苦参主要成分的分离提取及其抗犬流感病毒的研究

发布时间：2018-06-18 16:58

本文选题：犬流感病毒 + 苦参　；参考：《山东农业大学》2015年硕士论文

【摘要】：犬流感是由犬流感病毒(Canine influenza virus,CIV)引起的以体温升高、咳嗽、呼吸困难为特征的犬的急性传染性疾病,其特点是传播快,流行广。我国和韩国都在呼吸道严重感染犬中分离到H3N2亚型流感病毒,实验发现这种病毒可以在犬之间进行直接传播。在现代生活中,犬是人类的忠实朋友,近年来宠物犬数量急剧增加,这也为犬流感病毒的跨宿主传播提供了更多的机会,人们需要重视这一问题。西药在抗犬流感病毒的临床应用上尚存在极大的局限性,如降低畜禽免疫力,易产生抗药性、具有副作用等。中药毒副作用小、不易产生耐药性,近年来的研究逐渐增多。但是把中药的药理活性、有效成分与药物的基础功效结合起来进行综合性研究的报道仍较少见。本课题基于前期研究成果即中药苦参具有良好的体外抗犬流感病毒效果的结论,以苦参为原材料,进行苦参主要成分的抗流感病毒效果研究。本试验采用稀醇提取、大孔树脂纯化法,从苦参中获得了主要成分即生物碱类,黄酮类,皂苷类和多糖类化合物。用MDCK细胞建立体外实验模型,将苦参提取物进行有效处理,制成可用于MDCK细胞的药物制剂,以利巴韦林为阳性对照,应用细胞病变观察法和MTT法检测细胞活性,对苦参成分的抗犬流感病毒效果进行评价。首先测定各成分对MDCK的最大无毒浓度,然后在安全浓度范围内比较四种成分的抗犬流感病毒效果,通过改变给药时间及作用方式初步探讨有效成分抗犬流感病毒的作用环节,最后确定其最小有效浓度。结果表明:苦参四种主要成分的最大安全浓度在3.344μg/m L~1.229 mg/m L之间。四种主要成分均有显著的抗CIV效果(P0.05)且综合作用效果均优于利巴韦林。不同给药方式试验结果显示:(1)苦参四种成分均能阻止CIV吸附进入细胞,且效果均显著优于利巴韦林(P0.05),生物碱和皂苷类的效果优于其他两种成分;(2)苦参四种成分均能干扰CIV的合成与释放;(3)病毒和药物同时接触细胞时,苦参四种成分均能起到抑制CIV的作用,生物碱类与利巴韦林作用相近,但黄酮、皂苷、多糖类成分的效果显著低于利巴韦林(P0.05);(4)药物与病毒预先作用时,苦参四种成分均能起到显著的杀灭CIV的作用,皂类与利巴韦林的作用相近,但生物碱、黄酮、多糖成分的效果显著低于利巴韦林(P0.05)。苦参有效成分的最小有效浓度在0.2090μg/mL~0.0474 mg/m L之间。不同成分的治疗指数(TI)在16~128之间,其中生物碱和多糖的治疗指数最高,为128,皂苷的治疗指数为32,黄酮的治疗指数为16。
[Abstract]:Canine influenza is an acute infectious disease caused by canine influenza virus (influenza), which is characterized by elevated body temperature, cough and dyspnea. It is characterized by rapid transmission and widespread prevalence. H3N2 subtype influenza virus was isolated from dogs with severe respiratory tract infection in China and South Korea, and it was found that H3N2 subtype influenza virus can be transmitted directly from dog to dog. In modern life, dogs are the faithful friends of human beings. In recent years, the number of pet dogs has increased dramatically, which also provides more opportunities for the cross-host transmission of canine influenza virus. People should pay attention to this problem. Western medicine has great limitations in the clinical application of canine influenza virus, such as reducing the immunity of livestock and poultry, easily producing drug resistance, and having side effects, etc. The research of traditional Chinese medicine is increasing in recent years because of its low toxicity and resistance. However, the comprehensive study of pharmacological activity, active components and basic efficacy of traditional Chinese medicine is still rare. Based on the conclusion that Sophora flavescens has good antiviral effect in vitro, this paper studies the antiviral effect of Sophora flavescens as raw material. In this experiment, alkaloids, flavonoids, saponins and polysaccharides were obtained from Sophora flavescens by extraction of dilute alcohol and purification of macroporous resin. The experimental model of MDCK cells was established in vitro. The extract of Sophora flavescens was effectively treated to prepare the drug preparation for MDCK cells. Ribavirin was used as positive control. The cytopathic observation method and MTT method were used to detect the cell activity. The effect of Sophora flavescens against canine influenza virus was evaluated. First, the maximum nontoxic concentration of each component to MDCK was determined, and then the anti-canine influenza virus effect of four ingredients was compared within the safe concentration range, and the effect of the active ingredient on the canine influenza virus was preliminarily discussed by changing the administration time and the mode of action. Finally, the minimum effective concentration was determined. The results showed that the maximum safe concentration of four main components of Sophora flavescens was between 3.344 渭 g / mL and 1.229 mg/m / L. The four main components had significant anti-CIV effect (P0.05) and the comprehensive effect was superior to ribavirin. The results of different drug administration methods showed that all four components of Sophora flavescens could prevent CIV from adsorbing into cells. The effects were significantly better than that of ribavirin P0.05, alkaloids and saponins were superior to those of the other two components. All four components of Sophora flavescens could interfere with the synthesis and release of CIV, and when the virus and drugs were in contact with the cells at the same time, the four components of Sophora flavescens could interfere with the synthesis and release of CIV. The effects of alkaloids and ribavirin on CIV were similar, but the effects of flavonoids, saponins and polysaccharides were significantly lower than those of ribavirin (P0.05). The effects of soap and ribavirin were similar, but the effects of alkaloids, flavonoids and polysaccharides were significantly lower than that of ribavirin. The minimum effective concentration of active components of Sophora flavescens was 0.2090 渭 g / mL 0.0474 mg/m / L. Among them, alkaloids and polysaccharides had the highest treatment index (128), saponins (32) and flavonoids (16).
【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

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