【作者】 林彬彬；

【导师】 陈强； 黄志坚；

【作者基本信息】 福建农林大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 本研究课题依照疏毛白绒草民间记载和地方药物志中相关药理药效的记载和描述,对其水提物、95%乙醇提取物、乙醇提取物的石油醚、氯仿、乙酸乙酯和正丁醇萃取物及水层浓缩物的抗菌、抗炎、止咳、化痰、免疫和抗鸡新城疫病毒等药效进行研究,并通过各个萃取物之间作用的比较,确定其药效的有效萃取部位,为将来疏毛白绒草的有效成分的进一步分离、纯化和开发利用提供理论基础。1疏毛白绒草提取物的制备和化学成分预试验目的:制备试验所需的提取物、萃取物,初步了解其所含有的活性物质。方法:利用常规方法制备水提物和95%乙醇提取物,乙醇提取物再分别用石油醚、氯仿、乙酸乙酯、正丁醇依次萃取,并用化学方法进行有效成分的鉴别。结果:1000g干草获得水提物冷冻干燥粉末60.6g,得率为6.06%;2000g疏毛白绒草干草获得95%乙醇提取物浸膏141.27g,得率为7.06%。分别得到石油醚萃取物、氯仿萃取物、乙酸乙酯萃取物、正丁醇萃取物和水层浓缩物浸膏30g、17.1g、20g、34.8g、23.2g,得率分别为3.03%、1.73%、2.02%、3.51%、2.34%。其提取物含有甾体或三萜类、生物碱、黄酮类、香豆素与萜类内酯化合物、强心苷类化合物、酚类化合物、有机酸类、氨基酸、肽和蛋白质、糖、多糖和苷类等多种物质。结论:疏毛白绒草含有多种活性物质。2疏毛白绒草提取物抗菌活性研究目的:研究疏毛白绒草提取物和萃取物对金黄色葡萄球菌、大肠杆菌、鳗弧菌和枯草杆菌4种细菌的抗菌活性、抗菌活性有效萃取部位和最小抗菌浓度(MIC)。方法:应用打孔法研究疏毛白绒草水提物、95%乙醇提取物和各个萃取物对4种细菌抗菌作用;利用试管稀释法研究提取物和萃取物对4种细菌的MIC值。结果:其水提物、95%乙醇提取物在200mg/ml剂量浓度下,均对金黄色葡萄球菌有极强(P<0.01)的抑菌作用,其中水层浓缩物抑制金黄色葡萄球菌的作用极显著(P<0.01),乙酸乙酯萃取物、氯仿萃取物、正丁醇萃取物也有较为明显的抑菌作用。200mg/ml水提物除了对鳗弧菌有一定的抑制作用外,对其他2种革兰氏阴性菌没有显著的抑制作用,但95%乙醇提取物却对3种革兰氏阴性菌均表现出一定的抑制作用。通过疏毛白绒草提取物和萃取物对4种细菌的MIC值,可以发现其对金黄色葡萄球菌的最小抑菌浓度远远小于对大肠杆菌等3种革兰氏阴性菌的最小抑菌浓度。结论:疏毛白绒草有抑菌作用,其95%乙醇提取物的抗菌作用较水提物的抗菌效果突出,且初步推断其提取物对革兰氏阳性菌的抑制作用较革兰氏阴性菌的抑制作用显著;疏毛白绒草抑制金黄色葡萄球菌的有效成分主要分布在水层,部分分布在氯仿、乙酸乙酯和正丁醇萃取部位,而抗大肠杆菌等革兰氏阴性菌的有效成分主要集中于乙酸乙酯萃取部位。3疏毛白绒草水提物抗菌机制的研究目的:研究疏毛白绒草水提物对金黄色葡萄球菌和大肠杆菌的抗菌机制。方法:采用透射电镜观察300mg/ml水提物作用2h、6h后金黄色葡萄球菌和大肠杆菌的形态变化。结果:水提物作用后的2种细菌在透射电镜下出现了菌体变形,扭曲,细胞壁部分缺损,内容物外泄,部分菌体内出现空腔,菌体断裂,出现残片等异常的形态特征。结论:疏毛白绒草水提物能够使金黄色葡萄球菌和大肠杆菌的形态结构发生异常变化。4疏毛白绒草抗炎试验目的:研究疏毛白绒草抗炎作用及有效成分萃取部位。方法:采用疏毛白绒草对二甲苯导致的小鼠耳肿胀的抑制试验来测定其抗炎活性。结果:700mg/kg体重的95%乙醇提取物、300mg/kg体重的乙酸乙酯萃取物有极显著(P<0.01)的抑制小白鼠耳肿胀作用,600mg/kg的体重水提物、300mg/kg的体重正丁醇萃取物、300mg/kg体重的水层浓缩物有显著(P<0.05)的抑制小白鼠耳肿胀作用。结论:疏毛白绒草有较强的抗炎效果,其抗炎有效成分主要分布在乙酸乙酯萃取部位,部分分布于正丁醇和水层萃取部位。5疏毛白绒草免疫试验目的:研究疏毛白绒草提取物对机体免疫作用和免疫有效成分的萃取部位。方法:通过疏毛白绒草提取物和萃取物对小鼠免疫器官脾和胸腺指数的影响测定其免疫作用。结果:在一定浓度下,其95%乙醇提取物和乙酸乙酯萃取物能够显著(P<0.05)提高小鼠脾指数,水提物、95%乙醇提取物和石油醚萃取物能够显著(P<0.05)提高小鼠胸腺指数。结论:疏毛白绒草能够增强机体免疫能力,有效成分主要分布于乙酸乙酯和石油醚萃取部位。6疏毛白绒草化痰、止咳试验目的:研究疏毛白绒草提取物化痰、止咳的作用,及有效成分的萃取部位。方法:采用疏毛白绒草提取物和萃取物对浓氨水导致的小鼠咳嗽的止咳试验和气管段酚红法测定化痰作用。结果:在一定浓度下,其水提物、95%乙醇提取物、乙酸乙酯萃取物和水层浓缩物有明显的止咳作用。其止咳率(R)分别为134%、132%、135%、134%。其水层浓缩物和正丁醇萃取物有极显著的化痰作用(P<0.01),水提物和95%乙醇提取物、乙酸乙酯萃取物有显著的化痰作用(P<0.05)。结论:疏毛白绒草有较强的止咳、化痰作用;其止咳有效成分主要分布于乙酸乙酯和水层萃取部位;化痰有效成分主要分布于正丁醇和水层萃取部位,部分分布于乙酸乙酯萃取部位。7疏毛白绒草对鸡新城疫病毒抑制试验目的:研究疏毛白绒草对鸡胚感染鸡新城疫病毒(NDV)的抑制作用和有效成分的萃取部位。方法:采用加药后2h接种病毒(A组)、接种病毒2h后接药(B组)、药和病毒在体外孵育2h后同时接种(C组)三种途径研究疏毛白绒草提取物和萃取物对新城疫病毒的抑制作用和有效成分的萃取部位,并通过三种途径抗NDV效果的比较研究最优的抗NDV方式。结果:A组中,250mg/ml 95%乙醇提取物对鸡新城疫病毒有显著(P<0.05)的抑制作用;B组中,250mg/ml 95%乙醇提取物和150mg/ml乙酸乙酯萃取物对鸡新城疫病毒有极显著(P<0.01)的抑制作用,250mg/ml水提物和150mg/ml氯仿萃取物也有显著的抑制作用(P<0.05);C组中,水提物、95%乙醇提取物、乙酸乙酯萃取物对鸡新城疫病毒的抑制作用极显著(P<0.01),氯仿萃取物和水层浓缩物的抑制作用显著(P<0.05)。通过统计学比较可知,3组方式抗病毒效果没有显著区别。结论:疏毛白绒草能够抑制鸡胚感染鸡新城疫病毒,其有效成分分布于乙酸乙酯、氯仿和水层萃取部位。更多还原

【Abstract】 According to relative pharmacology and efficacy record in local Flora about Leucas mollissima Wall.Var.Chinensis Benth, we investigate the anti-bacterial action, anti-inflammatory action, immunization, the effects of removing the phlegm and relieving a cough, and anti-Newcastle disease virus action of its water extract, 95 % alcohol extract, the extracts of alcohol-petroleum ether, chloroform, ethyl acetate, n-butanol and water layer condensate. Then, by comparing the effects of each extract, we can determine the valid target of these drug actions. Above all, our work is a fundamental research for the further purification, isolation and provides theoretical principle to exploitation of Leucas mollissima Wall.Var.Chinensis Benth’s.1 Preparation of Leucas mollissima Wall.Var.Chinensis Benth’s extracts and the preliminary examination of its chemical compositionObjective: To prepare extracts for our research and get the initial message of the active ingredients. Methodology: A conventional method is used to prepare its water extract and 95 % alcohol extract. We choose petroleum ether, chloroform, Ethyl acetate, and N-butanol as the extracting solvents to separate and purify the chemical constituents in correct order. The system of preliminary examination is applied to study its chemical composition. Results: We get 60.6g frozen drying powder of water extract from 1000g dry herb, the yield is 6.06%; and 141.27g 95% alcohol extract from 2000g dry herb, the yield is 7.06 %; and 30g petroleum ether extract, 17.1g chloroform extract, 20g Ethyl acetate extract, 34.8g N-butanol extract, 23.2g water layer concentrate, their yield is 3.03%, 1.73%, 2.02%, 3.51%, and 2.34%, respectively. The examination shows that this herb contains a variety of materials: steroids or triterpenes, alkaloid, flavonoid, iridoid glycoside, terpenoid lactone, cardiac glycoside, phenols, organic acid, amino acid, peptides, proteins, polysaccharides and amylose, etc. Conclusion: The herb contains a number of active ingredients.2 Researches of antibacterial actionsObjective: To study antibacterial actions of this herb on Staphylococcus aureus, Escherichia coli, Vibrio anguillarum and Bacillus subtilis, get the minimal inhibitory concentration, and determine where the active ingredients are. Methodology: Our study on the herb’s antibacterial effects is based on punch method and the Minimal Inhibitory Concentration (hereafter: MIC)of all extracts which are determined by tube dilution method. Results: In the concentration of 200 mg/ml, the extracts of water and 95% alcohol have a significant antibacterial effect on Staphylococcus aureu(sP<0.01), while the water layer condensate has an extremely effect (p<0.01). The Ethyl acetate extract, chloroform extract, N-butanol extract also have the anti-Staphylococcus aureus effect. The water extract (200 mg/ml) only exerts some effects on Vibrio anguillarum but no effect on other 2 sorts of Gram-negative bacteria. However, the 95 % alcohol extract is proved to be functioned on three kinds of Gram-negative bacteria. Through the MIC of extracts on the 4 kinds of bacteria, it’s shown that the MIC of extracts on Staphylococcus aureus is much lower than the MIC on the 3 sorts of Gram-negative bacteria, e.g. E. coli. Conclusion: This herb has antibacterial action. The 95% alcohol extract has a little better antibacterial effect than water extract. Our initial inference is that the extracts have more effect on Gram-positive bacteria than on Gram-negative bacteria. The active ingredients of antibacterial upon Staphylococcus aureus are mainly in the water layer condensate, and partly in the extract of chloroform, Ethyl acetate and N-butanol. But the active ingredients of antibacterial on Gram-negative bacteria are in the extract of ethyl acetate.3 Study on antibacterial mechanism through Transmission Electron Microscopy (hearafter: TEM)Objective: To study the herb’s antibacterial mechanism on Staphylococcus aureus and Escherichia coli. Methodology: Observe the changes of the 2 kinds of bacteria’s structure and characteristics 2h or 6h by TEM dealing with water extract. Results: After initiation of water extract in 200mg/ml concentration, the structure and characteristics of the 2 sorts of bacteria under TEM show significant differences with the normal one. Some cell walls were defected, there was a leakage of contents, and some showed a cavity in the bacteria, etc. Conclusion: This herb has influence on the structure and characteristics of Staphylococcus aureus and Escherichia coli.4 Study on anti-inflammatory actionObjective: To study the herb’s anti-inflammatory action and determine where the active ingredients are. Methodology: The experiment is implemented by using anti-swelling of mouse’s ear caused by Xylene with the extracts. Results: 700mg/kg (body weight) of 95% alcohol extract , 300mg/kg (body weight) of Ethyl acetate extracts show significant effects on the swelling of mouse’s ear (p<0.01). 300mg/kg (body weight) of N-butanol extract, 600mg/kg (body weight) water extract and 300mg/kg (body weight) of Water layer condensate have great effects on the swelling of mouse’s ear (p<0.05). Conclusion: This herb has significant anti-inflammatory action. The active ingredients of anti-inflammatory action are mainly in ethyl acetate, and partly in the extracts of n-butanol and water layer.5 Study on immunization actionObjective: To study the herb’s immunization action and determine where the active ingredients are. Methodology: Observe the influence on the index of mouse’s immune organs such as spleen and thymus to determine its immune effect. Results: Under the given concentration, the 95% alcohol extract and Ethyl acetate extracts increase mouse’s spleen index (p<0.05). At the same time, the water extract, 95% alcohol extract and petroleum extract increase mouse’s thymus index (p<0.05). Conclusion: This herb can improve the body immunity. The active ingredients of immunization action are mainly in the extracts of Ethyl acetate and petroleum.6 Study on the effects of removing the phlegm and relieving a coughObjective: To study the herb’s effects of removing the phlegm and relieving a cough and determine where the active ingredients are. Methodology: The experiment of effect on mouse’s cough caused by strong ammonia is applied. Through the method of phenol red in trachea, the effect of anti-sputum is observed. Results: Under the given concentration, its extracts of water, 95%alcohol, ethyl acetate and water layer condensate show significant anti-cough effects. The percentage of relieving a cough (R) is 134 %, 132 %, 135 %, and 134 %, respectively. The water layer condensate and N-butanol extract have significant anti-sputum effect (p<0.01), Water extract and 95 % alcohol extract, Ethyl acetate extract show significant anti-sputum effect (p<0.05). The N-butanol extract and water layer condensate also have some anti-sputum effects. Conclusion: The herb shows remarkable effects of removing the phlegm and relieving a cough. The active ingredients of removing the phlegm effect are mainly in Ethyl acetate extract and water layer condensate. But the active ingredients of relieving a cough effect are mainly in N-butanol extract and water layer condensate, and partly in Ethyl acetate extract7 Study on anti-Newcastle disease virus actionObjective: To study the herb’s anti-Newcastle disease virus action and determine where the active ingredients are. Methodology: The anti-Newcastle disease virus action is studied in chick embryo through three ways which are A-way, B-way and C-way. Results: In A-way, only 95% alcohol extract (250mg/ml) has significant (P<0.05) anti-Newcastle disease virus action; In B-way, extracts of 95 % alcohol (250mg/ml) and Ethyl acetate (150mg/ml) show remarkable effect against NDV in embryo (P<0.01), and the water extract (250mg/ml) and chloroform extract(150mg/ml) also play obvious effects(P<0.05); In C-way, the extracts of water, 95 % alcohol and Ethyl acetate show very strong inhibition for NDV in vitro(P<0.01), while the chloroform extract and water layer condensate have some effects(P<0.05). By comparing the 3 ways through statistics, the anti- Newcastle disease virus effects have not much difference. Conclusion: This herb can inhibit NDV in chick embryos. The active ingredients of anti-Newcastle disease virus action are mainly in the extracts of ethyl acetate, chloroform and water layer.更多还原