鲟鱼皮GPC的制备及其对PC12细胞的保护作用

Preparation GPC from Sturgeon Skin and Its Protective Effect on PC12 Cells

目的:对比热解和酶解法所得明胶-肽复合物(GPC)的制备效率和活性。方法:以鲟鱼皮为原料,以GPC得率为指标,优化鲟鱼皮预处理条件及热解法制备GPC的条件。以热解法所得最大分子质量的GPC为底物,以三氯乙酸可溶性寡肽(TCA-SP)为指标,研究几种蛋白酶对该底物的水解效率。比较TCA-SP得率最高的热解法和酶解法制备GPC对正常和D-半乳糖致氧化损伤PC12细胞活力的影响。结果:碱处理对鲟鱼皮GPC提取无明显贡献,酸处理对GPC提取率影响较大,最适条件为0.25 mol/L HCl处理鲟鱼皮6 h。高温热解法制备GPC的最适条件为100℃提取2 h,此时GPC和TCA-SP得率最高,分别为34.72%,31.22%。低温热解法以60℃提取1 h所得GPC的黏度最大,TCA-SP最低,提示该产物分子质量最大,结构最完整。以GPC-60℃-1 h为底物,分别用几种蛋白酶单独水解,其中中性蛋白酶水解0.5 h所得GPC中的TCA-SP含量最高(28.74%)。GPC-100℃-2 h和GPC-N-0.5 h底物在质量浓度为50~1000μg/mL范围,对正常PC12细胞活力均无影响。在质量浓度为12.5~200μg/mL范围对D-半乳糖所致PC12细胞氧化损伤均有一定的保护作用,且两者间无明显差异。结论:高温热解法和中性蛋白酶水解法所得GPC中的TCA-SP含量及对PC12的保护作用相近。

Objective:To guide the rational use of sturgeon skin resources,this study aimed to compare the production efficiency and bioactivity between the gelatin-peptide complex(GPC)made from sturgeon skin by the method of pyrolysis and enzymatic hydrolysis,respectively.Methods:Using sturgeon skin as raw material and GPC yield as an indicator,pretreatment condition of sturgeon skin and the preparation of GPC by pyrolysis were optimized.On the other side,using the GPC with given maximum molecular weight as substrate,and trichloroacetic acid soluble oligopeptide(TCA-SP)was used as an index,the enzymatic hydrolysis efficiency of several kinds of protease on this substrate was investigated.Finally,the effects of GPCs prepared by pyrolysis and protease hydrolysis,with the highest TCA-SP yield,on the viability of normal and D-galactose-induced oxidative damage PC12 cells was compared.Results:Alkali treatment did not contribute significantly to the extraction of GPC from sturgeon skin,while acid treatment did.The optimized condition for acid treatment was incubating the sturgeon skin at 0.25 mol/L HCl for 6 h.The most suitable conditions for GPC preparation by high-temperature pyrolysis were found at 100℃for 2 h,with a highest GPC and TCA-SP yield of 34.72%and 31.22%,respectively.The GPC obtained by low-temperature pyrolysis at 60℃for 1 h had the highest viscosity and the lowest TCA-SP,suggesting it had maximum molecular weight and relatively complete structure.GPC-60℃-1 h was then used as the substrate to be hydrolyzed by several proteases respectively.A highest TCA-SP content of 28.74%was observed in the hydrolysate of neutral protease for 0.5 h.GPC-100℃-2 h and GPC-N-0.5 h had no cytotoxic effect on normal PC12 cell viability at 50-1000μg/mL.And both had a certain protective effect on PC12 cells against D-galactose-induced oxidative damage at 12.5-200μg/mL,and there seemed no significant difference between them.Conclusion:The TCA-SP content in the GPC obtained by high-temperature pyrolysis was similar to that prepared by neutral enzymatic hydrolysis,and their protective effect on PC12 was also similar.