Study Of Thorn Grape Seeds Unsaturated Fattyacid Extraction And Its Anti-apoptotic To Islet β-cell

Thorn grape (Vitis davidii Foex.) is one of eastern Asia’s species which is part of Vitis in the flowering plant family Vitaceae. It exists in a wild state in Hu-nan, Hu-bei, Guang-xi, Si-chuan province and exert good effects on Huoxue, Xingqi, Xiaoji. Modern researchers have found that UFA, Res, OPC, squalene, SOD and other bioactive substances are prolific in thron grape. Therefore, it maybe involve quite strongly antioxidant function and play critical roles in lowering blood fat, blood pressure, softening blood vessels. However, there is not any anti-apoptosis research has been reported about thron grape. In this study, thorn grape seeds were used as original materials, and response surface method was taken to analyze the content of thorn grape seeds UFA which was detected by the SFE-CO2technology. Pancreas islet beta-cells apoptosis models were constructed via high concentration of glucose. Furthermore, the function and mechanism of pancreas islet beta-cell anti-apoptosis on thorn grape seed UFA were studied at cellular and molecular level. The results are as follows:1. In the study, the content of fatty acid was major evaluation indicator. As extraction time, extraction pressure and extraction temperature were three influenced factors in SFE-CO2, they had been selected to optimize the extraction technology by response surface method. Result shows that the best conditions of thorn grape seed fatty acid by SFE-CO2is that extraction pressure of34.2Mpa, extraction time of95min, extraction temperature of39.3℃on the base of granularity of raw materials in40mesh, CO2flow of20～30L/h, separation pressure of12Mpa, separation temperature of55℃. In this case, the fatty acid yield was15.06%.2. While it was analyzed by GC-MS after methyl esterification. It shows that thorn grape seeds fatty acid includes unsaturated fatty acid mostly, and the yield was86.5%. Among them, linoleic acid account for76.5%.3. In this study, apoptotic model of β-cells was constructed by adding high glucose to the culture media of rattus Rin-m5F cells line. Forty-eight hours after thorn grape seeds UFA was added, the ratio of apoptosis was detected by flow cytometry. The result suggested a dose-dependent function of protection on rattus pancreatic β-Cells by adding50μg/ml-100μg/ml thorn grape seeds UFA.100μg/ml was the best anti-apoptosis concentration, the rate of apoptosis compared with model group decreased by64.7%.4. The index of insulin secretion was determined by ELISA. Result showed insulin secretion was promoted at concentration of100μg/ml thorn grape seeds UFA. Insulin concentration compared with the model group has increased8.0%.5. The mRNA expression of iNOS, ATF, Caspase-3, JNK, p38, NF-kB, Fas, Bcl-2, Bax and AKT were detected by Real-time quantitative PCR. The results showed that the high-glucose induced apoptosis of RIN-m5F were largely related to TNFR signal pathway, mitochondrial pathway and endoplasmic reticulum pathway. Among them, endoplasmic reticulum pathway was dominating. Result also indicated that thorn grape seeds UFA can protect β-cell from apoptosis by death receptor signal pathway, mitochondrial pathway and endoplasmic reticulum pathway.