大青叶水提物抗甲型流感病毒活性研究

Antiviral activity of Folium Isatidis aqueous extract on influenza A virus

目的观察大青叶水提物的抗甲型流感病毒(IAV)活性。方法用热回流法提取大青叶水提物;用细胞计数试剂盒(CCK-8)法检测大青叶水提物对狗肾传代细胞(MDCK)的毒性作用;将流感病毒反向遗传八质粒病毒包装系统共转染进混养的人类胚胎肾细胞(293T)和MDCK细胞中制备IAV,并测定IAV的半数细胞培养物感染量(TCID50)。分3组进行大青叶水提物抗IAV活性检测:第1组先分别用0.25、0.50、0.75、1.00、1.25 g·L^(-1)的水提物作用细胞4 h,再分别加10×TCID50病毒吸附细胞2 h;第2组用0.25、0.50、0.75、1.00、1.25 g·L^(-1)的水提物和10×TCID50病毒同时作用细胞4 h;第3组先用10×TCID50病毒吸附细胞2 h,再分别加0.25、0.50、0.75、1.00、1.25 g·L^(-1)的水提物作用细胞4 h;然后用CCK-8法测定大青叶水提物对IAV的增殖抑制率。结果大青叶水提物的细胞对照组、0.50、0.75、1.00、1.25、1.50、1.75、2.00、3.00、4.00、5.00 g·L^(-1)组的细胞存活率分别为(100.00±0.00)%、(99.97±1.08)%、(99.95±4.34)%、(98.62±1.45)%、(98.81±2.04)%、(74.65±6.29)%、(49.71±2.14)%、(39.86±4.31)%、(19.75±2.26)%、(7.86±3.74)%、(2.77±1.76)%,大青叶水提物对MDCK细胞的半数细胞毒性浓度为1.75 g·L^(-1),大青叶水提物对MDCK细胞的最大无毒浓度为1.25 g·L^(-1);第1、2、3组大青叶水提物各浓度亚组的病毒增殖抑制率均高于病毒对照组(P<0.01)。第2、3组大青叶水提物各浓度亚组的病毒增殖抑制率均显著低于第1组对应的大青叶水提物亚组(P<0.05)。第3组大青叶水提物各浓度亚组的病毒增殖抑制率显著高于第2组对应的大青叶水提物亚组(P<0.05)。第1、2、3组对IAV的半数抑制浓度分别为0.75、>1.25、1.25 g·L^(-1)。3组不同干预方法的抗病毒活性排序为:第1组>第3组>第2组。结论大青叶水提物有较好的抗IAV活性,其作用机制可能与增强机体免疫力相关。

Objective To study the antiviral activity of Folium [satidis aqueous extract on influenza A virus （IAV）. Methods Folium Isatidis aqueous extract was extracted by heat reflux. The cytotoxicity of Folium Isatidis aqueous extract on Madin-Darby canine kidney （MDCK） cells was detected by cell counting kit-8 （CCK-8） assay. The influenza virus reverse ge- nomic 8-plasmid virus packaging system was co-transfected into polyculture human embryonic kidney cells （293T） and MDCK cells to prepare IAV, then the 50% tissue culture infective dose （ TCIDs0 ） of IAV was detected. The experiment was divided into three groups to detect the antiviral activity of Folium Isatidis aqueous extract on IAV. The cells in the first group were treated with 0. 25 ,0. 50,0. 75 ,1. 00 and 1.25 g·L-l Folium Isatidis aqueous extract for 4 hours;then the cells were adsorbed by 10 x TCID50 virus for 2 hours respectively. The cells in the second group were treated with 0. 25,0. 50,0. 75,1. 00 and 1.25 g·L-1 Folium Isatidis aqueous extract and 10 ×TCID50 virus for 4 hours simultaneously. The cells in the third group were adsorbed by 10 × TCID50 virus for 2 hours ,then the cells were treated with 0. 25 ,0. 50,0. 75 ,1. 00 and 1.25 g·L -1 Foli- um Isatidis aqueous extract for 4 hours. The inhibition rate of Folium Isatidis aqueous extract on IAV proliferation was detected by CCK-8. Results The cell survival rate in control group and 0.50,0.75,1.00, 1.25,1.50, 1.75,2.00,3.00,4.00, 5.00 g·L-1 Folium Isatidis aqueous extract group was （ 100.00 ± 0.00）%, （99.97 ± 1.08）%, （99.95 ± 4.34）%, （98.62 ± 1.45）% ,（98.81 ±2.04）%, （74.65 ± 6.29）%, （49.71 ± 2.14）%, （39.86 ±4.31 ）%, （19.75 ± 2.26）%, （ 7.86 ± 3.74） % and （ 2.77 ± 1.76 ） % respectively. The median cytotoxic concentration of Folium Isatidis aqueous extract on MDCK cells was 1.75 g·L- 1, and the maximum non-toxic concentration was 1.25 g·L-1. The inhibition ratio of virus proliferation in the subgroups of the first, second and third groups was significantly higher than that in the virus control group（ P 〈 0. 01 ）. The inhibition ratio of virus proliferation in the subgroups of the second and third groups was significantly lower than that in the first group （ P 〈 0.05 ）. The inhibition ratio of virus proliferation in the subgroups of the third group was significantly lower than that in the second group （P 〈 0. 05 ）. The median inhibitory concentration of Folium Isatidis aqueous extract on IAV in the first, second, third groups was 0.75, 〉 1.25 and 1.25 g·L- 1 respectively. The antiviral activitiy of the three interven- tion methods from high to low was the first groups, the third groups and the second groups in tum. Conclusion Folium lsatidis aqueous extract has good anti IAV activity, and the mechanism may be related to the enhancement of immunity.