天麻多糖的分离纯化与抗氧化活性研究

Extraction,purification and antioxidant activity of polysaccharides from Gastrodia elata B1

目的从天麻中分离纯化出活性多糖并考查其抗氧化活性。方法用水提醇沉法提取天麻粗多糖,Sevage法脱去其中的蛋白质,依次通过D101大孔吸附树脂、DEAE-52离子交换色谱及Sephadex G-100凝胶色谱纯化,得到2个天麻多糖(GEP):GEP1-G和GEP2-G,测定其清除1,1-二苯基-2-三硝基苯肼(DPPH)自由基、超氧阴离子自由基和羟基自由基的能力,以评价其抗氧化作用。结果天麻中粗多糖提取率为5.6%,经D101大孔吸附树脂纯化后,粗多糖中多糖含量为65.7%;DEAE-52纯化后,有6个洗脱组分GEP 1~6;组分GEP1和GEP2再经Sephadex G-100纯化得到纯品多糖GEP1-G和GEP2-G,其多糖含量分别是97.3%和98.1%。在抗氧化活性实验中:当GEP1-G和GEP2-G浓度为1 mg·mL^(-1)时,两者对DPPH的清除率分别为44.50%和25.60%,对超氧阴离子抑制率分别为33.32%和21.55%,对羟自由基抑制率分别为39.50%和22.80%。结论通过大孔吸附树脂、离子交换色谱和凝胶过滤色谱得到了纯品天麻多糖,其具有一定的抗氧化作用。

Objective To extract,purify polysaccharides from Gastrodia elata and to determinate the antioxidant activity in vitro. Methods Fresh Gastrodia elata tuber was collected,cooked at normal pressure,sliced and dried. Crude Gastrodia elata polysaccharide（ GEP） was gained by boiling in hot-water,precipitating using ethanol,removing proteins with Sevage method. Then the crude polysaccharide was applied to macroporous resin D101,and further purified through DEAE-52 cellulose colume and Sephadex G-100 column,followed by dialysis,lyopilization. The total sugar and protein content were analyzed by phenol-sulfuric acid method and Bradford method. Then the scavenging activity and its antioxidant activity in vitro of the polysaccharides on the 1,1-two phenyl-2-three nitrophenyl hydrazine（ DPPH）,superoxide anion radical,Hydroxyl radical were evaluated. Results The yield of the polysaccharides extracted from the dried Gastrodia elata was 5. 6%. After the purification of macroporous resin D101,a large amount of pigment was removed and the polysaccharide content was increased to 65. 7%. Six polysaccharide fractions were eluted from DEAE-52 cellulose with water,5 × 10^（-2）,0. 1,0. 2,0. 5,1 mol·L^-1 NaCl,namely GEP 1-6. The major fractions,GEP1 and GEP2 were further purified by Sephadex-100 column chromatography namely GEP1-G and GEP2-G. The purified polysaccharides,GEP1-G and GEP2-G were found to contain almost no protein or nucleic acid with a purity more than 97%. Antioxidant activity tests revealed that GEP1-G and GEP2-G showed potential capability of scavenging free radical. At the concentration of 1 mg· mL^-1,the DPPH radical scavenging activities were 44. 50% and 25. 60%,superoxide radical scavenging activities were 33. 32% and 21. 55%,hydroxyl radical scavenging activities were 39. 50% and 22. 80%,for GEP1-G and GEP2-G,respectively. Overall,GEP1-G exhibited higher scavenging activities than GEP2-G. Conclusion The procedure was well separated and purified to obtain polysaccharides from Gastrodia elata. It had a certain antioxidant effect.