苦参主要成分体外抗H3N2亚型猪流感病毒作用及其机制研究
发布时间：2018-12-14 18:35
【摘要】：猪流感(Swine Influenza,SI)是由猪流感病毒(Swine Influenza Virus,SIV)引起的一种急性、热性、高度接触性呼吸道传染病,是规模化养猪场的常发病之一,以高致病率、低死亡率为特征。本病极易与其他呼吸道病原混合感染,使其死亡率急剧升高,给养猪业造成巨大的经济损失。猪是猪流感、人流感及禽流感等多种流感病毒共同的易感宿主,流感病毒易在其体内重组而产生新的流感病毒株。同时,猪流感病毒不仅能感染猪,也能感染其他动物。因此,对猪流感病毒的研究不但有重要的兽医传染病学意义,更有深远的公共卫生意义。流感病毒因血清型多、易变异以及易产生耐药性等,疫苗接种及抗流感西药的疗效甚微,因此当前尚无猪流感特效药。所以,研究及开发抗猪流感病毒天然药物及其有效成分迫在眉睫。目的:本研究旨在细胞水平上探讨苦参主要成分的抗H3N2亚型猪流感病毒效果,并对其作用机制进行初步探索。研究结果将为抗流感机制的深入研究提供基础,为兽医临床及医学临床用药提供科学依据和参考,更为抗流感病毒天然药物的开发及研制奠定基础。方法:采用传统有机溶剂回流提取法、传统水提醇沉法分别制备苦参生物碱、黄酮、皂苷和粗多糖成分,以利巴韦林作为阳性对照药,选择犬肾上皮细胞株(MDCK细胞)作为H3N2猪流感病毒作用的细胞体系,通过细胞病变(CPE)观察法和噻唑蓝盐微量酶反应比色法(MTT法)定性、定量测定细胞状态,从而对苦参生物碱、黄酮、皂苷、粗多糖成分抗H3N2猪流感病毒作用加以比较,然后通过改变给药方式等条件对其作用机制进行初步探索。结果显示:高浓度的苦参生物碱、黄酮、皂苷、粗多糖均会影响MDCK细胞的生长状态,其最大安全浓度分布在6.9μg/ml~1.5138 mg/ml之间,其中皂苷成分的毒性作用最小,浓度为1.5138 mg/ml时仍不表现细胞毒性作用。对四种成分体外抗SIV作用的研究发现,四种成分都能显著性抑制H3N2猪流感病毒,其中生物碱和粗多糖成分的效果优于利巴韦林。此外,⑴四种成分均能明显抵抗SIV的吸附,皂苷效果优于利巴韦林;⑵四种成分均能干扰病毒复制,生物碱的作用优于利巴韦林;⑶四种成分与病毒同时接触细胞时,均有显著抑制SIV作用,皂苷的效果优于利巴韦林;⑷四种成分均有体外直接杀灭病毒作用,生物碱的作用优于利巴韦林。苦参四种成分的最小有效剂量分布在0.1078μg/ml~19.2μg/ml之间,其中苦参黄酮的最小有效浓度最低;治疗指数在32~128之间,皂苷成分的治疗指数最高。苦参成分与病毒同时接触细胞时,各成分均呈现出明显的剂量效应关系。结论:苦参生物碱、黄酮、皂苷、粗多糖成分均有抗H3N2猪流感病毒作用,其中皂苷干扰病毒吸附的作用最强且能有效阻止病毒与细胞的结合,生物碱干扰病毒复制和直接杀病毒的作用最强。
[Abstract]:Swine flu (Swine Influenza,SI) is an acute, feverish and highly contact respiratory infectious disease caused by swine flu virus (Swine Influenza Virus,SIV. It is one of the common diseases in large-scale pig farms, characterized by high pathogenicity and low mortality. This disease is easy to be mixed with other respiratory pathogens, resulting in a sharp increase in mortality and a huge economic loss to the pig industry. Pigs are the common susceptible hosts of swine flu, human influenza and avian influenza. Influenza viruses are easy to recombine in their bodies to produce new strains of influenza viruses. At the same time, swine flu virus can infect not only pigs, but also other animals. Therefore, the study of swine flu virus not only has important veterinary infectious disease significance, but also has far-reaching public health significance. Due to the variety of serotypes, variability and drug resistance of influenza viruses, vaccination and anti-influenza drugs have little effect, so there is no specific swine flu drug. Therefore, the research and development of anti-swine flu virus natural drugs and their effective components are urgent. Objective: to investigate the effect of main components of Sophora flavescens against H3N2 subtype swine flu virus at cell level and to explore its mechanism. The results will provide a basis for the further study of the mechanism of anti-influenza, provide scientific basis and reference for the clinical use of veterinary medicine and medicine, and lay a foundation for the development and development of natural anti-influenza drugs. Methods: the alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavescens were prepared by traditional organic solvent reflux extraction method and traditional water extraction and alcohol precipitation method. Ribavirin was used as positive control drug. Canine renal epithelial cell line (MDCK cell) was selected as the cell system of H3N2 swine flu virus. The cytopathic (CPE) assay and MTT assay were used to determine the cell status quantitatively. The effects of alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavescens against H3N2 swine flu virus were compared. The results showed that high concentration of alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavescens affected the growth of MDCK cells. The maximum safe concentration was between 6.9 渭 g/ml~1.5138 mg/ml and the toxicity of saponins was the least. The concentration of 1.5138 mg/ml still showed no cytotoxicity. It was found that the four components could significantly inhibit H3N2 swine flu virus in vitro, and the effects of alkaloids and crude polysaccharides were superior to ribavirin. In addition, all the four components could resist the adsorption of SIV, and the effect of saponins was better than ribavirin, and the effect of alkaloids was superior to ribavirin. (3) when the four components were in contact with the virus at the same time, they all had significant inhibitory effects on SIV, and the saponins were superior to ribavirin in killing virus directly in vitro, and alkaloids were superior to ribavirin. The minimum effective dose of four components of Sophora flavescens was distributed in the range of 0.1078 渭 g/ml~19.2 渭 g/ml, in which the minimum effective concentration of flavone of Sophora flavone was the lowest, and the therapeutic index was between 32 ~ 128.The therapeutic index of saponins was the highest. When the components of Sophora flavescens and the virus were in contact with the cells at the same time, there was a significant dose-effect relationship between the components. Conclusion: the alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavone have the effect of resisting H3N2 swine flu virus, and the saponins have the strongest effect on interfering the virus adsorption and can effectively prevent the virus from binding to the cells. Alkaloids have the strongest effect on virus replication and direct virus killing.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7
本文编号：2379117
[Abstract]:Swine flu (Swine Influenza,SI) is an acute, feverish and highly contact respiratory infectious disease caused by swine flu virus (Swine Influenza Virus,SIV. It is one of the common diseases in large-scale pig farms, characterized by high pathogenicity and low mortality. This disease is easy to be mixed with other respiratory pathogens, resulting in a sharp increase in mortality and a huge economic loss to the pig industry. Pigs are the common susceptible hosts of swine flu, human influenza and avian influenza. Influenza viruses are easy to recombine in their bodies to produce new strains of influenza viruses. At the same time, swine flu virus can infect not only pigs, but also other animals. Therefore, the study of swine flu virus not only has important veterinary infectious disease significance, but also has far-reaching public health significance. Due to the variety of serotypes, variability and drug resistance of influenza viruses, vaccination and anti-influenza drugs have little effect, so there is no specific swine flu drug. Therefore, the research and development of anti-swine flu virus natural drugs and their effective components are urgent. Objective: to investigate the effect of main components of Sophora flavescens against H3N2 subtype swine flu virus at cell level and to explore its mechanism. The results will provide a basis for the further study of the mechanism of anti-influenza, provide scientific basis and reference for the clinical use of veterinary medicine and medicine, and lay a foundation for the development and development of natural anti-influenza drugs. Methods: the alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavescens were prepared by traditional organic solvent reflux extraction method and traditional water extraction and alcohol precipitation method. Ribavirin was used as positive control drug. Canine renal epithelial cell line (MDCK cell) was selected as the cell system of H3N2 swine flu virus. The cytopathic (CPE) assay and MTT assay were used to determine the cell status quantitatively. The effects of alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavescens against H3N2 swine flu virus were compared. The results showed that high concentration of alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavescens affected the growth of MDCK cells. The maximum safe concentration was between 6.9 渭 g/ml~1.5138 mg/ml and the toxicity of saponins was the least. The concentration of 1.5138 mg/ml still showed no cytotoxicity. It was found that the four components could significantly inhibit H3N2 swine flu virus in vitro, and the effects of alkaloids and crude polysaccharides were superior to ribavirin. In addition, all the four components could resist the adsorption of SIV, and the effect of saponins was better than ribavirin, and the effect of alkaloids was superior to ribavirin. (3) when the four components were in contact with the virus at the same time, they all had significant inhibitory effects on SIV, and the saponins were superior to ribavirin in killing virus directly in vitro, and alkaloids were superior to ribavirin. The minimum effective dose of four components of Sophora flavescens was distributed in the range of 0.1078 渭 g/ml~19.2 渭 g/ml, in which the minimum effective concentration of flavone of Sophora flavone was the lowest, and the therapeutic index was between 32 ~ 128.The therapeutic index of saponins was the highest. When the components of Sophora flavescens and the virus were in contact with the cells at the same time, there was a significant dose-effect relationship between the components. Conclusion: the alkaloids, flavonoids, saponins and crude polysaccharides of Sophora flavone have the effect of resisting H3N2 swine flu virus, and the saponins have the strongest effect on interfering the virus adsorption and can effectively prevent the virus from binding to the cells. Alkaloids have the strongest effect on virus replication and direct virus killing.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7
【参考文献】
相关期刊论文 前1条
1 陈亚军;苦参素治疗慢性乙型肝炎的疗效观察[J];现代中西医结合杂志;2004年09期
，本文编号：2379117
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