海胆壳多糖的提取及PMP柱前衍生化高效液相分析

Study on the extraction technology of the sea urchin shell polysaccharide and monosaccharide composition by Pre-column Derivatization HPLC

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 conditions and derivatization method of the sea urchin shell polysaccharide. Methods The polysaeeharides of shells were extracted through the water extraction and alcohol precipitation, which was optimized by orthogonal design. The shell polysaceharide was derivatized with 1 -phenyl- 3 -methyl -5 -pyrazolone （PMP） , and further detected by high performance liquid chromatography （ HPLC）. Spleen lymphocyte immune inhibitory activity of the shell polysaccharide was preliminarily explored. Results The best technological conditions of extraction of the sea urchin shell polysaccharides ： ratio of the raw material and water weight = 1：15 （g · ml^-1 ）, the extraction temperature for70℃ ,the extraction time for 1 h and the extraction times for 3. 100μl shell polysaecharide hydrolyzate was mixed with 50μl NaOH （0.2 mol · L^-1） and 40μl 1 - phenyl - 3 - methyl - 5 - pyrazolone （0.5 mol · L^-1 ） at a time, then the mixture was reacted in 70℃ water bath for half an hour. After cooled, 50μl HCl （0.2 mol · L^- 1 ）was added in the mixture, and extracted for three times by 200μl chloroform. Pharmacological experiments showed that the sea urchin shell polysaeeharide could restrain the proliferation of lymphocyte with the concentration range of 1 to 10 μg · ml^-1. Conclusion The extraction technology of the urchin shell polysaccharides and the derivatization conditions were successfully established.