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1 Evidene-Bsed Complementry nd Alterntive Mediine Volume 2013, Artile ID , 10 pges Reserh Artile Bet-Glun-Rih Extrt from Pleurotus sjor-ju (Fr.) Singer Prevents Oesity nd Oxidtive Stress in C57BL/6J Mie Fed on High-Ft Diet G. Kngspthy, 1,2 S. N. A. Mlek, 1,3 A. A. Mhmood, 1,2 K. H. Chu, 1,2 S. Vikineswry, 1,3 ndu.r.kuppusmy 1,2 1 Mushroom Reserh Centre, University of Mly, Kul Lumpur, Mlysi 2 Deprtment of Biomedil Siene, Fulty of Mediine, University of Mly, Kul Lumpur, Mlysi 3 Institute of Biologil Sienes, Fulty of Siene, University of Mly, Kul Lumpur, Mlysi Correspondene should e ddressed to U. R. Kuppusmy; umh@um.edu.my Reeived 14 Ferury 2013; Revised 10 April 2013; Aepted 14 April 2013 Ademi Editor: Menk C. Thounojm Copyright 2013 G. Kngspthy et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. Mushrooms hve een used in folk mediine for thousnds of yers. In this study, the effet of β-glun-rih extrt of P. sjor-ju (GE) on lipid lowering nd ntioxidnt potentil ws ssessed in C57BL/6J mie fed on high-ft diet. Oesity ws indued in C57BL/6J mie y feeding high-ft diet. The ontrol groups in this study were (for norml diet) nd (for high-ft diet). The treted groups were (for norml diet) (240 mg/kg.w) nd 60, 120, nd 240 (for high-ft diet), where the mie were dministrted with three dosges of GE (60, 120, nd 240 mg GE/kg.w). Metformin (2 mg/kg.w) served s positive ontrol. GE-treted groups showed signifintly redued ody weight, serum lipid, nd liver enzymes levels. GE lso ttenuted protein ronyl nd lipid hydroperoxide levels y inresing the enzymi ntioxidnts (SOD, CAT, nd GPx) tivities in the mie. GE-treted groups indued the expression of hormone sensitive lipse (HSL) nd dipose triglyeride lipse (ATGL) while downregulted the expression of peroxisome prolifertor-tivted reeptor gmm (PPAR-γ), sterol regultory inding protein-1 (SREBP-1), nd lipoprotein lipse (LPL). Hene, GE prevented weight gin in the mie y induing lipolysis nd my e vlule in the formultion of djuvnt therpy for oesity. 1. Introdution Oesity hs rehed epidemi proportions nd is mjor ontriutor to the glol urden of hroni disese nd disility euse of its inresing prevlene in ll ge groups, sex, nd re with the hnges of lifestyles nd dietry intke. A reent sttistil report y the World Helth Orgniztion showed tht one out of ten dults were overweight;hene,thereremorethnoneillionoverweight dults [1, 2]. Besides tht, ording to the Ntionl Helth nd Moridity Surveys (2011), 15.1% of Mlysins ged 18 ndovewereoesethusmlysihsthehighestrte ofoesityinsouthestasindthe6thinasi.oesity is hroni metoli disorder tht results from the disequilirium etween energy intke nd energy expenditure. It is hrterized y enlrging ft mss nd elevted lipid onentrtion in lood. The mount of ft mss is inresed when the numer nd size of dipoytes re inresed y prolifertionnd differentition [3]. The ovious lterntives for the tretment of oesity re diet, exerise, nd surgil intervention suh s ritri surgery, Roux-en-Y gstri ypss, gstri nding, nd sleeve gstretomy. However, it is proven to e suessful only in smll minority of the popultion [4, 5]. Drugs tht re urrently ville for the mngement of oesity, inlude orlistt (Xenil) whih redues intestinl ft sorption through inhiition of pnreti lipse nd siutrmine (Redutil), nd ppetite suppressnt [2] whih ws found to use numerous side-effets whih inlude vlvulr hert disese, high lood pressure, dry mouth, onstiption, nd hedhe [6]. Multiple risk ftor

2 2 Evidene-Bsed Complementry nd Alterntive Mediine syndrome or metoli syndrome suh s insulin resistne [7], dietes mellitus [8], rdiovsulr disese, stroke, hypertension [9], nd dyslipidemi [10] is growingmedil prolem in industrilized ountries. Oesity is the entrl nd usl omponent in this syndrome [11]. Furukw et l. [11] reported tht in oese individuls, elevted retive oxygen speies (ROS) upregultes the expression of NADPH oxidse, estlishing viious yle tht ugments oxidtive stress in dipoytes nd lood irultion. The ROS will inrese the expression of hemottrtnts suh s monohemottrtnt proteins-1 (MCP-1), y-produts of lipid oxidtion (lipid hydroperoxides nd mlondildehyde (MDA)), nd protein oxidtion (protein ronyl) [12, 13] whih re linked with systemi inflmmtion whih then led to the development of metoli syndrome. However, it is lso well reported tht ntioxidnts n intivte these ROS nd thus prevent metoli deregultion inluding metoli syndrome [14]. Mushrooms re well known for their mediinl properties nd hve een widely used in trditionl mediine. The mediinl effets of mushrooms inlude ntioxidnt, ntivirl, ntiteril, ntifungl, ntiprsiti, detoxifition, immunomodultory, ntitumor, rdil svengers, ntiinflmmtory, ntihyperlipidemi, or ntihyperholesterolemi, heptoprotetive, nd ntidieti [14]. In Mlysi, the genus Pleurotus (oyster mushroom) whih hs een shown to hve definite nutritive (high qulity proteins, vitmins, nd very little lipids or strh) nd mediinl vlues is widely ultivted. This mushroom is mostly populr in ountries suh s Indi, Chin, nd Jpn nd is reported to e le to redue the holesterol level in lood [15] nd prevent hyperglyemi, insulinresistne,ndinflmmtionindiposetissue[16]. Pleurotus mushroom is rih in fier yet low in lories nd ft, nd it hs een ited s potentil weight-loss id. The dietry fiers in the mushroom onsist of hitin, hemielluloses, mnnns, nd β-gluns. Bet-gluns re polyshrides with gluose residue linked y et glyosidi onds. The fermentility of β-gluns nd their ility to form highly visous solutions in the humn gut my onstitute the sis of their ntioesity enefits [17]. Nturl produts ontining βgluns hve een used for thousnds of yers, ut β-gluns were only identified s tive omponents reently. Therefore, this study ws undertken to investigte the effets of βglun-rih extrt (GE) from P. sjor-ju on prevention of oesity nd oxidtive stress in C57BL/6J mie fed on highft diet. 2. Mterils nd Methods 2.1. Mushroom Smples. All neessry permits nd permission for the olletion of mterils for the desried field study were otined, nd the prty involved is duly knowledged. Fresh fruiting odies of Pleurotus sjorju (10 kg) were grown nd olleted from Mr. Kun Kek How mushroom frm in Semenyih, Selngor Drul Ehsn, Mlysi. Authentition of P. sjor-ju ws rried out y the Mushroom Reserh Centre (MRC), University of Mly, nd vouher mteril (KUM 50082) for this study ws deposited t the MRC ulture olletion Isoltion nd Purifition of GE. The isoltion nd purifition of GE were rried out sed on the method desried y Roy et l. [18]. The β-glun level in GE ws estimted using the β-glun kit (speifi for mushroom nd yest) purhsed from Megzyme Interntionl (Irelnd). The enzyme kit ontins exo-1,3-β-glunse, βgluosidse, mylogluosidse nd invertse, gluose determintion regent (GOPOD-gluose oxidse, peroxidse, nd 4-minontipyrine), nd gluose stndrd solution. The estimtion of totl glun ontent ws done y hydrolysing GE with 37% hydrohlori id (v/v) for 45 minutes t 30 Cnd ontinued for 2 hours t 100 C. After neutrliztion with 2 M potssium hydroxide, gluose hydrolysis ws rried out using mixture of exo-β-(1-3)-d-glunse nd βgluosidse in sodium ette uffer (ph 5.0) for 1 hour t 40 C. To mesure the totl glun ontent, gluose oxidseperoxidse mixture ws dded to GE nd inuted for 20 minutes t 40 C. The sorne of the resulting olour omplex ws mesured using spetrophotometer (Bio- Tek Instruments In, USA) t 510 nm. The α-glun ontent ws estimted ording to the sme method s desried ove fter enzymti hydrolysis with mylogluosidse nd invertse. The β-glun ontent ws lulted y sutrting the α-glun from the totl glun ontent. Glun ontent ws expressed s perentge (w/w) of dry weight (DW) Animls nd Ethis Sttement. This study ws onduted in onformity with the poliies nd proedures of the Animl Cre nd Use Guidelines of Fulty of Mediine, University of Mly, with referene to the 8th edition of Guide for the Cre nd Use of Lortory Animls y the Institute of Lortory Animl Reserh, Ntionl Ademy of Siene, USA. The niml ethis pprovl ws otined from Animl Cre nd Use Committee of Fulty of Mediine, University of Mly (IACUC, UM) (pprovl numer: ISB/14/07/2010/GK [R]). Femle C57BL/6j mie (7 weeks old) were purhsed from BioLso Lortory, Tiwn. The nimls were mintined in stinless steel wire mesh ges in room kept t 21 Cwith stndrd ondition of 12-hour light/drk yle (light period: 8:00 20:00 hour) with free ess to food nd wter whih were provided fresh every dy Experimentl Design. After one week of limtistion, the mie were rndomly ssigned (sed on weight) into seven groups (n = 6). Tle 1 shows the type of diet nd onentrtion of GE dministered to eh group. On lori sis, the norml diet ontined 5% ft, 69.2% rohydrte, nd 25.8% protein wheres the high-ft diets (TestDiet, USA) omprised 45% of ft (46.1% ft from lrd, 35.8% rohydrte, nd 18.1% protein) nd 60% of ft (61.6% ft from lrd, 20.3% rohydrte, nd 18.1% protein). GE ws dministered thrie week vi epigstri route using feeding needle (size 20) to groups, 60, 120, nd 240 for 16 weeks. In this study, metformin (2 mg/kg.w) ws used s the positive ontrol (MET) sine metformin hs een reported to hve omprle effets with orlistt (ntioesity drug) [19], nditislsowidelyusedtotrettype2dietes whih is losely ssoited with oesity [11]. After 7 weeks

3 Evidene-Bsed Complementry nd Alterntive Mediine 3 Tle 1: Type of diet nd onentrtion of GE/metformin dministrted to eh group. Type of diet Groups Tretment Norml diet High-ft diet MET Norml diet only + sline Norml diet mg/kg of ody weight GE High-ft diet only + sline High-ft diet + 60 mg/kg of ody weight of GE High-ft diet mg/kg of ody weight of GE High-ft diet mg/kg of ody weight of GE High-ft diet + 2 mg/kg of ody weight of metformin of feeding with 45% of ft, the niml diet ws sustituted with 60% of ft for groups, 60, 120, 240 nd MET. The diet for groups nd ws not ltered throughout the experiment. For the norml diet group, only 240 mg/kg of ody weight of GE (highest dose) wsdministrtedtothemieinordertoreduetheusgeof mie Smple Colletion nd Anlytil Methods. Body weight nd food onsumption were monitored dily. During the experimentl period, urine ws olleted from eh group weekly (every Mondy morning t 10:00 hour). At the end of the 16 weeks, the mie were nesthetized with ether fter withholdingfoodfor12hoursndweresrifiedyorti exsnguintion. Blood smples were olleted in SST glss serumtuewithgoldbdhemogrdlosure(bdvutiner, USA). Serum smples were seprted fter entrifugtion t 2400 g for 15 minutes. The serum smples from eh mouse (within group) were pooled together in order to hve suffiient serum for further nlysis. The pooled serum smples were sent to the Clinil Dignosti Lortory Unit, University Mly Medil Centre, for the serum lipid nd liver nlysis. Immeditely fter lood olletion, the liver nd kidney were perfused in-situ with ie-old sline. The weight of the liver nd kidney of mie from eh group were reorded. Eight ml of ie-old phosphte uffer sline (PBS) ws dded to one grm of liver or kidney. The smples were then homogenized using homogenizer (WiseMix HG-15A, Germny). Adipose tissues were removed nd stored in RNAlter solution (Applied Biosystems, USA) ndrefrigertedt4 Covernight.Allsmpleswerethen stored t 80 C until further nlysis ws rried out Urinry Oxidtive Indies Mesurement. The protein ronyl ontent (AOPP) ws determined s previously desried [20]. Chlormine-T solution of known onentrtions (0 to 500 μm) ws used s stndrd for the estimtion of AOPP onentrtion, nd the result ws expressed s μm of hlormine-t. Lipid hydroperoxide level ws determined sed on the method desried y Esteruer nd Cheesemn [21] with modifitions. 1,1,3,3- Tetrethoxypropne (TEP) solution of known onentrtion (2.5 to 20 μm) ws used s stndrd for quntifition, nd the result ws expressed s μm oftep.thednadmge level ws quntified using 8-hydroxy-2-deoxy-Gunosine (8-OHdG) EIA kit (Cymn Chemil, USA). 8-Hydroxy- 2-deoxy-Gunosine hydroxyl EIA stndrd (10.3 pg/ml to 30 ng/ml) ws used for quntifition, nd the result ws expressed s pg/ml Enzymi Antioxidnt Ativity Mesurement. The kidney nd liver tissue homogentes were used to mesure the tivities of superoxide dismutse (SOD [EC ]), glutthione peroxidse (GPx [EC ]), nd tlse (CAT [EC ]). Commerilly ville kits were used for SOD, CAT, nd GPx ssys (Cliohem, Germny). The protein ontent of the homogentes ws determined using the Bio- Rd Protein Assy (Brelon, Spin) [22] with ovine serum lumin s stndrd. Enzyme tivities were expressed in units per milligrm of protein. One unit of SOD tivity ws defined s the mount of enzyme tht exhiited 50% dismuttion of the superoxide rdil. One unit of CAT tivity ws defined s the mount of enzyme tht used the formtion of 1.0 nmol formldehyde per min. The unit of GPx tivity ws expressed s nnomoles of NADPH per min (lulted using n extintion oeffiient of μm 1 ) Lipid Peroxidtion Assy (LPO). The LPO ssy ws determined ording to the modified method of Kuppusmy et l. [23] sed on thiorituri id retion in whih MDA ws used s n index of lipid peroxidtion. Trihloroeti id (15%) nd thiorituri id (1%) were dded to the tissue homogentes in triplites. The mixtures were inuted in oiling wter th for 10 minutes nd were entrifuged t 6000 g for10minutestoremovethe sediments. The sorne ws red t 532 nm using spetrophotometer (Bio-Tek Instrument In., USA). 1,1,3,3- Tetrethoxypropne (TEP) solution of known onentrtion (2.5 to 20 μm) ws used s stndrd for quntifition, nd the result ws expressed s mmol/l of TEP Gene Expression Using Rel Time: RT-PCR. The totl RNA ws isolted from the dipose tissue using Amion RNAqueous-Miro Kit (Applied Biosystems, USA). The purity of reovered totl RNA ws estimted y lulting the rtio of sorne reding of 260 nm nd 280 nm. The integrity of RNA ws estimted using Agilent 2100 Bionlyzer (Applied Biosystems, USA). Purified RNA with n A 260 /A 280 rtio etween nd RIN vlues 8 10 ws further used to synthesize omplementry DNA (DNA) y polymerse hin retion (PCR) pproh. High Cpity DNA Reverse Trnsription Kit (Applied Biosystems, USA) whih ontined ll regents needed (RT uffer, dntp mix, rndom primers, Multisrie reverse trnsriptse enzyme, ndnulesefreewter)forreversetrnsription(rt)oftotl RNA to single-strnded DNA ws used in this study. The mixture ws then loded into therml yler (Eppendorf, USA), nd PCR ws rried out ording to optimized

4 4 Evidene-Bsed Complementry nd Alterntive Mediine Tle 2: Genes investigted. Numer Gene nme nd revition Assy ID Aession numer 1 Adipose triglyerides lipse (ATGL/Pnpl2) Mm m1 NM Hormone sensitive lipse (HSL/Lipe) Mm m1 NM Lipoprotein lipse (LPL) Mm m1 NM Peroxisome prolifertor-tivted reeptor γ (PPAR-γ) Mm m1 NM Sterol regultory inding protein (SREBP-1) Mm m1 NM Generl revition of genes seleted for this study nd orresponding ssy ID nd ession numer ws otined from the Applied Biosystems wesite nd NCBI dtse. Assy ID refers to the Applied Biosystems Gene Expression Assys inventoried kits with proprietry primer nd TqMn proe mix. Assy ID with Mm is referred to s Mus musulus. All Gene Expression Assy kits indited re FAM/MGB proed. therml yling onditions provided y the mnufturer. Tle 2 shows the list of genes investigted in this study nd the orresponding ession numers. Endogenous ontrol used in this study ws eukryoti 18S rrna with FAM/MGB proe. All TqMn (Applied Biosystems, USA) proes used in this investigtion were leled with FAM reporter dye t the 5 end nd MGB quenher t the 3 end. The quntifition pproh used ws the omprtive CT method, lso known s 2 ΔΔCt method [24] Sttistil Anlysis. Dt re shown s men ± SD of triplite ssys. One-wy nlysis of vrine ws used to estimte the signifint differenes etween groups. Sttistil signifine ws epted t P < Dunn s multiple rnge tests (DMRT) ws used to determine the signifint differenes etween groups. Sttgrphis Plus softwre (version 3.0, Sttistil Grphis Corp., Prineton, NJ, USA) ws used for ll sttistil nlyses. All figures were drwn using GrphPd Prism 5 (GrphPd Softwre In., Cliforni, USA). 3. Results nd Disussion 3.1. Weight nd Estimtion of β-glun Conentrtion in GE. Fresh P. sjor-ju (5.5kg) ws oiled for 8 hours to otin g of GE. The onentrtion of totl glun in GE ws 85.95% (w/w) menwhile the onentrtions of αglun nd β-glun were 5.4% (w/w) nd 80.55% (w/w) whih orresponded to 0.01% nd 1.5% in fresh mushroom, respetively [17] Effets of GE on the Chnges in Body Weight nd Serum Lipid Levels. The test ompounds (GE/metformin/vehile) were only dministered thrie week to the mie in order to void physil stress. The men food onsumption ws not signifintly different etween high-ft diet-treted mie nd high-ft diet plus GE-treted mie. Figure 1 shows the effets of GE nd metformin on ody weight hnges in the mie. The ody weight in the group grdully inresed during the 16-week period. In ontrst, the ody weight of mie in the group showed rpid inrese of ody weight. The desending order of the perentges of weight gin in eh group ws > 60 > 120 > MET > > 240 >. The mie in 60, 120, nd 240 groups hd 27.55%, 36.69%, nd 39.76% lower ody Weight (g) Weeks P < MET Figure 1: Effets of GE nd metformin on ody weight hnges in C57BL/6J mie fed on high-ft diet or norml diet. The onentrtions of GE were 60, 120, nd 240 mg/kg/dy. Metformin (2 mg/kg/dy) ws used s positive ontrol. Vlues expressed re mens ± S.D of (n = 6per group) mesurements. weight, respetively, ompred to group. MET group showed 31.90% lower ody weight ompred to group; hene, the potentil weight lowering effet of GEtretedgroupswereomprletoMETgroup.Oesity hs een ssoited with inresed triglyerides (TG), very low-density lipoprotein (VLDL), totl holesterol (TC), nd deresed high-density lipoprotein holesterol (HDL-) nd thus is lso risk ftor of rdiovsulr disese [25]. Tle 3 shows the serum lipid profile whih inludes the levels of TG, TC, HDL-, low-density lipoprotein holesterol (LDL), nd therogeni index (AI). In ontrol group, the TGlevelwsinresedy33.3%,TCinresedy40%, HDL- inresed y 34.6%, nd LDL- inresed y 171.4% ompred to those in the group, thus the mie in were onsidered to e hyperlipidemi. Menwhile, mie in 60, 120, nd 240 groups showed onsiderly reduedlevelsoftg,tc,ndldl-ompredtothe group, nd this effet ws dose dependent. The perentges of redutionfortg,tc,ndldl-levelsin60were12.5%, 7.1%, nd 60.5%, respetively. The perentges of redution for TG, TC, nd LDL- levels in 120 were 25%, 10.7%, nd 81.6%, respetively. The perentges of redution for TG,

5 Evidene-Bsed Complementry nd Alterntive Mediine 5 Tle 3: Effets of GE nd metformin on lipid profile nd AI in C57BL/6J mie fed on high-ft diet or norml diet. Groups Serum onentrtion (mmol/l) TG TC HDL- LDL- AI 0.60 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± MET 0.50 ± ± ± ± Vlues expressed re mens ± S.D of (n = 6 per group) mesurements. For sme ssy with vrious tretment groups, supersripts in the different r with different lphets ( ) were signifintly different (P < 0.05). Supersripts with sme lphets were not signifintly different etween the treted groups (P > 0.05). TG is triglyerides; TC is totl holesterol; HDL- is high-density lipoprotein holesterol; LDL- is low-density lipoprotein holesterol; AI is therogeni index. TC, nd LDL- levels in 240 were 25%, 25%, nd 94.7%, respetively. However, there were no signifint differenes (P > 0.05) in the HDL- level etween the treted groups nd ontrol group. The MET group showed deresed levels of TG (37.5%), TC (7.1%), nd LDL- (52.65%) levels nd inresed level of HDL- (1.2%) ompred to the group. The AI nd rdi risk ftor were lulted sed on the mesurement otined from the lipid nlysis. The AI ws defined y TC minus HDL- divided y HDL-, whilst the rdi risk ftor ws lulted s TC divided y HDL[26]. In this study, the AI risk preditor indies for the group were inresed ompred to those in nd GE or metformin-treted groups. In ordne to the high AI risk ftor, the rdi risk ftor ws lso elevted in the group ompred to those in nd GE or metformintreted groups. The redutions in the therogeni nd rdi risk indexes in GE-treted groups indite deresed risk of rdiovsulr disese [27]. Bet-glun hs een shown to derese LDL- nd inrese HDL- to llevite possily dyslipidemi nd redue rdiovsulr disese [28]. Ots were first found to hve holesterol-lowering effet, nd the tive omponent ws identified s et-gluns [29]. Similr serum holesterol-lowering tivity ws lso oserved in Mitke, Shiitke, nd Enokitke mushrooms [30]. The mehnism for LDL- lowering y β-gluns is speulted toinvolveileidinding.theinresedexlusionofile ids tivtes holesterol 7α-hydroxylse nd upregultes low-density lipoprotein reeptor (LDLR) nd thus inreses the trnsport of LDL- into heptoytes nd the onversion ofholesterolintoileids[31] Effets of GE on Enzymes. Tle 4 shows the effets of GE nd metformin-treted groups on liver enzymes. Inresed liver enzyme onentrtions nd tivity in the serum re onventionlly interpreted s mrker of liver dmge. In this study, the lnine trnsminse (ALT), sprtte trnsminse (AST), nd lkline phosphte (ALP) levels of mie in the group were signifintly elevted ompred to the other groups. However, there were no hnges in the glutmyl trnsferse (GGT) level etween these groups. A reent study demonstrted tht oese ptients with inresed serum TG level showed rised levels of eh of the four liver enzymes [32]. Weight redutions hve een shown to redue the liver enzyme levels [33]. The present study shows tht GE onfers protetion ginst high-ft dietmedited liver dmge Effets of GE on the Urinry Oxidtive Indies. Oxidtion produts n e found in the urine nd re onsidered to reflet lol nd systemi oxidtive stress [34]. Figures 2() 2() show the AOPP, lipid hydroperoxide, nd 8-OHdG levels in eh group during the 16 weeks of experiment. The AOPP, lipid hydroperoxides, nd 8-OHdG levels in the group grdully inresed every week, however, these oxidtive stress indies were signifintly elevted in the group ompred with ll other groups. The mie in group were oese, nd this my hve ontriuted to the inresed level of oxidtive stress indies in the nimls [35]. The AOPP nd lipid hydroperoxide levels in GE-treted groups were lower ompred to the group, nd this effet ws dose dependent. Similrly, MET lso showed derese in AOPP nd lipid hydroperoxide levels ompred to the group. The 8-OHdG level ws elevted in group, however, no signifint differenes were oserved etween ll the groups tested (P > 0.05). Studies hve shownthtelevtedlevelsofmda[36], AOPP [37], nd 8- OHdG [38] in oese nimls or humns re ssoited with severl disese onditions inluding hypertension, dietes, rdiovsulr diseses, nd renl diseses [39] Effets of GE on Enzymti Antioxidnt Levels in nd Homogentes. Fruits, vegetles, spies, hers, nd mushrooms hve een studied for their ntioxidnt properties in-vitro extensively [40, 41]. However, the demonstrtion of the ntioxidnt properties of these omponents in-vivo is sre ut is gining importne nowdys. Previously, ntioxidnt pity hs een minly ssessed in serum or plsm fter n orl intke of food infusion. Nevertheless, numerous studies hve lso suggested tht oxidtive proesses ourring in vrious tissues nd orgns in the humn ody my e ruil in the onset of metoli diseses [42]. It is reported tht, fter sorption, the ntioxidnt

6 6 Evidene-Bsed Complementry nd Alterntive Mediine Protein ronyl ontent (μm) P < Weeks 60 e d d () d 8-OHdG ( 10 3 pg/ml) d MET P > 0.05 d Lipid hydroperoxides (μm) P < Weeks () Weeks MET d d d d () MET Figure 2: Effets of GE nd metformin on () AOPP () lipid hydroperoxide, nd () 8-OHdG levels in urine smples of C57BL/6J mie fed on high-ft diet or norml diet. Vlues expressed re mens ± S.D of triplite mesurements (n =6per group). For sme ssy with vrious tretment groups, supersripts in the different r with different lphets () (e) were signifintly different (P < 0.05). Supersripts with sme lphets were not signifintly different etween the treted groups (P > 0.05). There ws no signifint differene oserved in the 8-OHdG levels etween the groups tested (P > 0.05). Tle 4: Effets of GE nd metformin on liver enzymes in C57BL/6J mie fed on high-ft diet or norml diet. Groups enzymes (mmol/l) Alnine trnsminse (ALT) Asprtte trnsminse (AST) Alkline phosphte (ALP) G-glutmyl trnsferse (GGT) 45 ± ± ± 1.2 <3 29 ± ± ± 1.1 <3 48 ± ± ± 0.9 < ± ± ± 1.4 < ± ± ± 1.3 < ± ± ± 1.3 <3 MET 39 ± ± ± 1.5 <3 GE onentrtions were 60, 120, nd 240 mg/kg/dy. Metformin (MET) is used s positive ontrol. Vlues expressed re mens ± S.D of triplite mesurements. For sme enzyme level with vrious tretment groups, supersripts in the different r with different lphets ( ) were signifintly different (P < 0.05). Supersripts with sme lphets were not signifintly different etween the treted groups (P > 0.05).

7 Evidene-Bsed Complementry nd Alterntive Mediine 7 Tle 5: Effets of GE on enzymi ntioxidnts nd MDA levels in the kidney nd liver homogentes of C5BL/6J mie fed on high-ft diet MET Groups Antioxidnt tivity (nmol/min/mg protein) GPx CAT SOD (U/mg protein) LPO (mmol/l) ± 6.3 d ± 4.5 d 0.34 ± 0.0 d 0.89 ± 0.01 e ± ± ± ± ± 6.9 d ± ± 4.6 e ± 6.7 d 0.37 ± 0.0 d 0.32 ± 0.0 d 0.83 ± 0.04 d 0.7 ± ± ± ± ± 0.04 e ± ± ± ± ± ± ± ± 0.02 de ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.05 GE onentrtions were 60, 120, nd 240 mg/kg/dy. Metformin (MET) is used s positive ontrol. Vlues expressed re mens ± S.D of triplite mesurements. For sme ntioxidnt tivity with vrious tretment groups, supersripts in the different r with different lphets ( e) were signifintly different (P < 0.05). Supersripts with sme lphets were not signifintly different etween the treted groups (P > 0.05). GPx is glutthione peroxidse; CAT is tlse; SOD is superoxide dismutse; LPO is lipid peroxidtion. ompounds re trnsferred through the lood irultion to vrious orgns [43]. In the present study, the enzymi ntioxidnt tivities nd LPO level were ssessed in the liver nd kidney homogentes (Tle 5),sine these re the key orgns in the mmmlin oxidtive metolism. The nturl ntioxidnt system onsists of series of ntioxidnt enzymes nd numerous endogenous nd dietry ntioxidnt ompounds tht ret with nd intivte ROS. The primry ntioxidnt enzymes inlude SOD, CAT, nd GPx. Menwhile, the nonenzymti ntioxidnts inlude vitmin C, vitmin E, β-rotene, redued glutthione (GSH), nd numerous phytohemils. Cells must mintin their levels of ntioxidnts, often defined s their ntioxidnt potentil, through dietry intke nd/or de novo synthesis [44, 45]. Inresed levels of ROS in ells nd tissues my t s signl to enhne the tivity nd expression of ntioxidnt enzymes. A high-ft diet is known to inrese the superoxide nion (O 2 ) rdils in the ody. Superoxide dismutse onverts the O 2 rdils to hydrogen peroxide (H 2 O 2 )whih in turn is onverted to wter y CAT nd GPx. In this study, the group showed redued levels of SOD, CAT, nd GPx tivities in the kidney nd liver homogentes ompred to the group. Wheres, GE- nd metformin-treted groups showed inresed levels of SOD, GPx, nd CAT tivities ompred to the ontrol groups ( nd ) (Tle 4). Overll, the inresed level of ntioxidnt enzyme tivities in GE- nd metformin-treted groups onferred protetion ginst oxidtive dmges in the mie, nd this speultion is supported y the ttenuted levels of oxidtive stress indies suh s AOPP nd lipid hydroperoxide levels in the urine s well s MDA level in the kidney nd liver homogentes Effets of GE on the Expression of Differentition nd Lipolysis Genes in Adipose Tissue. Adiposetissueisomplex nd tive seretory orgn tht oth sends nd reeives signls tht modulte energy expenditure, ppetite, insulin sensitivity, endorine funtion, inflmmtion, nd immunity [46]. Tle 6 shows the expression of the seleted genes involved in the differentition nd lipolysis proesses in dipose tissue. The mie fed on high-ft diet ( group) weighed more nd developed sustntilly more dipose tissue thn the mie on norml diet ( group) (Figure 1). The mie eme hyperlipidemi, nd this is typilly ssoited with oesity [47] (Tle 3). PPAR-γ nd SREBP-1 genes re the key dipose trnsription ftors tht ply importnt roles in lipogenesis [48]. These genes t oopertively nd sequentilly to trigger terminl dipoyte differentition. The PPAR-γ is expressed seletively in the dipose tissues, nd it promotes the differentition nd prolifertion of the predipoytes therey using n inrese in ft mss [49], while SREBP-1 ontrols the prodution of endogenous lignds for PPAR-γ s mehnism for oordinting the tions of these dipogeni ftors [48] nd hs een implited s eing key regultor for ftty id nd triglyeride synthesis [50]. Menwhile, LPL is the key enzyme tht regultes the disposloflipidintheody,nditsroleistohydrolyse triglyeride irulting in the lipoprotein prtiles in order to filitte the uptke ftty ids into the ells [51]. GEtreted groups hd lower expression of PPAR-γ, SREBP-1, nd LPL ompred to group. PPAR-γ protein inds to the promoter regions of dipoyte-expressed LPL gene [52], nd the ttenution of PPAR-γ gene expression in GE-treted groups ould hve ttriuted to the redued expression of LPL s well. HSL nd ATGL genes re reported to ply n importnt role in the moiliztion of stored triylglyerol (TAG) [53]. The tivtion of these genes leds to moiliztion of TAG to form glyerol nd ftty ids where HSL minly reks down TAG to form diylglyerol (DAG) whilst ATGL reksdown DAG to form monoylglyerol (MAG). Susequently, MAG is onverted to free ftty ids

8 8 Evidene-Bsed Complementry nd Alterntive Mediine Tle 6: Effets of GE on the expression of genes in dipose tissue. Genes investigted MET Lipolysis ATGL 1.34 ± ± ± ± ± 0.98 HSL 1.98 ± ± ± ± ± 1.16 Differentition LPL 1.05 ± ± ± ± ± 0.99 PPAR-γ 1.69 ± ± ± ± ± 0.16 SREBP ± ± ± ± ± 1.13 Results re expressed s fold vrition over the pproprite ontrol groups; indites fold inrese over (norml diet ontrol group), nd 60, 120, 240, nd MET indite fold inrese over (high-ft diet ontrol group). Fold vritions less thn one were expressed s negtive numers (e.g., fold vrition of 0.50 is expressed s 2.00). Vlues expressed re mens ± S.D of triplite mesurements. Sttistil signifine ws lulted sed on the men ΔCT vlues y DMRT for only mie fed with high-ft diet with or without GE. For sme gene with vrious tretment groups, supersripts in the different r with different lphets ( ) were signifintly different (P < 0.05). Supersripts with sme lphets were not signifintly different etween the treted groups (P > 0.05). nd glyerol y monoylglyerol lipse (MGL) [54]. The GEtreted groups hd signifintly upregulted expressions of HSL nd ATGL genes, nd the effet ws dose dependent. Therefore, it is fesile to suggest tht the redued weight gin in the high-ft diet fed mie treted with GE ws due to the redued dipose differentition nd inresed lipolysis in dipoytes. 4. Conlusion Previous studies hve demonstrted tht the lipid lowering potentil of β-gluns ws minly medited y either ile id inding, dely in the digestion/sorption of ft, or suppressed ppetite. However, in this study, GE prevented weight gin nd hyperlipidemi in C57BL/6J mie fed on high-ft diet y induing lipolysis nd inhiiting the differentition of dipoytes. GE lso prevented oxidtive stress used y oesity y inresing the enzymi ntioxidnt tivities, hene, GE ould serve s potentil ndidte for the mngement of oesity. Conflit of Interests The uthors delre tht they hve no onflit of interests. Aknowledgments The uthors re grteful to the University of Mly nd the Ministry of Higher Edution (MOHE), Mlysi, for providing the following grnts: RG083-09AFR nd HIR F Referenes [1] B. M. Spiegelmn nd J. S. Flier, Oesity nd the regultion of energy lne, Cell, vol. 104, no. 4, pp , [2] J. W. Yun, Possile nti-oesity therpeutis from nture- review, Phytohemistry,vol.71,no.14-15,pp ,2010. [3] H.Choi,H.Eo,K.Prketl., AwtersoluleextrtfromCuruit mosht shows nti-oesity effets y ontrolling lipid metolism in high ft diet-indued oesity mouse model, Biohemil nd Biophysil Reserh Communitions, vol. 359, no. 3, pp , [4] D. Hslm, Oesity nd dietes: the links nd ommon pprohes, Primry Cre Dietes, vol. 4, no. 2, pp , [5] G. A. Kennett nd P. G. Clifton, New pprohes to the phrmologil tretment of oesity: n they rek through the effiy rrier? Phrmology Biohemistry nd Behvior, vol. 97, no. 1, pp , [6] L. Slovek, V. Pvlik, nd B. Slovkov, The effet of siutrmine therpy on ourrene of depression symptoms mong oese ptients, Nutrition, Metolism nd Crdiovsulr Diseses,vol.18,no.8,pp.e43 e44,2008. [7] C. Zou nd J. Sho, Role of dipoytokines in oesityssoited insulin resistne, Journl of Nutritionl Biohemistry,vol.19,no.5,pp ,2008. [8] Y.Ono,E.Httori,Y.Fuky,S.Imi,ndY.Ohizumi, Antioesity effet of Nelumo nuifer leves extrt in mie nd rts, Journl of Ethnophrmology,vol.106,no.2,pp , [9] Y.W.Hung,Y.Liu,S.Dushenkov,C.T.Ho,ndM.T.Hung, Anti-oesity effets of epigllotehin-3-gllte, ornge peel extrt, lk te extrt, ffeine nd their omintions in mouse model, JournlofFuntionlFoods, vol. 1, no. 3, pp , [10] A. Goly nd J. Yrr, The link etween oesity nd type 2dietes, Best Prtie & Reserh Clinil Endorinology & Metolism,vol.19,no.4,pp ,2005. [11] S. Furukw, T. Fujit, M. Shimukuro et l., Inresed oxidtive stress in oesity nd its impt on metoli syndrome, Journl of Clinil Investigtion, vol.114,no.12,pp , [12] M. Curzio, H. Esteruer, nd G. Poli, Possile role of ldehydi lipid peroxidtion produts s hemottrtnts, Interntionl Journl of Tissue Retions, vol.9,no.4,pp , [13] V. Witko-Srst, M. Friedlnder, T. N. Kho et l., Advned oxidtion protein produts s novel meditors of inflmmtion nd monoyte tivtion in hroni renl filure, Journl of Immunology, vol. 161, no. 5, pp , [14] S. P. Wsser, Current findings, future trends, nd unsolved prolems in studies of mediinl mushrooms, Applied Miroiology nd Biotehnology,vol.89,no.5,pp ,2001.

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11 MEDIATORS of INFLAMMATION The Sientifi World Journl Gstroenterology Reserh nd Prtie Journl of Dietes Reserh Interntionl Journl of Journl of Endorinology Immunology Reserh Disese Mrkers Sumit your mnusripts t BioMed Reserh Interntionl PPAR Reserh Journl of Oesity Journl of Ophthlmology Evidene-Bsed Complementry nd Alterntive Mediine Stem Cells Interntionl Journl of Onology Prkinson s Disese Computtionl nd Mthemtil Methods in Mediine AIDS Behviourl Neurology Reserh nd Tretment Oxidtive Mediine nd Cellulr Longevity