【作者】 陈度煌；

【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， 2008， 硕士

【摘要】 目的：从苦参、百部中提取生物碱，并优化提取条件；应用高速逆流色谱法对苦参、百部总生物碱进行分离提纯；检测苦参生物碱的抗菌作用和体外抗氧化活性，为其产品的开发和应用提供参考依据。方法：1．采用醇提法从苦参、百部中提取总生物碱，并用不同乙醇浓度对醇提法提取苦参生物碱的条件进行优化。2．应用HPLC测定优化高速逆流色谱法的溶剂系统的分配系数，并探讨流速、转速对苦参、百部生物碱分离的影响。在此基础进行生物碱的制备，并用红外光谱对苦参峰进行分析。3．采用杯碟法和对倍稀释法测定苦参生物碱对大肠杆菌、沙门氏菌、金黄色葡萄球菌、嗜水气单胞菌、枯草杆菌对抑菌效果和最低抑菌浓度（MIC）。应用扫描电镜方法观察苦参生物碱对大肠杆菌形态结构的影响。4．应用苦参生物碱体外对羟自由基（OH^-）和超氧阴离子（O₂（?））的清除、抑制H₂O₂诱导小鼠红细胞氧化溶血性等来研究苦参生物碱抗氧化活性。结果：1．苦参、百部的总生物碱得率分别为15．2％和16．8％；苦参醇提法的优化条件是：温度为80℃，提取时间为4 h，提取次数3次，料液比为1：10，乙醇浓度为85％。2．苦参、百部总生物碱的溶剂系统分别为氯仿-甲醇-pH5．4磷酸二氢钠水溶液（4：3：2）、氯仿-甲醇-水（4：3．5：2）体系，流速2mL／min，转速为650转／min。苦参峰2红外光谱分析具有典型生物碱图谱特征。3．20mg／mL苦参生物碱对大肠杆菌、金黄色葡萄球菌、嗜水气单胞菌的抑菌圈分别是18．8、12．6和6．4mm，MIC分别是5、8和20mg／mL，而对沙门氏菌、枯草杆菌无抑菌作用。扫描电镜观察，苦参醇提物使大肠杆菌的菌体形态结构发生明显变化，表现为菌体固缩变形，中间凹陷，呈规则元宝状，严重者细胞壁破损，内容物外泄。4．苦参生物碱对氧损伤的抑制作用均表现量效关系。在生物碱浓度为3．0mg／mL时，对超氧阴离子的清除率最高为53．7％；在生物碱浓度为5．0 mg／mL时，其对羟自由基清除率达42．10％，对H₂O₂诱导的小鼠红细胞氧化溶血的抑制率可达35．10％，这证实了苦参生物碱具有明显的体外抗氧化作用。结论：优化总生物碱的工艺与条件，总碱提取率高，条件温和，操作简单，易于实现产业化；HSCCC可以较好的分离苦参、百部总生物碱；苦参生物碱具有体外抗菌和抗氧化等活性功能。更多还原

【Abstract】 Objective: Choosing the optimum extraction conditions, the alkaloids extracted from Sophoraflavscens Ait and stemona, and extract and purify alkaloids from Sophoraflavscens Ait and stemona by using high speed countercurrent chromatography, detect antimicrobial and anti-oxidation properties of alkaloids from Sophoraflavscens Ait . According to it , we can exploitate new production by these biological activities.Method:1. Choosing the optimum extraction conditions by using different alcohol-density, and extracted the alkaloids from Sophoraflavscens Ait and stemona.2. The solvent system of high speed countercurrent chromatography should be measured by HPLC. It also talking about the separation factor of HSCCC which is include the flow-rate of mobile phase and the speed in this paper. Finally it used these methods to get some solvent systems and separated some alkaloids from Sophoraflavscens Ait and stemona, and analysis alkaloids by infrared spectrum.3. Determination of the antagonistic spectrum of alkaloids from Sophoraflavscens Ait and study on antimicrobial mechanism by using scanning electron microscope.4. The antioxidant properties of alkaloids from Sophoraflavscens Ait were evaluated by determining the ability of a alkaloids solution to scavenge hydroxyl and superoxide radicals, inhibit the level of lipid peroxidation and hemolyse of mouse.Result:1. Under the optimum extraction conditions, the content of total alkaloids is 15.2% and 16.8%, it prove to be alkaloids, the optimum alkaloids extraction conditions are as follows: temperature is 80℃, extraction time is 4 hours, the extraction times is 3 , solid-liquid ratio is 1:10, and the alcohol-density is 85%. 2. The solvent systems of HSCCC was CHCl₃ -MeOH-NaH₂PO₄（4:3:2）, CHCl₃ -MeOH-H₂O（4:3.5:2） respectively, the flow-rate of mobile phase is 2 mL/min; the speed is 850 r/min; the detection wavelength is 254 nm. IR spectrum of peak 2 have typical alkaloids characteristic.3. The extracts of Sophoraflavscens Ait had the obvious action on these three bacteriostasis, MIC was 5, 8 and 20mg/mL. Then the bacteriostatic action and mechanism and its effect on E.coli were studied. Observation under scanning electionmicroscope revealed that the E.coli had been constricted and distorted, depressed on center and just like the shoe-shaped gold ingots.4. With the dosage of 3.0 mg/mL, the inhibitory rate on inhibite superoxide anion was 53.70%. with the dosage of 5.0 mg/mL, the inhibitory rate on hydroxyl free radical was 42.10%, the inhibitory rate on the restraining auto-oxididation of erythrocytes was 35.10%. In the above experiments, the alkaloids inhibit oxidative damage in a dose-dependent manner and exhibit antioxidant activity.Conclusion: By using single factor tests and orthogonal design, we got the optimum conditions of extraction. From this we could get high extraction, and the extract conditions are mild, operation simple, easy to reach industrialization. We can extract alkaloids from Sophoraflavscens Ait and stemona by using HSCCC. And it have good antimicrobial and anti-oxidation ability .更多还原