Research Article J Ginseng Res Vol. 36, No. 2, 153-160 (2012) http://dx.doi.org/10.5142/jgr.2012.36.2.153
Increase in Insulin Secretion Induced by Panax ginseng Berry Extracts Contributes to the Amelioration of Hyperglycemia in Streptozotocininduced Diabetic Mice Eun-Young Park1#, Ha-Jung Kim2#, Yong-Kyoung Kim3, Sang-Un Park3, Jae-Eul Choi3, Ji-Young Cha2*, and Hee-Sook Jun1,4* 1
Laboratory of Beta Cell Biology and Autoimmunity, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon 406-840, Korea 2 Laboratory of Cell Metabolism and Gene Expression, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon 406-840, Korea 3 Division of Plant Science and Resources, Chungnam National University, Daejeon 305-764, Korea 4 College of Pharmacy, Gachon University of Medicine and Science, Incheon 406-840, Korea Panax ginseng has long been used as a traditional herbal medicine. More recently, it has received attention for its anti-diabetic and anti-obesity effects in humans and in animal models of type 2 diabetes. In the present study, we tested the hypoglycemic effects of ginseng berry extract in beta-cell-deficient mice and investigated the mechanisms involved. Red (ripe) and green (unripe) berry extracts were prepared and administered orally (100 or 200 mg/kg body weight) to streptozotocin-induced diabetic mice daily for 10 wk. The body weight was measured daily, and the nonfasting blood glucose levels were measured after 5 and 10 wk after administration. Glucose tolerance tests were performed, and the serum insulin levels were measured. The proliferation of betacells was measured in vitro. The administration of red or green ginseng berry extract significantly reduced the blood glucose levels and improved the glucose tolerance in beta-cell deficient mice, with the higher doses resulting in better effects. Glucose-stimulated insulin secretion was significantly increased in berry extract-treated mice compared with streptozotocin-induced diabetic control mice. Treatment with ginseng berry extract increased beta-cell proliferation in vitro. Both red berry and green berry extracts improved glycemic control in streptozotocin-induced diabetic mice and increased insulin secretion, possibly due to increased betacell proliferation. These results suggest that ginseng berry extracts might have beneficial effects on beta-cell regeneration. Keywords: Panax ginsneg, Red ginseng berry, Green ginseng berry, Diabetes, Beta-cell proliferation
INTRODUCTION Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia. It is believed that absolute or relative insulin deficiency due to the inadequate beta-
cell mass is the cause of hyperglycemia [1]. Type 1 diabetes results from the destruction of pancreatic beta-cells by beta-cell-specific autoimmune responses [2], and type
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Received 19 Oct. 2011, Revised 17 Jan. 2012, Accepted 17 Jan. 2012 # These authors contributed equally to this work. *
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J Ginseng Res Vol. 36, No. 2, 153-160 (2012)
2 diabetes results from the progressive loss of beta-cell mass and function [3]. Therefore, strategies that preserve or restore beta-cell mass and function are logical therapeutic approaches for the treatment of diabetes. Ginseng (Panax ginseng) has been used as an herbal medicine for thousands of years. Ginseng has pharmacological properties, including anti-cancer, anti-aging and anti-allergic effects [4-6], and is free from adverse effects, in contrast to chemical medicines [7]. Ginseng has also received increasing attention as a complementary and alternative medicine for the treatment of diabetes. Extracts of ginseng roots, berries and leaves have been reported to have hypoglycemic effects in animal models of type 1 and type 2 diabetes [8-10]. The anti-diabetic and anti-obesity effects of ginseng have been reported in animal experiments [11-16] and in clinical trials [17-19]. Previous studies have demonstrated that ginseng berry extract showed better anti-hyperglycemic activity than ginseng root extract when used at the same concentration [20], and berry extract has a higher content of total ginsenosides than root extract [9], with a distinct ginsenoside profile. It was reported that American ginseng (P. quinquefolius) berry extract reduced the blood glucose levels and body weight in ob/ob and db/db mice, models of type 2 diabetes [14,15]. However, the effects of ginseng berry extract on insulin-producing pancreatic beta-cells have not been specifically examined. In this study, we investigated the pharmacological effects of red (ripe) and green (unripe) ginseng berry extracts on betacells and insulin secretion using a streptozotocin (STZ)induced diabetic mouse model, in which pancreatic betacells are damaged and insulin production is deficient.
HPLC analysis Ginsenosides were analyzed as described previously [21] using an HPLC system (NS-4000; Futecs Co., Daejeon, Korea) equipped with a SofTA 300s evaporative light scattering detector (SofTA Co., Westminster, CO, USA) and a ProntoSIL fractionation column (250×4.66 mm) (Bischoff, Berlin, Germany). The flow rate was set to 0.8 mL/min. The identification and quantification of ginsenosides were carried out by comparing the retention times and the peak areas, respectively, with those of ginsenoside standards or by the direct addition of the ginsenoside standard into the sample (spike test). Sample aliquots were filtered through a 0.45 μm polytetrafluoroethylene filter prior to injection. All samples were run in triplicate. The ginsenoside standards (Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, and Rh1) were purchased from Canfo Chemical, Chengdu, China. Animals C57BL/6 mice were purchased from the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea). Mice were maintained under specific pathogen-free conditions in a temperature-controlled room (23±1°C) with a 12-h light/dark cycle and ad libitum access to food and water at the Animal Care Center, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Lee Gil Ya Cancer and Diabetes Institute. Induction of diabetes and treatment with ginseng berry extract At 8 wk of age, male mice were intraperitoneally injected with STZ (80 mg/kg body weight) in sterile citrate buffer, pH 4.0, for two consecutive days. Beginning 2 d after STZ injection, the glucose levels in the tail vein blood were monitored with a glucometer (One Touch Ultra; LifeScan Inc., Milpitas, CA, USA). Mice with blood glucose levels over 250 mg/dL were used for the subsequent experiments. Five days after the STZ treatment, animals were randomly divided into six groups (n=7 or 8 in each group): normal control mice, PBS-treated STZinduced diabetic mice, and STZ-induced diabetic mice given 100 or 200 mg/kg RB or GB extract. Berry extracts were given by oral intubation daily for 10 wk. After 5 and 10 wk of treatment, the glucose levels and body weights were measured following the removal of food for 3 h, and food consumption was measured weekly.
MATERIAL AND METHODS Preparation of ginseng berry extract Fresh green berries (GBs, unripe) or red berries (RBs, ripe) of P. ginseng were freeze-dried. Approximately 500 g of berries was extracted with 10 L of 70% ethanol at 37°C for three days in a shaking incubator, and this extraction was repeated three times. After evaporating the solvent, the extracts were dissolved in distilled water (30 mL) and extracted three times with 30 mL of methylene chloride in a separatory funnel. The residual aqueous phase was extracted two more times with ethyl acetate, and the ethyl acetate extracts were then mixed with n-butanol. The butanol phase was collected and evaporated. The dried powder was used for HPLC analysis and subsequent experiments. For administration to mice, the berry extracts were dissolved in phosphate-buffered saline (PBS).
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Intraperitoneal glucose tolerance tests After 4 and 8 wk of extract treatment, mice were not fed for 14 h, and then a glucose solution (2 g/kg body weight in PBS) was injected intraperitoneally. The blood glucose levels were measured at 0, 30, 60, 90, and 120 min after glucose injection.
GTTCTG-3’; and mouse cyclophilin: sense 5’-CCATGA ATTA CCTGGACCGTTT-3’ and antisense 5’-GCGCCCAGGAGCTGAAG-3’. The qRT-PCR results were evaluated using the comparative Ct method (User Bulletin No. 2; PerkinElmer Life Sciences, Waltham, MA, USA) with cyclophilin as the invariant control gene.
Measurement of the serum insulin levels After 10 wk of treatment, mice were not fed for 14 h, and then a glucose solution (2 g/kg body weight in PBS) was injected intraperitoneally. Blood samples were drawn into heparinized capillary tubes from the periorbital veins at 30 min after glucose injection. The blood was centrifuged (3,000 rpm, 15 min, 4°C), and the serum insulin levels were determined using a mouse insulin ultrasensitive EIA kit (Alpco Diagnostics, Salem, NH, USA).
Statistical analysis Data are presented as the mean±SE. The significance of the differences was analyzed with an unpaired Student’s t-test and ANOVA followed by a posteriori test. The values of statistical significance were set at p
