甘露消毒丹及其拆方体外抗肠道病毒71型作用

Anti-Enterovirus 71 Effect of Ganlu Xiaodu Micropills and Its Disassembled Formulas In Vitro

目的观察甘露消毒丹及其拆方体外抗肠道病毒71型(EV71)的作用。方法运用细胞培养技术,病毒毒力测定采用Reed-Muench法计算半数组织培养感染量(TCID50),确定实验用为100 TCID50。将甘全方(甘露消毒丹全方)、清残方(甘露消毒丹方中清热解毒药物组成)、利残方(甘露消毒丹方中化浊利湿药物组成)、病毒唑药液用维持液1∶8、1∶16、1∶32、1∶64、1∶96、1∶128、1∶256稀释,采用MTT法测定各浓度药物吸光度OD值,根据各药不同浓度的OD值计算其细胞存活率,以药物浓度为横坐标(g/ml)、细胞存活率(%)为纵坐标绘出非线性回归曲线,选择各药的90%细胞存活率浓度为药效学实验浓度。分别将90%细胞存活率浓度的甘全方、清残方、利残方、病毒唑药液和100 TCID50病毒各50μl混合,加到单层人恶性胚胎横纹肌瘤细胞(RD细胞)上,各设4复孔,每孔均设正常细胞对照、病毒对照,采用MTT法计算各药物对病毒生长的抑制率。结果根据Reed-Muench法计算得出TCID50=10-2.89/ml,从回归曲线获得各药90%细胞存活率浓度分别为甘全方:10-2.43=0.0037 g/ml;清残方:10-2.7=0.0020 g/ml;利残方:10-2.09=0.0081 g/ml;病毒唑:10-3.19=0.64 mg/ml。清残方EV71病毒抑制率为97.4%,甘全方为91.74%,利残方为75.64%,病毒唑为36.99%。结论甘露消毒丹全方及其拆方具有比病毒唑更强的抗EV71作用,清热解毒法在方中发挥了更强的抗EV71作用。

ABSTRACT Objective To observe the anti-enterovims 71 （EV71） effect of Ganlu Xiaodu Micropills and its disassembled formulas in vitro. Methods The cell culture technology was used and the virulence of virus was measured. The Reed-Muench method was used to calculate 50% tissue culture infective dose （TCIDs0） and 100 TCIDs0 was determined for the experiment. The drug solutions of Ganlu Xiaodu Micropills （ whole formual）, heat- clearing toxin-resolving formula （ heat-cleating and toxin-resolving medicinal ingredients of Ganlu Xiaodu Micropills）, turbidity-transforming dampness-disinhibiting formula （turbidity-transforming and dampness-disinhibiting medicinal ingredients of Ganlu Xiaodu Micropills） and Ribavirin were diluted with maintenance medium at a ratio of I ： 8, I ： 16, 1 ： 32, 1 ： 64, 1 ： 96, 1 ： 128 and 1 ： 256, respectively. The optical density （OD） value was measured by MTT assay and the cell viability was calculated according to the OD values of drugs at different concentrations. The linear regression curve was drawn with drug concentration as the horizontal axis （g/ml） and cell viability （ % ） as the vertical axis. A concentration of 90% cell viability was selected as the pharmacodynamic experimental concentration. The drug solutions of Ganlu Xiaodu Micropills, heat-clearing toxin-resolving formula, turbidity-transforming dampness- disinhibiting formula and Ribavirin at a concentration of 90% cell viability were mixed with 50p.1 100TCIDs0＂virus and added to the monolayer human malignant embryonic rhabdomyosarcoma cells （RD cells）. Four duplicate wells were established and equipped with normal cell control and virus control. The viral suppression rates of drugs were calculated by MTT assay. Results The TCIDso was 10-289/ml. The concentration of 90% cell viability was 10-243 = 0. 0037 g/ml, 10 -2.7 = 0. 0020 g/ml, 10 -2. 09 = 0. 0081 g/ml, and 10 -3 19 = 0. 64 mg/ml in Ganlu Xiaodu MicropiUs, heat-clearing toxin-resolving formula, turbidity-transforming dampness-disinhibiting formula, and ＇Ribavirin, respec- tively. The viral suppression rate of heat-clearing toxin-resolving formula, Ganlu Xiaodu Micropills, turbidity- transforming dampness-disinhibiting formula, and Ribavirin was 97.4% , 9l. 74% , 75.64% , and 36. 99% , respec- tively. Conclusion The anti-EV71 effects of Ganlu Xiaodu Micropills and its disassembled formulas are superior to that of Ribavirin, and heat-clearing toxin-resolving medicinal plays a stronger role.