Investigation of Gallic Acid in Stimulating Effect of Insulin Secretion by Microdialysis-High Performance Liquid Chromatography Analysis.

• Main Authors: Chih-Ju Yen, 顏志如
• Other Authors: Tung-Hu Tsai
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/07529887516970760015

碩士 === 國立陽明大學 === 傳統醫藥學研究所 === 96 === Gallic acid (3,4,5-trihydroxybenzoic acid) is a polyphenol structure divided from pomegranate. This compound has also been described as an excellent free radical scavenger. Due to this antioxidant effect, gallic acid containing plant extracts have showed the antidiabetic, antiangiogenic and antimelanogenic effect. It’s mechanisms for such effects are largely unknown, recent research suggests might via peroxisome proliferator-activated receptor-gamma binding. There has also been much effort to develop beneficial agents from medicinal plants to achieve antidiabetic effect. And treatment of diabetes with herbal remedies and the popularity of complementary medicine have also been increasing among practitioners. The beta-cells in the pancreatic islets of Langerhans synthesize and secrete insulin, the key regulator of glucose homeostasis. Decreased insulin secretion or impaired insulin action leads to one of the major world health problems, diabetes mellitus. It is a major cause of morbidity and mortality in patients despite significant advances in treatment. Promoting secretion of insulin by drugs is the current approach to improve the symptoms of diabetes. Though several new pharmacological agents have been developed for the management of diabetes, but the conventional therapies for diabetes have many shortcomings like side effects. On the other hand, herbal extracts are expected to have similar efficacy without side effects as that of conventional drugs. The objective of the present work is to develop an in vitro drug screen system; the application of the device is capable of taking samples for the extracellular compound with minimal disruption of the normal growth of cells in culture. This study evaluates the insulin-secreting stimulation effect of gallic acid on insulinoma cell. This procedure using microdialysis coupled with reverse-phase high performance liquid chromatography (HPLC). A Chromolith® Performance RP-18 endcapped column was used as stationary phase and mobile phase consisted of 70/30 volume ratio of water/acetonitrile with 0.12% trifluoroacetic acid. The eluent was monitored with a UV detector set at 215 nm, flow rate of 1 ml/min, and sample size of 50 µl, all over the study was carried out at room temperature. The method produced linear response over the concentration range of 0.25-10 µg/ml, with a intra- and inter-day variations were less than 11.96 % and 14.7% respectively. The microdialysis probes recoveries of insulin in the insulinoma cell culture medium were 3.18 ± 0.59 (means ± SD) based on the concentrations of 5, 10 and 20 μg/ml insulin standard. Unfortunately, due to the limitation of extremely low probe recovery and limitation of HPLC detection range, the study analysis insulin concentration by ELISA kit. In additional, the effect of candidate drugs regulate mRNA expression of insulin secretion-related gene has also been investigated. The study results shown the well effect of insulin-secretion and insulin mRNA upregulation of gallic acid compared with control group. To compare the in vitro and in vivo model condition, in animal model, the normal Sprague-Dawley rat was administrated with gallic acid (100 mg/kg/day),pioglitazone (10 mg/kg) and vehicle solution for one week, were divide as experiment, positive and negative controls group respectively. Measure the plasma glucose and insulin and then calculated insulin resistance value using the formula of homeostasis model assessment of insulin resistance. At the same time, the oral glucose tolerance test was performance as well. The new device provides one novel real-time sampling method for in vitro experiment, for other target molecular, various alternatives, modifications, and improvements could be developing and without departing from the spirit and scope of the invention. The in vitro and in vivo study findings suggest that gallic acid has the well effect in improving insulin level and insulin-resistance. All results could provide s new extension for diabetes treatment in the future.