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彭燕飞, 张俐娜,* 许艳霞, 许小娟
(武汉大学 化学与分子科学学院武汉 430072)
灵芝在我国作为传统中药已有两千多年的历史，是扶正固本，滋补强壮，延年益寿的良药。本工作从灵芝菌丝体培养液中分离出胞外多糖纯品, 确定不同级分的化学组成、分子量及其在溶液中链的构象，同时对它们进行抗肿瘤活性试验。
实验部分
Figure 1The DEAE-Sepharose CL-6B elution profile of the crude extracelluler polysaccharide from G. tsugae mycelium. The column was eluted stepwise with distilled water (EPF1,21-41mL ), 0.04 M NaCl (EPF2,164-224 mL) and 0.1 M NaCl aqueous solutions (EPF3, 338-369 mL).
胞外多糖分离与纯化：灵芝菌丝体在以麸皮提取液为主要成分的培养基中培养。培养液经脱色脱蛋白，透析、浓缩及冷冻干燥，即得到淡黄色的絮状胞外多糖粗品（简称CEP）。CEP经DEAE-Sepharose CL-6B(50 cm×2.0 cm)柱纯化得到三个级分EPF1、EPF2和EPF3。
由红外光谱仪（Nicolet FT-IR）、高效液相色谱（Shadox SUGAR SC1011色谱柱）、气相色谱（HP6890气相色谱仪，Hewlett Packard，色谱柱为DB-225毛细管柱）和KJELETC1030自动分析仪（Semi-micro Kjeldahl法测定蛋白质的含量）测试各多糖的化学组成。运用多角度激光光散射仪（LS，DAWN-DSP, Wyatt Technology Co.，波长为633nm）和尺寸排除色谱（SEC，P100泵、TSK-GEL G600PWXL 柱及RI-150示差折光检测器）和LS联用技术在25℃下测定各多糖的重均分子量。
体内抗肿瘤试验采用BALB/C清洁级（SPF）实验小鼠， S180腹水肿瘤瘤株，计算肿瘤抑制率ξ（%）和体重变化率f（%）。
结果与讨论
图1示出CEP 在DEAE-Sepharose CL-6B(50 cm×2.0 cm)柱上的淋洗曲线。由此得到两个纯化的级分EPF1和EPF2。它们的分子量、蛋白质含量以及单糖组成如表1所示。它们均为杂多糖，都以甘露糖为主，并含有少量葡萄糖、果糖、木糖、半乳糖和葡萄糖胺。EPF1的分子量高达92×104，远远高于EPF2的8.4×104。由表2可以看出三个胞外多糖试样均表现出对S180肿瘤的抑制性。尤其CEP和EPF2即使在很低的剂量，也表现出高的抑制肿瘤生长作用。从小鼠体重变化的数据可以看出，小鼠体重在用药期间均有明显增加，充分证明灵芝胞外多糖具有较高的肿瘤抑制作用和低的毒副作用。
Table 1. Components of monosaccharides，protein content and values of molecular mass of CEP
Structure and antitumor activity of extracellular polysaccharide from mycelium of Gendarme tsugae
Yanfei Peng, Lina Zhang, Yanxia Xu, Xiaojuan Xu
(Department of Chemistry Wuhan University, Wuhan, 430072)
Two polysaccharides coded as EPF1 and EPF2 were obtained from the CEP of G. tsugae mycelium by elution with DEAE-Sepharose CL-6B column. Sugar analysis indicated that the three samples were mainly composed with mannose and small amount of fucose, xylose, galactose and glucosamine. The Mw of samples EPF1, EPF2 and CEP determined with SEC-LLS were 92.0×104, 8.35×104 and 23.8×104, respectively. All the samples exhibited high inhibition ratio against Sarcoma 180 implanted in mice, the presence of bound protein and mannose, and relatively expanded chain would be benefit to increase antitumor activities of polysaccharides. |
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