天然产物化学英文版副本汇总.doc

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 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 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). 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, 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).
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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 (Figure 1).
However, high-fat feeding resulted in a significant increase in the glucose level of mice after 3 wk. The NC group also showed an increase in glucose content similar to that of the HF mice. On the final wk, the HF-O and HF-FA mice exhibited substantially lower glucose level compared with the HF and control groups.
2.3 Glycogen and insulin levels
The glycogen and insulin concentrations were considerably higher in HF-O and HF-FA mice than that of the control and HF ones (Table 3).

2.4 Hepatic glucose-regulating enzyme activities
The hepatic GK enzyme activity was significantly higher in mice fed with oryzanol and ferulic acid than that of the control and HF-fed ones (Figure 2).

High-fat feeding resulted in a marked increased in G6pase activity (Figure 3).
However, supplementation of oryzanol and ferulic acid in the diet suppressed the elevation and normalized the G6pase activity. Similarly, significantly lower PEPCK activities were observed in HF-O and HF-FA mice compared with that of the NC and HFones(Figure4).
3 Discussion
The strong antioxidant activities of oryzanol and ferulic acid have been well documented (Wang and others 2002; Srinivasan and others 2007). Since oxidative stress is considered to be a key factor in the development of diabetes, the effect of oryzanol and ferulic acid on the glucose metabolism in high-fat-fed mice was investigated. The significant decrease in the body weight of mice fed with oryzanol and ferulic acid relative to that of the high-fat-fed mice demonstrates that both compounds were able to control the increase in body weight of mice under high-fat diet condition. As expected, high-fat feeding resulted in an increase in the blood glucose level in mice. However, a similar rise in glucose was also observed in the control group, which could be attributed to the aging of the mice. Amrani and others (1998) previously reported that male C57BL/6 mice fed with standard food pellets exhibited increased blood glucose concentrations from 4 to6 wk of age. Nevertheless, results of the present study indicate that both oryzanol and ferulic acid were able to suppress the increase in blood glucose level in mice. This hypoglycemic action of oryzanol and ferulic acid is probably associated with the marked enhancement of GK activity and inhibition of G6pase and PEPCK in the liver. Hepatic GK enzyme is known to play a major role in the regulation of glucose homeostasis. An increase in the expression of hepatic GK can cause an increase in the utilization of blood glucose for energy production or glycogen storage in the liver (Iynedjian and others 1988), thereby resulting in reduced blood glucose level. G6pase and PEPCK, on the other hand, are the key enzymes that regulate gluconeogenesis and glucose output in the liver (Friedman and others 1997).
The activities of hepatic GK, G6pase, and PEPCK are reported to be partly regulated by insulin. High levels of insulin were shown to inhibit hepatic glucose production via stimulation of GK gene transcription and glycogen synthesis and inhibition of gluconeogenesis (Iynedjian and others 1988; Friedman and others 1997). In the present study, the changes in the hepatic glucose-regulating enzyme activities in mice fed with oryzanol and ferulic acid could be partly attributed to the significant elevation of insulin levels in HF-O and HF-FA mice relative to that of the NC and HF ones. Accordingly, enhanced rate of glycogenesis was also observed in HF-O and HF-FA mice, as evidenced by a marked increase in hepatic glycogen concentration.
The improvement of glucose metabolism in HF-O and HF-FA mice is likely associated with the antioxidative effect of oryzanol and ferulic acid. A high-fat diet negatively affects glucose metabolism and the regulation of blood glucose is essential in preventing the development of diabetes. Antioxidant is recognized as a means of treating diabetes as it can decrease blood glucose levels through improvement of insulin action (Kaneto and others1999). Ferulic acid has been shown to reduce blood glucose levels in streptozotocin induced diabetic animals (Sri Balasubashini and others 2003; Ohnishi and others 2004). The free radicals resulting from hyperglycemia causes cellular damage. Oryzanol and ferulic acid could inhibit oxidative stress in high-fat-fed mice by possibly scavenging excessive high-fat diet induced free radicals. The antioxidant property of ferulic acid is attributed to its aromatic phenolic ring that stabilizes and delocalizes the unpaired electron within its aromatic ring (Rice-Evans and others 1997), thereby acting as free-radical scavengers. Oryzanol, on the other hand, is a powerful inhibitor of iron-driven hydroxyl radical formation (Fardet and others 2008). Between oryzanol and ferulic acid, the latter is more readily consumed since it is found in various fruits and vegetables, while the former is abundantly present in rice bran only. Nevertheless, results of the present study showed that at equal dietary levels, both phenolic compounds have relatively similar effects on the glucose metabolism of high-fat-fed C 57BL/6N mice.
4 Conclusions
Results of the present study demonstrate that oryzanol and ferulic acid could improve the blood glucose metabolism in high-fat-fed C57BL/6N mice. The underlying mechanism responsible for lowering the blood glucose level i