Fractionation, Purification and Antioxidant, Antitumor Activity of Polysaccharides from Phellinus igniarius Mycelia
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摘要: 采用水提醇沉法制备桑黄菌丝体粗多糖，分别用终浓度为30%、45%和60%的硫酸铵溶液对桑黄菌丝体中粗多糖进行粗分离，并比较所得各多糖组分的抗氧化、抗肿瘤活性。对其中活性强的组分采用离子交换树脂柱色谱纯化，采用高效排阻色谱-折光示差检测器-多角度光散射仪对纯化多糖进行均一性分析和分子量测定。结果显示，45%硫酸铵分离多糖IPS45显示最强的抗肿瘤活性及较好的清除超氧阴离子活性，IPS45经DEAE-52纤维素离子交换柱纯化得到四个级分，经高效排阻色谱分析，其中蒸馏水洗脱级分（IPS45-W）为均一多糖，重均分子量为79.1 kDa；气相色谱分析其单糖组成为甘露糖:葡萄糖:半乳糖:未知单糖，摩尔比为1.57:8.38:1.09:1.00。IPS45-W可抑制HepG2细胞生长，当浓度为50 μg/mL时，对HepG2细胞的抑制率为56.74%，高于前期研究的乙醇沉淀分离所得桑黄菌丝体均一多糖同浓度下11%~35%的抑制率。该结果为不同组成的桑黄多糖的分离和应用提供依据。Abstract: The crude polysaccharide of Phellinus igniarius mycelia was prepared by the method of water extraction and ethanol precipitation. Then it was preliminarily fractionated by ammonium sulfate solution with the final concentration of 30%, 45% and 60%, respectively. The antioxidant and anti-tumor activities of the polysaccharides fractions were tested and compared with each other. The fraction which showed the strongest activity was further purified by ion exchange resin column chromatography. And the homogeneity and average molecular weight (MW) of purified polysaccharides were evaluated and determined by size-exclusion chromatography combined with multi-angel laser light scattering and refractive indexdetector (SEC-MALLS-RI). The results showed that 45% ammonium sulfate solution supernatant fraction (IPS45) had the strongest anti-tumor activity and better scavenging superoxide anion activity in fractions fractionated by different concentrations of ammonium sulfate. Four fractions were obtained from IPS45 by DEAE-52 cellulose ion exchange column purification. SEC-MALLS-RI analysis results showed that the ddH2O elution fraction (IPS45-W) was a homogeneous polysaccharide with a MW of 79.1 kDa. GC analysis indicated that IPS45-W was composed of mannose:glucose:galactose:unknown monosaccharide in a molar ratio of 1.57:8.38:1.09:1.00. The IPS45-W could inhibit the growth of HepG2 cells. Its inhibition rate was 56.74% at the concentration of 50 μg/mL. This inhibition rate of IPS45-W was higher than that of the homopolysaccharide (11%~35%) against HepG2 cells at same concentration, which from the Phellinus igniarius myceli polysaccharide fractionated by ethanol precipitation in previous studies. The results would provide the basis for the separation and application of different components of Phellinus igniariusmycelia polysaccharides.
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图 1 粗多糖及各上清液组分的抗氧化能力
Figure 1. Antioxidant capacity of crude polysaccharide and supernatant fractions
注：A：超氧阴离子清除能力；B：羟基自由基清除能力；C：还原能力。
图 2 各多糖组分对HepG2细胞的抑制作用
Figure 2. Inhibition of polysaccharides on HepG2 cells
注：*表示同一浓度不同样品间差异显著（P<0.05）。
图 4 IPS45-W的高效凝胶渗透色谱图
Figure 4. SEC-RI-MALLS chromatograms of IPS45-W
注：RI：折光示差；LS：光散射。
图 5 标准单糖（Ａ）和IPS45-W（B）的气相色谱图
Figure 5. GC chromatography of standard monosaccharide (A) and IPS45-W (B)
图 6 DEAE-52分离所得各多糖组分对HepG2细胞的抑制作用
Figure 6. Inhibition of polysaccharides fractionations from DEAE-52 on HepG2 cells
注：*表示差异显著（P<0.05）；**表示差异极显著（P<0.01）。
表 1 粗多糖各组分的得率、多糖和蛋白质的含量
Table 1. Yields and contents of carbohydrate and protein of fractions from crude polysaccharides
样品名称 IPS30 IPS45 IPS60 IPP30 IPP45 IPP60 得率（%） 16.72 7.86 6.5 − − − 多糖含量（%） 18.73 29.03 34.68 9.03 8.50 10.25 蛋白质含量（%） 3.03 2.47 2.00 5.02 5.17 5.41 注：“−”表示未检测。 
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表 2 IPS45经DEAE-52纯化后各级分的多糖与蛋白含量
Table 2. The content of polysaccharides and proteins of IPS45 fractions purified by DEAE-52
组分 得率（%） 多糖含量（%） 蛋白含量（%） IPS45-W 30.0 78.08 0.33 IPS45-1 7.0 47.89 0.65 IPS45-2 4.0 30.65 0.17 IPS45-3 1.6 27.94 0.57
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