【作者】 王国霞；

【导师】 马立保；

【作者基本信息】 华中农业大学 ， 动物营养与饲料科学， 2005， 硕士

【摘要】 选用黄芪、板蓝根、射干等二十种中草药和金刚烷胺、中性丫啶黄两种西药为试验药物进行体外抗病毒试验。中草药采用传统的水煮浸提法分别提取并浓缩成0.50g/ml溶液作为原液,金刚烷胺和中性丫啶黄分别配成160μg/ml的溶液作原液,高压灭菌处理备用。药物原液倍比稀释成不同浓度以测定其对Vero细胞的毒性作用,来确定药物的安全浓度。以安全浓度为准,稀释成四个不同浓度在Vero细胞上做抗病毒试验,采用三种作用方式:先加药物后加病毒,先加病毒后加药物,药物与病毒先作用一段时间后加入,以CPE、病毒滴度和MTT法测得的OD值为观测指标评价药物抗H9N2亚型禽流感病毒的活性,以探讨其抗病毒吸附、抗病毒复制或直接灭活病毒的作用方式。试验结果如下: 各药物对Vero细胞的安全浓度分别为黄芪31.25mg/ml、金银花3.90mg/ml、连翘15.62mg/ml、板蓝根31.25mg/ml、射干15.62mg/ml、穿心莲7.8lmg/ml、鱼腥草15.62mg/ml、大黄7.81mg/ml、贯众31.25mg/ml、黄柏31.25mg/ml、黄连3.90mg/ml、野菊花3.90mg/ml、麻黄31.25mg/ml、荆芥15.62mg/ml、柴胡15.62mg/ml、桂枝15.62mg/ml、黄芩3.90mg/ml、侧柏叶62.5mg/ml、百部31.25mg/ml、天花粉3.90mg/ml,金刚烷胺40μg/ml、中性丫啶黄0.625μg/ml。不同药物对同种细胞的毒性不一样。 三种不同的加药方式表明:黄芪、板蓝根、射干、穿心莲、野菊花、金刚烷胺、中性丫啶黄具有抗病毒吸附作用;金银花、板蓝根、射干、穿心莲、鱼腥草、野菊花、荆芥、柴胡、金刚烷胺、中性丫啶黄具有抗病毒复制作用,其中金银花、鱼腥草、荆芥作用极弱:连翘、板蓝根、射干、穿心莲、野菊花、鱼腥草、贯众、麻黄、荆芥、柴胡、侧柏叶、金刚烷胺、中性丫啶黄能够直接灭活病毒。不同作用方式表明其用药时间对抗病毒效果有影响。 综合三种加药方式的效果考虑,板蓝根、射干、穿心莲、野菊花、金刚烷胺、中性丫啶黄能够抑制病毒,对细胞的保护率较高,西药金刚烷胺和中性丫啶黄对细胞的保护率达80%以上,比中草药提取液的效果好。 药物以不同方式加入培养板培养72h后,取细胞培养基及细胞冻融物检测无血凝效价。药物原液与病毒体外37℃作用不同时间后测病毒滴度,其中连翘、板蓝根、射干、黄连、麻黄、荆芥、侧柏叶、大黄、贯众和百部能够降低病毒滴度。更多还原

【Abstract】 Twenty Chinese herbs such as Radix Astragali, Radix isatidis and rhizome belamcandae, and amantadine and neutral acriflavine were used in anti-virus experiment in vitro. The Chinese herbs were separately decocted and concentrated by heating to a final concentration of 0.50mg/ml respectively. The concentrations of amantadine and neutral acriflavine were 160μg/ml respectively. All the drug-liquids were sterilized by autoclave and stored at 4℃. In the Vero cell culture experiment in vitro, the cytotoxic influence of different concentrations diluted at multiple proportions was studied to determine the highest non-toxic concentration, and anti-virus mechanism of the effective anti-AIV was studied in three ways: adding medicine to the cultured Vero cell before adding virus, adding virus to the cultured Vero cell before adding medicine, and adding medicme and virus to the cultured Vero cell after they acted for a while. Antiviral activity against H9N2 AIV was estimated in terms of Cytopathic effect, viral titre yields and OD values of MTT colorimetnc method to determine the respective effects of anti-penetration activity against virus, restraint of virus propagation and direct destruction of virus, respectively. The results were as follows.The highest non-toxic concentrations of medicaments were: Radix Astragali, 31.25mg/ml; Lonicera Japonica, 3.90mg/ml; Forsythia suspense, 15.62mg/ml; Radix isatidis, 31.25mg/ml; Rhizome belamcandae, 15.62mg/ml; herba andrographis, 7.81mg/ml; Herba houttuyniae, 15.62mg/ml; Radix et rhizomarhei, 7.81mg/ml; Cyrotomium fortunei, 31.25mg/ml; Cortex phellodendri, 31.25mg/ml; Rhizoma coptidis, 3.90mg/ml; Flos chrysanthemi, 3.90mg/ml; Herba ephedrae, 31.25mg/ml; Herbaschizonepetae, 15.62mg/ml; Radix bupleuri, 15.62mg/ml; Ramulus cinnamomi, 15.62mg/ml; Radix scutellariae, 3.90mg/ml; Cacumen platycladi, 62.5mg/ml; Radix stamonae, 31.25mg/ml; i?a<fct fr/co santois, 3.90mg/ml; amantadine, 40/<g/ml; and neutral acriflavine, 0.625/ig/ml. Cytotoxicity of every medicament was different to the same cell.The results of the three different ways indicated that Radix Astragali, Radix isatidis, rhizome belamcandae, herba andrographis, Flos chrysanthemi, amantadine and neutral acriflavine had anti-penetration activity; and that Lonicera Japonica, Radix isatidis, rhizome belamcandae, herba andrographis, Flos chrysanthemi, Herba houttuyniae, Herba schizonepetae, Radix bupleuri, amantadine and neutral acriflavine could restrain virus propagation, but Lonicera Japonica, houttuyniae and herba schizonepetae had faint effect in restraining virus propagation. Forsythia suspense, Radix isatidis, rhizome belamcandae, herba andrographis, Flos chrysanthemi, houttuyniae, cyrotomium fortunei, ephedrae, schizonepetae, bupleuri, cacumen platycladi, amantadine and neutral acriflavine could directly destroy virus. The three ways indicated that time of adding medicine had influence on anti-virus action.The best medicines in the three ways were screened out. Radix isatidis, rhizome belamcandae, herba andrographis, Flos chrysanthemi, amantadine and neutral acriflavine could inhibit AIV and provided good protection for cell. The protection rates of amantadine and neutral acriflavine were over 80%, and this showed that they had better effect than Chinese herbal extracts.The Vero cell was cultured for 72h after medicine had been added in three ways. And then supemate and cell freezing substance were taken out to check hemagglutitin value of virus, but HA value was zero. Effect of original medicines on HA value of virus at 37’C in vitro showed that Forsythia suspense, Radix isatidis, Rhizoma belamcandae, Rhizoma coptidix, Herba ephedrae, Herba schizonepetae, Cacumen platycladi, Radix et rhizomarhei, Cyrotomium fortunei and Radix stemonae could reduce virus titre.更多还原