天然产物化学英文版副本汇总

Effect of Oryzanol and Ferulic Acid on the Glucose Metabolism of

Mice Fed with a High-Fat Diet

Myoung Jin Son, Catherine W. Rico, Seok Hyun Nam, and Mi Young

Kang

Abstract: The effects of oryzanol and ferulic acid on the glucose metabolism of high-fat-fed mice were investigated. Male C57BL/6N mice were randomly divided into 4 groups: NC group fed with normal control diet; HF group fed with high-fat (17%) diet; HF-O group fed with high-fat diet supplemented with 0.5% oryzanol; and HF-FA group fed with high-fat diet supplemented with 0.5% ferulic acid. All animals were allowed free access to the experimental diets and water for 7 wk. At the end of the experimental period, the HF-O and HF-FA groups exhibited significantly lower blood glucose level and glucose-6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (PEPCK) activities, and higher glycogen and insulin concentrations and glucokinase (GK) activity compared with NC and HF groups. The results of this study illustrate that both oryzanol and ferulic acid could reduce the risk of high-fat diet-induced hyperglycemia via regulation of insulin secretion and hepatic glucose-regulating enzyme activities. Keywords: diabetes, ferulic acid, high-fat-fed mice, hypoglycemic effect, oryzanol Introduction

Chronic consumption of a high-fat diet has been associated with the development of obesity and type 2 diabetes mellitus (Hill and others 1992; Bray and others 2004). Scientific studies have shown that excessive intake of dietary fat results in increased body weight and poor glucose regulation (Alsaif and Duwaihy2004; Petro and others 2004; Messier and others 2007). Diabetes is characterized by hyperglycemia that results in the generation of free radicals leading to oxidative stress (West 2000). Due to changes in lifestyle patterns, particularly poor eating habit and sedentary lifestyle, the incidence of diabetes has rapidly increased in epidemic proportions. Around 171 million cases of diabetes worldwide were reported in 2001 and it was projected that by 2030, 366 million people will have diabetes (Wild

1 / 20

and others 2004). With this increasing global prevalence of diabetes, the need for therapeutic measures against the disease has become stronger and more urgent. A wide range of oral medicines are currently being used for treating diabetes. However, various adverse effects and high rates of secondary failures have been associated with the available antidiabetic medicines (Inzucchi 2002). Thus, finding natural drugs with hypoglycemic activity has now become the focus of scientists and researchers.

At present, there is a considerable public and scientific interest in utilizing phytochemicals for the treatment and prevention of various diseases. Naturally occurring phenolic compounds, such as oryzanol and ferulic acid, are known to have strong antioxidant activities (Wang and others 2002; Srinivasan and others 2007). Oryzanol is a mixture of ferulic acid (4-hydroxy-3-methoxycinnamic acid) esters with phytosterols (Lerma-Garcia and others 2009) and primarily extracted from rice bran. Ferulic acid is commonly found in fruits and vegetables, including banana, broccoli, rice bran, and citrus fruits (Zhao and Moghadasian 2008). Both oryzanol and ferulic acid possess several physiological proper ties, such as reduction of serum cholesterol levels (Wilson and others 2007), inhibition of tumor promotion (Yasukawa and others 1998), and protective action against liver injury (Choti-markorn and Ushio 2008). Oxidative stress is regarded as the key factor in the development of diabetes and its associated health disorders. The high-fat diet fed C 57BL/6 mouse model has long been used by researchers in investigating the pathophysiology of impaired glucose tolerance and type 2 diabetes and for the development of new treatments (Surwit and others 1988; Surwit and other s 1991; Schreyer and others 1998; Winzell and Ahren 2004). Since diabetes is a free radical mediated disease, the strong antioxidant activity of oryzanol and ferulic acid may be useful in preventing the development of diabetic hyperglycemia under a high-fat diet. There are limited reports on the physiological functions of these phenolic compounds in relation to glucose metabolism in animal models. Thus, this study was conducted to investigate the effects of

2 / 20

dietary feeding of oryzanol and ferulic acid on the glucose metabolism in high-fat-fed C57BL/6 mice. 1. Materials and Methods 1.1 Animals and diets

Twenty-four male C57BL/6N mice of 4 wk of age, weighing 12 g, were obtained from Orient Inc. (Seoul, Korea). They were individually housed in stainless steel cages in a room maintained at 25?C with 50% relative humidity and 12/12 h light/dark cycle and fed with a pelletized chow diet for 2 wk after arrival. The mice were then randomly divided into 4 dietary groups (n = 6). The 1st and 2nd groups were fed with a normal and high-fat (17%, w/w) diets, respectively, while the other 2 groups were fed with high-fat diet supplemented with either 0.5% oryzanol or 0.5% ferulic acid (>98% pure, Tsuno, Osaka, Japan). The composition of the experimental diet (Table

1)

was

based

on

the

AIN-76

semisynthetic

diet.

The mice were fed for 7 wk and allowed free access to food and water during the experimental period. The body weight gain was measured weekly. At the end of the experimental period, the mice were anaesthetized with 60-μL Ketamine-HCl following a 12 h fast and sacrificed. Blood samples were collected and centrifuged at 1000 × g for 15 min at 4?C to obtain the plasma. The livers were removed, rinsed with physiological saline, and stored at ?70?C until analysis. The current study protocol was approved by the Ethics Committee of Kyungpook Natl. Univ. for anima studies. 1.2 Measurement of blood glucose level

The blood glucose level in mice was measured using Accu-Chek Active Blood Glucose Test Strips (Roche Diagnostics GmbH, Germany).

3 / 20

Blood samples were drawn from the tail vein of the mice before and after 3 and 7 wk of feeding the animals with experimental diets. 1.3 Determination of glycogen and insulin levels

The glycogen concentration in liver was determined using the method described by Seifter and others (1950)。 Fresh liver (100 mg) was mixed with 30% KOH and heated at 100?C for 30 min. The mixture was then added with 1.5 mL ethanol (95%) and kept over night at 4?C. The pellet was mixed with 4 mL distilled water. A 500 μL of the mixture was added with 0.2% anthrone (in 95% H2SO4) and the absorbance of the sample solution was measured at 620 nm. The results were calculated on the basis of a standard calibration curve of glucose. The insulin content was measured using enzyme-linked immunosorbent assay (ELISA) kits (TMB Mouse Insulin ELISA kit, Sibayagi, Japan). 1.4 Measurement of hepatic glucose-regulating enzyme activities

The hepatic enzyme source was prepared according to the method developed by Hulcher and Oleson (1973). The glucokinase (GK) activity was determined based from the method of Davidson and Arion (1987) with slight modification. A 0.98 mL of the reaction mixture containing 50 mM Hepes-NaOH(pH 7.4), 100 mM KCl, 7.5 m M MgCl2, 2.5 mM dithioerythritol, 10 mg/mL albumin, 10 mM glucose, 4 units of glucose-6-phosphate ( G6pase) dehydrogenase, 50 mM NAD+, and 10 μL cytosol was preincubated at 37?C for 10 min. The reaction was initiated with the addition of 10 μL of 5 mM ATP and the mixture was incubated at 37?C for 10 min. The G6pase activity was measured using the method described by Alegre and others (1988). The reaction mixture contained 765 μL of 131.58 mM Hepes-NaOH (pH 6.5), 100 μL of 18 mM EDTA ( pH 6.5), 100 μL of 265 mM G6pase, 10 μL of 0.2 M NADP+, 0.6 IU/mL mutarotase, and 0.6 IU/mL glucose dehydrogenase. the mixture was added with 5 μL microsome and incubated at 37?C for 4 min. The change in absorbance at 340 nm was measured. The phosphoenolpyruvate carboxykinase (PEPCK) activity was determined based from the method developed by Bentle and Lardy (1976). The reaction mixture consisted of 72.92 mM sodium Hepes (pH 7.0), 10 mM dithiothreitol, 500 mM NaHCO3, 10 mM MnCl2, 25 mM NADH, 100 mM IDP,

4 / 20

200 mM PEP, 7.2 unit of malic dehydrogenase, and 10μL cytosol. The enzyme activity was determined based from the decrease in the absorbance of the mixture at 350 nm at 25?C.在25?C 350nm。 1.5 Statistical analysis

All data are presented as the mean ± SE. The data were evaluated by 1-way ANOVA using a Statistical Package for Social Sciences software program (SPSS Inc., Chicago, Ill., U.S.A.) and the differences between the means we reassessed using Duncan’s multiple range test. Statistical significance was considered at P <0.05. 2. Results

2.1 Body weight gain

There was no significant difference in the body weight among the animal groups prior to feeding the mice with the experimental diets (Table 2).

。

The daily food intake of mice was constant (3 g/d) throughout the study. At the end of the experimental period, however, a significant increase was observed in animals fed with high-fat diet (HF group) relative to that of the control mice (NC group)。 While the mice fed with high-fat diet supplemented with oryzanol (HF-O group) or ferulic acid (HF-FA group) also showed higher weight gain compared with that of the NC group, their body weight gain was significantly lower than that of the HF group. Between HF-O and HF-FA groups, the latter exhibited lower final body weight. 2.2 Blood glucose levels

The initial blood glucose levels in mice prior to feeding with experimental diets did not significantly differ among the groups

5 / 20