112

Original Article

Anti-glycation Activity of Japanese Chestnut (Castanea crenata) Inner Skin Extract is Beneficial for Type 2 Diabetes in a Rat Model

Tomohiro Mizutani 1), Fujiko Shizuka 2), Tsunetomo Matsuzawa 3), Yoshihiko Amano 3), Yukihiko Arikawa 1)

1) Food Technology Department of Nagano Prefecture General Industrial Technology Center2) Nagano Prefectural College3) Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu University

AbstractObjective: The accumulation of advanced glycation end products (AGEs) contributes to the pathogenesis of diabetic complications. This study was performed to evaluate the in vitro and in vivo effects of Japanese chestnut (Castanea crenata) inner skin water extract (CWE) against AGE formation.Methods: In vitro, the activity against AGE formation in CWE was compared with that of commonly consumed agricultural products. In vivo, a diabetic model of Otsuka Long-Evans Tokushima Fatty (OLETF) rats were fed either a 1% CWE diet or control diet without CWE for 23 weeks. Long-Evans Tokushima Otsuka (LETO) rats, the normal counterparts of OLETF rats, were fed a control diet. The plasma and renal concentrations of Nε-(carboxymethyl) lysine (CML) and thiobarbituric acid reactive substances (TBARS) were measured.Results: CWE showed the highest inhibitory activity of AGEs among the 32 agricultural products tested. The inhibitory activity of CWE against the formation of CML was much higher than that of aminoguanidine, a well-known inhibitor of AGE formation. By feeding the control diet without CWE, plasma and renal cortex CML and plasma and renal TBARS concentrations in OLETF rats increased and were significantly higher than those in LETO rats. Feeding the CWE diet prevented an increase in renal cortex CML and renal TBARS concentrations of OLETF rats, with no significant difference between the OLETF and LETO rats.Conclusion: The results showed the potent anti-glycation property of chestnut inner skin in vitro and suggested an effective application for the extract in the prevention of AGEs-related disorders such as diabetic nephropathy.

KEY WORDS: Chestnut, Advanced glycation end products, Type 2 diabetes, Proanthocyanidin, Aminoguanidine

Received: Oct. 9, 2013 Accepted: Jan. 24, 2014Published online: Jan. 31, 2014

Anti-Aging Medicine 10 (6) : 112-119, 2014(c) Japanese Society of Anti-Aging Medicine

Tomohiro Mizutani,Food Technology Department of Nagano Prefecture General Industrial Technology Center,

205-1 Kurita, Nagano 380-0921, JapanTEL: 026-227-3131 / FAX: 026-227-3130 / E-mail: mizutani-tomohiro-r@pref.nagano.lg.jp

IntroductionDiabetes mellitus is a metabolic disorder characterized by

hyperglycemia resulting from insulin secretory dysfunction, insulin resistance, or both. Chronic hyperglycemia accelerates the reaction between glucose and proteins and leads to the formation of advanced glycation end products (AGEs), such as Nε-(carboxymethyl) lysine (CML) and pentosidine. It has been demonstrated that the accumulation of AGEs in tissues is considered to be one of the leading causes of age-related degeneration, atherosclerosis, and diabetic complications such as retinopathy, nephropathy, and neuropathy 1,2).

The inhibition of AGE formation is one of the therapeutic approaches for preventing the progression of diabet ic complications 3). From the pharmacological point of view, aminoguanidine, an inhibitor of AGE formation, has been shown to be effective for preventing retinopathy 4) and nephropathy 5) in diabetic rats. However, aminoguanidine has not yet been used in a clinical setting to treat retinopathy or nephropathy due to uncertainty regarding the occurrence and

nature of potential side effects 6). Accordingly, efforts have been directed at finding phytochemical compounds from edible plants, fruits, and herbs that are effective against the formation of AGEs 7-10).

In the present study, we evaluated the anti-glycation effects of Japanese chestnut (Castanea crenata) inner skin. Chestnuts have been used as a folk medicine in many countries for centuries and several studies have reported antioxidant 11), anti-allergic 12), and anti-amnesic properties 13) of chestnut inner skin and leaf extracts. Little is known about their inhibitory activity against the formation of AGEs. We, therefore, measured the inhibitory activity against AGE formation in relation to that of commonly consumed agricultural products: vegetables, fruits, and mushrooms. In addition to its in vitro effects, we investigated the in vivo effects of chestnut inner skin extract on AGE formation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes with obesity.

113

Anti-glycation activity of chestnut

113

Materials and Methods

Reagents Aminoguanidine hydrochloride was purchased from Sigma

(St. Louis, MO, USA). Anti-CML monoclonal antibody (MK-5A10) and CML-BSA were purchased from CycLex Co., Ltd. (Ina, Nagano, Japan). Ingredients of the experimental diet were purchased from Oriental Yeast Co. (Itabashi, Tokyo, Japan).

Preparation of sample extractsThirty-two agricultural products composed of 9 vegetables,

14 fruits, and 9 mushrooms were used for AGE formation assay. They were powdered after drying, and 5 g of dried sample were extracted in 50 mL water or ethanol for 4 h at room temperature. The solution was dried again. The dried extracts were then recovered in water (water extract) or DMSO (ethanol extract) and used for in vitro experiments. The concentration of the extracted dried samples was set at 100 μg/mL or 10 μg/mL for in vitro AGE or CML assay, respectively. The dried water extract of chestnut inner skin was used as the experimental diet in animal studies.

AGE formation assay by fluorospectrometry The AGE formation rate was assessed according to

the method described by Morimitsu et al. 14) with a slight modification. Briefly, 200 μL of extract solution were incubated for 2 weeks at 37 °C with a mixture of 700 μL of 10 mg/mL BSA in 50 mM phosphate buffer (pH 7.4) containing 0.02% sodium azide, 50 μL 0.2 M glucose aqueous solution, and 50 μL 0.2 M fructose aqueous solution (reaction mixture A). At the same time, a blank test sample was prepared by replacing the glucose and fructose solutions with distilled water (reaction mixture B). An extract-free sample was prepared as a control (reaction mixture C). At the same time, a blank test sample of the control was prepared by replacing the glucose and fructose solutions with distilled water (reaction mixture D). The fluorescent products in each of the 4 reaction mixtures (A, B, C, and D) were measured using a fluorescence spectrophotometer (RF-1500; Shimadzu Co., Kyoto, Japan) at excitation and emission wavelengths of 350 and 450 nm, respectively. The AGE formation rate (% of control) was estimated by using the following equation: AGE formation rate (% of control) = (A − B) / (C − D) × 100.

CML formation assay by ELISA The samples were incubated as described above. The

CML formation rate was assessed according to the method of Engvall 15). Briefly, each well of a 96-well microtiter plate was coated with 50 μL reaction mixture in 50 mM sodium carbonate buffer (pH 9.0), blocked with 1% BSA, and washed with PBS containing 0.05% Tween-20 (washing buffer). The wells were incubated with 50 μL anti-CML antibody (1:5,000 dilution) for 1 h. The wells were washed with the washing buffer and reacted with an HRP-conjugated anti-mouse IgG antibody (1:15,000 dilution), followed by reaction with 3,3',5,5'-tetramethylbenzidine. The reaction was stopped by the addition of 1 M sulfuric acid, and the absorbance at 450 nm was read using a microplate reader. The concentration of each test sample showing 50% inhibition of the activities

(IC50) was estimated from the least-squares regression line of the logarithmic concentration plotted against the remaining activity.

Measurement of proanthocyanidins The amount of proanthocyanidins, one of the anti-oxidative

polyphenol compounds known to be contained in chestnut inner skin, in the extracts was determined by the vanillin-H2SO4 method 16).

Animal and dietsFour-week-old male OLETF rats (n = 11) and Long-Evans

Tokushima Otsuka (LETO) rats, the normal counterparts of OLETF rats, were purchased from Japan SLC, Inc. (Shizuoka, Japan). After 2 weeks acclimation, half of the OLETF rats (n = 5) were fed a semipurified powdered experimental diet containing 1% dry chestnut inner skin water extract (CWE) for 23 weeks. The remaining OLETF rats (n = 6) and LETO rats (n = 5) were fed a control diet without CWE. The control diet contained the following (g/kg): α-cornstarch, 432; sucrose, 100; cellulose, 50; soybean oil, 70; lard, 100; casein, 200; L-cystine, 3; mineral mixture (AIN-93G), 35; and vitamin mixture (AIN-93), 10. The experimental diet containing CWE was prepared by subtracting the corresponding amounts of α-cornstarch from the control diet. Animals were allowed free access to the diets and water during the experiment. They were sacrificed by cervical dislocation at the end of the feeding period. Samples of heparin plasma, kidney, and thoracic aorta were stored at –80 °C until further analysis. The Animal Care and Use Committee of Nagano Prefectural College approved all experimental procedures. This study was conducted according to the Fundamental Guidelines for the Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions in Japan.

Measurement of plasma and tissue parameters The plasma glucose and insulin concentrations were

determined using the Glucose C2-test Wako Kit (Wako, Osaka, Japan) and the Rat Insulin ELISA Kit (Shibayagi, Gunma, Japan), respectively. The plasma adiponectin and pentosidine concentrations were determined using the Mouse/Rat High Molecular Weight Adiponectin ELISA Kit (Shibayagi, Gunma, Japan) and FSK Pentosidine ELISA Kit (Fushimi Pharmaceutical, Marugame, Kagawa, Japan), respectively. The plasma and renal TBARS concentrations were determined using the TBARS Assay Kit (Cayman Chemical, Ann Arbor, MI, USA).

Measurement of plasma, renal cortex, and thoracic aorta CML

Plasma samples were used directly in the assay. The renal cortex and thoracic aorta were homogenized in phosphate buffered saline containing 0.05% Tween-20 at 4 °C. The homogenates were centrifuged at 10,000 × g at 4 °C for 5 min. The supernatants were adjusted to 10 mg protein/mL and incubated with 1 mg/mL proteinase K for 3 h at 37 °C, and the enzymatic reaction was stopped by heating (80 °C, 10 min). The mixtures were used for the assay. Plasma, renal cortex, and

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Anti-glycation activity of chestnut

thoracic aorta CML concentrations were determined by ELISA using CML-BSA as a standard 17). Briefly, each well of a 96-well microtiter plate was coated with 50 μL of 0.5 μg/mL CML-BSA in 50 mM sodium carbonate buffer (pH 9.0), blocked with 1% BSA, and washed with the washing buffer. The wells were incubated with 25 μL sample and 25 μL anti-CML antibody for 1 h. The wells were washed with the washing buffer and reacted with an HRP-conjugated anti-mouse IgG antibody, followed by reaction with 3,3',5,5'--tetramethylbenzidine. The reaction was stopped by the addition of 1 M sulfuric acid, and the absorbance at 450 nm was read using a microplate reader.

Statistical analysis Data are expressed as the means ± SD. Data were analyzed

by one-way ANOVA followed by the Tukey-Kramer test. P-values less than 0.05 were considered statistically significant.

Results

In vitro inhibitory activity against AGE formation and the amounts of proanthocyanidins

The inhibitory activities of water and ethanol extracts of agricultural products on AGE formation are shown in Table 1. The inhibitory activities of water extracts on AGE formation in most of the fruit skins were higher than those of the corresponding pulp extracts. The inhibitory activity of chestnut inner skin water extract (CWE) was 72.0%, which was the highest among water extracts of all agriculture products tested. No inhibitory activities on AGE formation were detected in the vegetable and mushroom extracts. Among the water extracts of mushrooms, exceptionally higher values (220.1% of the value indicating accelerated AGE formation) were obtained in maitake (G. frondosa). The chestnut inner skin ethanol extract (CEE) showed notably higher inhibitory activity (31.5%), which was the highest activity among all of the extracts tested. However, the inhibitory activities of the ethanol extracts were not observed to be necessarily superior to those of water extracts. As shown in Fig. 1, the inhibitory activities of CWE and CEE were slightly weaker than those of aminoguanidine, an inhibitor of AGE formation used as the positive control. However, the inhibitory activities of CWE (IC50 value: 0.45 μg/mL) and CEE (IC50 value: 0.42 μg/mL) on CML formation were about 500-fold more potent than those of aminoguanidine (IC50 value: 220 μg/mL). The amount of proanthocyanidins and the inhibitory activities against CML formation of chestnut, apple, and grape extracts, which are known as proanthocyanidin-rich foods, are shown in Table 2. Skin extracts of the fruits contained higher proanthocyanidin content than the pulp extracts. Chestnut inner skin extracts contained extremely high concentrations of proanthocyanidins compared with the apple and grape skin extracts. The ethanol extract of chestnut inner skin contained notably higher proanthocyanidins content than the water extract. The water extracts of the fruit samples containing high amounts of proanthocyanidins tended to show high inhibitory activities. However, the ethanol extracts containing higher amounts of proanthocyanidins did not show higher inhibitory activities. Both the water and ethanol extracts of chestnut inner skin exhibited extremely high inhibitory activities against CML formation. On the other hand, the

Table 1 Effect of the extracts on the AGE formation rate

Vegetable Celery (Apium graveolens)

Asparagus (Asparagus officinalis) Cauliflower (Brassica oleracea var. botrytis) Broccoli (Brassica oleracea var. italic) Lettucc (Lactuca sativa) Leaf lettuce (Lactuca sativa var. Crispa) Watercress (Nasturtium officinale) Eggplant (Solanum melongena) Corn (Zea mays)

Fruit Peach (Amygdalus persica)

Japanese chestnut (Castanea crenata)

Persimmon (Diospyros kaki)

Apple (Malus pumila) “Fuji”

“Jonashan”

“Shinano Gold”

“Shuei”

“Tugaru”

Prune (Prunus domestica)

Japanese pear (Pyrus pyrifolia var. culta)

Grape (Vitis L.) “Kyoho”

“Nagano Purple”

“Niagara”

“Stuben”

Mushroom Sasakurehitoyotake (Coprimus comatus) Enoki (Flammulina velutipes) Maitake (Grifola frondosa) Bunashimeji (Hypsizygus marmoreus) Shiitake (Lentinula edodes) Nameko (Pholiota nameko) Eringi (Pleurotus eryngii) Bairingu (Pleurotus nebrodensis) Agitake (Pleurotus jp takizawa)

Common name(Scientific name) Part

90.391.986.990.182.391.283.886.794.895.1

92.188.3

113.972.091.587.2

100.280.5

100.372.399.892.698.780.2

104.974.797.086.0

100.491.1

91.174.189.880.785.589.598.875.3

98.7101.1220.1

93.2117.6103.3

96.799.097.3

±±±±±±±±±±

±±±±±±

±±±±±±±±±±±±±±

±±±±±±±±

±±±±±±±±±

0.92.92.13.33.04.12.53.44.97.7

3.12.11.01.53.30.8

2.43.21.10.32.24.12.20.40.80.35.20.91.51.4

0.81.21.41.32.92.12.01.4

1.10.410.81.13.14.71.22.92.1

102.379.5

101.1101.1101.1101.9

94.165.9

104.8103.6

87.282.477.131.5

105.098.0

100.390.094.684.8

101.785.692.185.690.491.395.692.2

104.290.8

102.195.797.491.899.992.2

108.289.6

96.699.198.999.5

101.7104.9104.1100.3100.0

±±±±±±±±±±

±±±±±±

±±±±±±±±±±±±±±

±±±±±±±±

±±±±±±±±±

1.82.42.73.60.31.22.74.52.81.4

1.30.60.50.32.12.4

1.31.22.27.30.64.70.92.62.00.21.11.20.31.9

0.92.66.56.51.75.21.21.4

4.90.51.40.31.60.81.41.20.4

lcafstem

pulpskinpulp

inner skinpulpskin

pulpskinpulpskinpulpskinpulpskinpulpskinpulpskinpulpskin

pulpskinpulpskinpulpskinpulpskin

AGE formation(% of control)

Ethanol extractWater extract

Inhibitory effects are shown as the relative AGE formation rate (%) against the control (0 µg/mL of extract). The concentration of the extracts used in the assay was 100 µg/mL. Values are the means ± SD (n = 3). AGE: advanced glycation end product.

115

Anti-glycation activity of chestnut

115

Fig. 1. Inhibitory effect of chestnut inner skin extracts against advanced glycation end products (AGEs) formation in vitroInhibitory effects are shown as the relative AGE or Nε-(carboxymethyl) lysine (CML) formation rate (%) against the control (0 μg/mL extract). (A) The rate of AGE formation measured by fluorospectrometry. (B) The rate of CML formation measured by ELISA. The open circles represent chestnut inner skin water extract (CWE), the closed circles represent chestnut inner skin ethanol extract (CEE), and the open squares represent aminoguanidine. Values are the means ± SD of three independent experiments.

Table 2 Proanthocyanidins contents and CML formation rate of thse extracts

Japanese chestnut (Castanea crenata)

Apple (Malus pumila) “Fuji”

“Jonashan”

“Shinano Gold”

“Shuei”

“Tugaru”

Grape (Vitis L.) “Kyoho”

“Nagano Purple”

“Niagara”

“Stuben”

Maitake (Grifola frondosa)

Common name(Scientific name) Part

380

< 13.9< 1

23.0< 12.2< 18.6< 17.0

< 113.8< 1

41.0< 12.2< 1

57.0

< 1

630

2.616.2

7.418.6< 1

13.810.224.6

7.06.6

< 18.6< 1

28.2< 1

21.0< 1

24.2

< 1

19.3

90.887.987.961.398.0

101.393.874.993.769.3

95.980.691.170.996.7

103.996.856.6

4330

15.9

109.8102.6108.2

99.3104.2110.8104.1105.1102.6109.0

113.9118.2115.3112.8107.3

95.3104.4116.5

114.9

inner skin

pulpskinpulpskinpulpskinpulpskinpulpskin

pulpskinpulpskinpulpskinpulpskin

Proanthocyanidin (mg/g)

Ethanol extractWater extract

CML formation(% of control)

Ethanol extractWater extract

Inhibiory effects are shown as the relative CML formation rate (%) against the control (0 µg/mL of extract). The concentration of the extracts used in the assay was 10 µg/mL.

116

Anti-glycation activity of chestnut

maitake water extract dramatically accelerated CML formation (Table 2).

General characteristics of the rats The general characteristics of the rats are shown in Table 3.

The total food intake of OLETF rats was significantly higher than that of LETO rats. Correspondingly, the retroperitoneal fat weight of OLETF rats was significantly higher than that of LETO rats. The fasting plasma glucose concentration of OLETF rats was higher, but not significantly, than that of LETO rats. The kidney and spleen weights of the OLETF rats fed the control diet were significantly higher than those of LETO rats, whereas there were no significant differences in the kidney and spleen weights between the OLETF rats fed the CWE diet and the LETO rats. The liver weight of OLETF rats was significantly higher than that of LETO rats. The plasma insulin concentration of OLETF rats was lower, but not significantly, than that of LETO rats. CWE administration tended to increase the plasma high molecular weight adiponectin concentration.

Table 3 Effect of chestnut inner skin water extract on growth and plasma parameters in OLETF rats

Body weight (g) Initial day Final dayTotal food intake (g)Organ weight (g/100g body weight) Liver Kidney SpleenFat weight (g) Mesenteric fat Epididymal fat Retroperitoneal fatPlasma parameters Glucose (mg/dL) Insulin (ng/mL) High molecular weight adiponectin (µg/mL)

LE(n=5)

154723

3952

3.510.500.18

12.924.881.6

3025.11.8

±±±

±±±

±±±

±±±

8b

155237b

0.25b

0.11b

0.03b

3.95.922.3b

913.01.0

126617

2728

2.070.350.13

10.518.036.0

1769.81.2

±±±

±±±

±±±

±±±

4a58195a

0.13a

0.01a

0.01a

2.65.210.1a

536.10.5

156756

3909

3.270.450.16

12.725.385.2

2816.42.8

±±±

±±±

±±±

±±±

6b

4992b

0.40b

0.06ab

0.02ab

2.22.911.8b

1472.41.3

OL+CWE(n=5)

OL(n=6)

Values are the mean ± SD. Labeled means in a row without a common letter differ, P < 0.05.LE, LETO rats fed the control diet; OL, OLETF rats fed the control diet; OL+CWE, OLETF rats fed the diet with 1% CWE (chestnut inner skin water extract).

Fig. 2. Concentration of plasma and tissue AGEs in rats(A) Plasma CML concentration. (B) Plasma pentosidine concentration. (C) Renal cortex CML concentration. (D) Thoracic aorta CML concentration. Values are the means ± SD. Values with different superscript letters are significantly different (P < 0.05). LE, Long-Evans Tokushima Otsuka (LETO) rats fed the control diet; OL, Otsuka Long-Evans Tokushima Fatty (OLETF) rats fed the control diet; OL+CWE, OLETF rats fed the diet with 1% CWE.

117

Anti-glycation activity of chestnut

117

Effects of CWE against AGE formation in rats The plasma CML concentration of OLETF rats was

significantly higher than that of LETO rats (Fig. 2A). The plasma pentosidine concentration of OLETF rats was higher, but not significantly, than that of LETO rats (Fig. 2B). The renal cortex CML concentration of OLETF rats fed the control diet was significantly higher than that of LETO rats. However, there was no significant difference in the renal cortex CML concentration between OLETF rats fed the CWE diet and LETO rats (Fig. 2C). CWE administration tended to decrease the thoracic aorta CML concentration (Fig. 2D).

Effects of CWE against oxidative stress in rats The plasma TBARS concentration of OLETF rats was

significantly higher than that of LETO rats (Fig. 3A). The renal TBARS concentration of OLETF rats fed the control diet was significantly higher than that of LETO rats. Similar to the renal cortex CML concentration, CWE administration tended to decrease the renal TBARS concentration (Fig. 3B).

DiscussionIn the present study, we measured the inhibitory activity

against AGE formation of chestnut inner skin extract in vitro. Our results showed that chestnut inner skin extracts had the highest inhibitory activity against AGE formation among the 32 agricultural products tested. CML, one of the major antigenic AGEs, is non-fluorescent and its concentration is known to be increased in diabetic patients with complications 18). Therefore, we further measured the inhibitory activity of chestnut inner skin extracts against CML formation using ELISA. Both CWE and CEE potently inhibited CML formation. Peng et al. 19) reported that proanthocyanidins, which are similar to

Fig. 3. Concentration of plasma and tissue thiobarbituric acid reactive substances (TBARS) in rats(A) Plasma TBARS concentration. (B) Renal TBARS concentration. Values are the means ± SD. Values with different superscript letters are significantly different (P < 0.05). LE, LETO rats fed the control diet; OL, OLETF rats fed the control diet; OL+CWE, OLETF rats fed the diet with 1% CWE.

aminoguanidine, scavenge reactive carbonyl species. It has also been reported that chestnut inner skin contains relatively high amounts of proanthocyanidins, which are well-known antioxidants 20). We measured proanthocyanidin content and conf irmed that both CWE and CEE contained high amounts of these compounds. Park et al. 21) reported that the DPPH radical-scavenging activity of proanthocyanidins was notably higher than that of aminoguanidine. CML is one of the oxidation-dependent AGEs 22). Therefore, one reason for the potent inhibitory activity against CML formation might be related to the high antioxidative activity of the proanthocyanidins contained in chestnut inner skin. Proanthocyanidins are generally considered to bind to a number of proteins nonspecifically 23). The protein binding ability of proanthocyanidins may also affect CML formation. Interestingly, the inhibitory activities against CML formation of Fuji and Shuei apple skin ethanol extracts, which contained higher amount of proanthocyanidins than the corresponding water extracts, were lower than those of their water extracts. The inhibitory activity might have been affected by the difference in structure and degree of polymerization of proanthocyanidins or some components other than the proanthocyanidins. In the present study, we observed potent AGEs promoting activity in maitake water extract. We further observed CML promoting activity in maitake water extract. The CML promoting activity was notably higher than that of the AGEs. It has been reported that natural compounds containing a catechol group, such as gallic acid and epicatechin, enhance CML formation by promoting oxidative stress 24). However, the promoting activities of natural compounds containing a catechol group were much lower than those of the maitake water extract. Therefore, it is suggested that maitake might contain some other compounds that enhance CML formation.

AGEs accumulate with age, and en hanced AGEs accumulation has been reported in patients with diabetes. Therefore, we investigated the effect of CWE against AGE formation in OLETF rats. OLETF rats are an animal model of spontaneously obese type 2 diabetes characterized by hyperglycemia, insulin resistance, hyperinsulinemia, and

118

Anti-glycation activity of chestnut

hypertriglyceridemia 25). By feeding a control diet without CWE, renal cortex CML and renal TBARS concentrations in OLETF rats increased and were significantly higher than those in LETO rats. Feeding the CWE diet prevented an increase in renal cortex CML and renal TBARS concentrations in OLETF rats, with no significant difference compared with the LETO rats. These results suggest that application of chestnut inner skin may be effective for the prevention or treatment of AGE-related diseases. Regarding mechanisms, the anti-oxidative properties of the abundant polyphenols in chestnut inner skin might be a factor. The absorbability and metabolism of dietary-consumed polyphenols, and hence their effects in the body, are known to be related to the chemical structures of these compounds. It has been reported that apple procyanidin oligomers, up to pentamers, are detected in rat plasma after oral administration 26). However, a number of studies have reported an opposite result, indicating that no polymeric proanthocyanidins are absorbed from the intestinal rumen 27). At present, the degree of polymerization of chestnut inner skin proanthocyanidins has not been elucidated. Regarding the mechanisms of the effect of polyphenols, there are two possibilities. One is related to the inhibition of α-amylase and the other is the inhibition of oxidative stress. Tsujita et al. 20) reported that chestnut inner skin contains high molecular weight polyphenols, including proanthocyanidins, which inhibit α-amylase and thereby suppress the elevation of blood glucose concentration after starch administration. We did not confirm hyperglycemia in OLETF rats, although the fasting blood glucose concentration was slightly higher than that of LETO rats. It is expected that the postprandial and average blood glucose concentrations of OLETF rats might be higher than those of LETO rats. Therefore, one reason for the reduction of CML accumulation may be related to the postprandial hypoglycemic effect of polymeric proanthocyanidins in CWE. The other possibility is as follows. Some AGEs, such as CML, interact with the receptor for AGEs (RAGE), which elicits oxidative stress in various types of cells 28). It is considered that AGE-RAGE-induced oxidative stress accelerates AGE formation 29). Lu et al. 30) and Cheng et al. 31) reported that grape seed proanthocyanidins, which contain monomers, oligomers, and polymers 32), reduced the expression of RAGE in diabetic rats. In addition, Mansouri et al. 33) reported that grape seed proanthocyanidins increased the activity of renal antioxidant enzymes including superoxide dismutase, glutathione peroxidase, and catalase. Therefore,

another reason for the reduction of CML accumulation may be the suppression of oxidative stress.

In conclusion, the administration of CWE inhibited the accumulation of AGEs in the renal cortex. The present findings suggest that application of chestnut inner skin may be effective for preventing AGEs-related diseases. Further research is needed to determine the active components and clarify the underlying mechanisms.

AbbreviationsAGEs, advanced glycation end products; CEE, Japanese

chestnut (Castanea crenata) inner skin ethanol extract; CWE, Japanese chestnut (Castanea crenata) inner skin water extract; CML, Nε-(carboxymethyl) lysine; LETO, Long-Evans Tokushima Otsuka; OLETF, Otsuka Long-Evans Tokushima Fatty.

AcknowledgementsWe are deeply grateful to Yuichi Hiraoka, Yoshiyasu

Nakamura, Tsuyoshi Segishita, Junichi Terashima, Yuko Nagasaki and Katsuyuki Hashizawa for helping with the preparation of chestnut inner skin extracts.

Conflict of Interest and Funding DisclosureThis study was partly supported by the Nagano Techno

Foundation.The authors declare no conflicts of interest.

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