三七粗多糖的分离纯化及其对人牙周膜干细胞、小鼠成骨细胞体外增殖活性的影响

Isolation and purification of Panax notoginseng polysaccharide and its effect on proliferation of human periodontal ligament stem cells and mice osteoblasts in vitro

目的从工业三七药渣中提取、分离及纯化三七多糖,测定三七粗多糖及三七多糖洗脱组分的含量、分子量和p H,并考察其对人牙周膜干细胞、小鼠成骨细胞体外增殖活性的影响。方法以提取过三七总皂苷的工业三七药渣为原料,采用水提醇沉法得到三七粗多糖,通过DEAE Sepharose Fast Flow阴离子交换层析柱分离纯化三七粗多糖;采用蒽酮-硫酸法测定三七粗多糖及三七多糖洗脱组分的含量,用高效凝胶渗透色谱法测定其分子量;采用MTT法测定三七粗多糖及三七多糖洗脱组分对人牙周膜干细胞、小鼠成骨细胞体外增殖活性的影响。结果水提醇沉法得到三七粗多糖的含量为69. 7%,得率2. 43%;通过DEAE Sepharose Fast Flow阴离子交换层析柱分离纯化三七粗多糖,得到水洗脱部分PNPSⅠ及Na Cl溶液梯度洗脱部分PNPSⅡ~Ⅴ;三七粗多糖、PNPSⅠ~Ⅴ重均分子量分别为38. 59、32. 00、96. 98、148. 60、154. 40、113. 60 k Da;在一定浓度范围内,三七粗多糖、PNPSⅠ~Ⅱ能促进人牙周膜干细胞的生长,PNPSⅢ~Ⅳ能抑制其生长;三七粗多糖、PNPSⅠ~Ⅳ具促进小鼠成骨细胞生长的作用。结论通过DEAE Sepharose Fast Flow阴离子交换层析柱分离纯化三七粗多糖,得到1种中性多糖和4种酸性多糖;三七粗多糖、PNPSⅠ~Ⅱ能促进人牙周膜干细胞的体外增殖,三七粗多糖、PNPSⅠ~Ⅳ具促进小鼠成骨细胞生长的作用。

OBJECTIVE To extract,separate and purify Panax notoginseng polysaccharide( PNPS) from industrial Sanqi dregs,and to determine the content,molecular weight and p H value of the elution components of P. notoginseng and PNPS,and to study the in vitro proliferation activity of human periodontal ligament stem cells and mice osteoblasts. METHODS The crude PNPS was obtained by water extraction and alcohol precipitation method,and the crude polysaccharide of P. notoginseng was separated and purified by DEAE Sepharose Fast Flow anion exchange chromatography column. The anthrone-sulfuric acid method was used to extract the crude PNPS from the crude P. notoginseng saponins. PNPS were determined,and the molecular weight was detected by high performance gel permeation chromatography. The MTT method was used to determine the effect of polysaccharides from P. notoginseng human mesenchymal stem cells. Effect of mice osteoblast proliferation activity in vitro was also tested. RESULTS The water extraction and alcohol precipitation method obtained the crude polysaccharide of P.notoginseng with the content of 69. 7%,and the yield was 2. 43%. The crude polysaccharide of P. notoginseng was separated and purified by DEAE Sepharose Fast Flow anion exchange chromatography column to obtain the water-eluting part PNPS Ⅰ,gradient elution of Na Cl solution,part of PNPS Ⅱ-Ⅴ. Weight average molecular weight of PNPS and PNPS Ⅰ-Ⅴ was 38. 59,32. 00,96. 98,148. 60,154. 40,113. 60 k Da,respectively. PNPS and PNPS Ⅰ-Ⅱ promoted the growth of human periodontal ligament stem cells,while PNPS Ⅲ-Ⅳ inhibited the growth. PNPS and PNPS Ⅰ-Ⅳ promoted the growth of mice osteoblasts.CONCLUSION Crude polysaccharides from P. notoginseng have been successfully separated and purified by DEAE Sepharose Fast Flow anion exchange chromatography column,obtaining a neutral polysaccharide and four acidic polysaccharides. PNPS and PNPS Ⅰ-Ⅱ can promote the proliferation of human periodontal ligament stem cells in vitro. PNPS and PNPS Ⅰ-Ⅳ can promote mice osteoblast growth.