【作者】 张建超；

【导师】 孙志良；

【作者基本信息】 湖南农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 地锦草(Euphorbia humifusa)是一种常见中草药,生于原野、荒地、路旁、田间,分布于全国各地。众多学者对其化学成分、药理作用、临床应用做了研究,但对其黄酮类提取物的研究尚浅。本文以地锦草为研究对象,确立了地锦草总黄酮含量测定方法,建立了提取和分离纯化的工艺路线;并对地锦草黄酮抑菌作用、免疫药理作用进行了研究,分别从整体水平、细胞水平和分子水平上探讨其免疫作用机制。研究结果如下:1、运用正交试验优化超声破碎地锦草细胞的最佳条件,采用超声-加热回流-大孔树脂吸附的工艺流程提取分离纯化地锦草总黄酮,紫外分光光度法在波长510nm处测吸光度测地锦草总黄酮含量。试验结果表明:超声破碎的最佳条件为以料液比为1∶20,70%的乙醇为提取溶剂,60℃超声提取35min。影响超声提取效果大小的因素依次为提取时间＞溶剂浓度＞料液比＞提取温度。100g地锦草干燥全草提取纯化得到3.679g黄褐色粉末,经多种化学试剂鉴定,均成明显的黄酮反应。在3.679g地锦草黄酮粉末中,含纯黄酮3.131g,总得率为3.131%。2、采用纸片法测定地锦草总黄酮体外抗菌效果,并采用试管倍比稀释法测定其最低抑菌浓度(MIC)和最小杀菌浓度(MBC)。结果表明:地锦草总黄酮对巴氏杆菌,沙门氏菌的体外抑菌作用较强,10mg/ml的总黄酮提取液对巴氏杆菌,沙门氏菌的抑菌直径分别达到22.0±1.0(mm),26.0±2.0(mm),随着黄酮浓度的增加,其抑菌作用也增强。本实验测得地锦草黄酮对巴氏杆菌,沙门氏菌的MIC分别为1.25mg/ml,0.625mg/ml,沙门氏菌的MBC浓度为1.25mg/ml。地锦草总黄酮提取液对金黄色葡萄球菌,大肠杆菌和链球菌也有一定的抑制作用。3、采用常规免疫药理学试验技术将地锦草总黄酮的3个剂量组与对照组进行比较,结果显示:脾指数、碳廓清指数(K)、吞噬指数(a)、足趾增厚值均显著高于空白组,说明一定剂量的地锦草黄酮具有提高免疫作用的能力。4、采用RT-PCR测定小鼠脾组织中IL-2、IL-12、IFN-γ、TNF-αmRNA的表达水平,试验结果表明:①H组和M组IL-2 mRNA表达水平均升高,与C组相比,H组和M组极显著差异(P＜0.01);②与对照组相比,三个剂量的地锦草黄酮对IL-12 mRNA表达水平稍有提高,但差异不是很显著(P＞0.05);③TNF-γmRNA表达水平均有提高,特别是H剂量组(P＜0.01);④与对照组相比,各个剂量的地锦草黄酮对TNF-αmRNA表达水平均有升高,且差异显著(P＜0.01)。说明地锦草黄酮三个剂量组均能提高IL-2、IL-12、IFN-γ、TNF-αmRNA的表达水平,从而提高机体的免疫功能与抗病能力。综上所述,超声-加热回流-大孔树脂吸附提取分离纯化地锦草总黄酮的方法操作简单,重复性好,提取的总黄酮含量高,纯度好。研究表明,地锦草黄酮具有体外抑菌活性;在在一定剂量范围内能有效提高小鼠的免疫功能及IL-2、IL-12、IFN-γ和TNF-αmRNA表达。更多还原

【Abstract】 Euphorbia humifusa was a common Chinese herbs which was distributed in the wildness,wasteland,roadsides,fields of almost every province of China.Many scholars researched in the chemical composition,pharmacological effects,clinical application of Euphorbia humifusa,but less involved in flavonoid extraction.In this paper,the author conducted a study to establish the method for determination of Euphorbia humifusa total flavonoids,and established the process line of extraction, separation and purification;furthermore,did some research in bacteriostasis,immune pharmacological effect of Euphorbia humifusa flavonoids,respectively,from the overall level,cell level and molecular level to explore the role in the immune mechanism.The results were as follows:1.To optimize the ultrasonic conditions with orthogonal test,the researcher extratced, separated and purified the total flavonoids with the method of Ultrasonication-Heating reflux-Macroreticular resin adsorbing.The UV-spectrophotometry was adopted with the detection wavelength at 510nm to determinate the content of Euphorbia humifusa total flavonoids.The experimental results showed that:the optimal ultrasonic conditions was solid-liquid ratio as 1:20,ethanol concentration as 70%,the temperature as 60℃,time as 35min.The factors which impact on the efficiency of ultrasonic extraction were as follows by the size of the factors:extraction time＞solvent concentration＞solid-liquid ratio＞temperature.3.679g brown powder was purified from the 100g whole dry Euphorbia humifusa,identified by a variety of chemical reagents,which showed clearly flavone reaction.3.679g extraction powder contained 3.131 g pure flavonoids,which yeild rate was 3.131%.2.The researcher tested the bacteriostasis effect of Euphorbia humifusa total flavonoids with paper disk method and tested the minimum inhibitory concentration (MIC) and minimum bactericidal concentration(MBC) with double dilution method. The experimental results showed that:The antimicrobial effect of 10mg/ml total flavonoids in vitro against Pasteurella and Salmonella was strong,with the diameter of inhibition zone as 22.0±1.0(mm) and 26.0±2.0(mm),respectively.The total flavonoids MIC was as follows:0.625mg/ml to Pasteurella,1.25mg/ml to Salmonella and MBC was 1.25mg/ml to Salmonella.Euphorbia humifusa total flavonoids extraction also had bacteriostasis effect on Staphylococcus aureus,E.coli and Streptococcus suis to a certain degree.3.The researcher compared three dose-groups to the control group with the routine immunological pharmacology experimental method.The results showed:mice spleen index,carbon clearance index(K),phagocytic index(a),toes thickening value were significantly higher than the control group,indicating that a certain dose of Euphorbia humifusa flavonoids had the ability to enhance immune function.4.Using RT-PCR way to test the IL-2,IL-12,IFN-γand TNF-αmRNA expression level in mice spleen tissue,we could know that:①The IL-2 mRNA expression level increased in H group and M group,difference was significant compared with C group(P＜0.01);②The IL-12 mRNA expression level slightly increased in three dose-groups,however,had not obvious difference compared with C group(P＞0.05);③The IFN-γmRNA expression level increased in in three dose-groups,especially H-dose group(P＜0.01);④Compared with C group,The TNF-αmRNA expression level increased in three dose-groups,and significantly different(P＜0.01).It was indicated that Euphorbia humifusa flavonoids were able to enhance the IL-2,IL-12, IFN-γand TNF-αmRNA expression levels,thereby enhancing the immune function and disease resistance.From the experiment results,we could know that the Ultrasonication-Heating reflux- Macroreticular resin adsorbing method was simple,stable and the total flavonoids extraction was pure and fruitful.Euphorbia humifusa total flavonoids had the bacteriostasis effect in vitro and could improve the immune function of mice and could increased IL-2,IL-12,IFN-γand TNF-αmRNA expression level to certain dose degree.更多还原