哈蟆油中免疫活性部位的提取工艺与药效学研究

Study on Pharmacodynamics and the Extraction Process of the Immune Active Site of Rana temporaria chensinensis

目的:研究哈蟆油中免疫活性部位的最佳提取工艺,并通过药效学实验验证其免疫调节作用。方法:采用正交试验,以酶解液的水解度、收率为指标,利用酶法对哈蟆油中蛋白质进行提取;并通过腹腔注射不同剂量的环磷酰胺复制免疫低下和免疫增强模型小鼠,以脏器指数、吞噬指数为指标,验证哈蟆油中免疫活性部位的免疫调节作用。结果:哈蟆油中蛋白质分2次水解出,第1次水解的最佳工艺为加木瓜蛋白酶2000u·g-1,调pH值至6.0,在45℃下提取4.5h;第2次水解的最佳工艺为固液比为1∶20(V/V),调pH值至6.0,在40℃下提取4.5h;药效学实验证明0.1g·kg-1哈蟆油提取物对免疫低下模型小鼠免疫功能有显著增强作用(P<0.01);0.2g·kg-1哈蟆油提取物对免疫增强模型小鼠免疫功能有显著抑制作用(P<0.01),并对免疫器官有一定的保护作用。结论:所选工艺合理、可行,提取的蛋白质有一定的免疫调节作用。

OBJECTIVE： To study the optimum extraction process of the immune active site of Rana temporaria chensinensis, and to validate immune regulation of it by pharmacodynamic experiment. METHODS： Orthogonal experiment was adopted. The protein was extracted from R. temporaria chensinensis by aqueous enzymatic extraction method using the degree of hydrolysis and yield as index. Low immunity and immunoenhancement model were induced by intraperitoneal injection of different doses of cyclo- phosphamide, and immunity regulation effects of active site of R. temporaria chensinensis were validated with visceral index and phagocytic coefficient as index. RESULTS： The optimum extraction process of R. temporaria chensinensis for the first time was as follows：that papain was 2 000 u·g^-1,pH value adjusted to 6.0, enzymolysis temperature was 45 ℃, enzymolysis time was 4.5 h. For the second time, optimal extraction process of R. temporaria chensinensis was that： the result indicated that ratio of solid to liq- uid was 1 ： 20（V/V）, pH value adjusted to 6.0, enzymolysis temperature was 40℃, enzymolysis time was 4.5 h. Pharmacodynamic experiment indicated that R. temporaria chensinensis 0.1 g·kg^-1 promoted immune function of low immunity model mice （P〈 0.01） ;R. temporaria chensinensis 0.2 g·kg^-1 significantly inhibited immune function of immunoenhancement model mice （P〈 0.01） and protected immune organs. CONCLUSION：The process is reasonable and feasible, the extracted proteins have immuno- modulatory effects.