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目的建立海胆壳多糖提取工艺及柱前衍生化条件。方法水提醇沉法提取壳多糖,采用正交实验优化提取工艺;柱前衍生化后,利用高效液相色谱法检测单糖组分,对海胆壳多糖的脾淋巴细胞免疫抑制活性实验进行了初步探索。结果海胆壳多糖最佳提取工艺为15倍的水,70℃水浴回流1 h,提取3次;100μl壳多糖水解液,加入50 ml Na OH(0.2 mol·L-1),40μl l-苯基-3-甲基-5-吡唑啉酮(0.5 mol·L-1),混匀后70℃水浴30 min,取出冷却,加入50μl HCl(0.2 mol·L-1)中和,200μl氯仿萃取3次。药理学实验表明海胆壳多糖溶液在浓度1~10μg·ml-1范围时显示出一定的免疫活性。结论本次实验成功建立海胆壳多糖的提取工艺和衍生化条件。
OBJECTIVE To establish the extraction process of sea buckthorn chitin and pre-column derivatization conditions. Methods The chitosan was extracted by water-alcohol precipitation method and optimized by orthogonal experiment. After pre-column derivatization, the components of monosaccharide were detected by high performance liquid chromatography (HPLC), and the immunosuppressive activity of spleen lymphocytes of chitosan Preliminary Study. Results The optimal extraction process of polysaccharides from sea urchin was 15 times of water, refluxed in water bath at 70 ℃ for 1 h and extracted 3 times. 100 μl chitosan hydrolyzate was added to 50 ml NaOH (0.2 mol·L-1), 40 μl l-phenyl (0.5 mol·L-1). The mixture was homogenized and then was subjected to a water bath at 70 ° C for 30 min. After cooling, the mixture was neutralized with 50 μl of HCl (0.2 mol·L-1) and extracted with 200 μl of chloroform 3 times. Pharmacological experiments show that the sea urchin chitin solution in the concentration range of 1 ~ 10μg · ml-1 showed some immunocompetence. Conclusion This experiment successfully established sea buckthorn polysaccharide extraction process and derivatization conditions.