重楼皂苷的制备及其抗A型流感病毒活性

Preparation of polyphylla saponins and their antiviral effect on influenza A virus

目的研究重楼皂苷的制备及体内外对A型流感病毒(IAV)活性的影响。方法 采用柱层析和反相液相色谱分离技术,从重楼中提取重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ,应用高效液相色谱法测定其纯度。采用MTT比色法测定重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ的细胞毒性,并检测其体外对IAV的直接灭活作用、对IAV吸附和侵入细胞的阻断作用及对IAV在靶细胞内增殖的抑制作用。用IAV滴鼻法制备IAV感染小鼠模型,模型制备4 h后分别每天ig给予重楼皂苷Ⅰ5和10 mg.kg-1及阳性对照药奥司他韦3 mg.kg-1,每天分2次给药,连续5 d,自给药后每天观察并记录小鼠发病情况和死亡数,共观察14 d,计算小鼠死亡率和生命延长率,评价其体内抗IAV作用。结果 提取分离得到的重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ的纯度分别为95.3%,90.2%,92.4%和86.8%。重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ浓度分别低于50,12.5,6.25和12.5 mg.L-1时,未见对靶细胞MDCK的细胞毒性。重楼皂苷Ⅰ6.25,12.5,25和50 mg.L-1,重楼皂苷Ⅱ6.25和12.5 mg.L-1,重楼皂苷Ⅵ3.13和6.25 mg.L-1,重楼皂苷Ⅶ6.25和12.5 mg.L-1及奥司他韦40 mg.L-1对IAV均有显著的直接灭活作用(P<0.01),对IAV吸附和侵入靶细胞亦具有一定的阻断作用(P<0.01),对IAV在靶细胞内的增殖具有抑制作用(P<0.01)。重楼皂苷Ⅰ5和10 mg.L-1可明显降低IAV感染小鼠的死亡率(P<0.01),由流感病毒感染组的8/10均下降到2/10;并可延长IAV感染小鼠的存活时间(P<0.01),存活时间由(8.5±0.3)d分别延长到12.7±0.4和(13.2±0.5)d(P<0.01),生命延长率分别为49.4%和55.3%,效果与奥司他韦近似。结论 从植物重楼中分离到高纯度的重楼皂苷Ⅰ,Ⅱ,Ⅵ和Ⅶ,重楼皂苷Ⅱ,Ⅵ和Ⅶ在体外具有较好的抗IAV活性,重楼皂苷Ⅰ在体内外均具有较好的抗IAV活性。

OBJECTIVE To study the preparation of polyphylla saponins and their antiviral activity on influenza A virus （IAV） in vitro and in vivo. METHODS The column chromatography and reversed phase liquid chromatography separation technology in extraction and purification were used to prepare polyphylla saponins Ⅰ , Ⅱ, Ⅵ and Ⅶ, and their purity was assayed with HPLC. MTT method was used for determination of their cell toxicity, direct inactivation, prevention from adsorbing MDCK cells and inhibitory effect on reproduction of IAV in MDCK cells in vitro. The model mice were prepared by an intranasal inoculation of IAV. Four hours after the mice infected with IAV, they were orally administered polyphylla saponin I and oseltamivir twice daily for 5 d. The doses of polyphylla saponin I were 5 and 10 mg.kg-1, and oseltamivir was 3 mg.kg-1. The parameters for the anti-virus efficacy including mortal- ity, mean survival time, and life extension rate were determined on the 14th day after the first drug treat- ment. RESULTS The purity of polyphylla saponins Ⅰ , Ⅱ, Ⅵ and Ⅶ was 95.3% ,90.2% ,92.4% and 86.8%, respectively. The polyphylla saponins Ⅰ , Ⅱ, Ⅵ and Ⅶ did not show toxicity for MDCK cells when they were below 50, 12.5, 6.25 and 12.5 mg. L-1 , respectively. Polyphylla saponin I 6.25, 12.5,25 and 50 mg. L-1 , polyphylla saponin Ⅱ 6.25 and 12.5 mg. L-1 , polyphylla saponinVI 3.13 and 6.25 mg. L-l, polyphylla saponinVI 6.25 and 12.5 mg. L-1 and oseltamivir 40 mg. L-1 showed remarkable inactivation effect on IAV （ P 〈 0.01 ）, prevention effect from adsorbing MDCK cells （ P 〈 0.01 ）, and inhibitory effect on reproduction of IAV in MDCK cells. Polyphylla saponin I 5 and 10 mg· kg-1 could significantly reduce the mortality of mice infected with IAV, which was dropped to 2/10 from 8/10 （ P 〈 0.01 ）. They could prolong the survival time of mice, which was extended to 12.7 ±0.4 and （13.2 ± 0.5）d from （8.5 ±0.3） d（ P 〈0.01 ）. The life extension rate was 49.4% and 55.3%, respectively. CONCLUSION Polyphylla saponin Ⅰ , Ⅱ, Ⅵ and Ⅶ are prepared from Paris polyphylla. Polyphylla saponins Ⅱ, Ⅵ and Ⅶ show obvious antiviral activity on IAV in vitro, and polyphylla saponin I has an obvious antiviral activity on IAV in vitro and in vivo.