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彭燕飛, 張俐娜,* 許艷霞, 許小娟
(武漢大學(xué) 化學(xué)與分子科學(xué)學(xué)院武漢 430072)
靈芝在我國作為傳統(tǒng)中藥已有兩千多年的歷史，是扶正固本，滋補(bǔ)強(qiáng)壯，延年益壽的良藥。本工作從靈芝菌絲體培養(yǎng)液中分離出胞外多糖純品, 確定不同級分的化學(xué)組成、分子量及其在溶液中鏈的構(gòu)象，同時對它們進(jìn)行抗腫瘤活性試驗。
實驗部分
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).
胞外多糖分離與純化：靈芝菌絲體在以麩皮提取液為主要成分的培養(yǎng)基中培養(yǎng)。培養(yǎng)液經(jīng)脫色脫蛋白，透析、濃縮及冷凍干燥，即得到淡黃色的絮狀胞外多糖粗品（簡稱CEP）。CEP經(jīng)DEAE-Sepharose CL-6B(50 cm×2.0 cm)柱純化得到三個級分EPF1、EPF2和EPF3。
由紅外光譜儀（Nicolet FT-IR）、高效液相色譜（Shadox SUGAR SC1011色譜柱）、氣相色譜（HP6890氣相色譜儀，Hewlett Packard，色譜柱為DB-225毛細(xì)管柱）和KJELETC1030自動分析儀（Semi-micro Kjeldahl法測定蛋白質(zhì)的含量）測試各多糖的化學(xué)組成。運(yùn)用多角度激光光散射儀（LS，DAWN-DSP, Wyatt Technology Co.，波長為633nm）和尺寸排除色譜（SEC，P100泵、TSK-GEL G600PWXL 柱及RI-150示差折光檢測器）和LS聯(lián)用技術(shù)在25℃下測定各多糖的重均分子量。
體內(nèi)抗腫瘤試驗采用BALB/C清潔級（SPF）實驗小鼠， S180腹水腫瘤瘤株，計算腫瘤抑制率ξ（%）和體重變化率f（%）。
結(jié)果與討論
圖1示出CEP 在DEAE-Sepharose CL-6B(50 cm×2.0 cm)柱上的淋洗曲線。由此得到兩個純化的級分EPF1和EPF2。它們的分子量、蛋白質(zhì)含量以及單糖組成如表1所示。它們均為雜多糖，都以甘露糖為主，并含有少量葡萄糖、果糖、木糖、半乳糖和葡萄糖胺。EPF1的分子量高達(dá)92×104，遠(yuǎn)遠(yuǎn)高于EPF2的8.4×104。由表2可以看出三個胞外多糖試樣均表現(xiàn)出對S180腫瘤的抑制性。尤其CEP和EPF2即使在很低的劑量，也表現(xiàn)出高的抑制腫瘤生長作用。從小鼠體重變化的數(shù)據(jù)可以看出，小鼠體重在用藥期間均有明顯增加，充分證明靈芝胞外多糖具有較高的腫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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