Nutrition Research and Practice 2015;9(3): ; doi: /nrp ; pissn eissn

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1 Nutrition Reserch nd Prctice 215;9(3): c215 The Koren Nutrition Society nd the Koren Society of Community Nutrition Dul effects of mixture of grpe pomce (Cmpell Erly) nd Omij fruit ethnol extrcts on lipid metolism nd the ntioxidnt defense system in diet-induced oese mice Hye Jin Hn 1,2, Un Ju Jung 1, Hye-Jin Kim 3, Byoung Seok Moon 3, Su-Jung Cho 1,2, Yong Bok Prk 4, Dong Gun Lee 4 Myung-Sook Choi 1,2 1 Deprtment of Food Science nd Nutrition, Kyungpook Ntionl University, 8 Dehkro, Bukgu, Degu 72-71, Kore 2 Center for Food nd Nutritionl Genomics Reserch, Kyungpook Ntionl University, Degu 72-71, Kore 3 Food R&D, CJ Cheiljedng Corp., Seoul , Kore 4 School of Life Science & Biotechnology, Kyungpook Ntionl University, Degu 72-71, Kore nd BACKGROUND/OBJECTIVES: We investigted the effects of comintion of grpe pomce (Vitis lrusc, Cmpell Erly) nd Omij fruit (Schizndr chinensis, Billon) ethnol extrcts on lipid metolism nd ntioxidnt defense system in diet-induced oese mice. MATERIALS/METHODS: Forty mle C57BL/6J mice were divided into four groups nd fed high-ft diet (control group, CON) or high-ft diet dded.5% grpe pomce extrct (GPE),.5% Omij fruit extrct (OFE) or.5% GPE plus.5% OFE (GPE+OFE) for 12 weeks. RESULTS: In contrst to the GPE- or OFE-supplemented groups, the GPE+OFE group showed significntly lower ody weight nd white dipose tissue weights thn the CON group. Moreover, GPE+OFE supplementtion significntly decresed plsm totl cholesterol nd incresed the plsm HDL-cholesterol/totl-cholesterol rtio (HTR) compred to the control diet. The heptic triglyceride level ws significntly lower in the GPE+OFE nd GPE groups y incresing β-oxidtion nd decresing lipogenic enzyme compred to the CON group. Furthermore, GPE+OFE supplementtion significntly incresed ntioxidnt enzyme ctivities with simultneous decrese in liver H 2O 2 content compred to the control diet. CONCLUSIONS: Together our results suggest tht supplementtion with the GPE+OFE mixture my e more effective in improving diposity, lipid metolism nd oxidtive stress in high-ft diet-fed mice thn those with GPE nd OFE lone. Nutrition Reserch nd Prctice 215;9(3): ; doi:1.4162/nrp ; pissn eissn Keywords: Grpe pomce, Omij fruit, high-ft diet, lipid-regulting enzyme, ntioxidnt enzyme INTRODUCTION 2) Oesity is defined s disese in which excess ody ft hs ccumulted to the extent tht it my impir helth. In prticulr, oesity cuses or excertes dietes mellitus, dyslipidemi, hypertension, coronry hert disese, nd certin forms of cncer [1]. It is lso ssocited with the development of nonlcoholic ftty liver disese [2]. Although some synthetic ntioesity drugs effectively reduce ody weight nd the risk of comoridities, phrmcologicl tretment strtegies hve een limited y their undesirle side effects [3]. For this reson, there hs recently een growing interest in nutrceuticls, which hve een used since ncient times [4]. A nutrceuticl is food contining helth-promoting ingredients or nturl components tht my provide desirle helth enefits nd contriute to the prevention of chronic diseses [5]. It hs een suggested tht nutrceuticls hve the potentil to regulte imlnces etween energy intke nd output nd to meliorte the development of oxidtive stress nd inflmmtion in oesity [6]. Grpe pomce, which is n industril wste from wine production nd consists of grpe seeds, skins, nd stems, contins lrge numer of polyphenolic compounds [7]. The mjor phenolics found in grpe skin include nthocynin, ellgic cid, myricetin, quercetin, kempferol, nd trns-resvertrol. Gllic cid, ctechin, nd epictechin re found in grpe seeds, while grpe stems contin rutin, quercetin 3-O-glucuronide, trns-resvertrol, nd stilin [8,9]. A previous study hs reported tht grpe pomce hs ntioxidnt nd nti-inflmmtory effects in diet-induced oese mice [1]. Schizndr chinensis, This work ws supported y the High Vlue-dded Food Technology Development Progrm (No ), the Ministry for Food, Agriculture, Forestry nd Fisheries nd y the Ntionl Reserch Foundtion of Kore (NRF, 212M3A9C & 215R1A5A6196), Seoul, Kore. Corresponding Author: Myung-Sook Choi, Tel , Fx , E-mil. mschoi@knu.c.kr Received: Septemer 18, 214, Revised: Jnury 8, 215, Accepted: Ferury 5, 215 This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution Non-Commercil License ( which permits unrestricted non-commercil use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 228 Effects of mixture of grpe pomce nd Omij Billon is known s Omij in South Kore, which literlly mens five-flvor (slty, sweet, sour, pungent nd itter) erry. Its seeds nd fruits hve een used in trditionl medicine for dry cough, sthm, night swets, nocturnl seminl emissions, nd chronic dirrhe [11-13]. A recent rticle hs reported tht phenolic phytochemicls, including nthocyns nd flvonols, from Omij displyed high ntioxidnt ctivity nd nti-inflmmtory effects in in vitro nd in vivo models [14,15]. Further, the ethnol extrct of Omij not only inhiited predipocyte differentition nd dipogenesis in 3T3-L1 cells, ut lso reduced ody weight gin, diposity, nd plsm triglyceride nd totl cholesterol levels in high-ft diet-induced oese rts [16]. There re mny different grpe vrieties round the world. The mjor vrieties of grpe produced in Kore re the Musct Biley A nd Cmpell Erly. Choi et l. hve reported tht the Cmpell Erly vriety hs higher levels of cynidin, peonidin, pelrgonidin, gllic cid, ctechin, nd epictechin in its skins nd seeds, nd the Cmpell Erly seeds exhiit more effective free rdicl scvenging effects thn those of the Musct Biley A vriety [17]. In previous study, we demonstrted tht Musct Biley A grpe pomce extrct did not significntly ffect ft ccumultion nd heptic stetosis in high-ft diet-induced oese mice, ut, interestingly the comined extrcts of Musct Biley A grpe pomce nd Omij fruit improved diposity, heptic stetosis, nd inflmmtion [18]. However, there re no reports on the nti-oesity nd ntioxidnt effects of Cmpell Erly grpe pomce comined with Omij in diet-induced oese mice. Accordingly, in the present study, we exmined the effects of ethnol extrcts of Cmpell Erly grpe pomce nd Omij fruit, lone or in comintion, on diposity, lipid metolism, nd the ntioxidnt defense system in mice fed high-ft diet. MATERIALS AND METHODS Preprtion of grpe pomce nd Omij fruit extrcts The grpes (Vitis lrusc, Cmpell Erly) nd Omij fruit (Schizndr chinensis, Billon) were purchsed from Gimcheon nd Sngjusi, Gyeonsnguk-do, Kore nd Mungyeong-si, Gyeongsnguk-do, Kore, respectively. In this study, grpe pomce nd Omij fruit (Fructus Schisndre) hrvested in 213 were used. Smples were prepred y dding 1 L of 8% or 5% ethnol to 1 g of dried grpe pomce or Omij fruit, respectively, followed y extrction t 8 C for 2 h nd then cooling. The solution ws filtered (Whtmn pper no. 2), concentrted with rotry vcuum evportor, nd stored t -7 C. The finl weight of the lyophilized powder of grpe pomce ethnol extrct (GPE) ws 17.9 g (17.9%) nd of Omij fruit ethnol extrct (OFE) ws 39.7 g (39.7%). The GPE included 1 mg/g resvertrol, 3 mg/g flvonoids, 64 mg/g polyphenol, 1.35 mg/g ctechin, nd 17 mg/g gllic cid. The OFE contined 8 mg/g schizndrin, 7 mg/g flvonoids, nd 17 mg/g polyphenol. Animls nd diets Mle mice (strin C57BL/6J) were purchsed from the Jckson Lortory (Br Hror, ME, USA) t 4 weeks of ge. The nimls were individully housed with constnt temperture (24 C) nd 12 h light/drk cycle, nd fed pelletized commercil nonpurified diet for 1 week fter rrivl. The mice were then Tle 1. Composition of experimentl diets (g of diet, w/w) Groups 1) Ingredients Csein D, L-Methionine Sucrose Cellulose AIN-76 minerl mixture AIN-76 vitmin mixture Choline itrtrte Corn Strch Lrd Corn oil Cholesterol tert-butylhydroquinone Grpe extrct 5 5 Omij extrct.5.5 Totl ) CON: high-ft diet (2% ft, w/w) control; GPE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w); OFE: high-ft diet+omij fruit ethnol extrct (.5%, w/w); GPE+OFE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w)+omij fruit ethnol extrct (.5%, w/w) rndomly divided into 4 groups (n = 1) nd fed experimentl diets for 12 weeks: high-ft diet (2% ft, w/w) control (CON), high-ft diet with.5% (w/w) grpe pomce extrct (GPE), high-ft diet with.5% (w/w) Omij fruit extrct (OFE), nd high-ft diet with.5% (w/w) GPE plus.5% (w/w) OFE (GPE+OFE). Bsed on previous pulished reports, we chose the dose of experimentl mterils [18,19]. The composition of ech diet is presented in Tle 1. The mice hd free ccess to food nd distilled wter during the experimentl period. Their food intke nd ody weight were mesured dily nd weekly, respectively. This niml study ws pproved y the Ethics Committee for niml studies t Kyungpook Ntionl University, Repulic of Kore (No ). Preprtion of smples At the end of the experimentl period, mice were nesthetized with diethylether nd scrificed fter 12 h of fsting. Blood ws tken from the inferior ven cv nd then centrifuged t 1 g for 15 min t 4 C, nd the plsm ws seprted to nlyze plsm iomrkers. After lood collection, the liver nd white dipose tissues (s), including the epididyml, perirenl, retroperitonel, mesenteric, sucutneous, nd interscpulr s, were promptly removed, rinsed, weighed, frozen in liquid nitrogen, nd stored t -7 C. The enzyme source frction from the liver ws prepred nd nlyzed ccording to Hulcher nd Oleson with slight modifictions in order to mesure the heptic lipid-regulting nd ntioxidnt enzyme ctivities [2]. A 2% (w/v) homogente ws prepred in uffer contining.1 mol triethnolmine,.2 mol ethylenediminetetrcetic cid (EDTA) nd 2 mmol dithiothreitol (ph 7.) nd then centrifuged t 6 x g for 1 min to remove ny cell deris. The superntnt ws centrifuged t 1, x g nd gin t 12, x g for 2 min t 4 C to remove the mitochondril pellet. Susequently, the superntnt ws

3 Hye Jin Hn et l. 229 ultrcentrifuged twice t 1, x g for 6 min t 4 C to otin the cytosolic superntnt. The mitochondril nd microsoml pellets were then dissolved in 8 μl of homogeniztion uffer nd the protein content ws determined using the Thermo Scientific Pierce BCA Protein Assy Kit. Plsm nd heptic lipids Plsm totl cholesterol, triglyceride, nd high-density lipoprotein (HDL)-cholesterol concentrtions were determined using commercilly ville kits (Asn, Seoul, Repulic of Kore). The heptic lipids were extrcted using the procedure developed y Folch et l. [21]. The dried lipid residues were dissolved in 1 ml of ethnol for the cholesterol nd triglyceride ssys. Triton X-1 nd sodium cholte solution in distilled wter were dded to 2 μl of the dissolved lipid solution to produce finl concentrtions of 5 g/l nd 3 mmol/l, respectively. The heptic cholesterol nd triglyceride were nlyzed using the sme enzymtic kit used in the plsm nlysis. Levels of plsm lnine minotrnsferse (ALT) nd sprtte minotrnsferse (AST) were determined using enzymtic kits (Asn, Seoul, Repulic of Kore). Heptic lipid-regulting enzyme ctivity Mlic enzyme (ME) ctivity ws mesured ccording to the method of Ocho y monitoring the production of NADPH t 34 nm, where ctivity ws represented y the formtion of nicotinmide denine dinucleotide phosphte (NADPH) nmol/ min/mg protein [22]. Glucose-6-phosphte dehydrogense (G6PD) ctivity ws ssyed using spectrophotometric methods ccording to the procedures descried y Pitknen et l. where the ctivity ws expressed s the reduced NADPH nmol/min/mg protein [23]. Ftty cid synthse (FAS) ctivity ws determined using spectrophotometric ssy ccording to the method y Crl et l. [24]; one unit of FAS ctivity represented the oxidtion of 1 nmol of NADPH per minute t 3 C. Phosphtidte phosphohydrolse (PAP) ctivity ws determined using the method of Wlton nd Possmyer [25]. The ftty cid β-oxidtion ws determined using the method of Lzrow y monitoring the reduction of NAD to NADH t 34 nm, where the ctivity ws expressed s the reduced NAD nmol/min/mg protein [26]. Heptic ntioxidnt enzyme ctivity nd hydrogen peroxide content The superoxide dismutse (SOD) ctivity ws estimted ccording to the method of Mrklund, which is sed on the color chnge due to the uto-oxidtion of pyrogllol [27]. The ctlse (CAT) ctivity ws mesured using Aei's method, in which the decrese of hydrogen peroxide ws monitored spectrophotometriclly t 24 nm for 5 min using spectrophotometer [28]. The glutthion peroxidse (GSH-Px) ctivity ws mesured using spectrophotometric ssy, s descried previously y Pgli nd Vlentine with slight modifiction [29]. The rection mixture included 3 mmol glutthione, 6 mmol NADPH, nd hydrogen peroxide (H 2O 2) in.1 mol Tris-HCl (ph 7.2) uffer. The rection ws initited y dding the enzyme source nd the sornce ws mesured t 34 nm for 5 min. The glutthione reductse (GR) ctivity ws mesured y monitoring the oxidtion of NADPH t 34 nm [3]. The H 2O 2 levels in the liver were mesured using Wolff s method [31]. FOX 1 (ferrous oxidtion) regent ws prepred from.25 mol H 2SO 4, 1 mol soritol, 2.5 mmol mmonium iron (II), nd 1 mmol xylenol ornge, nd H 2O 2 levels were determined t 56 nm sornce. Sttisticl nlysis Dt were expressed s the men ± stndrd error of the men. Significnt differences mong the groups were determined using one-wy nlysis of vrince (ANOVA) in SPSS (version 11., SPSS Inc., Chicgo, IL, USA). Duncn s multiplernge test ws performed if differences were identified etween the groups t P <.5. RESULTS Effect on ody weight, ody weight gin, food intke, nd food efficiency rtio (FER) The time courses for chnges in ody weights re shown in Fig. 1. Compred with the control, neither GPE nor OFE lone significntly ltered ody weight in high-ft diet-fed mice. The GPE nd OFE lone groups lso showed no difference in ody weight gin nd FER (Tle 2). However, the ody weight of Fig. 1. Effects of supplementtion with grpe pomce nd Omij fruit ethnol extrcts for 12 weeks on chnges in ody weight in C57BL/6J mice fed high-ft diet. Dt re men±se. Superscripts not shring common letter re significntly different mong groups t P <.5. CON: high-ft diet (2% ft, w/w) control; GPE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w); OFE: high-ft diet+omij fruit ethnol extrct (.5%, w/w); GPE+OFE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w)+omij fruit ethnol extrct (.5%, w/w). Tle 2. Effects of supplementtion with grpe pomce nd Omij fruit ethnol extrcts on ody weight gin, food intke, nd food efficiency rtio in C57BL/6J mice fed high-ft diet Groups 1) Body weight gin (g) ± ± ± ± 1.29 Food intke (g/dy) 3.25 ± ± ± ±.11 FER 2).6 ±.4.6 ±.4.7 ±.4.5 ±.6 Dt re men±se. Superscripts not shring common letter within the sme row re significntly different mong groups t P <.5. 1) CON: high-ft diet (2% ft, w/w) control; GPE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w); OFE: high-ft diet+omij fruit ethnol extrct (.5%, w/w); GPE+OFE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w)+omij fruit ethnol extrct (.5%, w/w) 2) FER: food efficiency rtio

4 23 Effects of mixture of grpe pomce nd Omij Ft weight (g/kg ody weight) Tle 3. Effects of supplementtion with grpe pomce nd Omij fruit ethnol extrcts on plsm lipid profiles in C57BL/6J mice fed high-ft diet Groups 1) Plsm Triglyceride (mmol/l) 1.53 ± ± ± ±.9 Totl-C 2) (mmol/l) 5.6 ± ± ± ±.26 HDL-C 3) (mmol/l) 1.14 ± ± ± ±.6 HTR 4) (%) 2.36 ± ± ± ±.51 Liver Triglyceride (mmol/g liver) 7.12 ± ± ± ±.29 Cholesterol (mmol/g liver) 3.2 ± ± ± ±.32 Dt re men ± SE. Superscripts not shring common letter within the sme row re significntly different mong groups t P <.5. 1) CON: high-ft diet (2% ft, w/w) control; GPE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w); OFE: high-ft diet+omij fruit ethnol extrct (.5%, w/w); GPE+OFE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w)+omij fruit ethnol extrct (.5%, w/w) 2) Totl-C: totl cholesterol 3) HDL-C: high-density lipoprotein cholesterol 4) HTR: HDL-C/Totl-C rtio ([HDL-C]/[Totl-C] 1) CON GPE OFE GPE+OFE Epididyml Perirenl Retroperitonel Mesenteric Sucutneous Interscpulr Fig. 2. Effects of supplementtion with grpe pomce nd Omij fruit ethnol extrcts on white dipose tissue () weights in C57BL/6J mice fed high-ft diet. Dt re men±se. Superscripts not shring common letter re significntly different mong groups t P <.5. CON: high-ft diet (2% ft, w/w) control; GPE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w); OFE: high-ft diet+omij fruit ethnol extrct (.5%, w/w); GPE+OFE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w)+omij fruit ethnol extrct (.5%, w/w). Totl Tle 4. Effects of supplementtion with grpe pomce nd Omij fruit ethnol extrcts on heptic lipid-regulting enzyme ctivities in C57BL/6J mice fed high-ft diet Groups 1) FAS 2) ME 3) G6PD 4) PAP 5) β-oxidtion 6) nmol/min/mg protein CON ± ± ± ± ± 1.51 GPE 9.91 ± ± ± ± ± 3.53 OFE ± ± ± ± ± 2.31 GPE+OFE 1.87 ± ± ± ± ± 1.82 Dt re men ± SE. Superscripts not shring common letter within the sme column re significntly different mong groups t P <.5. 1) CON: high-ft diet (2% ft, w/w) control; GPE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w); OFE: high-ft diet+omij fruit ethnol extrct (.5%, w/w); GPE+OFE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w)+omij fruit ethnol extrct (.5%, w/w) 2) FAS: ftty cid synthse 3) ME: mlic enzyme 4) G6PD: glucose-6-phosphte dehydrogense 5) PAP: phosphtidte phosphohydrolse 6) Ftty cid β-oxidtion the GPE+OFE group tended to e lower thn tht of the CON group etween 8 nd 11 weeks of the experimentl period, nd t 12 weeks, ut finl ody weight ws significntly lower in the GPE+OFE group thn in the CON group (Fig. 1). Moreover, the GPE+OFE group showed significntly lower ody weight gin thn the CON group (Tle 2). FER ws significntly lower in the GPE+OFE group thn in the control group, lthough food intke ws not different etween these 2 groups. Diet supplementtion with OFE led to slightly lower food intke thn tht with GPE or GPE+OFE. Effect on white dipose tissue weights The weights of the s re shown in Fig. 2. Supplementtion with GPE or OFE lone only tended to reduce the perirenl nd totl weights from those seen in the CON group. However, in the GPE+OFE group the weights of epididyml, perirenl, retroperitonel nd totl s were significntly lower thn those in the CON group. The mesenteric, sucutneous, nd interscpulr weights were not significntly different mong groups.

5 Hye Jin Hn et l. 231 (A) (A) SOD (unit/mg protein) (B) (B) CAT (μmol/min/mg protein) c c (C) (C) GSH-Px (nmol/min/mg protein) (D) (D) GR (nmol/min/mg protein) (E) (E) 3 H 2 O 2 (mmol/mg protein) Fig. 3. Effects of supplementtion with grpe pomce nd Omij fruit ethnol extrcts on heptic ntioxidnt enzyme ctivities nd hydrogen peroxide content in C57BL/6J mice fed high-ft diet. Dt re men±se. Superscripts not shring common letter re significntly different mong groups t P <.5. CON: high-ft diet (2% ft, w/w) control; GPE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w); OFE: high-ft diet+omij fruit ethnol extrct (.5%, w/w); GPE+OFE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w)+omij fruit ethnol extrct (.5%, w/w). (A) SOD: superoxide dismutse, (B) CAT: ctlse, (C) GSH-Px: glutthion peroxidse, (D) GR: glutthione reductse, (E) H 2O 2: hydrogen peroxide Effect on plsm nd heptic lipid levels As shown in Tle 3, there ws no significnt difference in plsm triglyceride concentrtions mong groups. However, plsm totl cholesterol concentrtion ws significntly lower in the GPE+OFE group thn in the CON group. Although plsm HDL-cholesterol levels were not significntly different mong groups, the HDL-C/Totl-C rtio (HTR) ws significntly higher in the GPE lone nd GPE+OFE groups thn in the CON group. Heptic triglyceride levels were lso significntly lower in the GPE lone nd GPE+OFE groups thn in the CON group, ut there were no significnt differences in heptic totl cholesterol levels mong the groups. Effect on lipid-regulting enzyme ctivities in the liver The ctivities of lipogenic enzymes nd β-oxidtion in the liver re shown Tle 4. The GPE group showed significntly lower ctivities of heptic lipogenic enzymes, including ME, G6PD, FAS nd PAP, thn the CON group. The heptic ME nd PAP ctivities were significntly lower in the GPE+OFE group thn in the CON group. In contrst to the lipogenic enzyme ctivities, ftty cid β-oxidtion ws significntly higher in the GPE lone nd GPE+OFE groups thn in the CON group. The OFE group exhiited no significnt differences in lipid-regulting enzyme ctivities. Effect on ntioxidnt enzyme ctivities nd hydrogen peroxide content in the liver When exmining the ntioxidnt enzyme ctivities, heptic SOD, CAT, GSH-Px, nd GR ctivities were significntly higher in the GPE+OFE group thn in the CON group (Fig. 3). Compred to the control group, the group with GPE supplementtion lso showed significntly incresed heptic CAT ctivity nd tended to hve incresed heptic SOD ctivity. The heptic SOD nd CAT ctivities oserved in the OFE group tended to e higher

6 232 Effects of mixture of grpe pomce nd Omij Tle 5. Effects of supplementtion with grpe pomce nd Omij fruit ethnol extrcts on plsm minotrnsferse ctivities in C57BL/6J mice fed high-ft diet Groups 1) AST 2) ALT 3) IU/L CON 43.7 ± ± 7.76 GPE ± ± 7.36 OFE ± ± 6.75 GPE+OFE 4.33 ± ± 8.63 Dt re men ± SE. 1) CON: high-ft diet (2% ft, w/w) control; GPE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w); OFE: high-ft diet+omij fruit ethnol extrct (.5%, w/w); GPE+OFE: high-ft diet+grpe pomce ethnol extrct (.5%, w/w)+omij fruit ethnol extrct (.5%, w/w) 2) AST: sprtte minotrnsferse 3) ALT: lnine minotrnsferse thn those of the CON group, ut these differences were not sttisticlly significnt. The heptic mitochondril hydrogen peroxide content ws the lowest in the GPE+OFE group. Effect on plsm minotrnsferse ctivities The AST nd ALT ctivities in plsm re shown in Tle 5. There were no significnt differences in plsm AST nd ALT ctivities mong groups. DISCUSSION Our study ws crried out to clrify whether plnt mixture contining grpe pomce nd Omij fruit ethnol extrcts synergisticlly ffects lipid nd ntioxidnt metolism in dietinduced oese mice. Compred with the control, supplementtion with GPE+OFE significntly decresed ody weight, ody weight gin, ody ft ccumultion, nd FER in mice fed high-ft diet, lthough supplementtion with GPE or OFE lone did not influence these prmeters. Similrly, previous studies reported no significnt effects of GPE on ody weight gin nd ft mss in high-ft diet-induced oese mice or fructose-fed oese rts [18,32,33], ut GPE+OFE supplementtion significntly lowered ody weight gin in high-ft diet-fed mice [18]. On the other hnd, in contrst to our results, Prk et l. demonstrted tht supplementtion with OFE (5 mg/kg ody weight nd 2 mg/kg ody weight) for 6 weeks decresed ody weight in dose-dependent mnner in high-ft diet-fed rts, which my e due to higher doses of OFE nd different niml models [16]. Adipose tissue is not simply n energy storge orgn ut it lso regultes lipid metolism loclly s well s t distnt sites such s the liver, muscle, nd rin [34]. Oesity is ssocited with n incresed risk of dyslipidemi nd nonlcoholic ftty liver disese [35,36]. In the present study, we found tht compred with the control, supplementtion with GPE+OFE led to significnt reductions in the levels of plsm totl cholesterol nd heptic triglyceride nd mrked increse in the plsm HTR, good predictor of crdiovsculr risk [37]. Compred with the control, GPE supplementtion lso significntly incresed the plsm HTR nd mrkedly decresed heptic triglyceride content, wheres OFE supplementtion did not lter plsm nd heptic lipid profiles. Consistent with the decresed heptic triglyceride content, GPE with or without OFE, significntly decresed the ctivities of heptic ftty cid synthesis-relted enzymes (FAS, G6PD, nd/or ME) from those seen in the CON group. FAS ctlyzes the reductive synthesis of long-chin ftty cids, nd its inhiition reduced food intke nd ody weight in mice [38]. Cellulr NADPH, which is required for the iosynthesis of ftty cids nd cholesterol, cn e produced y enzymtic rection of ME nd G6PD. A previous study oserved tht the enzyme ctivity nd expression levels of G6PD were significntly elevted in oese mice models [39]. In our previous study in which Musct Biley A ws used insted of the Cmpell Erly grpe vriety, the mrna expression of heptic lipogenic genes (FAS, cetyl-coa croxylse, or ME) nd their trnscription fctor (PPAR γ) ws significntly down-regulted y mixture of GPE nd OFE extrcts [18]. In the present study, we lso found tht the ctivity of PAP, centrl enzyme in the synthesis of triglycerides, ws significntly lower in the GPE nd GPE+OFE groups thn in the CON group, nd heptic ftty cid β-oxidtion ws higher in the GPE nd GPE+OFE groups thn in the CON group, which supports the finding of lower heptic triglyceride content. A study y Furukw et l. reported ft ccumultion correlted with oxidtive stress in oese mice [4]. Oxidtive stress contriutes to tissue injury y rective oxygen species (ROS) ttck, nd tissues re protected from ROS dmge y ntioxidnt enzymes [41]. Among the ntioxidnt enzymes, SOD ctlyzes the dismuttion of the superoxide nion to hydrogen peroxide, nd CAT nd GSH-Px cn rek down hydrogen peroxide. GSH-Px lso oxidizes glutthione to glutthione disulfide (GSSG). The reduction of GSSG to GSH cn e ctlyzed y GR gin. In the present study, heptic SOD, CAT, GSH-Px, nd GR ctivities were significntly higher in the GPE+OFE group thn in the CON group, wheres heptic H 2O 2 content ws lower. It is well known tht GPE contins sustntil mounts of ntioxidnt phenolic compounds, in prticulr nthocynins, ctechin, epictechin, nd severl phenolic cids [1]. In rts fed high-ft diet, grpe skin supplementtion ws effective in protecting ginst oxidtive dmge cused y lipid peroxidtion [42]. Phenolic compounds from Omij dose-dependently promote ntioxidnt ctivities such s electron donting ility, SOD-like ility, hydroxyl rdicl scvenging ility, nd nitrite scvenging ility in vitro [43]. In the present study, the eneficil effects on heptic ntioxidnt cpcity s well s diposity were greter with the GPE+OFE mixture thn with GPE or OFE lone. This suggests synergistic effect of the ioctive phytochemicl compounds in grpe pomce nd Omij. Anthocynins from grpe nd Omij improved oesity induced y high-ft feeding [44]. Resvertrol present in grpes is lso effective for protecting ginst diet-induced oesity in vivo [45,46]. Thus, phytochemicl comintions hve greter dditive nd synergistic effects on helth enefits thn single preprtions [5]. It is supposed tht some flvonoids nd polyphenols in GPE+OFE my hve synergistic effects vi their interction, lthough we did not determine it in the present study. This hypothesis is supported y previous our study in which the effects of GPE+OFE were compred with those of resvertrol nd schizndrin mixture (RS), which is mixture of the mjor index components of GPE+OFE [47]. GPE+OFE hve more protective effect on hyperglycemi, diposity nd heptic

7 Hye Jin Hn et l. 233 stetosis in type 2 dietic mice thn RS [47]. Another possiility is tht other unknown ctive components present in extrcts of GPE+OFE lso ply importnt in their iologicl effects. To confirm the sfety of the experimentl supplements in the liver, AST nd ALT were mesured in plsm, ecuse incresed AST nd ALT ctivities hve een oserved in liver injury [48]. The ethnol extrcts used did not exhiit heptic toxicity. In conclusion, supplementtion with GPE+OFE mixture, in which the Cmpell Erly grpe vriety ws used, improves diposity, lipid metolism, nd ntioxidnt cpcity in high-ft diet-fed mice y suppressing heptic lipogenic enzyme ctivities nd simultneously incresing heptic ntioxidnt enzyme ctivities. The nti-diposity nd ntioxidnt effects of GPE+OFE supplementtion ppered to e more potent thn tht of GPE nd OFE lone. These results suggest tht GPE+OFE is more eneficil for the prevention of oesity thn GPE or OFE lone. REFERENCES 1. Kopelmn PG. Oesity s medicl prolem. 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