绞股蓝内生真菌多糖对α-葡萄糖苷酶的抑制作用

Inhibitory Effect of Endophytic Fungal Polysaccharide from Gynostemma pentaphyllum on α-Glycosidase

目的:探究绞股蓝内生真菌JY25多糖对α-葡萄糖苷酶的抑制作用,为绞股蓝内生真菌多糖的应用提供基础数据。方法:确定4-硝基酚-α-D-吡喃葡萄糖苷(4-N-trophenyl-α-D-glu-copyranoside,PNPG)比色法检测α-葡萄糖苷酶活力的最佳条件,以阿卡波糖为阳性对照,采用优化后的最佳条件测定JY25多糖对α-葡萄糖苷酶的抑制作用,并确定抑制类型、计算抑制常数;以Caco-2细胞作为体外产α-葡萄糖苷酶模型,测定JY25多糖的抑制效果。结果:反应时间30 min、反应温度44℃、pH 6.8、底物(PNPG)浓度5 mmol/L为PNPG比色法的最佳反应条件。以此优化条件进行比色,当JY25多糖质量浓度为6.15 mg/mL时,对α-葡萄糖苷酶抑制率达到最大(68.21%);JY25多糖的半数抑制浓度(IC_(50))为(2.46±0.42)mg/mL,抑制常数Ki为1.15 mg/mL,JY25多糖为竞争性抑制剂。在JY25多糖质量浓度为10.00 mg/mL时,对培养16 d的Caco-2细胞所产α-葡萄糖苷酶的抑制率达到16.48%。结论:体外实验证实JY25多糖具有竞争性抑制α-葡萄糖苷酶的作用。

The inhibitory effect of the polysaccharide secreted by the endophytic fungus Chaetomium JY25 isolated from Gymostamma pentaphyllum on α-glucosidase was explored to provide basic data for the application of this endophytic fungal polysaccharide. To this end, the conditions for colorimetrically detecting α-glycosidase activity using 4-N-trophenyl-α-Dglu-copyranoside（PNPG） as a substrate were optimized and the evaluation of α-glucosidase inhibitory effect was performed using acarbose as a positive control. At the same time, the inhibition type and inhibition constant were determined, and the inhibitory effect of the fungal polysaccharide on α-glycosidase derived from Caco-2 cells was measured in vitro. The results showed the optimum conditions for colorimetric determination of α-glycosidase activity were found to be reaction for 30 min at 44 ℃ with an initial p H of 6.8 and a substrate（PNPG） concentration of 5 mmol/L. The polysaccharide secreted by JY25 at 6.15 mg/mL showed the strongest inhibition against α-glucosidase with a percentage inhibition of 68.21%. The IC50 value of the polysaccharide was（2.46 ± 0.42） mg/mL, and the inhibition constant was 1.15 mg/mL in a competitive manner. The percentage inhibition of α-glycosidase derived from Caco-2 cells cultured for 16 days reached 16.48% in the presence of 10.00 mg/mL JY25 polysaccharide. Therefore, the exopolysaccharide produced by the endophytic fungus JY25 has inhibitory activity on α-glycosidase in vitro in a competitive manner.