Anti-Diabetic Effects of Actinidia arguta Polyphenols on Rats and KK-A y Mice

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1 Food Sci. Technol. Res., 17 (2), , 2011 Anti-Dibetic Effects of Actinidi rgut Polyphenols on Rts nd KK-A y Mice Shizue Kurkne 1,2*, Noriko Ymd 2, Hideyo Sto 3 nd Kihru Igrshi 3 1 Course of the Science of Bioresource, The United Grdute School of Agriculturl Science, Iwte University, Moriok, Iwte , Jpn 2 Deprtment of Helth nd Nutrition, Yonezw Women s Junior College of Ymgt Prefecture, Yonezw, Ymgt , Jpn 3 Deprtment of Bioresource Engineering, Fculty of Agriculture, Ymgt University, Tsuruok, Ymgt , Jpn Received September 7, 2010; Accepted November 24, 2010 The polyphenol frction of Actinidi rgut (AP) inhibited α-glucosidse ctivity in vitro. The orl dministrtion of AP with mltose nd strch suppressed postprndil hyperglycemi ssocited with the intke of the respective sugrs by rts. The re under the curve (AUC) of blood glucose in the orl glucose tolernce test (OGTT) of type 2 dibetic KK-A y mice, fed AP for 6 weeks, significntly decresed when compred with tht of the control group. The frction contining isoquercitrin nd hyperoside, which ws prepred from AP, showed stronger inhibitory ctivity for mltse. The blood glucose levels in OGTT of KK-A y mice fed isoquercitrin for 4 weeks tended to be lower t 60 min fter the dministrtion of glucose. These results suggested tht AP hs ntidibetic effects, nd isoquercitrin, component of AP, my be useful in preventing type 2 dibetes mellitus. A decrese in the G6Pse ctivity nd increse in the ACO or CPT ctivities in the liver of KK-A y mice fed AP or isoquercitrin suggested tht the suppression of gluconeogenesis nd the enhncement of β-oxidtion of lipids, s well s the inhibition of mltse by AP nd isoquercitrin, might lso be relted to their nti-dibetic effects. Keywords: Actinidi rgut, isoquercitrin, α-glucosidse, dibetes, glucose tolernce Introduction Type 2 dibetes mellitus is component of metbolic syndrome, defined s bnorml glucose tolernce with glucose/lipid metbolism bnormlity due to impired insulin secretion or insulin resistnce. In those with metbolic syndrome, glucose/lipid metbolism bnormlity my be ttributed to insulin resistnce resulting from n ccumultion of viscerl ft (Reven, 1995). Enlrged dipocytes secrete tumor necrosis fctor-α nd free ftty cid decreses insulin sensitivity in peripherl orgns nd thereby induces hyperglycemic stte (Dresner et l., 1999; Hotmisligil et l., 1994; Reven, 1988), which in turn further stimultes insulin secretion, inititing vicious cycle tht leds to dibetes mellitus. As the number of dibetes mellitus nd metbolic syndrome sufferers is incresing every yer, the effective prevention of dibetes mellitus is n importnt issue to be ddressed on worldwide bsis. One pproch in the prevention or tretment of dibetes *To whom correspondence should be ddressed. E-mil: kurknes@yhoo.co.jp mellitus is the inhibition of postprndil hyperglycemi. More specificlly, the bsorption of crbohydrte in food is inhibited or delyed to prevent n elevtion in the blood glucose level, nd the rte of the secretion of insulin is slowed down to prevent the development or worsening of dibetes mellitus. To inhibit postprndil hyperglycemi, α-glucosidse inhibitors such s crbose or voglibose re used in clinicl setting (Hnefeld, 1998; Mtsumoto et l., 1998; Okd et l., 1996; Puls et l., 1977). One study reported tht 48-week tretment with voglibose resulted in 40.5% prevention of type 2 dibetes mellitus in those with bnorml glucose tolernce (Kwmori et l., 2009). Since guv lef polyphenols nd ycon polyphenols were found to inhibit α-glucosidses (Aybr et l., 2001; Deguchi nd Miyzki, 2010; Miur, 2006), the demnd for food-derived components free of dverse effects, s opposed to the use of phrmceuticls, is incresing to reduce glucose bsorption in dily life. The inhibition of glucose bsorption lso contributes to the inhibition of lipid synthesis nd thus the prevention of obesity nd dibetes. Actinidi rgut belongs to the fmily Actinidicee, nd

2 94 grows in intermedite nd mountinous res. There hve been few reports on the function of A. rgut, except for the ntillergic nd nti-inflmmtory effects of A. rgut extrct PG102 (Kim et l., 2009; Prk et l., 2005) nd for the prolifertion promoting effects of (+) ctechin nd ( ) epictechin from the stems of A. rgut on bone mrrow cells (Tkno et l., 2003). In determining the functionlity of A. rgut, we focused on polyphenols nd investigted whether they could inhibit glucose bsorption nd/or the elevtion of postprndil blood glucose levels. As vriety of polyphenols re contined in A. rgut, the mjor components with α-glucosidse inhibitory ctivity nd ntidibetic effects were lso investigted. Additionlly, heptic enzyme ctivities were ssyed to further consider the mechnistic ction of AP nd its components. Mterils nd Methods Preprtion of AP A. rgut ws extrcted with hot methnol (MeOH) nd concentrted in vcuo. The concentrted solution ws dded to chloroform, H 2 O, nd MeOH (1/2/1, v/v/v), nd then shken. The queous MeOH lyer ws evported to remove MeOH, plced on Diion HP-20 column (Mitsubishi Ksei, Tokyo, Jpn), nd subsequently wshed with distilled wter. Polyphenols dsorbed on the Diion HP-20 were eluted with MeOH, evported in vcuo, nd then used s the polyphenol frction (AP). Determintion of mltse nd sucrse inhibitory ctivities The ssy procedure ws performed ccording to the method described by Deguchi et l. (Deguchi et l., 1998) with modifictions. Rt intestinl cetone powder (Sigm Chemicl Co., St. Louis, USA) ws the source of mltse nd sucrse. The rt intestinl cetone powder ws homogenized with 50 mm mlete buffer t ph 6.9 (0.1 g/ml) nd then centrifuged t 1,750 g for 10 min t 4. The superntnt ws used s crude enzyme solution nd diluted 20-fold with 50 mm mlete buffer (ph 6.9) for the mesurement of mltse ctivity or 2-fold for the mesurement of sucrse ctivity. One hundred microliters of 2% mltose or 2% sucrose solution ws pre-incubted with 100 µl of smple solution for 5 min t 37. Ech enzyme solution (100 µl) ws dded to the mixture to strt the rection. The enzymtic rection ws llowed to proceed t 37 for 90 min, before being stopped by heting t 100 for 10 min. The solution ws then kept in n ice bth. The glucose relesed ws determined using commercil ssy kit (Glucose C II - test, Wko Pure Chemicls, Osk, Jpn). The finl concentrtion of n inhibitor in the rection mixture tht inhibited 50% of the enzyme ctivity under the foregoing ssy conditions ws defined s the IC 50 vlue. Identifiction of the ctive components nd nlysis of S. Kurkneet l. AP by HPLC AP ws dissolved in MeOH nd nlyzed by HPLC with diode rry detection (HPLC-DAD) on Develosil C30-UG-5 column (4.6 i.d. 250 mm, Nomur Chemicl, Aichi, Jpn). A liner grdient of 0 100% of solvent B (40% cetonitrile) in solvent A (5% cetonitrile/1% cetic cid) ws used over 180 min t flow rte of 0.8 ml/ min. Compounds corresponding to peks were identified by compring their reltive retention times nd UV spectr with those of the uthentic smples previously mentioned. The elementl composition of the compounds corresponding to the mjor peks ws confirmed by LC/MS nlyses, using Xevo QTof MS (Wters Co., Milford, USA). Isoquercitrin (quercetin 3-Ο-glucoside) nd hyperoside (quercetin 3-Ο-glctoside) s stndrds were purchsed from Extrsynthese, Geny, Frnce. For the determintion of compounds showing mltse inhibitory ctivity, AP ws frctionted by Sephdex LH- 20 column (25 i.d. 400 mm, GE Helthcre, Tokyo, Jpn) using 50% EtOH s the developing solvent. Two-grm frctions were obtined nd nlyzed by the previously described method for the mesurement of mltse inhibitory ctivity (Deguchi et l., 1998). Animls Mle KK-A y mice, type 2 dibetic model, nd C57BL/6J mice, non-dibetic control model of KK-A y mice, were purchsed from Cle Jpn, Tokyo, Jpn. Mle Sprgue-Dwley (SD) rts were obtined from Cle Jpn for single-dose test. The mice nd rts were mintined t 22 ± 2 with reltive humidity of 55 ± 5% with 12 h light-drk cycle (light cycle: 08:00 20:00). The diet nd tp wter were given d libitum. Experiments, except for experiment 1, were conducted following the Guidelines of Animl Experimenttion (No. 105) nd Notifiction (No. 6) implemented by the Ministry of Eduction, Culture, Sports, Science nd Technology, Jpn. All protocols for niml experiment 2 were pproved by the Animl Use Committee of Ymgt University. Effects of single AP dose on the postprndil blood glucose level in crbohydrte-loded rts Mle SD rts (7-week-old) were fed commercil diet (CE-2, Cle Jpn) for 1 week. The rts were deprived of food overnight nd were orlly dministered strch, mltose, or glucose t dose level of 1 g/kg body weight with or without 0.2 g/kg of AP, dissolved in 1 ml of distilled wter. Blood ws collected from the til vein t 0, 15, 30, 60, nd 120 min fter orl dministrtion to mesure blood glucose levels. The blood glucose levels were mesured with blood glucose test meter (Snw kgku kenkyusyo, Aichi, Jpn). Effects of AP or isoquercitrin on type 2 dibetic KK- A y mice Experiment 1: KK-A y mice nd C57BL/6J mice (5-week-old) were fed commercil diet (CE-2, Cle Jpn)

3 Anti-Dibetic Effects of A. rgut Polyphenols tion. The ctivities of cyl-coa oxidse (ACO) (Hshimoto et l., 1981), nd of ftty cid synthse (FAS) (Kelley et l., 1986), glucose 6-phosphtse (G6Pse) (Lnge et l., 1986) nd glucokinse (GK) (Dvidson et l., 1987) were mesured using the superntnt obtined by centrifugtion t 500 g nd tht obtined by centrifugtion t 9,000 g, respectively. Experiment 2: KK-A y mice (6-week-old), which hd been fed commercil diet (CE-2, Cle Jpn) for 1 week, were divided into 2 groups, then fed either the bsl diet (control group) or the bsl diet contining 0.15% isoquercitrin (Q3G group) for 4 weeks. The composition of ech experimentl diet is shown in Tble 1. OGTT ws performed, s previously described, t dy 18 (3rd week) during the feeding period. Serum glucose, triglyceride, insulin, nd liver lipid levels were mesured by methods similr to those of Experiment 1. To mesure the heptic enzyme ctivities, portion of the liver (0.5 g) ws homogenized by the method previously described. After precipitting the mitochondril frction, the superntnt ws centrifuged t 100,000 g for 60 min to precipitte microsomes, nd the obtined superntnt ws used s the cytosol frction. The ctivities of crnitine plmitoyltrnsferse (CPT) (Mrkwell et l., 1973) nd of FAS nd G6Pse were mesured using the homogente nd the cytosol frction, respectively. Sttisticl nlysis All dt re expressed s men ± SEM. Significnt differences between the two groups were determined by n unpired student s t-test, wheres significnt differences between multiple groups ws nlyzed by One-Wy Anlysis Of Vrince (ANOVA) followed by Tukey s test. The vlues of p < 0.05 were considered sttistifor 1 week, divided into 2 groups, nd were fed either the bsl diet (control group) or the bsl diet contining 0.5% AP (AP group) for 6 weeks. C57BL/6J mice, s negtive control, were fed the bsl diet (C57 group). The composition of ech experimentl diet is shown in Tble 1. The orl glucose tolernce test (OGTT) ws crried out t dy 21 nd 36 (3rd nd 5th weeks) during the feeding period. All mice were fsted for 12 h before OGTT. The blood glucose levels in the OGTT were determined using blood smples collected from the til vein t 0 min (just before orl dministrtion), nd 30, 60, nd 120 min fter orl glucose dministrtion (1 g/kg body weight). At the end of the feeding period, the mice were nesthetized with sodium pentobrbitl 12 h fter fsting, blood ws collected by hert puncture nd then the liver ws detched. Serum ws prepred by centrifuging the blood t 1,000 g for 15 min. The serum nd liver were stored t 80 until required for nlysis. The serum glucose nd triglyceride levels were mesured using Glucose C-II Test nd Triglyceride E-Test kits (Wko Pure Chemicl Industries, Osk, Jpn), respectively. Serum insulin ws mesured with ELISA kits (Levis mouse insulin kit, Sibygi Co., Gunm, Jpn). Liver totl lipids were extrcted by the method of Folch et l. (Folch et l., 1957), nd the triglyceride level ws mesured using the sme kit s bove. To mesure the heptic enzyme ctivities, portion of the liver (0.3 g) ws homogenized in 10 volumes of 0.25 M sucrose solution contining 1 mm EDTA in 3 mm Tris-HCl buffer (ph 7.2). After centrifugtion t 500 g for 10 min, the obtined superntnt ws re-centrifuged t 9,000 g for 10 min to precipitte the mitochondril frc- 95 Tble 1. Composition of the experimentl diets (%). Experiment 1 Experiment 2 Bsl AP Q3G Csein Cornstrch α-cornstrch Sucrose Cellulose powder Soyben oil Minerl mixture (AIN-93G-MX) Vitmin mixture (AIN-93-VX) b L-Cystine Choline bitrtrte AP 0.50 Q3G 0.15 AIN-93G-MX nd b AIN-MX were obtined from Cle Jpn, Tokyo, Jpn.

4 96 S. Kurkneet l. clly significnt. Results α-glucosidse inhibitory ctivity of AP The inhibitory effects of AP on mltse nd sucrse ctivities in vitro re shown in Fig. 1. AP inhibited mltse nd sucrse ctivities in concentrtion-dependent mnner, mltse ws prticulrly strongly inhibited. The IC 50 of AP ws clculted s mg/ml for mltse nd 7.84 mg/ml for sucrse. Effects of AP on the postprndil blood glucose level in crbohydrte-loded rts The chnge in blood glucose level following the orl dministrtion of AP with strch, mltose, or glucose in rts is shown in Fig. 2. After the dministrtion of strch, the mximum increse in the blood glucose level of the control group ws observed 15 min fter dministrtion. The blood glucose level ws significntly lower in the AP group (Fig. 2A). When mltose ws dministered, the blood glucose level t 15 nd 30 min post-dose ws significntly lower in the AP group, compred with the control group (Fig. 2B). The blood glucose level fter glucose dministrtion did not differ between the AP nd control groups (Fig. 2C). Antidibetic effect of AP in type 2 dibetic KK-A y mice (Experimentl 1) C57BL/6J mice (norml mouse, C57 group), nd KK-A y mice (type 2 dibetic model) for the control nd AP groups were fed the respective diets for 6 weeks. Body weight gin, food intke, wter intke, fsting blood glucose, insulin levels, nd the serum triglyceride level me- Inhibition of α-glucosidse ctivity (%) Concentrtion (mg/ml) Fig. 1. Effects of the polyphenol frction of Actinidi rgut (AP) on mltse nd sucrse ctivities in vitro. The ctivities of mltse ( ) nd sucrse ( ) were determined over 120 min fter the ddition of AP to the rection mixture. The percentge inhibition is defined s tht compred with the control rection being 100%. 100 A B C Blood glucose (mg/dl) * * * Control AP -20 Time (min) Fig. 2. Effects of the co-dministrtion of the polyphenol frction of Actinidi rgut (AP) with sugrs on postprndil blood glucose levels in rts. Eight-week-old mle SD rts were orlly dministered either strch (A), mltose (B) or glucose (C) t 1 g/kg of body weight, nd distilled wter or AP t 0.2 g/kg of body weight. The blood glucose level ws mesured t 0, 15, 30, 60 nd 120 min fter the sugr ws dministrted. Ech vlue is expressed s men ± SEM (n=6). *Significntly different from the control t ech time-point (p < 0.05).

5 Anti-Dibetic Effects of A. rgut Polyphenols sured t the end of the feeding period re shown in Tble 2. All prmeters except for the heptic enzyme ctivities, shown in Tble 2, were significntly higher in the control group thn in the C57 group. No significnt differences were observed in the level of food intke when the AP nd control groups were compred, indicting tht AP hs no effect on food intke. The blood glucose level collected t the end of the feeding period tended to be lower in the AP group, but the insulin level did not differ between the two groups. The serum triglyceride level ws significntly lower in the AP group in comprison with the control group. The liver totl lipid nd triglyceride levels did not differ between the AP nd control groups. The results of OGTT in mice t dy 21 nd 36 (3rd nd 5th week) re shown in Fig. 3. The re under the curve (AUC) of the blood glucose level clculted from the results of OGTT is shown in Tble 2. An increse in the blood glucose level t 30 min fter glucose loding tended to be suppressed by AP dministrtion (0.05 < p < 0.1), nd the AUC ws significntly lower in the AP group thn the control 97 group on the 5th week, but ws not significntly different when compred to the 3rd week. The ctivities of heptic crbohydrte- nd lipid-metbolizing enzymes re shown in Tble 2. In the AP group, both ctivities of G6Pse, rte-determining enzyme in gluconeogenesis, nd GK, rte-determining enzyme in the glycolytic pthwy, were significntly lower in the AP group thn in the control group. The ctivity of FAS, involved in lipid metbolism, tended to be suppressed in the AP group, nd the ctivity of ACO, key enzyme in mitochondril ftty cid β-oxidtion, tended to be incresed in the AP group compred with the control group, but not to significnt level. Components of AP The HPLC chromtogrm of the MeOH-soluble fction of AP is shown in Fig. 4. The mjor peks of the MeOH-soluble frction were identified s hyperoside nd isoquercitrin by co-injection with the uthentic smples nd their UV spectrl chrcteristics, mesured by DAD. Their chemicl structures were lso supported by n elementl composition clculted from the mss number, mesured by HRESI-Tof-MS equipment (XEVO QTof-MS, Tble 2. Effects of AP on body weight gin, nd serum glucose, insulin nd triglyceride levels, liver lipids, nd heptic enzyme ctivities in KK-A y mice (Experiment 1). KK-A y C57 Con AP Body weight gin (g) 3.8 ± 0.3 b 8.2 ± ± 0.8 Food intke (g) ± 3.1 b ± ± 0.9 Wter intke (g) ± 24.8 b ± ± 27.4 AUC for OGTT ( 10 3 mg min/dl) At dy 21 (3rd week) 3.51 ± 0.57 b 10.9 ± ± 1.35 At dy 36 (5th week) 4.82 ± 0.63 b 11.5 ± ± 1.16 b Serum Glucose (mg/dl) ± 2.3 b ± ± 28.4 b Insulin (ng/ml) 0.54 ± 0.14 b 3.64 ± ± 0.78 Triglyceride (mg/dl) 67.0 ± 9.0 c ± ± 14.2 b Liver lipids (mg/g liver) Totl lipid 40.5 ± 1.4 b 57.8 ± 5.9 b 58.8 ± 5.3 Triglyceride 12.7 ± 1.0 b 24.4 ± ± 3.7 b Heptic enzyme ctivities (nmol/min/mg protein) G6Pse ± ± 8.0 b 72.9 ± 13.1 c GK ± 0.07 b 1.09 ± ± 0.05 b FAS ± 0.69 b 6.22 ± ± 0.46 b ACO ± 0.35 b 2.51 ± 0.45 b 3.44 ± 0.52 All of the dt, except for AUC, corresponds to mesurements concerning the mice, which were fed the diet for 6 weeks. Ech vlue is expressed s men ± SEM (n = 7). Vlues without common letter differ significntly (p < 0.05). 1 Ftty cid synthse (FAS), glucose 6-phosphtse (G6Pse) nd glucokinse (GK) ctivities were mesured with the 9,000 g superntnt of the liver homogente. 2 Acyl-CoA oxidse (ACO) ctivity ws mesured with the 500 g superntnt of the liver homogente.

6 98 S. Kurkneet l. A B Blood glucose (mg/dl) b b Time (min) Blood glucose (mg/dl) b b Time (min) C57 Con AP b Fig. 3. Effects of dietry AP on blood glucose levels during n orl glucose tolernce test (OGTT) in KK-A y mice (Experiment 1). OGTT ws crried out on dy 21 (A) nd dy 36 (B) of the feeding period (3rd nd 5th weeks). All mice were fsted for 12 h before OGTT. The blood glucose level ws determined using blood collected from the til vein t 0, 30, 60, nd 120 min fter the orl dministrtion of glucose (1 g/kg body weight). Ech vlue is expressed s men ± SEM (n=7-8).vlues without common letter differ significntly (p < 0.05). A OH OH HO O OH O O R Hyperoside (R=Glctose) Isoquercitrin (R=Glucose) B Hyperoside Isoquercitrin Retention time (min) Fig. 4. Chemicl structures of hyperoside nd isoquercitrin (A), nd HPLC chromtogrm of AP (B). Column, Develosil C-30-UG-5 ( mm); Solvent A, 5% MeCN/1% AcOH; Solvent B, 40% MeCN. Progrm: A liner grdient of 0-100% of B in A over the course of 180 min t flow rte of 0.8 ml/min. Detection, 320 nm.

7 Anti-Dibetic Effects of A. rgut Polyphenols Wters). Pseudomoleculr ions ([M-H] ) t m/z ( clculted for C 21 H 19 O 12 ) nd t m/z ( clculted for C 21 H 19 O 12 ) indicted tht their moleculr formul is C 21 H 20 O 12, respectively. The contents of hyperoside nd isoquercitrin were 2.17 nd 1.99 g/100 g of AP. In ddition, the polyphenol content in AP ws bout 60% when determined s gllic cid by the Folin-Denis method (Folin et l., 1915), suggesting tht the other polyphenols re lso contined in AP s reltively lrge mount. Mltse-inhibiting ingredients in AP AP ws frctionted by Sephdex LH-20 chromtogrphy to determine components showing mltse inhibitory ctivity. Frction No. 120 hd the most potent inhibitory ctivity. Anlysis of this frction by HPLC with DAD detection showed tht this frction contined isoquercitrin nd hyperoside. The ntihyperglycemic effect of isoquercitrin in type 2 dibetic KK-A y mice (Experimentl 2) Body weight gin, food intke, wter intke, serum insulin nd triglyceride levels mesured t the end of the feeding period re shown in Tble 3. No significnt differences were observed in ny of the prmeters between Q3G nd control groups; however, the fsting blood glucose level tended to be lower in the Q3G group (0.05 < p < 0.1). The results of OGTT in KK-A y mice t dy 18 (3rd week) re shown in Fig. 5. In Q3G groups, the blood glucose level of the Q3G group tended to be lower thn in the control group t 60 min fter glucose loding (0.05 < p < 0.1). AUC ws significntly lower in the Q3G group thn in the control Blood glucose (mg/dl) Time (min) Con Q3G Fig. 5. Effects of isoquercitrin on blood glucose levels during OGTT in KK-A y mice (Experiment 2). 99 OGTT ws crried out on dy 18 of the feeding period (3rd week). All mice were fsted for 12 h before OGTT. The blood glucose level ws determined using blood collected from the til vein t 0, 30, 60, nd 120 min fter the orl dministrtion of glucose (1 g/kg body weight). Ech vlue is expressed s men ± SEM (n = 7-8). Tble 3. Effects of dietry isoquercitrin on body weight gin, fsting blood glucose levels, serum insulin nd triglyceride levels, liver lipids, nd heptic enzyme ctivities in KK-A y mice (Experiment 2). Con Q3G Body weight gin (g) 7.7 ± ± 0.7 Food intke (g) ± ± 3.5 wter intke (g) ± ± 31.9 AUC for OGTT ( 10 3 mg min/dl) At dy ± ± 3.0 ** Serum Glucose (mg/dl) ± ± 8.9 Insulin (ng/ml) 4.29 ± ± 0.78 Triglyceride (mg/dl) ± ± 14.3 Liver lipids (mg/g liver) Totl lipid 60.9 ± ± 2.5 * Triglyceride 16.3 ± ± 1.2 * Heptic enzyme ctivities (nmol/min/mg protein) G6Pse ± ± 0.24 FAS ± ± 0.01 * CPT ± ± 0.60 All of the dt, except for AUC, corresponds to mesurements in mice, which were fed the diet for 4 weeks. Ech vlue is expressed s men ± SEM (n = 7-8). Significntly different from the control group (Con), *p < 0.05, **p < Ftty cid synthse (FAS) nd glucose 6-phosphtse (G6Pse) ctivities were mesured with the 100,000 g superntnt of the liver homogente. 2 Crnitine plmitoyltrnsferse (CPT) ws mesured with the whole liver homogente.

8 100 group (Tble 3). Liver totl lipid nd triglyceride levels were significntly lower in the Q3G group thn in the control group, respectively. Additionlly, the FAS ctivity ws significntly lower, nd CPT ctivity tended to be higher in the Q3G group (0.05 < p < 0.1). The G6Pse ctivity tended to be suppressed in the Q3G group, compred to tht of the control group (Tble 3). Discussion In the present study, the effects of AP on in vitro α-glucosidse ctivity nd on the elevtion of postprndil blood glucose levels were exmined in n effort to evlute the ntidibetic effect of AP. We observed n dditionl effect of isoquercitrin, one of the mjor flvonoids in AP, on the progress of dibetes in KK-A y mice. AP inhibited α-glucosidse ctivity, prticulrly strongly in vitro (Fig. 1). Elevtion of postprndil blood glucose, following strch or mltose dministrtion, ws suppressed when AP ws codministered to rts, with the strongest effect observed when dministered with mltose (Fig. 2). These results suggest tht AP hs the bility to inhibit α-glucosidse nd/or the bsorption of glucose through the inhibition of mltse ctivity. As postprndil blood glucose levels were not ltered when AP ws dministered with glucose, it ws thought tht AP ws not involved in the bsorption of glucose from the smll intestine; wheres tht the inhibitory ctivity of AP towrds α-glucosidse suppressed increses in blood glucose levels observed fter the dministrtion of mltose nd strch. When AP ws fed to type 2 dibetic KK-A y mice for 6 weeks, the AUC from the OGTT for the AP group tended to decrese t 3 weeks nd ws significntly decresed t 5 weeks, compred with the control group. This result my suggest tht glucose tolernce is improved by longer tretment with AP (Fig. 3). The lower ctivity of G6Pse, heptic crbohydrte-metbolizing enzyme, in the AP group my indicte tht suppression of gluconeogenesis ws prtly involved in lowering the AUC of the OGTT, nd the serum glucose levels t the end of the feeding period (Tble 2). Moreover, the decrese in the serum triglyceride level in the AP group indicted AP lters lipid metbolism. The decrese in FAS ctivity nd the increse in ACO ctivity in the APfed mice, in comprison with the control mice, suggest tht the inhibition of ftty cid synthesis nd stimultion of β-oxidtion my be cuse of the decrese in serum triglyceride (Tble 2). Among the frctionted AP, the frction contining isoquercitrin nd hyperoside inhibited mltse ctivity most strongly. These two components were the mjor flvonoids in A. rgut, lthough the presence of these compounds hs S. Kurkneet l. not yet been reported in A. rgut (Fig. 4). Extrcts from vrious plnts such guv leves (Deguchi nd Miyzki, 2010), Slci reticulte (Yoshino et l., 2009), nd mulberry leves (Asno et l., 2001; Prk et l., 2009) hve been shown to inhibit α-glucosidse ctivity, often with the involvement of polyphenols. Isoquercitrin, which ws isolted from dyflower (Commelin communis L.), ws found to be component tht inhibits α-glucosidses with potency close to tht of 1-deoxynojirimycin, potent α-glucosidse inhibitor (Shibno et l., 2008). In rts with lloxn-induced type 1 dibetes mellitus, isoquercitrin is known to decrese fsting blood glucose levels nd to improve dibetes mellitus by reducing insulin synthesis nd/or secretion, nd/or by suppressing G6Pse ctivity (Pnd nd Kr, 2007). In ddition, it ws reported tht the frction contining isoquercitrin hd hypoglycemic effect in mice with streptozotocin-induced type 1 dibetes mellitus (Hntyszyn et l., 2002). These reports support the contention tht AP-ssocited isoquercitrin my be useful in preventing dibetes, especilly through the regultion of glucose metbolism nd/or the effect of insulin secretion; however, no studies hve been conducted to evlute the efficcy of isoquercitrin in type 2 dibetes mellitus, which ccounts for 95% of Jpnese dibetic ptients. The present study with isoquercitrin in type 2 dibetic KK-A y mice showed the possibility tht isoquercitrin is cpble of decresing fsting glucose levels (glucose level t 0 min on OGTT, Fig. 5) nd improving glucose tolernce in type 2 dibetes (AUC for OGTT, Tble 3). The decrese in liver triglyceride levels, suppression of FAS ctivity nd enhncement of CPT ctivity in KK-A y mice fed isoquercitrin suggests tht the regultion of these enzymes by isoquercitrin nd/or its metbolites my lso be relted to decresed triglycerides, resulting in the mitigtion of dibetes. Reports indicting tht triglyceride ccumultion my be closely ssocited with the enhncement of dibetes, through decreses in diponectin levels (Arit et l., 1999; Hott et l., 2000; Ymuchi et l., 2001; Ye et l., 2003), my suggest tht the decrese in triglyceride by isoquercitrin my n importnt fctor in its mechnism of ction in mitigting dibetes. As the isoquercitrin content in AP ws 2.0%, portion of the efficcy of AP might be due to other components, such s hyperoside or the other minor components: (+)-ctechin, ( )-epictechin, nd procynidin B2. As the polyphenol content of AP ws bout 60%, it cn not be excluded tht other components besides polyphenols lso prticipted in the mitigtion of dibetes. In this report it ws suggested tht AP improves glucose tolernce in type 2 dibetes mice, presumbly by suppressing the progress of dibetes through inhibition of α-glucosidse nd G6Pse ctivity, s well s through the down-regultion

9 Anti-Dibetic Effects of A. rgut Polyphenols 101 of FAS nd up-regultion of CPT, resulting in lowering of triglyceride levels. The experiment with isoquercitrin s component of AP indicted tht isoquercitrin is one of the ctive components in AP, suggesting tht AP my contin other ctive components. As the ctivity of isoquercitrin hs not been exmined in KK-A y mice, the results obtined in this study re of high interest when it is considered tht isoquercitrin is contined in mny food products, s well s in Actinidi rgut, nd is ctive in mitigting type 2 dibetes. Acknowledgment This work ws supported by Grnts-in-Aid for Scientific Reserch (project no ) from the Jpn Society for the Promotion of Science. References Arit, Y., Kihr, S., Ouchi, N., Tkhshi, M., Med, K., Miygw, J., Hott, K., Shimomur, I., Nkmur, T., Miyok, K., Kuriym, H., Nishid, M., Ymshit, S., Okubo, K., Mtsubr, K., Murguchi, M., Ohmoto, Y., Funhshi, T. nd Mtsuzw, Y. (1999). 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