板栗壳多糖的提取、分离纯化及抗氧化活性研究

论文编号:SW135  论文字数:10747,页数:26

摘要： 本实验以板栗壳为原料，用水煮醇沉法提取了板栗壳粗多糖，测得其提取率为1.3%；用考马斯亮蓝G-250法对提取的板栗壳粗糖蛋白的蛋白质含量作了测定，结果为2.73%；得到的粗糖蛋白用酶+Sevag法脱蛋白，处理了12次后无明显的蛋白层；紫外光谱检测脱蛋白后的板栗壳粗多糖，显示在280nm处已无吸收峰，说明蛋白质已经基本除尽；脱蛋白后得到的板栗壳粗多糖用苯酚-硫酸法测糖含量，测得粗多糖中己糖含量为74%。
 用DEAE-Sepadax Fast Flow阴离子交换层析柱对板栗壳粗多糖进行了分离纯化，经蒸馏水和不同浓度的NaCl溶液梯度洗脱后得到两个组分cnps1，cnps2，用Sephacryl 100 HR层析柱对组分cnps2作了纯度检验，结果显示cnps2并不是单一的均多糖，表明经一次阴离子交换柱层析后所得到的板栗壳多糖并不是一个单一的均多糖，需要调整分离条件，或改变填料类型，或改变洗脱剂种类进行进一步的纯化研究。通过红外光谱检测层析前后的板栗壳多糖的组成变化，发现虽然分离纯化没有得到精多糖，但是粗多糖中的某些组分已经被分离并富集，从而产生了不同的吸收峰。
    最后，采用Fenton法对板栗壳粗多糖进行了抗氧化活性的研究，结果显示板栗壳粗多糖在剂量为1-20mg/ml范围内，对羟基自由基具有一定的清除效果。但在同等剂量下弱于阳性对照Vc，20 mg/ml的板栗粗多糖的清除率为35.1%，而阳性对照Vc(20mg/ml)的清除率为66.08%。
关键词：板栗壳；多糖；提取；分离纯化；抗氧化活性

Study on the extraction, purification and antioxidant activity of polysaccharide in Castanea mollissima shell
         

Abstract: In this study, the Castanea mollissima shell was extracted with water and Castanea mollissima shell crude polysaccharide was obtained with a productivity of 1.3%. The protein content of the Castanea mollissima shell crude polysaccharide measured with Coomassie brilliant blue G-250 method was 2.73%. The crude polysaccharide was deproteined with enzyme and Sevag, after handling 12 times there was no obvious protein layer left. UV detection showed that there was no absorption peak at 280 nm, that is to say ,the protein in the crude polysaccharide had been removed entirely. The polysaccharide content of the deproteined  crude polysaccharide was measured with phenol and sulfuric acid, hexose had a content of 74%.
      Then using DEAE - Sepadax Fast Flow anion exchange chromatography column to purify chestnut shell crude polysaccharides. Two components cnps1, cnps2 were abtained with the mobile phase distilled water and NaCl solution of different concentration. The purity of components cnps2 was measured by differential refractive index detector with Sephacryl 100 HR column, results showed that it was not uniform. That is to say, the crude polysaccharidea handled one time by anion-exchange column chromatography was not uniform. Thus, separation conditions, the filling or the eluting agent is need to change for further purification. Characterized the Chestnut polysaccharide before and after the separation and purification by infrared spectroscopy, we found that though uniform polysaccharide could not been abtained, some components of the crude polysaccharides had been isolated and enriched, thereby different absorption signal was observed.
 Finally, the antioxidant activity of chestnut shell crude polysaccharide was studied by Fenton reaction. The results proved that chestnut shell crude polysaccharide in the dose range from 1mg/ml to 20mg/ml had scavenging effects on hydroxyl radical. However, it is weaker than the positive control Vitamin C in the same dose , the clearance rate of the former in the dose of  20 mg / ml was 35.1%, while that of the later was 66.08%.
 Key Words：Castanea mollissima shell; Polysaccharides; Extraction; Purification; Antioxidant activity

目 录
中文摘要 I
英文摘要 II
目 录 III
1 绪 论 1
1.1 板栗壳概述 1
1.2 板栗壳研究现状 1
1.3 多糖概述 2
1.4 多糖研究现状 2
1.5 本章小结 3
2 实验部分 4
2.1 材料与设备 4
2.2 实验主要试剂 5
2.3 实验方法 6
2.4 本章小结 10
3 结果和讨论 11
3.1 板栗壳粗多糖提取 11
3.2 粗糖蛋白中蛋白质含量的测定 11
3.3 板栗壳粗糖蛋白紫外光谱检测 12
3.4 脱蛋白处理 12
3.5 粗多糖中己糖含量的测定 13
3.6 板栗壳粗多糖的紫外光谱检测 14
3.7 Fenton法测定板栗壳粗多糖对羟基自由基的清除率 14
3.8 板栗壳粗多糖红外光谱分析 15
3.9 板栗壳粗多糖的分离纯化 16
3.10 板栗壳多糖的纯度检测 17
3.11板栗壳多糖cnps2红外光谱分析 17
3.12 本章小结 18
4 总结与展望 19
致 谢 20
参考文献 21

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