铁皮石斛多糖提取及对羟自由基诱导的SH-SY5Y细胞凋亡的抑制作用

Extraction of Polysaccharides from Dendrobium officinale and Their Inhibition Effect on Hydroxyl Radical-Induced Apoptosis of SH-SY5Y Cells

以酶解超声提取法提取铁皮石斛多糖,通过对料液比、超声时间、复合酶质量分数的单因素试验和正交试验,确定铁皮石斛多糖的最佳提取条件;以高效液相色谱法对铁皮石斛多糖进行组成成分分析;以香豆素-3-羧酸荧光法测定铁皮石斛多糖的羟自由基清除能力;以噻唑蓝实验和细胞流式实验研究铁皮石斛多糖提取物对SH-SY5Y细胞的保护作用。结果表明,最优铁皮石斛多糖提取条件为料液比1∶100(g/mL)、超声时间2 h、复合酶(纤维素酶和果胶酶1∶1等质量混合)质量分数50%,最优提取条件下铁皮石斛多糖得率为25.69%,其主要单糖组分为甘露糖和葡萄糖。在质量浓度为20~80μg/mL范围内铁皮石斛多糖提取物能有效清除Cu2+催化H2O2产生的羟自由基并有效抑制SH-SY5Y细胞的凋亡(凋亡率从56.97%下降到17.97%),且其作用效果成质量浓度依赖性。因此,铁皮石斛多糖可通过清除羟自由基从而抑制Cu2+催化H2O2产生羟自由基诱导的SH-SY5Y细胞凋亡。

The ultrasonic-assisted enzymatic extraction of polysaccharides from Dendrobium officinale was optimized using one-factor-at-a-time and orthogonal array design methods. Solid-to-liquid ratio, ultrasonication time, and enzyme concentration were considered as independent variables. High performance liquid chromatography(HPLC) was used for analyzing the monosaccharide composition of D. officinale polysaccharides. Coumarin-3-carboxylic acid(CCA) fiuorescence method was employed for determining the antioxidant activity of D. officinale polysaccharides. Methyl thiazolyl tetrazolium(MTT) and flow cytometry were employed for investigating the inhibition effect of the polysaccharides on hydroxyl radical-induced apoptosis of SH-SY5 Y cells. The results showed that the optimum extraction conditions were as follows: solid-to-solvent ratio 1:100(g/mL), ultrasonication time 2 h, and enzyme(a 1:1 mixture of cellulase and pectinase, m/m) concentration 50%, and the yield of polysaccharides was 25.69% under these conditions. The monosaccharide composition analysis showed that D. officinale polysaccharides were mainly composed of mannose and glucose. D. officinale polysaccharides at concentrations ranging from 20 to 80 μg/mL could effectively scavenge hydroxyl radicals produced from hydrogen peroxide with Cu2+ as a catalyst and consequently decreased hydroxyl radical-induced apoptosis of SH-SY5 Y cells(the apoptotic rate decreased from 56.97% to 17.97%) in a concentration-dependent manner.