复合酶-微波辅助萃取结合超滤纯化的灰树花子实体多糖的免疫活性研究

Immunoactivity of Grifola frondosa Fruit Body Polysaccharides Prepared by Microwave-assisted Extraction followed by Multi-enzyme Hydrolysis and Purified by Ultrafiltration

目的:研究经酶解-微波辅助萃取、超滤分级纯化的灰树花子实体多糖的免疫活性。方法:采用复合酶解结合微波辅助萃取方法提取灰树花子实体多糖,采用超滤工艺对提取多糖进行分级纯化;采用MTT法研究多糖对小鼠脾淋巴细胞、ConA/LPS诱导的小鼠脾淋巴细胞的增殖作用;建立荷瘤小鼠模型,研究灰树花多糖对肿瘤的抑制作用。结果:获得分子质量范围为<50kD的GFP-I、50～100kD的GFP-II和100～200kD的GFP-III;与RPMI-1640对照组相比,GFP-I、GFP-II和GFP-III对淋巴细胞增殖率分别增加26.558%～74.167%(P<0.001)、20.175%～152.075%(P<0.001)和26.473%～334.797%(P<0.001);对ConA诱导的淋巴细胞增殖率分别增加31.408%～122.894%(P<0.001)、53.075%～235.693%(P<0.001)和83.347%～367.241%(P<0.001);对LPS诱导的淋巴细胞增殖率分别增加20.526%～97.075%(P<0.001)、38.288%～211.348%(P<0.001)和54.454%～344.883%(P<0.001);相对于生理盐水(NS)组,在实验剂量范围内,GFP-I、GFP-II和GFP-III对S180肿瘤的抑制率分别达到10.15%～22.94%(P<0.001)、12.79%～30.10%(P<0.001),23.86%～51.65%(P<0.001)。结论:经复合酶-微波辅助萃取、超滤分级纯化后多糖具有显著免疫活性和抗肿瘤活性,其中GFP-III具有比GFP-I和GFP-II更强的免疫活性和抗肿瘤活性,而且活性与其浓度正相关,多糖可能以对T淋巴细胞增殖的促进作用为主。

Objective：To explore the immunoactivity of Grifola frondosa fruit body polysaccharides（GFP） with different relative molecular weights.Methods：Three polysaccharide fractions from Grifola frondosa fruit body named as GFP-I（ 50 kD）,GFP-II（50-100 kD） and GFP-III（100-200 kD） were prepared by microwave-assisted extraction followed by multi-enzyme hydrolysis and purified by ultrafiltration.The proliferation of murine lymphocytes with and without Con A or LPS induction in virto was evaluated by MTT assay.A S180 tumor-bearing mouse model was established,and the inhibitory effect of the three GFP fractions at different concentrations on tumor growth was also explored.Results：Lymphocyte proliferation rate without induction was increased by 26.558%-74.167%（P0.001）,20.175%-152.075%（P0.001） and 26.473%-334.797%（P0.001）,respectively in group GFP-I,GFP-II and GFP-III as compared with RPMI-1640 group,Con A-induced lymphocyte proliferation rate exhibited respective increases of 31.408%-122.894%（P0.001）,53.075%-235.693%（P0.001） and 83.347%-367.241%（P0.001） for group GFP-I,GFP-II and GFP-III,and LPS-induced lymphocyte proliferation rate was increased by 20.526%-97.075%（P0.001）,38.288%-211.348%（P0.001） and 54.454%-344.883%（P0.001）,respectively.In comparison with NS group,the inhibitory rates of GFP-I,GFP-II and GFP-III on tumor growth in S180 tumor-bearing mice were 10.15%-22.94%（P0.001）,12.79%-30.10%（P0.001） and 23.86%-51.65%（P0.001）,respectively.Conclusion：GFP-I,GFP-II and GFP-III have notable immunoactivity and anti-tumor activity and the activity of GFP-III is the strongest and positively related to its concentration.Further,these three polysaccharides might mainly promote the T lymphocyte proliferation.