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目的探讨葛根芩连汤中有效部位总生物碱类体外干预对人轮状病毒(rotavirus,RV)的药理作用。方法分离自临床RV患者粪便的RV,采用乙醇加热回流法提取葛根芩连汤方剂原4味药材中总生物碱,采用大孔树脂吸附法进行纯化,采用紫外分光光度计在350nm处测定总生物碱吸光度值(optical density,OD),绘制标准曲线,计算葛根芩连汤中总生物碱浓度。在恒河猴肾细胞(MA104)培养液中分别加入总生物碱0.32、0.16、0.08、0.04、0.02、0.01、0g/L,继续培养72h后采用MTT法检测细胞存活率。将MA104细胞分为药物处理组(64、32、16、8、4、2mg/L系列浓度组)、病毒对照组和正常对照组,其中药物处理组再分为药物处理1组、药物处理2组和药物处理3组。药物处理1组采用各浓度总生物碱与RV作用后再接种于MA104细胞,药物处理2组采用各浓度总生物碱与MA104细胞作用后再接种RV,药物处理3组采用RV先接种于MA104细胞再加各浓度总生物碱。正常对照组不加药物且不接种RV;病毒对照组接种RV但不加药物。采用MTT法检测各组细胞存活率、病毒抑制率及细胞半数抑制浓度(half maximal inhibitory concentration,IC50),采用治疗指数(treatment index,TI)评价不同浓度总生物碱对轮状病毒的抑制作用。结果乙醇加热回流法提取全方有效部位的总生物碱含量为10%;总生物碱致MA104细胞的半数存活浓度为0.09g/L;药物处理1组各总生物碱浓度下和病毒对照组细胞OD值明显低于正常对照组(P<0.05),药物处理1组各总生物碱浓度下与病毒对照组比较差异无统计学意义(P>0.05);药物处理2组随总生物碱浓度增加,病毒抑制率增加,呈剂量-效应关系(Z=6.235,P=0.000),IC50=0.025g/L,TI=3.661;药物处理3组随总生物碱浓度增加,病毒抑制率明显增加,存在剂量-效应关系(Z=-4.833,P=0.000),IC50=0.005g/L,TI=17.304;药物处理3组TI值明显高于药物处理2组,差异有统计学意义(P<0.05)。结论葛根芩连汤中有效部位总生物碱对人RV吸附宿主细胞有一定干预作用,但主要通过抑制RV在恒河猴胚胎肾细胞内生物合成过程发挥体外抗RV作用。
Objective To investigate the pharmacological effects of total alkaloids from Gegen Qinlian Decoction on rotavirus (RV) in vitro. Methods The total alkaloids of the four medicinal herbs of Gegen Qinlian decoction were isolated from RV in feces of patients with clinical RV. The total alkaloids were extracted from the four medicinal herbs of Gegen Qinlian Decoction by ethanol heating. The total alkaloids were purified by macroporous resin adsorption method. Total organism was determined by UV spectrophotometer Alkali optical density (OD), draw a standard curve, calculate the total alkaloid concentration of Gegen Qinlian Decoction. The total alkaloids 0.32,0.16,0.08,0.04,0.02,0.01,0 g / L respectively were added to the culture medium of rhesus monkey kidney (MA104), and the cell survival rate was measured by MTT method after cultured for 72h. MA104 cells were divided into drug treatment group (64,32,16,8,4,2 mg / L series concentration group), virus control group and normal control group, the drug treatment group was further divided into drug treatment group 1, drug treatment 2 Group and drug treatment 3 groups. The drug-treated group 1 was treated with total alkaloids and RV, and then inoculated into MA104 cells. The drug-treated group 2 was treated with MA104 cells with various concentrations of total alkaloids and then inoculated with RV. The drug-treated groups were inoculated with MA104 cells Together with the total alkaloids concentration. Normal control group without drugs and not vaccinated RV; virus control group vaccinated RV but without drugs. The cell viability, virus inhibitory rate and half maximal inhibitory concentration (IC50) of each group were determined by MTT assay. The inhibitory effect of total alkaloids with different concentrations on rotavirus was evaluated by treatment index (TI). Results extract effective part of the whole side of the reflux of ethanol was heated total alkaloid content of 10%; total alkaloids half the concentration of viable cells was induced MA104 0.09g / L; at each drug-treated group and a total alkaloid concentration of viral control cells OD value was significantly lower than the control group (P <0.05), virus control and drug treatment groups was not statistically significant (P 1 at each concentration of total alkaloid groups> 0.05); 2 drug-treated group increased with the concentration of total alkaloids , viral suppression rate increased, in a dose - dependent (Z = 6.235, P = 0.000), IC50 = 0.025g / L, TI = 3.661; 3 drug-treated group increased with the concentration of total alkaloids, viral suppression was significantly increased presence Dose-effect relationship (Z = -4.833, P = 0.000), IC50 = 0.005g / L, TI = 17.304; TI value of drug treatment group 3 was significantly higher than that of drug treatment group 2, the difference was statistically significant (P < . Conclusion The total alkaloids in Gegen Qinlian Decoction can interfere with human RV-adsorbed host cells, but play an anti-RV effect in vitro mainly by inhibiting RV biosynthesis in rhesus embryo kidney cells.