复方人参免疫增强方配比优选及其对小鼠的免疫活性和急性毒性研究

Optimization of Composition Proportion of Compound Ginseng Immune-enhancing Formula and Study of Its Immunomodulatory Activity and Acute Toxicity on Mice

目的:优选复方人参免疫增强方4味组方药材(人参、黄芪、黄精、枸杞)的最佳配伍比例,并对最佳配比提取物进行免疫活性及急性毒性研究。方法:在采用细胞活性试验筛选4味药材生药量浓度范围的基础上,以不同生药量浓度的药材提取物作用后小鼠RAW264.7巨噬细胞中的一氧化氮(NO)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)含量为指标,采用均匀设计法确定复方人参免疫增强方各药材的最佳配比。取小鼠240只,随机分为4批,每批60只,均随机分为空白组(生理盐水)、模型组(生理盐水)、阳性药物组[左旋咪唑,4 mg/(kg·d)]和复方人参免疫增强方最佳配比提取物低、中、高剂量组[0.952 8、1.905 6、2.858 4g/(kg·d)],每组 10 只,灌胃给药,qd,连续给药 30 d;除空白组外,其余组小鼠于首次给药后第 24 天时腹腔注射环磷酰胺[40mg/(kg·d)],qd,连续注射3 d以建立免疫低下模型。通过测定各组小鼠脏器系数,脾淋巴细胞转化能力,血清溶血素、IL-2、IgM、IgG、IgA含量,腹腔巨噬细胞吞噬功能,来评价最佳配比提取物的免疫活性。另取小鼠20只,灌胃最佳配比提取物20 mL/kg,给药2次,以经口最大耐受剂量(MTD)考察其急性毒性。结果:复方人参免疫增强方的最佳配比为人参、黄芪、黄精、枸杞生药质量比 1 ∶ 2 ∶ 2 ∶ 4。免疫活性实验结果显示,该方最佳配比提取物可显著提高小鼠的脏器指数,脾淋巴细胞增殖能力,血清中溶血素、IL-2、IgM、IgG、IgA含量以及腹腔巨噬细胞吞噬功能(P<0.05或P<0.01);急性毒性实验结果显示,其经口MTD均大于15 g/kg,口服无毒性。结论:复方人参免疫增强方最佳配比提取物可显著增强小鼠的免疫功能,且口服无毒性。

OBJECTIVE:To optimize the optimal composition proportion of 4 ingredients (Panax ginseng,Astragalus membranaceus,Polygonatum sibiricum,Lycium chinensis) in Compound ginseng immune-enhancing formula (CGIF),and to study immune activity and acute toxicity of the extracts with the optimal ratio. METHODS:The cell activity test was used to screen the crude drug concentration range of 4 ingredients. After treated with different crude drug concentrations of each medicinal material,using the contents of NO,IL-6 and TNF-α as indexes,uniform design was used to determine the optimal ratio of each ingredient in CGIF. Totally 240 mice were taken and randomly divided into 4 batches,with 60 mice in each batch. Each batch of mice was randomly divided into blank group(normal saline),model group(normal saline),positive drug group [levamisole,4 mg/(kg·d)],and optimal proportion extract of CGIF low-dose,medium-dose and high-dose groups [0.952 8,1.905 6,2.858 4 g/(kg·d)],with 10 mice in each group;they were given medicine intragastrically,qd,for consecutive 30 d. Except for blank group,mice in the other groups were intraperitoneally injected with cyclophosphamide [40 mg/(kg·d)] on the 24th day after first administration,qd,for consecutive 3 d to induce immunocompromised model. The immune activity of the optimal proportion extract was evaluated by determining visceral coefficients, spleen lymphocyte transformation capacity, serum contents of hemolysin,IL-2,IgM,IgG and IgA,phagocytosis function of peritoneal macrophages. Another 20 mice were collected and given the optimal proportion extract 20 mL/kg intragastrically,twice;acute toxicity of the formula was investigated with oral maximum tolerated dose(MTD). RESULTS:The optimal ratio of CGIF was that crude drug mass ratio of P. ginseng, A.membranaceus,P. sibiricum,L. chinensis was 1 ∶ 2 ∶ 2 ∶ 4.The immunological activity experiment showed that the optimal proportion extract can significantly improve visceral indexes of mice, spleen lymphocyte proliferation ability, serum contents of hemolysin,IL-2,IgM,IgG and IgA as well as macrophage phagocytosis ability (P<0.05 or P<0.01). The acute toxicity test indicated that oral MTD was over 15 g/kg,which was non-toxic. CONCLUSIONS:The optimal proportion extract of CGIF can significantly enhance the immune function of mice and are non-toxic.