黄芩苷提取工艺优化及其抗马立克氏病病毒的作用研究

发布时间：2018-03-25 15:52

本文选题：黄芩苷　切入点：马立克氏病病毒　出处：《聊城大学》2017年硕士论文

【摘要】：病毒性肿瘤病是一种自然性的常发病,严重的危害畜禽健康的重要疾病。肿瘤性疾病导致了畜禽的生产性能的下降,甚至死亡,对养殖户的经济造成了严重的损失。家禽T细胞的淋巴瘤就是主要由马立克氏病所引起,主要是对家禽体内的病毒提取出的样品进行诊断,做出判断。病毒是不断变异并且发展着的,马立克氏病的毒性也随之增强,疫苗的注射作用也是有局限性,所以,马立克氏病的防控以后还会遇到更多挑战。抗病毒方面,中药有不易出现耐药性、组分多、途径多、靶点也多的独特优势。据药理学研究报道,具有抗微生物、抗病毒、保肝、利胆和镇静等作用的黄芩苷,具有很好的临床应用价值。为了深入探讨黄芩苷的生物学活性,本研究拟探讨黄芩苷的提取工艺,建立黄芩苷的质量标准;以鸡马立克氏病为模型,进行黄芩苷体外、体内抗马立克氏病病毒试验,系统研究黄芩苷的抗病毒活性,并进一步阐明其作用机理,为临床获取能有效控制病毒所致肿瘤性疾病的黄酮类化合物提供依据,试验结果如下:1、黄芩苷含量测定方法建立:采用紫外分光光度法,以黄芩苷的标准品为原料,278nm处测定其吸光度,建立吸光度和浓度的标准曲线。黄芩苷标准曲线为A=0.0625C+0.005,R~2=0.9995。精密性、稳定性、重复性、加样回收均符合要求。2、正交设计法优选黄芩苷的提取工艺:采用热水煎煮提取法,通过三水平三因素的正交试验优化其提取因素,方差分析结果显示,提取次数为2次,料液比为1:25,煎煮时间为50 min时,黄芩苷的提取率最高,达到20.36%。3、黄芩苷对鸡胚成纤维细胞(CEF)最大安全浓度的测定:黄芩苷在中、低浓度能够促进CEF细胞的贴壁和生长且对CEF细胞的作用都是具有时效性的,应用了细胞病变的观察法和MTT法,测得了黄芩苷对CEF最大的安全浓度为312.5μg/mL。4、MDV细胞半数感染量(TCID_(50))测定:MDV的半数感染量TCID_(50)为10~(-4.5)/0.1 mL。5、黄芩苷体外抗MDV作用结果:增值抑制、感染阻断和直接杀灭三种不同给药途径下,黄芩苷浓度在78.13~312.5μg/mL范围时都具有较好的抗病毒感染作用,并且感染阻断途径下黄芩苷浓度为156.25~312.5μg/mL时能够促进细胞的生长、直接灭活途径下,黄芩苷在浓度312.5μg/mL的时候能够促进细胞的生长。6、黄芩苷体内抗MDV作用结果:(1)通过利巴韦林组,黄芩苷高、中、低剂量组,病毒感染对照组的统计,正常对照组的死亡日龄数、肿瘤发生率、试验前后鸡体重变化情况等,明显得出黄芩苷高、中剂量组对病毒延缓发生优于阳性对照组和黄芩苷的低剂量组;(2)通过观察各组器官组织切片,也能直观的说明黄芩苷组能够降低MDV感染的病变程度。综上所述,正交法优化的黄芩苷提取工艺方法更加合理,得到了较高提取率的黄芩苷,用于临床上的延缓马立克氏病病毒的感染,并且通过将马立克氏病作为研究动物肿瘤病的模型进行研究,中药黄芩苷对动物肿瘤病的治疗是有开发前景的。
[Abstract]:Viral tumor disease is a natural common diseases, diseases of livestock and poultry. Serious health hazards of neoplastic diseases has decreased, the production performance of livestock and poultry and even death, causing serious losses to the farmers economy. Poultry T cells lymphoma is mainly caused by Marek's disease, mainly the poultry virus extracted samples for diagnosis, judge. The virus is constantly variation and development of Marek's disease, the toxicity also increases, injection of vaccine effect is limited, therefore, the prevention and control of Marek's disease will encounter more challenges. Traditional Chinese medicine has antiviral, not prone to drug resistance multi component, multi way, and also the unique advantages of targets. According to pharmacological research reports, antimicrobial, antiviral, liver, gallbladder and sedative effects of baicalin, has good clinical should Use value. In order to study the biological activity of baicalin, this study intends to explore the extraction process of baicalin, to establish a quality standard of baicalin; in Marek's disease model of Baicalin in vitro and in vivo anti Marek's disease virus test system of baicalin antiviral activity, and to further elucidate its mechanism and provide the basis of flavonoids obtained can effectively control the virus induced tumor disease for clinical and experimental results are as follows: 1, establish the method to determine the content of baicalin: by UV spectrophotometry, using standard baicalin as raw material, 278nm absorbance, establishing a standard curve of absorbance and concentration of glycosides. The standard curve of Baicalin was A=0.0625C+0.005, R~2=0.9995. precision, stability, repeatability and recovery are in line with the requirements of.2, the extraction process of baicalin by orthogonal design: the use of hot water boiling extraction The method, by optimizing the extraction factors of orthogonal test of three factors and three levels, the variance analysis results showed that the extraction times is 2, the ratio of liquid to 1:25, decoction time was 50 min, the extraction rate of baicalin is the highest, reaching 20.36%.3, baicalin in chick embryo fibroblast (CEF) determination of the maximum safe concentration the baicalin in low concentration can promote CEF cell adhesion and growth of CEF cells and the role of timeliness of the application, the cellular pathological observation method and MTT method, obtained CEF safety of Baicalin on the maximum concentration of 312.5 g/ mL.4, MDV cells (MID50 TCID_ (50): MDV) by MID50 TCID_ (50) 10~ (-4.5) /0.1 mL.5, the anti MDV effect of Baicalin in vitro proliferation, infection blocking and killing three different routes of administration, concentrations of baicalin has better antiviral in the range of g/mL 78.13~ 312.5 Effect of infection, and the infection blocking concentrations of baicalin was 156.25~312.5 ~ g/mL pathway can promote cell growth, direct inactivation pathway, baicalin can promote the growth of.6 cells when the concentration of 312.5 g/mL, the anti MDV effect of Baicalin in vivo results: (1) by Leigh Bhave Lin group, baicalin, in the low dose group, control group, statistical virus infections, death age number of normal control group, the incidence of tumors before and after testing chicken weight changes, baicalin was obtained in high dose group delay than the positive control group and baicalin low dose group of virus; (2) through the observation each organ section, can also be that baicalin group can reduce the severity of MDV infection. In conclusion, the orthogonal method to optimize the extraction process of baicalin was more reasonable, high extraction rate of baicalin, for clinical. In order to delay the infection of Marek's disease virus and study Marek's disease as a model of animal tumor, baicalin has potential for the treatment of animal tumor.

【学位授予单位】：聊城大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S853.74

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