蟛蜞菊活性成分分离及抑菌作用研究
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摘要
蟛蜞菊是一味资源丰富且在民间广泛应用的中草药,三裂蟛蜞菊是一种由于绿化而引进的南美植物。本文从抑菌的角度,研究比较了蟛蜞菊和三裂蟛蜞菊挥发油、水提物、95%乙醇提取物、石油醚、氯仿、乙酸乙酯、正丁醇萃取物对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、葡萄球菌、沙门氏菌、鳗弧菌、嗜水气单胞菌、小球菌和耶尔森氏菌9种细菌的抑制作用和抑菌机制,初步研究并探讨了蟛蜞菊和三裂蟛蜞菊作为抑菌草药的开发和应用前景。主要研究内容如下:
1蟛蜞菊和三裂蟛蜞菊各提取物的制备和化学成分的初步测定
目的:制备试验所需的各种提取物、萃取物,初步了解其所含有的活性物质。方法:利用常规方法制备挥发油、水提物和95%乙醇提取物,95%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,得萃取物,再用化学方法对其进行化学成分的初步测定。结果:三裂蟛蜞菊、蟛蜞菊水提物主要含有甾体或三萜类、挥发油和酯类、生物碱、香豆素与萜类内酯化合物、强心苷类化合物、酚类化合物、鞣质、氨基酸、肽和蛋白质、糖、多糖和苷类等多种物质;三裂蟛蜞菊、蟛蜞菊95%乙醇提取物主要含有甾体或三萜类、挥发油和酯类、生物碱、黄酮类、醌类、香豆素与萜类内酯化合物、强心苷类化合物、酚类化合物、鞣质、有机酸类、糖、多糖和苷类以及甾体皂苷类等多种物质。结论:蟛蜞菊和三裂蟛蜞菊各提取物中含有多种主要化学成分,二者同属不同种,在成分上仍存在一定的差异。
2蟛蜞菊和三裂蟛蜞菊挥发油成分测定
目的:测定分析蟛蜞菊和三裂蟛蜞菊挥发油的化学成分,并比较二者的不同点。方法:通过气相—质谱联用法对蟛蜞菊和三裂蟛蜞菊挥发油进行成分分离与扫描,再结合化学工作站检索Nist05版标准质谱图库初步确认挥发油中的化学成分。结果:蟛蜞菊挥发油中含有52种化合物,三裂蟛蜞菊挥发油中含有48种,其中以烯萜类化合物为主,其次为脂肪族化合物和其他化合物。结论:蟛蜞菊和三裂蟛蜞菊挥发油中只有17种共同成分,仍有三分之二的成分不相同。
3蟛蜞菊和三裂蟛蜞菊提取物和萃取物抑菌活性研究
目的:研究蟛蜞菊和三裂蟛蜞菊各提取物和萃取物对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、葡萄球菌、沙门氏菌、鳗弧菌、嗜水气单胞菌、小球菌和耶尔森氏菌9种细菌的抑制作用和最小抗菌浓度(MIC)以及最小杀菌浓度(MBC)。方法:采用滤纸片扩散法研究蟛蜞菊和三裂蟛蜞菊挥发油、水提物、95%乙醇提取物和各个萃取物对9种细菌的抑制作用;利用试管稀释法研究提取物和萃取物对9种细菌的MIC和MBC值。结果:采用不同方法和不同极性溶剂分离得到的蟛蜞菊和三裂蟛蜞菊各提取物中,挥发油、石油醚和乙酸乙酯萃取物的抑菌作用较强,95%乙醇提取物、氯仿和正丁醇萃取物的次之,水提物的抑菌作用最弱,对沙门氏菌、嗜水气单胞菌和葡萄球菌的抑制作用甚至不明显。结论:除了水提物外,蟛蜞菊和三裂蟛蜞菊挥发油、95%乙醇提取物、石油醚萃取物、氯仿萃取物、乙酸乙酯萃取物、正丁醇萃取物都具有广谱的抑菌活性,且两种植物同种提取物的抑菌效果相当,差异不显著(P>0.05)。
4蟛蜞菊和三裂蟛蜞菊挥发油和醇提物抑菌机制的研究
目的:研究蟛蜞菊和三裂蟛蜞菊挥发油和95%乙醇提取物对大肠杆菌和金黄色葡萄球菌的抑菌机制。方法:通用透射电镜观察50mg/mL蟛蜞菊和三裂蟛蜞菊挥发油和200mg/mL95%乙醇提取物作用2h、6h后的大肠杆菌和金黄色葡萄球菌的形态变化。结果:两种药物用后的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,部分菌体内呈空腔状,菌体断裂成残片等异常的形态特征。结论:蟛蜞菊和三裂蟛蜞菊挥发油和95%乙醇提取物主要通过使大肠杆菌和金黄色葡萄球菌的形态结构发生异常变化来抑制细菌。
Wedelia chinensis (Osbeck.) Merr. is a Chinese herbal medicine which is an abundant resource in China and is widely used in folk.Wedelia trilobata(L.) Hitchc. is a South Americ- an plant which was introduced into China as a sprigging operation plant.The antibacterial acti- on of the water extracts,95% alcohol extracts,the extracts of alcoholpetroleum ether,chorofo- rm,ethyl acetate,n-butanol from Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. on Escherichia coli,Staphyloccocus aureus Rosenbach,Staphylococcus,Bacillussubti- lis,Yersinia enterocolitica,V.anguillarum,Aeromonas hydrophila,Salmonella,Micrococci was studied.Exploitations and prospects of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilo- bata(L.) Hitchc. as antibacterial agent were discussed.The main contents were as follows:
1 Preparation of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,s extracts and the preliminary examination of their chemical composition
Objective: To prepare extracts for our research and get the initial message of the active ingre- dients.Methodology:the conventional method was used to prepare their water extracts and 95% alcohol extracts.Petroleum ether,chloroform,Ethyl acetate, and N-butanol were choosed as the extracting solvents to separate and purify the chemical constituents in correct order.The system of preliminary examination was applied to study their chemical compositions.Results: The examinations showed the water extracts of Wedelia chinensis (Osbeck.) Merr. and Wedel- ia trilobata(L.) Hitchc. contain a variety of ingredients:steroids or triterpenes,volatile oil and esters,alkaloid,iridoid glycoside,terpenoid lactone,cardiac glycoside,phenols,tannin,amino ac- id,peptides,proteins, polysaccharides and amylose,etc; the 95% alcohol extracts contain a var- iety of ingredients:steroids or triterpenes,volatile oil and esters,alkaloid,flavonoid,quinine,iri- doid glycoside,terpenoid lactone,cardiac glycoside,phenols,tannin,organic acid, polysacchari- des and amylose,steroidal saponin etc.Conclusion:Wedelia chinensis (Osbeck.) Merr. and W- edelia trilobata(L.) Hitchc. contain many chief chemical compositions, although they are sibling, they are not homogeneity,so there are some differences between them.
2 The the components identification of the volatile oils from Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.
Objective: To define the chemical compositions of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,s volatile oils and compare with the differences between them. Methodology: The method of GS-MS was applied to isolate and analyze the components of extraeted Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,s volatile oils. Results: 52 components were detected from Wedelia chinensis (Osbeck.) Merr. ,s volatile oi- ls,48 components were detected from Wedelia trilobata(L.) Hitchc.,s volatile oils. Among th- em,the main ingredients are terpenes,aliphatic compounds and other compounds are less.Con- clusion:There are only 17 components in common between the volatile oils of Wedelia chine- nsis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,still have two-thirds of the ingredients are different.
3 Researches of antibacterial activity
Objective: To study the antibacterial actions and MIC,MBC of extracts from Wedelia chinen- sis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. on Escherichia coli,Staphyloccocus aur- eus Rosenbach,Staphylococcus,Bacillus subtilis,Yersinia enterocolitica,V.anguillarum, Aerom- onas hydrophila,Salmonella,Micrococci.Methodology:the filter paper diffusion method was applied to study the antibacterial actions of extracts from Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. on all bacterias,and the dilution method was applied to stud- y the MIC and MBC of them. Results:The volatile oils,petroleum ether extracts and ethyl ac- etate extracts of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. had rel- atively strong antimicrobial effect,the effect of the 95%ethanol extracts,chloroform extracts a- nd butanol extracts was weaked.The inhibitory effect of water extracts was weakest relatively, and the inhibitory effect on Staphyloccocus aureus Rosenbach, Aeromonas hydrophila and S- almonella wasn,t obvious.Conclusion: In addition to the water extract,the volatile oils,95% ethanol extracts,petroleum ether extracts,chloroform extracts, ethyl acetate extracts,butanol extracts of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. had a broad spectrum of antibacterial activity.The difference of antimicrobial effect between Wedelia chine- nsis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. wasn,t significant (P>0.05).
4 Study on antibacterial mechanism through Transmission Electron Microscopy (hearafter: TEM)
Objective:To study the Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,s antibacterial mechanism on Escherichia coli and Staphyloccocus aureus Rosenbach.Method- ology:TEM was applied to observe the changes of the two kinds of bacteria’s structure and characteristics after 2h or 6h by dealing with the volatile oils and 95% ethanol extracts of We- delia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc..Results:After initiation of the volatile oils in 50mg/mL and 95% ethanol extracts in 200mg/mL of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,the structures and characteristics of the two sorts of bacteria under TEM showed significant differences with the normal one.Some cell w- alls were defected,there was a leakage of contents, and some showed a cavity in the bacteria, etc.Conclusion:The volatile oils and 95% ethanol extracts of Wedelia chinensis (Osbeck.) M- err. and Wedelia trilobata(L.) Hitchc. had great influence on the structures and characterristi- cs of Escherichia coli and Staphyloccocus aureus Rosenbach.
1蟛蜞菊和三裂蟛蜞菊各提取物的制备和化学成分的初步测定
目的:制备试验所需的各种提取物、萃取物,初步了解其所含有的活性物质。方法:利用常规方法制备挥发油、水提物和95%乙醇提取物,95%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,得萃取物,再用化学方法对其进行化学成分的初步测定。结果:三裂蟛蜞菊、蟛蜞菊水提物主要含有甾体或三萜类、挥发油和酯类、生物碱、香豆素与萜类内酯化合物、强心苷类化合物、酚类化合物、鞣质、氨基酸、肽和蛋白质、糖、多糖和苷类等多种物质;三裂蟛蜞菊、蟛蜞菊95%乙醇提取物主要含有甾体或三萜类、挥发油和酯类、生物碱、黄酮类、醌类、香豆素与萜类内酯化合物、强心苷类化合物、酚类化合物、鞣质、有机酸类、糖、多糖和苷类以及甾体皂苷类等多种物质。结论:蟛蜞菊和三裂蟛蜞菊各提取物中含有多种主要化学成分,二者同属不同种,在成分上仍存在一定的差异。
2蟛蜞菊和三裂蟛蜞菊挥发油成分测定
目的:测定分析蟛蜞菊和三裂蟛蜞菊挥发油的化学成分,并比较二者的不同点。方法:通过气相—质谱联用法对蟛蜞菊和三裂蟛蜞菊挥发油进行成分分离与扫描,再结合化学工作站检索Nist05版标准质谱图库初步确认挥发油中的化学成分。结果:蟛蜞菊挥发油中含有52种化合物,三裂蟛蜞菊挥发油中含有48种,其中以烯萜类化合物为主,其次为脂肪族化合物和其他化合物。结论:蟛蜞菊和三裂蟛蜞菊挥发油中只有17种共同成分,仍有三分之二的成分不相同。
3蟛蜞菊和三裂蟛蜞菊提取物和萃取物抑菌活性研究
目的:研究蟛蜞菊和三裂蟛蜞菊各提取物和萃取物对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、葡萄球菌、沙门氏菌、鳗弧菌、嗜水气单胞菌、小球菌和耶尔森氏菌9种细菌的抑制作用和最小抗菌浓度(MIC)以及最小杀菌浓度(MBC)。方法:采用滤纸片扩散法研究蟛蜞菊和三裂蟛蜞菊挥发油、水提物、95%乙醇提取物和各个萃取物对9种细菌的抑制作用;利用试管稀释法研究提取物和萃取物对9种细菌的MIC和MBC值。结果:采用不同方法和不同极性溶剂分离得到的蟛蜞菊和三裂蟛蜞菊各提取物中,挥发油、石油醚和乙酸乙酯萃取物的抑菌作用较强,95%乙醇提取物、氯仿和正丁醇萃取物的次之,水提物的抑菌作用最弱,对沙门氏菌、嗜水气单胞菌和葡萄球菌的抑制作用甚至不明显。结论:除了水提物外,蟛蜞菊和三裂蟛蜞菊挥发油、95%乙醇提取物、石油醚萃取物、氯仿萃取物、乙酸乙酯萃取物、正丁醇萃取物都具有广谱的抑菌活性,且两种植物同种提取物的抑菌效果相当,差异不显著(P>0.05)。
4蟛蜞菊和三裂蟛蜞菊挥发油和醇提物抑菌机制的研究
目的:研究蟛蜞菊和三裂蟛蜞菊挥发油和95%乙醇提取物对大肠杆菌和金黄色葡萄球菌的抑菌机制。方法:通用透射电镜观察50mg/mL蟛蜞菊和三裂蟛蜞菊挥发油和200mg/mL95%乙醇提取物作用2h、6h后的大肠杆菌和金黄色葡萄球菌的形态变化。结果:两种药物用后的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,部分菌体内呈空腔状,菌体断裂成残片等异常的形态特征。结论:蟛蜞菊和三裂蟛蜞菊挥发油和95%乙醇提取物主要通过使大肠杆菌和金黄色葡萄球菌的形态结构发生异常变化来抑制细菌。
Wedelia chinensis (Osbeck.) Merr. is a Chinese herbal medicine which is an abundant resource in China and is widely used in folk.Wedelia trilobata(L.) Hitchc. is a South Americ- an plant which was introduced into China as a sprigging operation plant.The antibacterial acti- on of the water extracts,95% alcohol extracts,the extracts of alcoholpetroleum ether,chorofo- rm,ethyl acetate,n-butanol from Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. on Escherichia coli,Staphyloccocus aureus Rosenbach,Staphylococcus,Bacillussubti- lis,Yersinia enterocolitica,V.anguillarum,Aeromonas hydrophila,Salmonella,Micrococci was studied.Exploitations and prospects of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilo- bata(L.) Hitchc. as antibacterial agent were discussed.The main contents were as follows:
1 Preparation of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,s extracts and the preliminary examination of their chemical composition
Objective: To prepare extracts for our research and get the initial message of the active ingre- dients.Methodology:the conventional method was used to prepare their water extracts and 95% alcohol extracts.Petroleum ether,chloroform,Ethyl acetate, and N-butanol were choosed as the extracting solvents to separate and purify the chemical constituents in correct order.The system of preliminary examination was applied to study their chemical compositions.Results: The examinations showed the water extracts of Wedelia chinensis (Osbeck.) Merr. and Wedel- ia trilobata(L.) Hitchc. contain a variety of ingredients:steroids or triterpenes,volatile oil and esters,alkaloid,iridoid glycoside,terpenoid lactone,cardiac glycoside,phenols,tannin,amino ac- id,peptides,proteins, polysaccharides and amylose,etc; the 95% alcohol extracts contain a var- iety of ingredients:steroids or triterpenes,volatile oil and esters,alkaloid,flavonoid,quinine,iri- doid glycoside,terpenoid lactone,cardiac glycoside,phenols,tannin,organic acid, polysacchari- des and amylose,steroidal saponin etc.Conclusion:Wedelia chinensis (Osbeck.) Merr. and W- edelia trilobata(L.) Hitchc. contain many chief chemical compositions, although they are sibling, they are not homogeneity,so there are some differences between them.
2 The the components identification of the volatile oils from Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.
Objective: To define the chemical compositions of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,s volatile oils and compare with the differences between them. Methodology: The method of GS-MS was applied to isolate and analyze the components of extraeted Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,s volatile oils. Results: 52 components were detected from Wedelia chinensis (Osbeck.) Merr. ,s volatile oi- ls,48 components were detected from Wedelia trilobata(L.) Hitchc.,s volatile oils. Among th- em,the main ingredients are terpenes,aliphatic compounds and other compounds are less.Con- clusion:There are only 17 components in common between the volatile oils of Wedelia chine- nsis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,still have two-thirds of the ingredients are different.
3 Researches of antibacterial activity
Objective: To study the antibacterial actions and MIC,MBC of extracts from Wedelia chinen- sis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. on Escherichia coli,Staphyloccocus aur- eus Rosenbach,Staphylococcus,Bacillus subtilis,Yersinia enterocolitica,V.anguillarum, Aerom- onas hydrophila,Salmonella,Micrococci.Methodology:the filter paper diffusion method was applied to study the antibacterial actions of extracts from Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. on all bacterias,and the dilution method was applied to stud- y the MIC and MBC of them. Results:The volatile oils,petroleum ether extracts and ethyl ac- etate extracts of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. had rel- atively strong antimicrobial effect,the effect of the 95%ethanol extracts,chloroform extracts a- nd butanol extracts was weaked.The inhibitory effect of water extracts was weakest relatively, and the inhibitory effect on Staphyloccocus aureus Rosenbach, Aeromonas hydrophila and S- almonella wasn,t obvious.Conclusion: In addition to the water extract,the volatile oils,95% ethanol extracts,petroleum ether extracts,chloroform extracts, ethyl acetate extracts,butanol extracts of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. had a broad spectrum of antibacterial activity.The difference of antimicrobial effect between Wedelia chine- nsis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc. wasn,t significant (P>0.05).
4 Study on antibacterial mechanism through Transmission Electron Microscopy (hearafter: TEM)
Objective:To study the Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,s antibacterial mechanism on Escherichia coli and Staphyloccocus aureus Rosenbach.Method- ology:TEM was applied to observe the changes of the two kinds of bacteria’s structure and characteristics after 2h or 6h by dealing with the volatile oils and 95% ethanol extracts of We- delia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc..Results:After initiation of the volatile oils in 50mg/mL and 95% ethanol extracts in 200mg/mL of Wedelia chinensis (Osbeck.) Merr. and Wedelia trilobata(L.) Hitchc.,the structures and characteristics of the two sorts of bacteria under TEM showed significant differences with the normal one.Some cell w- alls were defected,there was a leakage of contents, and some showed a cavity in the bacteria, etc.Conclusion:The volatile oils and 95% ethanol extracts of Wedelia chinensis (Osbeck.) M- err. and Wedelia trilobata(L.) Hitchc. had great influence on the structures and characterristi- cs of Escherichia coli and Staphyloccocus aureus Rosenbach.
引文
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