摘要
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 explore the pharmacological effect of direct intervention in vitro of total alkaloids in Gegen Qinlian decoction on the human rotavirus. Methods Rotavirus from stool was isolated from the patients with rotavirus. The total alkaloids were extracted from four kinds of the prescriptions of Gegen Qinlian decoction by ethanol heating reflux extraction, and were purified by macroporous resin adsorption method. The optical density of total alkaloids was detected by ultraviolet spectrophotometer at 350 nm. The standard curve was drawn and the concentration of total alkaloids in the decoction was calculated. The culture media in rhesus monkey kidney cells （MA104） was added 〈3.32, 0.16, 0.08, 0.04, 0.02, 0.01, and 0 mg/mL total alkaloids, respectively, which was continuously cultured for 72 h. The cell survival rate was determined by MTT method. MAI04 cells were divided into drug group （64, 32, 16, 8, 4 and 2 mg/mL series concentration groups）, virus control group and normal control group. The drug group was subdivided into drug 1 group, drug 2 group and drug 3 group. Drug 1 group was inoculated by MA104 cells after treated by total alkaloids in various concentrations and rotavirus. Drug 2 group was inoculated by rotavirus after treated by total alkaloids in various concentrations and MA104 cells. Drug 3 group was inoculated by rotavirus-inoculated MA104 cells and cultured with total alkaloids in various concentrations. Normal control group received no treatment. Virus control group was inoculated by rotavirus but not added any drug. MTT method was used to detect cell survival rates, virus inhibitory rates and half maximal inhibitory concentration （ICs0） among groups. Treatment index （TI） was used to evaluate the inhibitory effect of total alkaloids in various concentrations on rotavirus. Results The content of total alkaloids of effective part of Gegen Qinlian decoction was 10~ by ethanol heating reflux extraction. The median toxic concentration of MA104 by total alkaloids was 0.09 g/L. OD values were obviously lower in various concentrations of total alkaloids in drug 1 group and virus control group than those in normal control group （P〈0.05）, and showed no significant differences between drug 1 group and virus control group （P〈0.05）. In drug 2 group, the virus inhibitory rate increased along with the increase of total alkaloid, showing a dose-effect relation （Z=6. 235, P=0. 000）, ICs0 was 0. 025 g/L and TI was 3. 661. In drug 3 group, the virus inhibitory rate increased along with the increase of total alkaloid, showing a dose effect relation （Z=-4. 833, P〈0. 000）, IC50 was 0. 005 g/L and TI was 17. 304. TI value was significantly higher in drug 3 group than that in drug 2 group （P〈0.05）. Conclusion The total alkaloids in the effective part of Gegen Qinlian decoction can inhibit the human rotavirus adsorbed host cells, and play a role of anti-rotavirus in vitro by inhibiting the biosynthesis of rotavirus in rhesus monkey embryo kidney cells.
出处
《中华实用诊断与治疗杂志》
2016年第10期968-971,共4页
Journal of Chinese Practical Diagnosis and Therapy
基金
陕西省青年人才自然基金(C0305)
关键词
轮状病毒
葛根芩连汤
生物碱
体外
Rotavirus
Gegen Qinlian decoction
alkaloids
in vitro
