Research Article Antidiabetic Effect of Morindacitrifolia (Noni) Fermented by Cheonggukjang in KK-A y Diabetic Mice

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1 Evidence-Bsed Complementry nd Alterntive Medicine Volume 2012, Article ID , 8 pges doi: /2012/ Reserch Article Antidietic Effect of Morindcitrifoli (Noni) Fermented y Cheonggukjng in KK-A y Dietic Mice So-Young Lee, So-Lim Prk, Jin-Tek Hwng, Sung-Hun Yi, Young-Do Nm, nd Seong-Il Lim Fermenttion nd Functionlity Reserch Group, Kore Food Reserch Institute, Gyeonggi-Do, Sungnm-Si , Repulic of Kore Correspondence should e ddressed to Seong-Il Lim, silim@kfri.re.kr Received 19 Mrch 2012; Accepted 17 July 2012 Acdemic Editor: Pierre Kmtchouing Copyright 2012 So-Young Lee et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Antidietic effects of Morind citrifoli (k Noni) fermented y Cheonggukjng (fst-fermented soyen pste) were evluted using T2DM (type 2 dietes mellitus) murine model. Six-week-old KK-Ay/TJcl mice were rndomly divided into four groups: (1) the dietic control (DC) group, provided with norml mouse diet; (2) the positive control (PC) group, provided with functionl helth food diet; (3) the M. citrifoli (MC) group, provided with n MC-sed diet; (4) the fermented M. citrifoli (FMC) group, provided with n FMC-sed diet. Over testing period of 90 dys, food nd wter intke decresed significntly in the FMC nd PC groups compred with the DC group. Blood glucose levels in the FMC group were mg/dl fter 90 dys, while those in the control group were over 400 mg/dl fter 20 dys. In ddition, FMC supplementtion reduced glycosylted hemogloin (HA1c) levels, enhnced insulin sensitivity, nd significntly decresed serum triglycerides nd low-density lipoprotein (LDL) cholesterol. Furthermore, fermented M. citrifoli 70% ethnolic extrct (FMCE) ctivted peroxisome prolifertor-ctivted receptor-(ppar-) γ nd stimulted glucose uptke vi stimultion of AMP-ctivted protein kinse (AMPK) in cultured C2C12 cells. These results suggest tht FMC cn e employed s functionl helth food for T2DM mngement. 1. Introduction Dietes mellitus (DM) is chronic disorder chrcterized y hyperglycemi, with disturnces in crohydrte, lipid, nd lipoprotein metolism stemming from defects in insulin secretion nd insulin ction, or oth. Aout 90 95% of ll cses of dietes mellitus cn e ttriuted to type 2 dietes mellitus (T2DM, or dult-onset dietes). T2DM is cused y the nonresponsiveness of cells to insulin rther thn y defective insulin production. If not treted, T2DM cn result in numerous severe complictions, including coronry rtery disese, peripherl vsculr disese, nd even deth due to hyperglycemi [1]. T2DM currently ffects more thn 246 million people worldwide, nd it is estimted tht the numer of ptients with dietes mellitus will increse to 360 million y 2030 [2]. Indeed, out 5 10% of the totl helth cre udget hs een used for tretment nd mngement of T2DM in mny countries [3]. Mngement of T2DM usully egins with chnge in diet nd exercise. Nonetheless, most ptients ultimtely require phrmcotherpy, such s injected insulin nd/or orl ntidietic drugs. Mny orl ntidietic drugs re ville for the tretment nd control of the symptoms of T2DM, such s sulfonylure gents, igunides (e.g., metformin), thizolidinedione (TZD) drugs (lso known s glitzones),α-glucosidse inhiitors, nd glucgon-like peptide-1 (GLP-1) inhiitors. However, these drugs cn cuse serious dverse effects, mong them hypoglycemi, heptic toxicity, weight increse, physconi (enlrgement of the domen), nd lctic cidosis [4]. Thus, there is n incresing demnd for nturl products (nd in prticulr, nutrceuticls) tht hve fewer dverse effects nd higher ntidietic efficcy. Mny studies hve een conducted on nturl products tht efficiently nd sfely reduce lood glucose levels in dietic ptients, nd severl dietes mellitus hundred plnts hve reported eneficil effects. For exmple, Lgerstroemi specios [5], Gymnem sylcestre [6], Mori folium [7], nd Psidiium gujv [8] re currently sold commercilly s complementry nd lterntive medicinl mterils for the tretment of dietes.

2 2 Evidence-Bsed Complementry nd Alterntive Medicine Weighing of MC powder Heting (hot kneder) 82.5 ± 2.5 C, 4 h Addition of Fermenttion 1% Cheonggukjng 1) 25 powder nd wter C, 40 dys Pckging Second drying (Moisture <3%) 90 C, 2 h First drying (Moisture <8%) 82.5 ± 2.5 C, 2 h Figure 1: Process of fermenttion. MC powder ws first weighed nd heted. Next, Cheonggukjng ws dded to initite fermenttion. Cheonggukjng is type of fst-fermented soyen pste tht is mde y cooking the legumes until they re tender nd then incuting them with diverse Bcillus species in wrm plce. In this study, the dominnt Bcillus microes were Bcillus sp. (KCTC 11351BP), Bcillus sutilis (KCTC 11352BP), Bcillus sonolensis (KCTC 11354BP), nd Bcillus circulns (KCTC 11355BP). Morind citrifoli, lso clled Noni, is tropicl plnt tht grows widely throughout the Pcific. M.citrifolhs trditionlly een used for the prevention, nd tretment of rthritis, circultory wekness, dietes, cncer, nd skin inflmmtion [9 12], nd is widely known s helth food nd dietry supplement. A growing numer of phrmcologicl studies on M. citrifoli hve een pulished in recent yers, ut mny of the reports re only ccessile s congress strcts. In ddition, few peer-reviewed reserch mnuscripts re ville tht discuss the ntidietic ctivity of M. citrifoli, even though M. citrifoli hs een customrily used to tret dietes. In fct, only single study hs presented evidence, limited t est, for the ntidietic effect of M. citrifoli fruit juice in n niml model of T2DM [13]. The present study therefore focused on the ntidietic properties of M. citrifoli nd fermented M. citrifoli (FMC) in KK-Ay oese/dietic mice, which re fflicted with geneticlly induced form of dietes tht mnifests itself s hyperinsulinemi, result of insulin resistnce [14].M. citrifoli ws fermented using Cheonggukjng, trditionl fstfermented Koren soyen pste tht ws itself fermented using vrious Bcillus spp. After demonstrting the efficcy of FMC, we then imed to elucidte the ntidietic mechnisms of FMC. 2. Mterils nd Methods 2.1. Preprtion of FMC. Fifteen kilogrm of dried M. citrifoli (MC) fruitpowder (Bosewoo Seoul, Kore) ws heted in hot kystion ( L, Dehn Food Mchine, Inc., Gimpo, South Kore) t 82.5±2.5 Cfor4h.Cheonggukjng (1%, employed to initite fermenttion) contining soyens, Bcillus sp. (KCTC 11351BP), Bcillus sutilis (KCTC 11352BP), Bcillus sonolensis (KCTC 11354BP), Bcillus circulns (KCTC 11355BP), nd wter (40% w/w) were dded to the MC powder. For ripening, the mixture ws moved to rrels (Jpnese cedr, 10 L (40 35 cm)) tht contined nine holes for ventiltion. The powder ws then fermented t 25 C for 40 dys. Fermented MC (FMC) powder ws dried t 82.5±2.5 C for 2 h, nd then dried t 90 C for nother 2 h to decrese the moisture content of the finl product to <3% nd to inctivte microorgnisms relted with fermenttion. The flow digrm for this process is shown in Figure 1.Dried FMC ws pckged nd stored t 20 C prior to experimenttion Extrction of FMC. ThedriedndmilledMCndFMC were dded to wter or 70% ethnol (10X the smple mount w/v) with stirring for 24 h t 25 C. The extrcts were centrifuged for 10 min t 3000 rpm, nd the superntnts were filtered through Whtmn no. 5 filter pper. The filtrtes were evported y rotry evportor (RE200; Ymto Co., Tokyo, Jpn) under reduced pressure t temperture lower thn 40 C. Next, the resulting concentrtes were freeze-dried ndstoredt 20 C prior to experimenttion Animls, Diets, nd Tretments. Twenty-eight KK-Ay/ TJcl mice (5 weeks old, mle) with genetic oese type II dietes were purchsed from Cle Jpn Inc. (Tokyo, Jpn). After cclimting for 1 week, the mice were divided into four groups of seven nimls ech, s follows: dietic control nimls (DC group), n-fed nimls (positive control (PC) group), MC-fed nimls (MC group), nd FMCfed nimls (FMC group). Bn (Lgerstroemi specios) leves re commercilly sold s functionl helth food intended for the regultion of lood glucose levels. Dietic control nimls were freely fed on norml mouse diet (AIN-93G food). PC, MC, nd FMC groups were freely fed on AIN-93G lended with freeze-dried n, MC, or FMC powder (0.4%). All mice were mintined in the sme room under conventionl conditions with regulr 12 h light/drk cycle nd temperture nd reltive humidity mintined t 23 ± 2 Cnd50± 5%, respectively. At the end of the experimentl period (90 dys), the nimls were fsted overnight, nesthetized with n intrperitonel injection of zoletil 50 (30 mg/kg), nd scrificed for the procurement of tissue smples. The study ws pproved y the Ethics Committee of the Experimentl Animl Reserch Lortory, Kore Food Reserch Institute (pprovl code, KFRI-M-10020) Anlysis of Blood Glucose Levels. Whole lood ws smpled from the til vein of mice without fsting t fixed time

3 Evidence-Bsed Complementry nd Alterntive Medicine 3 every 10 dys over period of 90 dys, nd the lood smple ws nlyzed using the glucose oxidse method, which utilizes lood glucose sensor strip (ACCU-CHEK Sensor, Roche, Bsel, Switzerlnd) Anlysis of Glycosylted Hemogloin (HA1c) Levels. At the end of the 90-dy experimentl period, mice were sujected to fsting overnight. Next, fsting whole lood smple (4 μl) ws otined vi retro-oritl sinus puncture. The lood smple ws injected into n A1c test crtridge nd then nlyzed y using Hemogloin A1c Testing Anlyzer (Asn Phrmceuticl, Seoul, Kore) Serum Anlysis. After90dysofdietrytretment,KK- Ay/TJcl mice were scrificed, nd fsting lood smples were collected nd centrifuged t 3000 rpm for 10 min to otin serum smples for serum chemicl nlysis. Triglyceride, totl cholesterol, high-density lipoprotein (HDL)- cholesterol, nd low-density lipoprotein (LDL) cholesterol were mesured y n ADVIA Automted Hemtology Anlyzer (ADVIA 1650, Byer, USA). Fsting serum glucose levels were ssessed using glucometer, which employed the glucose oxidse/peroxidse rection. This rection llows the colorimetric detection of H 2 O 2 for the estimtion of free glucose in the serum. Fsting serum insulin levels were mesured with murine insulin TMB (tetrmethyl enzidine) enzyme-linked immunosorent ssy (ELISA) kit (Shiygi, Gunm, Jpn). A homeostsis model ssessment of insulin resistnce (HOMA-IR), n index of insulin resistnce, ws clculted using fsting insulin nd glucose concentrtions ccording to the following eqution: HOMA-IR ( ( ) ) fsting insulin μiu/ml fsting glucose (nmol/l) = (1) 2.7. Muscle Differentitionnd Glucose Uptke Assy. C2C12 cells were grown in Dulecco s modified egle medium (DMEM) supplemented with 10% fetl ovine serum (FBS), penicillin (120 unit/ml), nd streptomycin (75 μg/ml) in 5%CO 2 tmosphere. Differentition medium (DMEM medium with 1% horse serum) ws dded to confluent C2C12 cells for 4 dys. The cells were then incuted overnight in low-glucose, serum-free medium followed y 24-h tretment in the presence of 2-[N-(7-nitroenz-2-ox-1, 3-dizol-4-yl) mino]-2-deoxy-d-glucose (2-NBDG). The fluorescence ws red t excittion nd emission wvelengths of 485 nd 535 nm Western Blot Anlysis. C2C12-derived myotues were hrvested with lysis uffer (50 mm Tris HCl, 1% Triton X- 100, 0.5% sodium deoxycholte, 150 mm NCl, 1 mm EDTA, 1 mm PMSF, 1 mm sodium orthovndte, 1 mm NF nd 0.2% protese inhiitor cocktil; ph 7.2). Proteins (40 μg) were sujected to sodium dodecyl sulfte polycrylmide gel electrophoresis (SDS-PAGE) followed y trnsfer onto nitrocellulose memrne. Protein phosphoryltion or expression ws detected y Western lot nlysis using primry ntiodies specific for phospho-amp protein kinse (AMPK) nd β-ctin (Cell Signling Technology, Beverly, MA) Peroxisome Prolifertor-Activted Receptor (PPAR)-γ Trnscriptionl Activity Assy. Plsmids for PPAR-γ or retinoid X receptor (RXR)-α, s well s luciferse reporter vectors contining PPAR-response elements (PPREs), were the generous gift of Dr. Je Bum Kim (Seoul Ntionl University, South Kore). For the PPAR-γ ssy, humn emryonic kidney (HEK) 293 cells were cotrnsfected with PPAR-γ or RXRα plsmids nd PPAR-PPRE luciferse reporter vectors y using trnsfection regent (Qigen, Vlenci, CA) for 18 h. Cells were then treted with fermented Morind citrifoli ethnol extrct (FMCE) for 24 h, nd PPAR-γ ctivity ws mesured vi Luciferse Assy System (Promeg, Mdison, WI) Sttisticl Anlysis. Numericl dt re expressed s mens ± the stndrd devition using SAS 9.1softwre. The significnce of differences etween experimentl conditions ws nlyzed using one-wy nlysis of vrince (ANOVA) followed y Duncn s multiple rnge test. Vlues of P<0.05 were considered significnt. 3. Results 3.1. Blood Glucose nd HA1c Levels. Figure 2 shows nonfsting lood glucose levels mesured every ten dys over the experimentl period of 90 dys. Blood glucose levels were remrkly incresed in the DC group compred with the PC (n fed) group fter 20 dys, nd were mintined t more thn 400 mg/dl throughout the remining 70 dys of the experiment. Blood glucose levels in the MC group lso incresed to the sme extent s tht oserved for the DC group during the first 40 dys of dietry evlution. After 40 dys, the levels grdully decresed to out 300 mg/dl t 70 dys nd were then mintined t this level for the next 20 dys. In contrst, lood glucose levels did not increse in the FMC group reltive to the PC group, nd the levels were mintined t mg/dl during the experimentl period. The lood glucose levels in the FMC group were ctully lower thn those in the PC group. Figure 3 shows HA1c levels t 90 dys. The men H1Ac level in the FMC group ws 7.62%, which ws significntly lower thn the H1Ac levels in the other three groups. The corresponding vlues were 9.45% in the DC group, 8.96% in the PC group, nd 8.96 in the MC group Serum Glucose nd Insulin Levels. Serum glucose (Figure 4) nd insulin (Figure 5) levels were significntly decresed in the PC, MC, nd FMC groups t 90 dys reltive to the DC group. The FMC group in prticulr showed decresed serum glucose nd insulin levels y out 36% nd 45%, respectively (Figures 4 nd 5). The HOMA-IR index for

4 4 Evidence-Bsed Complementry nd Alterntive Medicine 600 Blood glucose level (mg/dl) Dys DC PC MC FMC Figure 2: Effect of FMC on lood glucose levels in KK-Ay mice during the 90 dys of experimenttion. DC: dietic control, PC: positive control (n), MC: Morind citrifoli,fmc:fermentedmorind citrifoli. Ech dt point represents the men ± SD (n = 7) HA1c (%) Fsting glucose levels (mg/dl) c 6 DC PC MC FMC Figure 3: Effect of FMC on HA1c levels (%) in KK-Ay mice fter 90 dys. DC: dietic control, PC: positive control (n), MC: Morind citrifoli,fmc:fermented Morind citrifoli. Ech r represents the men ± SD (n = 7). Brs with different letters represent vlues tht re significntly different from ech other sed on onewy ANOVA nd the Duncn s multiple rnge test (P <0.05). 100 DC PC MC FMC Figure 4: Effect of FMC on serum fsting glucose levels in KK-Ay mice fter 90 dys. DC: dietic control, PC: positive control (n), MC: Morind citrifoli, FMC:fermentedMorind citrifoli. Ech r represents the men ± SD (n = 7). Brs with different letters represent vlues tht re significntly different from ech other sed on one-wy ANOVA nd the Duncn s multiple rnge test (P <0.05). the PC group, MC group, nd FMC group were lso ll significntly decresed compred with the control. This result ws especilly evident in the FMC group (Figure 6) Serum Lipid Profiles. Triglyceride levels were significntly decresed in the MC ( mg/dl) nd FMC ( mg/dl) groups compred with the DC ( mg/ dl) nd PC ( mg/dl) groups (Tle 1). Triglyceride levels in the MC nd FMC groups decresed y out 29% nd 37%, respectively, reltive to the DC group. No significnt differences were oserved in totl cholesterol etween the groups; however, HDL-cholesterol levels in the MC nd FMC groups were significntly higher thn those oserved in the DC group, wheres LDL-cholesterol levels in the FMC group were significntly lower thn those oserved in the other three groups (Tle 1) Glucose Uptke. To estlish the potentil ntidietic mechnisms of FMC, we evluted the effect of n ethnol

5 Evidence-Bsed Complementry nd Alterntive Medicine 5 Tle 1: Effect of FMC on totl serum cholesterol, HDL-cholesterol, LDL-cholesterol nd triglyceride levels (mg/dl) in KK-Ay mice fter 90 dys. Totl cholesterol HDL-cholesterol LDL- Cholesterol Triglycerides Dietic control (DC) ± ± ± ± Positive control (PC) ± ± ± ± MC ± ± ± ± FMC ± ± ± ± Ech vlue represents the men ± SD (n = 7). - Mens with different superscripts in the sme column re significntly different from ech other (P < 0.05, Duncn s multiple rnge test). Serum insulin (μ IU/mL) c HOMA-IR c c 50 DC PC MC FMC Figure 5: Effect of FMC on serum insulin levels in KK-Ay mice fter 90 dys. DC: dietic control, PC: positive control (n), MC: Morind citrifoli, FMC:fermentedMorind citrifoli. Ech rrepresentsthemen± SD (n = 7). Brs with different letters represent vlues tht re significntly different from ech other sed on one-wy ANOVA nd the Duncn s multiple rnge test (P <0.05). 0 DC PC MC FMC Figure 6: Effect of FMC on the insulin resistnce index HOMA score (HOMA-IR) fter 90 dys. DC: dietic control, PC: positive control (n), MC: Morind citrifoli, FMC: fermented Morind citrifoli. Echrrepresentsthemen± SD (n = 7). Brs with different letters represent vlues tht re significntly different from ech other sed on one-wy ANOVA nd the Duncn s multiple rnge test (P <0.05). extrct of FMC (FMCE) on 2-NBDG uptke. FMCE significntly stimulted the uptke of 2-NBDG into C2C12-derived myotues in dose-dependent mnner (Figure 7). When the cells were treted with FMCE t concentrtions of 200 nd 400 μg/ml, FMCE stimulted 2-NBDG uptke y 1.6-fold nd 2.2-fold, respectively, compred with untreted control cells. On the other hnd, FMCW (FMC wter extrct), MCW (MC wter extrct), nd MCE (MC ethnol extrct) did not stimulte 2-NBDG uptke. Rosiglitzone ws employed s control ecuse it hs een shown to stimulte glucose uptke; however, it ws less effective thn FMCE in this effect ssy PPAR-γ Agonist Activity. As shown in Figure 8, FMCE stimulted the trnscriptionl ctivity of PPAR-γ in dosedependent mnner s ssessed y using luciferse ssy, ut FMCW, MCW nd MCE did not. The PPAR-γ gonist ctivity of FMCE ws comprle or even etter thn tht of rosiglitzone, known commercil PPAR-γ gonist Phosphoryltion of AMPK. To exmine whether FMCE cn increse the phosphoryltion of AMPK, western lotting nlysis ws performed. As shown in Figure 9, FMCE mrkedly stimulted the phosphoryltion of AMPK in C2C12-derived myotues in dose-dependent mnner. In contrst, FMCW did not induce AMPK phosphoryltion (dt not shown). 4. Discussion The results of this study clerly indicte tht the ntidietic effects of FMC were stronger thn those of the commercilly ville, functionl helth food, n. For exmple, lood glucose levels re n importnt indictor of dietic sttus, nd n FMC-sed diet ws more effective in reducing lood (Figure 2)nd serum(figure 4) glucose levels in KK-Ay mice thn norml diet. As reported in previous findings [13], MC lso grdully reduced lood glucose levels fter 40 dys of experimenttion; however, FMC ws fr more efficient in this regrd compred with MC. It cn e presumed tht the ntidietic ctivity of M. citrifoli ws mrkedly improved through solid sustrte fermenttion, ioconversion technique employed to increse the function of nturl products. This result would e in greement with recent studies [15, 16] demonstrting tht the phrmcologicl effects of medicinl plnts re incresed through solid sustrte fermenttion.

6 6 Evidence-Bsed Complementry nd Alterntive Medicine NBDG uptke (fold induction) PPAR-γ trnscriptionl ctivity (fold induction) None Rosi 80 μm FMCW FMCE MCW MCE 0 None Rosi 20 μm FMCW FMCE MCW MCE 50 μg/ml 100 μg/ml 200 μg/ml 400 μg/ml Figure 7: Effect of FMC extrct on glucose uptke in C2C12 cells. FMCW: fermented Morind citrifoli wter extrct, FMCE: fermented Morind citrifoli 70% ethnol extrct, MCW: Morind citrifoli wter extrct, MCE: Morind citrifoli 70% ethnol extrct. Rosi: rosiglitzone. Ech r represents the men ± SD (n = 7). 50 μg/ml 100 μg/ml 200 μg/ml 400 μg/ml Figure 8: PPAR-γ gonist ctivity of FMCE compred with FMCW, MCE nd MCW. FMCW: fermented Morind citrifoli wter extrct, FMCE: fermented Morind citrifoli 70% ethnol extrct, MCW: Morind citrifoli wter extrct, MCE: Morind citrifoli 70% ethnol extrct. Rosi: rosiglitzone. Ech r represents the men ± SD. Glycosylted hemogloin is considered to e the goldstndrd indictor for the ccurte nd relile mesurement of fsting glucose. Fsting glucose levels re strongly ssocited with the extent of mient glycemi during the 3-month period prior to fsting, s well s with the degree of protein glyction. Prlleling the results for lood glucose levels, the mount of glycosylted hemogloin in the FMC group ws lower thn tht in the other test groups (Figure 3). Hence, FMC cn ssist in the mintennce of low lood glucose levels nd the control of lood glucose over the long term. This hypoglycemic ction of FMC ws ssocited with decrese in oth insulin content (Figure 5) nd insulin resistnce (Figure 6). In the control group, insulin content in ddition to lood glucose levels ws high reltive to the other groups, prticulrly the FMC group. This is indictive of hyperinsulinemi nd hyperglycemi cused y insulin resistnce in DC mice. Notly, the HOMA-IR index (n index of insulin resistnce) ws significntly decresed in the FMC group compred with the DC group. The HOMA-IR index provides simple method to mesure insulin sensitivity, nd its use is widespred in oth clinicl nd niml studies [17]. Insulin resistnce, or the filure of norml insulininduced ctions in mjor tissues including the liver, ft, nd skeletl muscle, plys key role in the pthophysiology of T2DM. A primry feture of insulin resistnce is the reduction of glucose uptke into muscle cells. We therefore used C2C12 skeletl muscle cells to evlute whether FMC increses glucose uptke. Our results indicte tht FMCE stimulted uptke of 2-NBDG into terminlly differentited C2C12-derived myotues in dose-dependent mnner (Figure 7). Thus, stimultion of glucose uptke my e one mechnism y which FMC improved insulin resistnce in KK-Ay mice. MCW nd MCE lso incresed glucose uptke into muscle cells; however, the effects of these extrcts were lower thn the effect of FMCE. These results indicte tht the glucose disposl rte medited y MC in muscle cells ws incresed y solid sustrte fermenttion. Recently, mny reserchers hve reported tht vrious plnt-derived flvonoids, nthrquinones, nd terpenes stimulte glucose uptke in cells [18 20], reducing insulin resistnce. In ddition, insulin resistnce hs een reported to e ssocited with inflmmtion nd oxidtive stress [21]. Consequently, compounds such s flvonoids nd lignns, which possess powerful ntiinflmmtory nd ntioxidnt properties, my e useful in the control of T2DM. It hs een reported tht M. citrifoli contins numerous nthrquinones, flvonol glycosides nd terpenoids, which possess ntioxidnt, ntiinflmmtory nd ntidyslipidemic effects [22 24]. Therefore, these compounds were likely incresed in FMC y fermenttion of MC, leding to t lest some of the ntidietic ctions of FMC. However, further detiled study will e required to identify ntidietic compounds in FMC. AMPK functions s sensor of cellulr energy tht cn e ctivted y glucose deprivtion nd high AMP-to-ATP rtios [25]. There re indictions tht the ctivtion of this enzyme is eneficil for the tretment nd prevention of T2DM nd metolic syndrome [26, 27], ecuse its phosphoryltion leds to incresed glucose uptke nd lipid oxidtion in muscle cells [28, 29]. To investigte whether the effect of FMC on glucose uptke ws medited through AMPK ctivtion, C2C12-derived myotues were treted with FCME t vrious concentrtions. As shown in Figure 9, FMCE dose-dependently incresed the phosphoryltion of AMPK. The ctions of FMCE were similr to those

7 Evidence-Bsed Complementry nd Alterntive Medicine 7 P-AMPK FMCE (μg/ml) AICAR strong support for the use of FMC for the tretment of dietes. However, high-qulity clinicl studies re essentil to determine the optiml conditions for complementry or lterntive tretment in dietic ptients. β-ctin Figure 9: Effect of FMCE on the stimultion of AMPK ctivity s ssessed y levels of P-AMPK. β-ctin ws used s loding control. FMCE: fermented Morind citrifoli 70% ethnolextrct. Acknowledgments This study ws supported y Kore Institute for Plnning nd Evlution for Technology in Food (ipet), Kore Ministry for Food, Agriculture, Forestry nd Fisheries, nd GeonNong Nture Co. Ltd. (Mun, Kore). References of AICAR (5-mino-4-imidzolecroxmide rioside), n AMPK-specific ctivtor. These dt re in greement with recent reports y Lee et l. [30] nd Benhddou-Andloussi et l. [31], which demonstrted n increse in cellulr glucose uptke s result of AMPK ctivtion. PPAR-γ is trnscription fctor tht regultes gene expression in the liver, dipose tissue, vsculr endothelium, nd muscle [32]. PPAR-γ cn improve insulin sensitivity nd glucose tolernce vi the regultion of lipid storge, glucose homeostsis, nd dipokine production [33, 34]. We found tht FMCE functioned s PPAR-γ gonist to increse PPAR-γ-dependent luciferse ctivity in vitro. This suggests tht FMC cts on PPAR-γ s well s AMPK to ugment glucose uptke. FMCE (400μg/mL) showed enhnced PPARγ gonist reltive to the TZD drug rosiglitzone (20 μm). TZDs re widely used s PPAR-γ gonists in the tretment of dietes, nd TZD-medited ctivtion of PPAR-γ results in mrked improvement in lood glucose levels nd insulin sensitivity [35]. PPAR-γ gonists lso significntly decrese totl cholesterol nd triglyceride in type 2 dietic ptients nd model nimls [36, 37]. The current results indicte tht FMCcnnoweconsiderednewtypeofPPAR-γ gonists. Hypertriglyceridemi nd hypercholesterolemi re centrl components of dietic dyslipidemi, occurring in 20 60% of ll dietic ptients. Dyslipidemi chrcterized y high plsm levels of totl cholesterol nd triglycerides, in ddition to low plsm levels of HDL cholesterol, is mong the mny risk fctors for therosclerotic crdiovsculr disese. The ltter is in turn mjor cuse of moridity in T2DM ptients [38]. In this study, dietry supplementtion with FMC cused significnt decrese in serum triglycerides nd LDL-cholesterol nd concomitnt increse in HDLcholesterol. This improvement of the lipid profile my e ttriuted to the PPAR-γ gonist ctivity of FMC, t lest in prt. In summry, the decrese in lood glucose level medited y FMC ws ssocited with significnt reduction in insulin resistnce, s reveled y the plsm insulin content nd lood glucose levels in vivo in KK-Ay mouse model of T2DM. This enhnced insulin sensitivity ws relted to elevted glucose disposl rtes in C2C12 myotues vi the ctivtion of PPAR-γ nd AMPK. 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9 MEDIATORS of INFLAMMATION The Scientific World Journl Gstroenterology Reserch nd Prctice Journl of Dietes Reserch Interntionl Journl of Journl of Endocrinology Immunology Reserch Disese Mrkers Sumit your mnuscripts t BioMed Reserch Interntionl PPAR Reserch Journl of Oesity Journl of Ophthlmology Evidence-Bsed Complementry nd Alterntive Medicine Stem Cells Interntionl Journl of Oncology Prkinson s Disese Computtionl nd Mthemticl Methods in Medicine AIDS Behviourl Neurology Reserch nd Tretment Oxidtive Medicine nd Cellulr Longevity