白首乌甾苷复合酶法提取工艺优化及保肝活性分析

Optimization of complex enzymatic extraction process of steroidal glycosides from Cynanchum auriculatum Royle ex Wight and its hepatoprotective activity

目的:为开发白首乌保肝高值化产品提供依据。方法:以多年生白首乌为原料,采用单因素试验及响应面法,考察酶解时间、酶解温度、复合酶用量和酶解pH对白首乌甾苷得率的影响;通过乙醇诱导HepG2肝细胞损伤模型研究谷丙转氨酶(Alanine transaminase,ALT)、乳酸脱氢酶(Lactate dehydrogenase,LDH)及谷胱甘肽(Glutathione,GSH)的含量和细胞形态变化。结果:在酶解时间100 min、温度46℃、酶用量1.30%和pH 5.0的条件下,实际得率为(2.80±0.04)%。与模型组相比,白首乌可明显抑制肝损伤细胞中ALT、LDH含量,显著提高细胞内的GSH水平,恢复细胞损伤。结论:复合酶法可有效提取白首乌甾苷;白首乌甾苷具有保肝作用。

Objective:Response surface methodology was used to optimize the extraction process of steroidal glycosides from Cynanchum auriculatum Royle ex Wight by complex enzymatic method,and its effects on alcohol dehydrogenase activity and human hepatoma cell HepG2 were analyzed.Methods:Four experimental factors;enzymolysis duration,temperature,compound enzyme dose and pH were used as responses to a single-factor experiment of the Response Surface Methodology on the steroidal glycoside yields,using perennial C.auriculatum as the target material.The alcohol-induced hepatocyte damage model of HepG2 was used to investigate the contents of ALT,LDH,and GSH and changes in cell shape.Results:The results show that the optimum extraction conditions of steroidal glycoside enzyme were obtained at an enzymolysis duration of 100 min,a temperature of 46℃,enzyme dose of 1.30%and pH of 5.0.Consequently,the actual extraction rate of(2.80±0.04)%was achieved using the optimized process conditions,which was close to the expected value.Compared with the model group,it significantly suppresses the growth of ALT and LDH while increasing the amount of intracellular GSH,and the cell damage was restored.Conclusion:The compound enzyme method can effectively extract the steroidal glycosides of Cynanchum auriculatum Royle ex Wight;steroidal glycosides of C.auriculatum have hepatoprotective effect.