灰树花多糖的分离纯化及其体外抗肿瘤活性

Isolation and Purification of Polysaccharide from Grifola frondosa and Its in vitro Antitumor Activity

探究低温提取的灰树花多糖的结构及其体外抗肿瘤活性。利用低温(4℃)水提醇沉法提取灰树花粗多糖,采用Sevage法除去粗糖中的蛋白,透析除去盐类、寡糖等小分子物质,得到多糖组分命名为GFP,其得率为2.13%;离子色谱检测其主要单糖为葡萄糖、半乳糖和甘露糖;高效液相色谱分析结果表明GFP为混合物,含有两个主要的大分子群体,分子量分别为1700.00 ku和34.12ku,其组分峰面积分别为66.57%和28.23%;红外光谱显示GFP可能为α-吡喃多糖。GFP的抗肿瘤活性研究表明:在一定的浓度范围内,GFP能显著抑制人肝癌HepG2细胞的增殖;通过JC-1染色检测线粒体膜电势的变化发现GFP会引起线粒体膜电位降低,这说明GFP诱导的细胞凋亡伴随着膜电位的改变;经过GFP处理的HepG2细胞出现了典型的细胞凋亡的形态变化,细胞收缩,形成微核。以上均说明GFP诱导HepG2细胞凋亡。

The structure and in vitro anti-tumor activity of polysaccharides extracted from Grifola frondosa at low temperature were investigated in this study. Grifola frondosa polysaccharides were extracted by alcohol precipitation at low temperature（4℃）, and the proteins in crude polysaccharides were removed by Sevage method. The small molecule substances such as salts and oligosaccharides were removed by dialysis to obtain the polysaccharide fraction named as GFP. The yield was 2.13%. The main monosaccharides were glucose, galactose and mannose detected by ion chromatography. The results of HPLC analysis showed that GFP was a mixture containing two major macromolecules with molecular weights of 1700.00 ku and 34.12 ku, respectively. The peak areas of the components were 66.57% and 28.23%, respectively. Infrared spectra showed that GFP might be α-pyran polysaccharide. The anti-tumor activity of GFP showed that GFP could significantly inhibit the proliferation of human hepatic hep G2 cells within a certain concentration range; Detection of mitochondrial membrane potential by JC-1 staining revealed that GFP caused a decrease in mitochondrial membrane potential, indicating that GFP-induced apoptosis was accompanied by changes in membrane potential. Morphological observation showed that the GFP-treated cells showed the typical morphological changes of apoptosis, and the cells contracted to form micronuclei. All of the above showed that GFP induced HepG2 cell apoptosis.