rts fe on fe on hyperholesterolemi iet s ompre to other hyperholesterolemi iet (AC) ws greter thn the groups (Tle 4). Also, nimls fe on iet supplemente other groups initing tht the PSO, PSR n s repler with pomegrnte see oil (PSO) whih PSOR-supplemente iets signifintly prevente the ontine polyunsturte ftty is signifintly lesser rise of TG in rts plsm fe on hyperholesterolemi iet. the oy weight gin mong ll groups ue to the On the other hn, plsm HDL-C ws signifintly lower reution of ominl ft umultion s reporte y in rts reeiving AC iet ontrry to PSO, PSR n Aro et l. [29] menwhile, this iet enhne the weight PSOR-supplemente iets (Tle 5). The plsm totl gin ontrry to AC group. On the other hn, rts fe on holesterol/hdl-holesterol rtio signifintly iets supplemente with pomegrnte see resiue (PSR) inrese in nimls fe on AC iet while it erese n the PSOR mixture use signifintly higher signifintly in nimls fe on PSO, PSR n PSOR - inrese in oy weight gin ompre to NC, AC n supplemente iets n ontrol group. For this reson, PSO groups. These results hypothesize tht the therogeni inex (AI) ws signifintly higher in pomegrnte see resiue my improve ppetite n the AC rts group in omprison to the NC n PSO, enhne weight gin. The reltive weight of herts ws PSR n PSOR groups (Tle 5). The risk rtio n not signifintly ffete y the type of iets fe to rt therogeni inex re lso preitors of oronry risk [30]. groups (Tle 4). The signifint inrese in totl holesterol/hdlholesterol n therogeni inex oserve in AC rts Lipi Profile Prmeters: The present stuy showe hs n effet on riovsulr iseses. PSO, PSR n signifint inrese in the onentrtion of plsm totl PSOR my ply n importnt protetive role ginst holesterol (TC) n low ensity lipoprotein holesterol theroslerosis n riovsulr isese in mmmls, (LDL-C) in the therogeni ontrol (AC) rts group fe on whih ws eue from the reution of the risk rtio hyperholesterolemi iet ompre to other groups. n therogeni inex in PSO, PSR n PSOR niml While, the PSO, PSR n PSOR-supplemente iets groups (Tle 5). 88

5 Worl J. Diry & Foo Si., 7 (1): 85-92, 2012 Tle 6: Effet of experimentl iets on plsm MDA n GSH in rts fe on hyperholesterolemi iets. Diets MDA (nmol/ml) GSH (mg/l) NC 4.47± ±0.08 AC 5.30± ±0.05 PSO 3.85± ±0.04 PSR 4.21± ±0.05 PSOR 3.76± ±0.06 Mens hving ifferent letters in the sme olumn re signifintly ifferent (P * 0.05). Oxitive Stress: Lipi peroxition (MDA) is n utotlyti proess, whih is ommon onsequene of ell eth. This proess my use peroxitive tissue mge in inflmmtion n ging [31]. In the present stuy, plsm Lipi peroxition onentrtions ws signifintly higher in therogeni ontrol (AC) group fe on hyperholesterolemi iet s ompre to other groups (Tle 6). Menwhile, feeing on PSO, PSR n PSOR-supplemente iets signifintly reue the level of MDA. Chnges in reue glutthione (GSH) level s n ntioxint iomrker ontent of ifferent groups re shown in Tle 6.The levels of non-enzymti ntioxint (GSH) were signifintly lowere in therogeni ontrol (AC) group fe on hyperholesterolemi iet y out 53.5% s ompre to those of norml ontrol group (NC). In ontrst, feeing on PSO, PSR n PSOR-supplemente iets signifintly improve the level of glutthione ompre with AC trete group. Furthermore, the reution in the levels of MDA n the enhnement of reue glutthione inite strong ntioxint properties of pomegrnte see oil, pomegrnte see resiue n their mixture. Histopthologil Exmintion: Mirogrph (1) to (10) revels the histopthologil exmintion of semi-thin setions of hert n ort of rts stine with hemtoxylin n eosin. The group fe on norml ontrol iet (NC) h no histopthologil ltertion in the myorium (m), Mirogrph (1); s well s in the tuni intim (i), mei (m) n tuni ventiti () of the ort (Mirogrph 2). Tretment with therogeni ontrol group (AC) use severe ongestion in myoril loo vessels (Mirogrph 3), ssoite with thikening n slerosis in the vsulr wll n ft eposition with mssive numer of inflmmtory ells infiltrtion were etete in the perivsulr re of ort (Mirogrph 4). (1) (2) (3) (4) (5) (6) Mirogrph 1: Hert of norml ontrol rt (H & E, 160X) Mirogrph 2: Aort of norml ontrol rt (H & E, 64X) Mirogrph 3: Hert of therogeni rt (H & E, 80X) Mirogrph 4: Aort of therogeni rt (H & E, 64X) Mirogrph 5: Hert of rt trete with pomegrnte see oil (H & E, 64X) Mirogrph 6: Aort of rt trete with pomegrnte see oil (H & E, 64X) 89

6 Worl J. Diry & Foo Si., 7 (1): 85-92, 2012 (7) (8) (9) (10) Mirogrph 7: Hert of rt trete with pomegrnte see resiue (H & E, 40X) Mirogrph 8: Aort of rt trete with pomegrnte see resiue (H & E, 80X) Mirogrph 9: Hert of rt trete with mixture of pomegrnte see oil n its resiue (H & E, 40X) Mirogrph 10: Aort of rt trete with mixture of pomegrnte see oil n its resiue (H & E, 64X) Rts fe on pomegrnte see oil supplemente iet h ACKNOWLEDGMENT severe ongestion notie in the myoril loo vessels ssoite with oeem in etween the The uthors woul like to thnk Prof. Dr. Ael Bkeer myoril musle unles, (Mirogrph 5). There ws Kholoussy, Professor of Pthology, Fulty of Veterinry hemorrhge in the perivsulr re of the ort Meiine, Ciro University, for interprettion the slies of (Mirogrph 6). Rt fe on pomegrnte see resiue histopthology. (PSR) supplemente iet showe ongestion in the myoril loo vessels (Mirogrph 7). Ft eposition REFERENCES ws etete in the perivsulr tissue of the ort (Mirogrph 8). Rts fe on omintion of pomegrnte 1. Bimon, L., G. Vilhur n T. Pro, see oil n its resiue (PSOR) supplemente iet h Nutreutils n Atheroslerosis: Humn Trils, mil ongestion oserve in the myoril loo vessels Review. Criovsulr Therpeutis, 28: (Mirogrph 9). There ws no histopthologil ltertion 2. Viu-Mrtos, M., J. Fern nez-l opez n oserve in the ort, s reore in (Mirogrph 10). J.A. P erez- Alvrez, Pomegrnte n its Mny Funtionl Components s Relte to Humn Helth: CONCLUSION A Review. Comprehensive Reviews in Foo Siene n Foo Sfety, 9: Bse on the ove results, it oul e onlue 3. Tehrnifr, A., M. Zrei, Z. Nemti, B. Esfniyri tht the pomegrnte sees oil n its resiue re rih n M.R. Vzifeshens, Investigtion of soures of phytohemil n their supplemente iets physio-hemil properties n ntioxint tivity signifintly hinere the inresing in TC, TG, LDL-C, of twenty Irnin pomegrnte (Puni grntum L.) risk rtio, therogeni inex n Lipi peroxition levels. ultivrs. Sienti Hortiulture, 126: Therefore, the hypolipiemi n ntioxitive effet of 4. Mohgheghi, M., K. Rezei, M. Lfi n pomegrnte see oil n pomegrnte see resiue S.M.E. Mousvi, Pomegrnte see oil s oul e utilize in foo pplitions s nutreutil funtionl ingreient in everges. Eur. J. Lipi Si. resoure. Tehnol., 113:

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