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【目的】研究木瓜蛋白酶对条斑紫菜R-藻红蛋白的抗氧化性和肿瘤增殖抑制活性的影响。【方法】采用超声波破壁法提取条斑紫菜R-藻红蛋白,用DEAE柱层析法纯化后进行木瓜蛋白酶酶解,通过正交试验设计,以还原力A700为考察指标确定酶解反应的最佳工艺参数,进一步测定获得的R-藻红蛋白及其酶解物的还原力、清除羟自由基能力和对人肉瘤细胞U2O及人肝癌细胞HepG-2的肿瘤增殖抑制活性。【结果】R-藻红蛋白的最佳酶解工艺条件为:木瓜蛋白酶添加量25 000 U.g-1,pH 7.0,温度50℃,酶解时间4 h。在此条件下,R-藻红蛋白酶解物还原力为0.573,较未酶解的R-藻红蛋白提高了2.35倍;清除羟自由基能力为51.03%,较未酶解的R-藻红蛋白活性提高了3.22倍。随着浓度的增加,R-藻红蛋白及其酶解物对人肉瘤细胞U2O和人肝癌细胞HepG-2抑制生长作用增强。R-藻红蛋白对人肉瘤细胞U2O抑制作用的IC50值为2 431.32μg.mL-1,其酶解物IC50值降低为1 271.46μg.mL-1。R-藻红蛋白对肝癌细胞HepG-2抑制作用的IC50值为1 593.61μg.mL-1,其酶解物IC50值降低为512.05μg.mL-1。【结论】经木瓜蛋白酶酶解后,R-藻红蛋白酶解物具有较强的抗氧化和抑瘤活性。酶解技术可作为进一步提高R-藻红蛋白生物活性的有效手段。
【Objective】 The objective of this study was to investigate the effect of papain on the antioxidant activity and tumor growth inhibitory activity of R-phycoerythrin. 【Method】 R-phycoerythrin was extracted by sonication and purified by DEAE column chromatography. After orthogonal design, the reducing enzyme A700 was used as the indicator to determine the enzymatic reaction The optimum technological parameters were determined. The reducing power of R-phycoerythrin and its hydrolyzate, hydroxyl radical scavenging ability and tumor growth inhibitory activity on human sarcoma cell U2O and HepG-2 were further determined. 【Result】 The optimal conditions for enzymatic hydrolysis of R-phycoerythrin were as follows: papain at 25 000 U.g-1, pH 7.0, temperature 50 ℃, hydrolysis time 4 h. Under these conditions, the reducing power of R-phycoerythrin hydrolyzate was 0.573, which was 2.35 times higher than that of R-phycoerythrin which was not enzymatically hydrolyzed. The ability of eliminating hydroxyl radical was 51.03% Protein activity increased by 3.22 times. With the increase of concentration, R-phycoerythrin and its hydrolyzate enhanced the inhibitory effect on human sarcoma cell U2O and human hepatoma cell HepG-2. The IC50 value of R-phycoerythrin on U2O inhibition of human sarcoma cells was 2 431.32μg.mL-1, and the IC50 value of enzymolysis decreased to 1 271.46μg.mL-1. The IC50 value of R-phycoerythrin on HepG-2 cells was 1 593.61μg.mL-1, and its IC 50 value was reduced to 512.05μg.mL-1. 【Conclusion】 After papain digestion, R-phycoerythrin hydrolyzate has strong anti-oxidation and anti-tumor activity. Enzymatic hydrolysis technology can be used as an effective means to further improve the biological activity of R-phycoerythrin.