抗流感病毒天然产物的筛选及其抗病毒活性的评价
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摘要
流感病毒在病毒学分类上属于正粘病毒科(Orthomyxoviridae),根据病毒粒核蛋白(NP)和膜蛋白(MP)抗原特性及其基因特性的不同,流感病毒分为甲(A)、乙(B)、丙(C)三型。甲型流感病毒根据其表面抗原的(H和N)结构及其基因特性的不同又可分成许多亚型,至今甲型流感病毒已发现血凝素有15个亚型(H1～15),神经氨酸酶9个亚型(N1～9)。流感的显著特点之一就是发病率高,死亡率低,但所造成的总死亡人数是相当可观的。而且由于流感病毒的抗原性,尤其HA蛋白的抗原性,能经常不断的发生变异,这不仅给疫苗的制备增加了难度,更主要的是能很快使疫苗免疫接种效果下降,甚至无效;同时它与其它病毒性疾病一样,到目前为止还未找到理想的防治药物。因此,流感病毒是当前严重影响人类健康的重要病原之一。本研究的目的在于筛选具有抗流感病毒作用的样品,并从中选择抑制效果显著的天然产物样品分离其有效组分,研究结果将为进一步分离提取有效成分,以开发有效、特异的防治流感病毒感染的药物奠定实验基础。
首先在细胞病变抑制实验的基础上,结合CPE观察指标、MTT和血凝实验结果3种评价方法,建立一种快速、可靠的多种环节筛选抗流感病毒药物的方法。采用上述方法检测了160种中药和复方的样品的3种作用方式(对病毒的直接灭活作用、对病毒吸附的抑制作用和对病毒核酸复制的抑制作用),获得了抗流感病毒A/PR/8/34(H1N1)效果较好的提取物21种,其中鸡血藤样品水提物的IC50为74.5μg/mL,TI为13.3,将其做为下一步研究的对象。
采用体内、外两种方法对鸡血藤样品水提物体抗流感A/PR/8/34H1N1病毒效果进行评价。体外实验包括对3种流感病毒:A/PR/8/34(H1N1)、A/京科/12/64(H3N2)、B/京科/1/87体外细胞实验、直接血凝实验和鸡胚增殖实验3个部分。其实验结果显示,鸡血藤样品对3种流感病毒在细胞水平均有较好的抑制作用;鸡血藤在直接血凝实验和抑制鸡胚增殖实验的结果中表现出对A/PR/8/34(H1N1)病毒的抑制作用。体内实验结果显示:昆明小鼠口服鸡血藤水提物的LD50为800.49±38.75mg/kg;并在A/PR/8/34(H1N1)病毒小鼠病毒性肺炎模型的基础上,进行了一系列实验。实验结果显示,灌注鸡血藤样品水提物,能够明显降低小鼠的5d和9d死亡率、感染病毒小鼠肺组织的病毒滴度和小鼠肺病理切片坏死和浸润的级别并可抑制A/PR/8/34(H1N1)病毒核酸在小鼠肺组织的复制。说明鸡血藤能明显延长感染小鼠的生存时间,保护感染病毒的肺组织。
从体外细胞水平的抗病毒作用环节、抗流感病毒A/PR/8/34(H1N1)侵染后的RNA复制、免疫作用(体内免疫器官和体外免疫细胞测定)等三个水平初步探讨了鸡血藤样品水提物体外抗流感病毒A/PR/8/34(H1N1)的机制。细胞水平显示:鸡血藤样品对流感病毒A/PR/8/34(H1N1)有较好的灭活作用;RT-PCR结果显示:鸡血藤样品能明显抑制侵染细胞后流感病毒A/PR/8/34(H1N1)RNA的复制;对小鼠免疫器官指数和体外免疫细胞测定结果显示:鸡血藤样品可提高小鼠的免疫器官(胸腺和脾脏)指数;有直接促进T、B淋巴细胞增殖的作用,并且对ConA和LPS刺激淋巴细胞增殖有促进作用。
为进一步探明鸡血藤样品抗流感A/PR/8/34(H1N1)病毒有效组分,我们以抗A/PR/8/34(H1N1)病毒药效学活性为指标进行了初步的分离。采用热水浸提、聚酰胺柱层析分离的方法,获得了不同组分,并通过体外药效学活性评价。结果显示,鸡血藤水提物的50%-70%乙醇聚酰胺分离部位,体外抗病毒实验证明了其对A/PR/8/34(H1N1)有明显的抑制作用,EC50<10.0μg/mL,说明乙醇洗脱提纯物中有效成分比较集中。经初步化学分析,该提纯物经一系列化学反应检测呈阳性,可确定其活性成分为黄酮类化合物。核磁共振检测其1H谱、13C谱,结合谱库检索表明,主要的成分为黄酮类,其总回收率为83.56%,TI为15.17。
综上所述,本研究得出以下结论:
1.建立了一种快速、可靠的筛选抗流感病毒药物的方法,并对160种样品进行筛选,获得21种对A/PR/8/34(H1N1)病毒有不同抑制作用的样品。其中鸡血藤样品对3种流感病毒均有很好的抑制作用。
2.鸡血藤提取物对A/PR/8/34(H1N1)病毒在体内和体外均有很好的抑制作用。
3.鸡血藤提取物抗A/PR/8/34(H1N1)病毒机制与其直接灭活A/PR/8/34(H1N1)病毒,抑制A/PR/8/34(H1N1)病毒复制和增加免疫功能等综合作用有关。
4初步确定.鸡血藤提取物中抗A/PR/8/34(H1N1)病毒有效组分为黄酮类物质。
Influenza virus, the largest genus of Orthomyxoviridae family, based on the virus particle nucleoprotein (NP) and membrane protein (MP) antigen characteristics and genetic characteristics of different influenza viruses are divided into A, B, C. Influenza A virus in accordance with its surface antigen (H and N) and its genetic characteristics can be divided into many different subtypes. It has been found 15 known hemagglutinin subtypes (H1~15) and neural 9 NA subtypes (N1~9). The distinctive feature of influenza is high incidence, low mortality rate. But the total number of deaths caused by it is considerable. And as a result of influenza virus antigen, particularly the HA protein, which is mutate constantly, not only hard to the preparation the vaccines so that the effect of vaccine immunization will decline quickly, or even invalid. Until now, there are no influenza virus specific drugs available for clinical use, which is the same as the other viral diseases. Thus, new and more effective antiviral agents for future therapy in influenza virus infection are desired. In this study, we will screen the antiviral natural products against influenza virus A/PR/8/34(H1N1) in vitro and in vivo model and separate active site, for the aim of supplying references to the discovering new antiviral drugs.
In the first part of the study, we established a rapid and reliable method for screening potential antiviral agents against influenza virus base on the cytopathogenic effect inhibition assay; MTT-based colorimetric assay and the hemagglutination assay. We have detected the anti-influenza virus A/PR/8/34(H1N1) activity of 160 natural products, including effect on viral replication, effect on viral adsorption and subsequent replication, and viral inactivation. The results showed that there were 21 aqueous extracts exhibited anti-influenza virus A/PR/8/34(H1N1) by the three different antiviral assays. Among of them, the aqueous extract from Spatholobus suberectus Dunn. showed potent antiviral activity against A/PR/8/34(H1N1) with its IC50 was 74.5μg/mL and TI was 13.3.
In order to evaluate the effective of the aqueous extract from Spatholobus suberectus Dunn. inhibiting the influenza virus , we designed different assays in vitro and in vivo. In vitro: we determined the antiviral effects against three types of influenza virus on MDCK cells, which result show that the aqueous extract from Spatholobus suberectus Dunn.could inhibit all three virus. And the directly hemagglutinin assays and the reproduction of chick embryo assays showed that the A/PR/8/34 (H1N1) virus could be inactivation by the aqueous extract from Spatholobus suberectus Dunn. In vivo: The aqueous extract of Spatholobus suberectus Dunn. on acute toxicity in Kunming mice showed the oral LD50 is 800.49±38.75mg/kg. In the use of A/PR/8/34 (H1N1) virus strains of the establishment of appropriate mouse pneumonia virus mouse model based on a series of experiments. Experimental results show that the aqueous extract from Spatholobus suberectus Dunn. perfusion were able to significantly reduce the 5d and 9d mouse mortality in mice infected with a virus titer of lung tissue and necrosis in mouse lung slices and the level of infiltration and to inhibit A / PR/8/34 (H1N1) virus replication in lung tissue of mice. Description the aqueous extract from Spatholobus suberectus Dunn. could significantly prolong the survival time of infected mice, the protection of infected lung tissue.
In order to investigate the mechanism of the aqueous extract from Spatholobus suberectus Dunn. inhibiting the infection of A/PR/8/34 (H1N1) we designed three different assays. Firstly, a assays design to investigate the time course effect of the virus infection at different concentration of the aqueous extract of Spatholobus suberectus Dunn. revealed it can inactivated A/PR/8/34 (H1N1) in vitro. Secondly the RT-PCR results showed that: the aqueous extract from Spatholobus suberectus Dunn. could inhibit the A/PR/8/34 (H1N1) virus replication. Effected of the aqueous extrat from Spatholobus suberectus Dunn. on immune organ of mice is assayed, the results showed that immune organ coefficient, such as thymus and spleen, augment obviously, burst coefficient is significant compared with control group. In vitro, the aqueous extract from Spatholobus suberectus Dunn. can enhance proliferation of control group and extract-hemolymph T cell stimulated by Con A. At the same time, it could promote proliferation clearly of T lymphocyte induced by Con A and B lymphocyte induced by LPS.
The Spatholobus suberectus Dunn. antiviral properties were worth further investigated to identify the active sites. In summary, the active site was tracing by antiviral pharmacology assay in vitro. Firstly, the Spatholobus suberectus Dunn. powders were extracted with heated water. The extract was further purified by system extract assay, which used polyamide column chromatography methods. The results showed that the 50% -70% ethanol polyamide separation of the Spatholobus suberectus Dunn. parts were the active part. The characters of active site were identified by physics character experiments. The major ingredient of the alcohol was flavonoid, which were the most bio-active ingredient of the Spatholobus suberectus Dunn..
In conclusion, the main results are as follows:
1. Establishing a rapid and reliable method for screening potential antiviral agents against the influenza virus A / PR/8/34 (H1N1) and aquiring 21 aqueous anti- influenza virus A / PR/8/34 (H1N1) strongly.
2. The aqueous extract from Spatholobus suberectus Dunn. could inhibit the influenza virus A / PR/8/34 (H1N1) in vitro and in vivo.
3. The aqueous extract from Spatholobus suberectus Dunn. could inhibit influenza virus A / PR/8/34 (H1N1) RNA sysnthesis selectively and hence inhibit virus replication, also stimulated the immune organ coefficient and the proliferation of T lymphocyte and B lymphocyte.
4. The aqueous extract from Spatholobus suberectus Dunn. could inhibit influenza virus A / PR/8/34 (H1N1) RNA sysnthesis selectively and hence inhibit virus replication, also stimulated the immune organ coefficient and the proliferation of T lymphocyte and B lymphocyte.
5. The major anti-influenza virus A / PR/8/34 (H1N1) site of the Spatholobus suberectus Dunn. was mostly flavonoids.
首先在细胞病变抑制实验的基础上,结合CPE观察指标、MTT和血凝实验结果3种评价方法,建立一种快速、可靠的多种环节筛选抗流感病毒药物的方法。采用上述方法检测了160种中药和复方的样品的3种作用方式(对病毒的直接灭活作用、对病毒吸附的抑制作用和对病毒核酸复制的抑制作用),获得了抗流感病毒A/PR/8/34(H1N1)效果较好的提取物21种,其中鸡血藤样品水提物的IC50为74.5μg/mL,TI为13.3,将其做为下一步研究的对象。
采用体内、外两种方法对鸡血藤样品水提物体抗流感A/PR/8/34H1N1病毒效果进行评价。体外实验包括对3种流感病毒:A/PR/8/34(H1N1)、A/京科/12/64(H3N2)、B/京科/1/87体外细胞实验、直接血凝实验和鸡胚增殖实验3个部分。其实验结果显示,鸡血藤样品对3种流感病毒在细胞水平均有较好的抑制作用;鸡血藤在直接血凝实验和抑制鸡胚增殖实验的结果中表现出对A/PR/8/34(H1N1)病毒的抑制作用。体内实验结果显示:昆明小鼠口服鸡血藤水提物的LD50为800.49±38.75mg/kg;并在A/PR/8/34(H1N1)病毒小鼠病毒性肺炎模型的基础上,进行了一系列实验。实验结果显示,灌注鸡血藤样品水提物,能够明显降低小鼠的5d和9d死亡率、感染病毒小鼠肺组织的病毒滴度和小鼠肺病理切片坏死和浸润的级别并可抑制A/PR/8/34(H1N1)病毒核酸在小鼠肺组织的复制。说明鸡血藤能明显延长感染小鼠的生存时间,保护感染病毒的肺组织。
从体外细胞水平的抗病毒作用环节、抗流感病毒A/PR/8/34(H1N1)侵染后的RNA复制、免疫作用(体内免疫器官和体外免疫细胞测定)等三个水平初步探讨了鸡血藤样品水提物体外抗流感病毒A/PR/8/34(H1N1)的机制。细胞水平显示:鸡血藤样品对流感病毒A/PR/8/34(H1N1)有较好的灭活作用;RT-PCR结果显示:鸡血藤样品能明显抑制侵染细胞后流感病毒A/PR/8/34(H1N1)RNA的复制;对小鼠免疫器官指数和体外免疫细胞测定结果显示:鸡血藤样品可提高小鼠的免疫器官(胸腺和脾脏)指数;有直接促进T、B淋巴细胞增殖的作用,并且对ConA和LPS刺激淋巴细胞增殖有促进作用。
为进一步探明鸡血藤样品抗流感A/PR/8/34(H1N1)病毒有效组分,我们以抗A/PR/8/34(H1N1)病毒药效学活性为指标进行了初步的分离。采用热水浸提、聚酰胺柱层析分离的方法,获得了不同组分,并通过体外药效学活性评价。结果显示,鸡血藤水提物的50%-70%乙醇聚酰胺分离部位,体外抗病毒实验证明了其对A/PR/8/34(H1N1)有明显的抑制作用,EC50<10.0μg/mL,说明乙醇洗脱提纯物中有效成分比较集中。经初步化学分析,该提纯物经一系列化学反应检测呈阳性,可确定其活性成分为黄酮类化合物。核磁共振检测其1H谱、13C谱,结合谱库检索表明,主要的成分为黄酮类,其总回收率为83.56%,TI为15.17。
综上所述,本研究得出以下结论:
1.建立了一种快速、可靠的筛选抗流感病毒药物的方法,并对160种样品进行筛选,获得21种对A/PR/8/34(H1N1)病毒有不同抑制作用的样品。其中鸡血藤样品对3种流感病毒均有很好的抑制作用。
2.鸡血藤提取物对A/PR/8/34(H1N1)病毒在体内和体外均有很好的抑制作用。
3.鸡血藤提取物抗A/PR/8/34(H1N1)病毒机制与其直接灭活A/PR/8/34(H1N1)病毒,抑制A/PR/8/34(H1N1)病毒复制和增加免疫功能等综合作用有关。
4初步确定.鸡血藤提取物中抗A/PR/8/34(H1N1)病毒有效组分为黄酮类物质。
Influenza virus, the largest genus of Orthomyxoviridae family, based on the virus particle nucleoprotein (NP) and membrane protein (MP) antigen characteristics and genetic characteristics of different influenza viruses are divided into A, B, C. Influenza A virus in accordance with its surface antigen (H and N) and its genetic characteristics can be divided into many different subtypes. It has been found 15 known hemagglutinin subtypes (H1~15) and neural 9 NA subtypes (N1~9). The distinctive feature of influenza is high incidence, low mortality rate. But the total number of deaths caused by it is considerable. And as a result of influenza virus antigen, particularly the HA protein, which is mutate constantly, not only hard to the preparation the vaccines so that the effect of vaccine immunization will decline quickly, or even invalid. Until now, there are no influenza virus specific drugs available for clinical use, which is the same as the other viral diseases. Thus, new and more effective antiviral agents for future therapy in influenza virus infection are desired. In this study, we will screen the antiviral natural products against influenza virus A/PR/8/34(H1N1) in vitro and in vivo model and separate active site, for the aim of supplying references to the discovering new antiviral drugs.
In the first part of the study, we established a rapid and reliable method for screening potential antiviral agents against influenza virus base on the cytopathogenic effect inhibition assay; MTT-based colorimetric assay and the hemagglutination assay. We have detected the anti-influenza virus A/PR/8/34(H1N1) activity of 160 natural products, including effect on viral replication, effect on viral adsorption and subsequent replication, and viral inactivation. The results showed that there were 21 aqueous extracts exhibited anti-influenza virus A/PR/8/34(H1N1) by the three different antiviral assays. Among of them, the aqueous extract from Spatholobus suberectus Dunn. showed potent antiviral activity against A/PR/8/34(H1N1) with its IC50 was 74.5μg/mL and TI was 13.3.
In order to evaluate the effective of the aqueous extract from Spatholobus suberectus Dunn. inhibiting the influenza virus , we designed different assays in vitro and in vivo. In vitro: we determined the antiviral effects against three types of influenza virus on MDCK cells, which result show that the aqueous extract from Spatholobus suberectus Dunn.could inhibit all three virus. And the directly hemagglutinin assays and the reproduction of chick embryo assays showed that the A/PR/8/34 (H1N1) virus could be inactivation by the aqueous extract from Spatholobus suberectus Dunn. In vivo: The aqueous extract of Spatholobus suberectus Dunn. on acute toxicity in Kunming mice showed the oral LD50 is 800.49±38.75mg/kg. In the use of A/PR/8/34 (H1N1) virus strains of the establishment of appropriate mouse pneumonia virus mouse model based on a series of experiments. Experimental results show that the aqueous extract from Spatholobus suberectus Dunn. perfusion were able to significantly reduce the 5d and 9d mouse mortality in mice infected with a virus titer of lung tissue and necrosis in mouse lung slices and the level of infiltration and to inhibit A / PR/8/34 (H1N1) virus replication in lung tissue of mice. Description the aqueous extract from Spatholobus suberectus Dunn. could significantly prolong the survival time of infected mice, the protection of infected lung tissue.
In order to investigate the mechanism of the aqueous extract from Spatholobus suberectus Dunn. inhibiting the infection of A/PR/8/34 (H1N1) we designed three different assays. Firstly, a assays design to investigate the time course effect of the virus infection at different concentration of the aqueous extract of Spatholobus suberectus Dunn. revealed it can inactivated A/PR/8/34 (H1N1) in vitro. Secondly the RT-PCR results showed that: the aqueous extract from Spatholobus suberectus Dunn. could inhibit the A/PR/8/34 (H1N1) virus replication. Effected of the aqueous extrat from Spatholobus suberectus Dunn. on immune organ of mice is assayed, the results showed that immune organ coefficient, such as thymus and spleen, augment obviously, burst coefficient is significant compared with control group. In vitro, the aqueous extract from Spatholobus suberectus Dunn. can enhance proliferation of control group and extract-hemolymph T cell stimulated by Con A. At the same time, it could promote proliferation clearly of T lymphocyte induced by Con A and B lymphocyte induced by LPS.
The Spatholobus suberectus Dunn. antiviral properties were worth further investigated to identify the active sites. In summary, the active site was tracing by antiviral pharmacology assay in vitro. Firstly, the Spatholobus suberectus Dunn. powders were extracted with heated water. The extract was further purified by system extract assay, which used polyamide column chromatography methods. The results showed that the 50% -70% ethanol polyamide separation of the Spatholobus suberectus Dunn. parts were the active part. The characters of active site were identified by physics character experiments. The major ingredient of the alcohol was flavonoid, which were the most bio-active ingredient of the Spatholobus suberectus Dunn..
In conclusion, the main results are as follows:
1. Establishing a rapid and reliable method for screening potential antiviral agents against the influenza virus A / PR/8/34 (H1N1) and aquiring 21 aqueous anti- influenza virus A / PR/8/34 (H1N1) strongly.
2. The aqueous extract from Spatholobus suberectus Dunn. could inhibit the influenza virus A / PR/8/34 (H1N1) in vitro and in vivo.
3. The aqueous extract from Spatholobus suberectus Dunn. could inhibit influenza virus A / PR/8/34 (H1N1) RNA sysnthesis selectively and hence inhibit virus replication, also stimulated the immune organ coefficient and the proliferation of T lymphocyte and B lymphocyte.
4. The aqueous extract from Spatholobus suberectus Dunn. could inhibit influenza virus A / PR/8/34 (H1N1) RNA sysnthesis selectively and hence inhibit virus replication, also stimulated the immune organ coefficient and the proliferation of T lymphocyte and B lymphocyte.
5. The major anti-influenza virus A / PR/8/34 (H1N1) site of the Spatholobus suberectus Dunn. was mostly flavonoids.
引文
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