Borapetoside C from Tinospora crispa improves insulin sensitivity in diabetic mice.
Glucose transporter-2 (GLUT2)
Diabetes mellitus (DM) often leads to disability from vascular complications and neurological complications. Tinospora crispa has been widely used in Asia and Africa as a remedy for diabetes and of her diseases. In this study, we investigated the hypoglycemic actions of borapetoside C isolated from T. crispa. and the mechanisms underlying its actions. Acute treat ment with borapetoside C (5 mg/kg, i.p.) attenuated the elevated plasma glucose induced by oral glucose in normal and type 2 DM (T2DM) mice. Compared to the effect of injected insulin (0.5 IU/kg). borapetoside C caused a more prominent increase of glycogen content in skeletal muscle of T2DM mice, but a less increase in type 1 DM (T1 DM) mice. Combined treatment of a low dose borapetoside C (0.1 mg/kg, i.p.) plus insulin enhanced insulin-induced lowering of the plasma glucose level and insulin-induced increase of muscle glycogen content. Continuous treatment with 5 mg/kg borapetoside C (twice daily) for 7 days increased phosphorylation of insulin receptor (IR) and protein kinase B (Akt) as well as the expression of glucose transporter-2 (GLUT2) in T1 DM mice. Combined treatment of a low dose borapetoside C (0.1 mg/kg, twice daily) plus insulin for 7 days enhanced insulin-induced IR and Akt phosphorylation and GLUT2 expression in the liver of T1 DM mice. This study proved that borapetoside C can increase glucose utilization, delayed the development of insulin resistance and enhanced insulin sensitivity. The activation of IR-Akt-GLUT2 expression and the enhancement of insulin sensitivity may contribute to the hypoglycemic action of borapetoside C in diabetic mice.
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Diabetes mellitus (DM) is associated with the pathological progression in various organs such as the liver and skeletal muscle. The epidemiological studies characterize obesity to he highly associated with DM. About 80% of individuals with type 2 DM (T2DM) are overweight (Bays et al. 2007). Since the onset of metabolic disease starts to develop earlier in obese people, studies have focused on the development of pharmaceuticals that can be used in patients with obesity and DM (Bays 2009).
There are several studies showing that diabetes is associated with abnormal insulin secretion and insulin sensitivity. Since insulin is the most important substance in regulating glucose metabolism, impaired insulin secretion results in an increase in hepatic glucose production and reduction of glucose uptake in muscle (Kahn et al. 2006). On the other hand, increased insulin resistance is a key feature in T2DM. It is characterized with a remarkable decrease in tissue glucose utilization in response to insulin (Granberry et al. 2007).
Tinospora crispa (family Menispermaceae) has been widely used in Asia and Africa as a herbal remedy for a long time. In traditional medicine, a decoction from the sterns of T. crispa has been used for anti-inflammation, reducing thirst, increasing appetite, antipyretics, and maintaining good health (Messmer 1961; Zafinindra et al. 2003). The chemical constituents of T. crispa extracts have been extensively studied since the 1980s. The major active ingredients of crispa are identified as terpenoids and terpenoid glycosides. The terpenoid glycosides are mainly composed of borapetosides A, B, C, D, E and F (Cavin et al. 1998; Choudhary et al. 2010a,b; Kongkathip et al. 2002; Martin et al. 1996; Pachaly et al. 1992; Pathak et al. 1995; Ragasa et al. 2000; Yonemitsu et al. 1993). Most of these substances are yet to be investigated for their pharmacological activities.
Our previous study showed that borapetoside C from T crispa could decrease serum glucose via enhancing insulin secretion in both normal and T2DM mice, whereas it reduced glucose level without changing the insulin level in type 1 DM (T1 DM) mice (Lam et al. 2012). The molecular mechanism for the increase of glucose utilization, inhibition of hepatic gluconeogenesis, and associated lowering of plasma glucose by borapetoside C in diabetic mice remain to be investigated. In this study, we demonstrated that borapetoside C attenuated the elevation of plasma glucose induced by oral glucose in normal and diabetic mice. Continuous treatment with borapetoside C at 5 mg/kg (twice day) for 7 days induced phosphorylation of insulin receptor (IR), protein kinase B (Akt) and resulted in enhancement of glucose transporter subtype 2 (GLUT2) expression in the liver ofT1 DM mice. Combined treatment with insulin and low dose of borapetoside C (0.1 mg/kg) increased insulin sensitivity and enhanced insulin induced IR/Akt/GLUT2 signaling in the liver of T1 DM mice. On the basis of the study, we propose that borapetoside C is a potential therapeutic agent that can be further explored and developed as an alternative remedy for managing diabetic disorders in the future.
Material and methods
Animals and treatment protocol
This study was conducted by following the University ethical guidelines on animal experimentation and complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Taiwan University (IACUC No. 20070004). The animal facility was well controlled for temperature (22 [+ or -] 1 [degrees]C), and humidity (60 [+ or -] 5%) and a 12 h/12 h light-dark cycle was maintained with access to food and water ad libitum. Four-week-old male ICR mice were acquired from BioLasco Taiwan Co., Ltd. and maintained at College of Medicine Experimental Animal Center, National Taiwan University. The study was conducted on 8-10 week-old male ICR mice.
T1DM mice were induced by following a modified protocol after the acclimatizing period (Hayashi et al. 2006). In brief, an intraperitoneal injection of streptozotocin (STZ; Sigma Chemical Co.; St. Louis, MO) at 150 mg/kg was performed in mice that were fasted for 48 It The induction of T1DM was assessed and confirmed when the mice had plasma glucose levels [greater than or equal to] 350 mg/dl, accompanied with polyuria, hyperphagia and decreased body weight. The control mice group received an injection of vehicle and then carried out for 4 weeks. T2DM mice were induced by maintaining on a fat-rich chow diet and fructose-sweetened water for 4 weeks from the age of 4-5 weeks according to previous methods (Huang et al. 2004; Weng et a1. 2010). The induction T2DM mice were assessed by measuring fasting plasma glucose levels and confirmed when plasma glucose level was [greater than or equal to] 150 mg/dl after a 4-week induction.
Measurement of plasma glucose and insulin levels
Blood samples were collected from the orbital vascular plexus of mice under anesthesia with sodium pentobarbital (80 mg/kg, intraperitoneal, Sigma Chemical Co., St. Louis, MO, USA). Blood samples were then centrifuged at 13,000 rpm for 5 min, and the plasma was kept on ice prior to the assay (Park et al. 2005). The plasma glucose concentration was measured using commercial kits following manufacturer's instructions (BioSystems S.A., Barcelona, Spain).
Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)
To perform OGTT, mice were fasted overnight, divided into 2 groups and then administered a vehicle control and 5 mg/kg borapetoside C. This was followed by administration of a glucose solution at 2 g/kg via tube feeding. Blood samples were withdrawn from the orbital vascular plexus at intervals of 30, 60, 120, and 150 min after glucose administration (Kuftinec and Mayer 1964). For the ITT, mice were fasted for 3 h. Human insulin (Insulin Actrapid[R] HM: Novo Nordisk, Denmark) was injected intraperitoneally after an intraperitoneal administration of 0.1 mg/kg borapetoside C for 30 min. Blood samples were collected from the orbital vascular plexus at the timed intervals mentioned above (Kuftinec and Mayer 1964).
Glycogen content assay
Glycogen content of skeletal muscles was measured according to an earlier established method (Sadasivam and Manickam 1996). In brief, mice were injected with borapetoside C (5.0 mg/kg, interperitoneal) for 60 min or insulin (0.5 IU/kg, interperitoneal.) for 30 min, and the soleus muscle was isolated from anesthetized mice. About 40 mg of muscle sample was dissolved in 1 N KOH at 75[degrees]C for 30 min. The dissolved homogenate was neutralized by glacial acetic acid and then incubated overnight in acetate buffer (0.3 M sodium acetate, pH 4.8) containing amyloglucosidase (Sigma, St. Louis, MO). The mixture was then neutralized with 1 N NaOH to stop the reaction (Chou et al. 2005). The glycogen contents in the tissue samples were determined as p.g of glucose per mg of tissue (wet weight).
Collection of liver tissue
After treatment, the mice were sacrificed by cervical dislocation under anesthesia. The liver was immediately frozen in liquid nitrogen. The liver tissue was preserved at -80[degrees]C before they were used for further assays.
Western blot analysis
Tissues were homogenized in T-PER[R] Tissue Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL) with Halt[TM] Protease Inhibitor Single-Use Cocktail (Pierce Biotechnology, Rockford, IL). Liver homogenates were prepared by mechanical homogenization (Polytron PT3100, Luzernerstrasse, Switzerland). After centrifuging the homogenates at 10,000 x g for 30 min, the supernatants were collected and frozen at -80[degrees]C for further use. The protein concentrations were determined by using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). For western blot analysis, about 60[micro]g of protein preparations were applied on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked with 5% (w/v) non-fat dry milk in phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBS-T). After blocking, the blotted membrane was incubated with anti-phospho-insulin receptor [beta] (IR[beta]) ([Tyr.sup.1345], phospho-Akt ([Ser.sup.473] (Cell Signaling Technology, Beverly, MA), anti-IR[beta], Akt, [beta]-actin (Santa Cruz Biotechnology, Santa Cruz, California), and anti-GLUT2 antibodies (Abcam, Cambridge, UK) in presence of 3% bovine serum albumin (BSA) in PBS-T buffer. Following the incubation, the membranes were washed 3 times with PBS-T for 15 min each and then incubated with the appropriate peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, USA) in PBS-T. After removal of the secondary antibody, blots were washed and developed using the enhanced chemiluminescence (ECL) western blotting system (Millipore, Billerica, MA). The density of the protein bands were quantified using ImageQuant (Chi et al. 2007).
Results were presented as mean [+ or -] SEM for the number (n) of animals in the group as indicated in the tables and figures. Statistical difference between the means of the various groups were analyzed using one way analysis of variance (ANOVA) followed by Turkey's multiple test with Prism 5.0 demo software (GraphPad Software Inc., La Jolla, CA). Data were considered statistically significant at * p < 0.05.
Effect of borapetoside Con oral glucose tolerance test (0G7T) in the non-diabetic and diabetic mice
To determine the effect of borapetoside C on glucose tolerance, OGITs were performed in non-diabetic and T2DM mice. In non-diabetic mice, the basal plasma glucose concentrations in the borapetoside C-treated and vehicle-treated groups were 115 [+ or -] 3.5 mg/dl and 121.8 [+ or -] 1.5 mg/dl respectively as shown in Fig. 1 A. At 60 min after oral administration of glucose, the plasma glucose concentration was elevated to 406.8 [+ or -] 28.0 mg/dl in the vehicle-treated mice, and to 312.8 [+ or -] 21.3 mg/dl in the borapetoside C-treated mice. The plasma glucose level of borapetoside C-treated mice was significantly lower than that of vehicle-treated mice after oral administration of glucose. The plasma glucose levels of borapetoside C-treated mice were maintained significantly lower than those of the vehicle-treated mice at 120 min, and 150 min after treatment. In T2DM mice (Fig. 1B), the basal plasma glucose concentrations of the vehicle-and borapetoside C-treated groups were 200.0 [+ or -] 6.8 mg/dl and 206.8 [+ or -] 19.0 mg/d1, respectively. Sixty min after oral glucose administration, the plasma glucose concentration elevated to 411.1 [+ or -] 11.7 mg/dl in vehicle-treated mice, and to 338.2 [+ or -] 24.5 mg/dl in borapetoside C-treated mice. The plasma glucose levels in borapetoside C-treated mice at 60. 120, and 150 min after oral administration of glucose were significantly lower than those of vehicle-treated mice at the same time points. These results indicate that borapetoside C significantly enhanced glucose utilization in T2DM mice. Borapetoside C also decreased the area under the curve (AUC) by 22% and 16% of that of the vehicle-treated group in normal and T2DM mice, respectively (Fig. 1C). These data indicate that glucose tolerance improved in the borapetoside C-treated group.
Borapetoside C stimulated glycogen synthesis in skeletal muscle of the normal and diabetic mice
The glycogen content in skeletal muscle of the normal, T1 DM, and T2DM mice, were determined at 30 min after intraperitoneal injection with borapetoside C (5 mg/kg) and Actrapid (0.5 IU/kg; a short-acting insulin provided by Novo Nordisk). As shown in Table 1, borapetoside C significantly increased glycogen synthesis in both normal and diabetic mice. Relative to the insulin effect, borapetoside C caused a more dramatic increase in glycogen content in T2DM mice, but a less dramatic change in T1 DM mice.
Table 1 Glycogen synthesis in skeletal muscles in normal and diabetic mice. Vehicle Borapetoside C Insulin Normal mice 21.5 [+ or -] 25.1 [+ or -] 30.2 [+ or -] 0.4 1.3 * 2.0 *** T1DM mice 16.7 [+ or -] 23.2 [+ or -] 30.0 [+ or -] 0.9 0.7 *** 1.0 *** T2DM mice 19.2 [+ or -] 23.3 [+ or -] 20.2 [+ or -] 0.8 0.4 *** 0.6 Values expressed as mean [+ or -] SEM from six animals for each group. T1DM. T2DM and normal mice treated with vehicle at the same volume. * P<0.05 represents the level of significance compared with the values with vehicle-treated mice. *** P<0.005 represents the level of significance compared with the values with vehicle-treated mice.
Effect of borapetoside Con the phosphorylation state of insulin signaling pathway in the liver of TI DM mice
To characterize the mechanistic effect of borapetoside C on glucose utilization of liver, western blot analyses for the levels of IR and Akt/PKB phosphorylation states and GLUT2 expression in the liver of T1 DM mice were examined after a 7-day treatment of borapetoside C at 5.0 mg/kg or insulin at 0.5 IV/kg twice daily were performed. The results showed that borapetoside C treatment increased the levels of phosphorylated IR, phosphorylated Akt and the expression of GLUT2 in the liver of T1 DM mice (Fig. 2). Compared with the effect of insulin, borapetoside C induced more phosphorylation of IR, but less phosphorylation of Akt and GLUT2 expression than insulin. The density ratios of phosphor-[Tyr.sup.1345]-IR to IR, phosphor-[Ser.sup.473]-Akt to Akt and GLUT2 to [3-actin were calculated and plotted in the lower panel of Fig. 2.
Effect of borapetoside Con insulin tolerance test OM in the non-diabetic and diabetic mice
In a previous study, borapetoside C showed a hypoglycemic effect beyond the dose of 0.1 mg/kg both in normal, T1 DM, and T2DM mice. Therefore, the effects of borapetoside Con insulin tolerance were tested in this study. The non-diabetic, T1 DM, and T2DM mice were injected with insulin at various doses (i.e., 0.1 IU/kg, 0.5 IU/kg, and 1.0 IU/kg) in conjunction with either borapetoside C (0.1 mg/kg) or a vehicle control (Table 2), and their plasma glucose levels were examined before and 30 min after insulin injection. The activity of lowering plasma glucose levels ([A.sub.L]) was calculated by the formula: ([G.sub.i] - [G.sub.t])/[G.sub.i] x 100%, where the [G.sub.i] was the initial glucose concentration and [G.sub.t] was the plasma glucose concentration after 30-min treatment with insulin. Insulin reduced the plasma glucose levels in both normal and diabetic mice, and the glucose levels were further decreased when insulin was co-administered with borapetoside C to the mice. Since the dose of borapetoside C at 0.1 mg/kg did not alter the plasma glucose levels, these results indicate that borapetoside C significantly increased the sensitivity of diabetic mice to exogenous insulin.
Table 2 Plasma glucose reduction in mice treated with insulin and borapetoside C. Insulin [A.sub.L] (%) (a) 0.1 IU/kg 0.5 IU/kg 1.0 IU/kg Normal Vehicle 14.6 [+ or -] 37.5 [+ or -] 56.8 [+ or -] mice 0.8 1.1 1.9 Borapetoside 17.2 [+ or -] 45.7 [+ or -] 64.7 [+ or -] C 1.2 3.7 1.8 * T1DM Vehicle 8.7 [+ or -] 11.5 [+ or -] 24.0 [+ or -] mice 1.6 0.6 1.3 Borapetoside 10.5 [+ or -] 18.9 [+ or -] 32.2 [+ or -] C 1.4 2.1 2.1 ** T2DM Vehicle 19.5 [+ or -] 27.5 [+ or -] 32.0 [+ or -] mice 1.5 2.6 1.1 Borapetoside 24.0 [+ or -] 31.6 [+ or -] 42.5 [+ or -] C 1.1 1.6 0.8 *** (a.) Values shown in table are mean [+ or -] SEM of [A.sub.L]. * P < 0.05 represents the level of significance compared with the values vehicle-treated mice. ** P<0.01 represents the level of significance compared with the values with vehicle-treated mice. *** P<0.005 represents the level of significance compared with the values with vehicle-treated mice.
In combination with insulin, low dose of borapetoside C stimulates glycogen synthesis in skeletal muscle in the non-diabetic and diabetic mice
Both non-diabetic and diabetic mice were subjected to various doses of insulin injections at 30 min after intraperitoneal injections with borapetoside C (0.1 mg/kg) or with the vehicle. The glycogen contents in skeletal muscles were then determined at 30 min after insulin injections (Table 3). The ratios of increased glycogen content in the other groups were normalized to those of vehicle-treated group. The ratio was 119.8%, 125.3%, and 108.5% in normal, T1 DM, and T2DM mice of insulin-treated group. In borapetoside C combined with insulin-treated group, it was 130.2%, 162.8%, and 122.6% in normal, T1 DM, and T2DM mice. Administration of borapetoside C followed by insulin injection significantly enhanced glycogen synthesis in normal mice, T1 DM mice, and T2DM mice by 52.5%, 148.2%, and 165.9% as compared to that in mice injected only with insulin. The increase of insulin sensitivity by borapetoside C is more prominent in diabetic mice than normal mice.
Table 3 Glycogen content in skeletal muscle in mice treated with insulin and boraperoside C. Vehicle BoC Insulin BoC + insulin Normal 31.8 [+ or -] 32.7 [+ or -] 38.1 [+ or -] 41.4 [+ or -] mice 0.4 0.7 0.3 *** 0.6 ***, ### T1DM 30.4 [+ or -] 30.0 [+ or -] 38.1 [+ or -] 49.5 [+ or -] mice 1.6 1.2 2.3 * 3.5 ***, # T2DM 32.7 [+ or -] 30.7 [+ or -] 35.5 [+ or -] 40.1 [+ or -] mice 1.0 1.3 1.1 1.4 **, # Values shown in table are mean [+ or -] SEM from six animals for each group. * P<O.05 represents the level of significance compared with the values with vehicle-treated mice. ** P<0.01 represents the level of significance compared with the values with vehicle-treated mice. *** P<0.005 represents the level of significance compared with the values with vehicle-treated mice. # P < 0.05 represents the level of significance compared with the values with insulin-treated mice. ### P < 0.005 represents the level of significance compared with the values with insulin-created mice.
Effect of combining borapetoside C and insulin on the protein level in the liver of TI DM mice
After a continuous 7-day treatment, the degree of tyrosine phosphorylation of IR, serine phosphorylation of Akt and GLUT2 in the liver was not significantly increased in the borapetoside C-treated (0.1 mg/kg twice daily) group. However, under the combination of insulin stimulation (1.0 IU/kg twice daily), the level of tyrosine phosphorylation of IR, serine phosphorylation of Akt and GLUT2 were elevated to 1.4, 3.0 and 1.3-fold of their vehicle-treated counterparts (Fig. 3).
In the condition of insulin resistance, certain intracellular signaling pathways become more resistant to insulin stimulation (Shulman 2000: Vollenweider 2003). The normalization of insulin sensitivity is important for the body to ingest nutrients, in particular. dietary carbohydrates (Bessesen 2001). In our previous unpublished study. high caloric diet comprising of high-fat and high-fructose reduced insulin sensitivity in peripheral tissues and resulted in a remarkable elevation of circulating glucose. To characterize how borapetoside C may regulate insulin sensitivity, the effect of borapetoside C on the glucose utilization was verified by OM' test in the present study. The results showed that borapetoside C significantly accelerated the glucose uptake and utilization in peripheral tissues in both non-diabetic and T2DM mice (Fig. 1) after oral administration of glucose. Moreover, borapetoside C (5 mg/kg) also increased glycogen content in skeletal muscle (Table 1).
In addition to the skeletal muscle, liver is another important metabolic organ for glucose metabolism. Hepatic insulin resistance is well accepted to be the primary leading cause for developing DM (Taniguchi et al. 2005). However, many of the cellular processes including glucose homeostasis, fat metabolism, and cell growth are regulated by the insulin signaling pathway. Defects in cellular processes mediated by insulin signaling pathways are central to the development of obesity and related diseases, such as insulin resistance, diabetes, and cancer (Jackson 2006). Although the molecular events are not fully elucidated yet, it is likely that Akt/PKB kinase activity plays a crucial role in the hepatic insulin action, at least in terms of glycogen synthesis (Eklar-Finkelman et al. 1999; Lavoie et al. 1999). Thus, we focused on the pathway leading to the activation of Akt/PKB kinase from IR. Moreover, the diabetogenic action of STZ in the pancreas is preceded by its rapid selective uptake by pancreatic [beta] cells through the low-affinity GLUT2 (Hosokawa et al. 2001). Peripherally this transporter is a component of the signaling pathway involved in glucose sensing and regulation of insulin secretion from pancreatic insulin secretion from pancreatic cells (Thorens et al. 1988). As shown in Fig. 2, we found that borapetoside C treatment increased phosphorylation of IR and Akt as well as the protein levels of GLUT2 in liver. In addition to the enhancement of insulin sensitivity, borapetoside C could induce IR phosphorylation and consequently lead to Akt phosphorylation and GLUT2 expression in T1 DM mice. Compared with the effect of insulin, borapetoside C induced more IR phosphorylation but less Akt phosphorylation and GLUT2 expression than insulin. This finding indicates that borapetoside C may bind to different site of insulin receptor and result in less efficient activation of Akt/GLUT2 signaling.
The other method developed to evaluate insulin sensitivity in vivo involved the use of the insulin tolerance test, which is based on the change of plasma glucose level after a bolus injection of regular insulin (Ginsberg 1977), and its results reflected the insulin sensitivity. The extract from T. crispa has been shown to reduce the plasma glucose level in moderately diabetic rats (Noor and Ashcroft 1989). It was also reported that the hypoglycemic effect of the extract is probably due to its stimulus efficacy on [beta]-cell insulin release (Noor et al. 1989). Our previous study found that a bolus intraperitoneal injection of borapetoside C from 0.5 to 5 mg/kg significantly lowered plasma glucose concentrations in a dose-dependent manner in the non-diabetic, T1 DM, and T2DM mice (Lam et al. 2012). Borapetoside Cat the ineffective dose (0.1 mg/kg) combined with insulin (1.0 IU/kg) could enhance insulin-induced lowering of plasma glucose (Table 2) and insulin-induced increase of glycogen content in skeletal muscle of diabetic mice and normal mice (Table 3). The increase of insulin sensitivity by borapetoside C is more prominent in diabetic mice than normal mice. Corresponding to the enhancement of insulin sensitivity, insulin co-administered with low-dose borapetoside C enhanced insulin-induced IR phosphorylation and consequent Akt phosphorylation and GLUT2 expression in liver of T1 DM mice (Fig. 3). These results suggest that borapetoside C is not only a hypoglycemic agent, but can also act as an adjuvant for insulin function.
In conclusion, we showed that borapetoside C delayed the development of insulin resistance in association with increased insulin sensitivity. The activation or the enhancement of insulin stimulation of the IR/Akt/GLUT2 pathway may contribute to the plasma glucose-lowering effect of borapetoside C in T1DM mice. Borapetoside C at the ineffective dose co-administrated with insulin to diabetic animals not only enhanced plasma glucose lowering action of insulin, but also increased the glycogen storage in skeletal muscles. Although the underlying mechanisms for the hypoglycemic activities of borapetoside C remains to be determined, its well-characterized antidiabetic activity suggests that the use of borapetoside C alone or in conjunction with insulin may provide a novel strategy for managing diabetes in the future.
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Chi-Tun Ruan (a), Sio-Hong Lam (b), Tzong-Cherng Chi (c), Shoei-Sheng Lee (b), Ming Jai Su (a), *
(a.) Institute of Pharmacology, College of Medicine, National Taiwan University. Taipei. Taiwan
(b.) School of Pharmacy. National Taiwan University, Taipei, Taiwan
(c.) Graduate Institute of Medical Sciences. Chang Jung Christian University. Tainan County. Taiwan
* Corresponding author at: Institute of Pharmacology, College of Medicine. National Taiwan University. Room 1150. No. 1, Sec. 1, Jen-Ai Rd., Taipei. Taiwan. Tel.: +886 2 23123456x88317: fax: +886 2 23971403.
E-mail address: email@example.com (M.-J. Su).
0944-7113/$--see front matter [c] 2012 Elsevier GmbH. All rights reserved.
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|Author:||Ruan, Chi-Tun; Lam, Sio-Hong; Chi, Tzong-Cherng; Lee, Shoei-Sheng; Su, Ming-Jai|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jun 15, 2012|
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