北虫草多糖提取工艺优化及其细胞氧化损伤保护作用

Optimization of Extraction Process of Cordyceps militaris Polysaccharide and Its Protective Effect on Cellular Oxidative Damage

目的:优化北虫草多糖的提取工艺,研究北虫草多糖对血管平滑肌细胞氧化损伤的保护作用。方法:采用L9(34)正交实验法优化多糖提取工艺,利用羟自由基法、DPPH自由基法和FRAP法检测北虫草多糖清除自由基的能力。在细胞水平上构建过氧化氢诱导细胞氧化损伤模型,采用MTT法、ROS细胞染色法评估北虫草多糖对过氧化氢诱导大鼠主动脉平滑肌细胞氧化损伤的保护作用。结果:北虫草多糖的最佳提取条件为:提取温度90℃,料液比1∶30 g/mL,提取次数3次,提取时间3.0 h,最高得率为7.94%±0.16%。在多糖浓度为1.6 mg/mL时对羟自由基和DPPH自由基的清除能力以及总抗氧化能力分别为75.57%±1.39%、80.23%±2.75%和(0.22±0.01)mmol/mg。细胞实验表明北虫草多糖可显著抑制过氧化氢诱导细胞内ROS生成,提高细胞存活率。结论:成功优化北虫草多糖提取工艺,北虫草多糖具备良好的体外清除自由基的活性,对由氧化应激所导致的心血管疾病中起到很好的防治意义。

Objective:To optimize the extraction process of Cordyceps militaris polysaccharides and study the protective effect of Cordyceps militaris polysaccharides on oxidative damage of vascular smooth muscle cells.Methods:L9(34)orthogonal experiment was used to optimize the extraction parameters of polysaccharides.Hydroxyl radical method,DPPH free radical method and FRAP method were used to test the radical scavenging ability of Cordyceps militaris polysaccharides.The model of cell oxidative damage induced by hydrogen peroxide was constructed at the cellular level.MTT assay and ROS cell staining were used to evaluate the protective effect of Cordyceps militaris polysaccharide on oxidative damage induced by hydrogen peroxide in rat aortic smooth muscle cells.Results:The optimal extraction conditions of Cordyceps militaris polysaccharides were:extraction temperature 90℃,ratio of material to liquid 1∶30 g/mL,extraction time 3.0 h,extraction times 3 times,the highest yield was 7.94%±0.16%.The scavenging ability of hydroxyl radicals and DPPH free radicals and total antioxidant capacity at the concentration of 1.6 mg/mL were 75.57%±1.39%,80.23%±2.75% and(0.22±0.01)mmol/mg,respectively.Cell experiments showed that Cordyceps militaris polysaccharides significantly inhibited hydrogen peroxide-induced intracellular ROS production and increased cell viability.Conclusion:The technology of extracting polysaccharides from Cordyceps militaris was successfully optimized.Cordyceps militaris polysaccharide has good activity of scavenging free radicals in vitro and plays a very good role in prevention and treatment of cardiovascular diseases caused by oxidative stress.