川牛膝、麻牛膝及杂交牛膝多糖的分离、纯化和对猪小肠上皮细胞活性的比较研究

Comparing study of separation, purification and porcine intestinal epithelial cells activity of polysaccharide from Cyathula officinalis, Cyathulae capitatae and C.officinalis×C.capitata

研究旨在比较川牛膝及其常见伪品的多糖含量、组分和对猪小肠上皮细胞活性的差异。用乙醇除杂,水加热回流法得粗多糖;用阴离子交换层析法和凝胶柱层析法分离、纯化多糖,并测定多糖的相对分子量;用CCK-8试剂盒系统测定多糖对猪小肠上皮细胞活性的影响;用荧光定量PCR检测多糖作用后炎症细胞因子IL-6表达情况。试验结果:①川牛膝粗多糖得率为7.40%;麻牛膝粗多糖得率为1.67%;杂交牛膝粗多糖得率为0.38%;②川牛膝酸性多糖得率为94.40%,中性多糖得率为1.10%;麻牛膝酸性多糖得率为30.74%,中性多糖得率为23.00%;杂交牛膝酸性多糖得率为11.20%,中性多糖得率为10.00%;③川牛膝多糖相对分子量主要分布在7 500 Da左右、200 000～400 000 Da和1 200 000～1 700 000 Da;麻牛膝多糖的相对分子量主要集中在7 500 Da左右;杂交牛膝多糖相对分子量主要集中在6 000 Da左右;④川牛膝多糖和麻牛膝多糖的低剂量和高剂量组对猪小肠上皮细胞活性均无明显影响;川牛膝多糖和麻牛膝多糖均能显著抑制炎症细胞因子IL-6的表达。川牛膝多糖含量显著高于麻牛膝和杂交牛膝多糖,川牛膝和麻牛膝多糖对猪小肠上皮细胞活性无明显影响,但均能降低炎症细胞因子IL-6的表达量。

This study was conducted to compare content,component and porcine intestinal epithelial cells viability of polysaccharide of Cyathula officinalis and its' common spurious breed. Digestion with ethanol, use heating reflux method extract crude polysaccharide;use anion exchange chromatography and gel column chromatography to separate and purify the crude polysaccharide, determinate the molecular weight of the purified polysaccharide;use Cell Counting Kit-8 to measure the effect of polysaccharides on cell viability of IPEC-J2;use quantitative real-time PCR to measure the relative expression of inflammatory cytokine IL-6 after the treatment of polysaccharides.① The crude polysaccharide yield of Cyathula officinalis is 7.40%;The crude polysaccharide yield of Cyathulae capitatae is 1.67%;The crude polysaccharide yield of C.officinalis × C.capitata is 0.38%.② The acidic polysaccharide yield of Cyathula officinalis is 94.40%, the neutral polysaccharide yield is 1.10%;The acidic polysaccharide yield of Cyathulae capitatae is 30.74%, the neutral polysaccharide yield is 23.00%;The acidic polysaccharide yield of C.officinalis×C.capitata is 11.20%, the neutral polysaccharide yield is 10.00%.③ The relative molecular weight of polysaccharide of Cyathula officinalis mainly distribute in 7 500, 200 000~400 000 Da and 1 200 000~1 700 000 Da;The relative molecular weight of polysaccharide of Cyathulae capitatae mainly distribute in 7 500 Da;The relative molecular weight of polysaccharide of C.officinalis × C.capitata mainly distribute in 6 000 Da.④ Both low dose and high dose of polysaccharides of Cyathula officinalis and Cyathulae capitatae have no obvious effect on cell viability of IPEC-J2;Polysaccharides of Cyathula officinalis and Cyathulae capitatae can significantly inhibit the expression of inflammatory cytokine IL-6. Polysaccharides content of Cyathula officinalis is much higher than Cyathulae capitatae and C.officinalis×C.capitata, polysaccharides of Cyathula officinalis and Cyathulae capitatae have no obvious effect on cell viability of IPEC-J2 and they both can significantly inhibit the expression of inflammatory cytokine IL-6.