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(O_2(?))的清除、抑制H_2O_2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H_2O_2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。
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 CHCl_3 -MeOH-NaH_2PO_4(4:3:2), CHCl_3 -MeOH-H_2O(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 .
引文
[1]马养民,傅建熙.生物碱的研究概况[J].陕西林业科技,1997,1:75.
[2]吕梅香.农药用生物碱的研究进展[J].农药,2004,6(6):249-253.
[3] Newman, Steven E, Michael J Roll, Ronald J. Harkrader A. Naturally Occurring Compond for Controling Powdery Mildew of Greenhouse Roses[J]. Hortscience, 1999, 14(4):686.
[4] Shi G F,Li Q.Effects of oxymatrine on experimental hepatic fibrosis and its mechanie [J].World J Gastroenterol,2005,11(2):268-270.
[5]董常青,周莹.广西地不容总生物碱的含量测定方法[J].中国中药杂志,2004,29(9):915-916
[6]喻朝阳,王晓琳.生物碱提取与纯化技术应用进展[J].化工进展,2006,25(3):259-263.
[7]于腾,20种中药及其复方抗真菌实验与临床研究[D].山东中医药大学硕士研究生学位论文, 2003.
[8]马玲娣,中药苦参碱抗肿瘤作用的实验研究[D].重庆医科大学博士研究生学位论文,2005.
[9]崔建国,白毛藤生物碱抗菌抗病毒及免疫药理学研究[D].湖南农业大学硕士研究生学位论 文,2005.
[10]国家药典委员会.中华人民共和国药典(一部)[s].北京:化学工业出版社,2000:161
[11]薛小红,沈立荣,金永昌.百部栽培技术[J].现代应用药学,1992,9(4):64-71.
[12] Gonzalez-coloma A, Cabera R, Monzon A R S. Persea indicant as a natural source of the insecticide ryanodol[J].Phytochemistry,1993,34(2):397-400.
[13]张大佑,逆流色谱技术[M].北京科学技术出版社,1991,274-303.
[14]陈月圆,李典鹏,高江林.黄柏中总生物碱的提取及测定方法研究[J].广西植物,2003,23 (6):565-567.
[15]孙丽艳,魏雄辉,韦宇,等. 博落回中总生物碱提取方法的研究[J].药物分析杂 志,2003(23):15-18.
[16]吴智慧,黄绳武.苦参生物碱的药理研究进展[J].中南药学,2006,4(5):380-382.
[17]冯骥良,黄高升,张永清,等.苦参碱抑制JM细胞株增殖和诱导凋亡的研究[J].中国中药 杂志,2003,28(5):437-442
[18]王晓燕,黄敏仁.微波辅助提取石蒜属植物忽地笑中加兰他敏的研究[J].南京中医药大学学 报,2005,21(6):374-375.
[19]王玉芹,李晓静.峨眉千里光生物碱乙醇提取工艺研究[J].中成药,2004,26(10):附2
[20]赵维民,张天佑翻译,制备色谱技术[M].北京:科学出版社,2000,207-208.
[21]袁黎明,傅若农,张大佑.高速逆流色谱在植物有效成分分离中的应用[J].药物分析杂志, 1998,18(1):60-64
[22]M,吉伯尔迪,D.佩里尔.药物动力学(第二版)[M].朱家壁译.北京科学出版社,1987, 381-390.
[23]汪洪武,汤敏燕,孙凌峰.具有抗肿瘤活性的生物碱类化合物[[J].江西师范大学学报,1996, 20(3):233.
[24]高鹏华,杨绍娟.君子兰总生物碱抗肿瘤作用的研究[[J].吉林中医药,1998,4:53.
[25]李继强,李超群,曾民德,等.氧化苦参碱治疗慢性丙型病毒性肝炎的初步研[J].中国中西医 结合杂志,1998,18(4):227-229.
[26]郝乃斌,戈巧英.中国植物源杀虫剂的研制与应用[J].植物学通报,1999,6(5):495-503
[27]苗抗立,张建中.苦参的化学成分及药理[J].天然产物研究与开发,2001,13(2):69
[28]杨立新,阎西艳,王民宪等.黄连素治疗高血压53例疗效观察[J].临床荟萃,1990,5(10):454.
[29]胡发光.异喳琳生物碱对心血管药理作用的研究与临床[J].中国药理学通报,1992, 8(5):331.
[30]刘斌.苦豆子生物碱对中枢神经系统和肿瘤作用研究进展[[J].基层中药杂志,1995,9(1):44.
[31]邓敏,宋秀媛,王家富.普洛托品的药理作用研究进展[J].中草药,2001,32(3):275-277.
[32]陈小松,王国俊,蔡雄,等.氧化苦参碱对乙型肝炎病毒转基因小鼠乙肝抗原表达的影响[J]. 第二军医大学学报,1999,20(10):746-748.
[33]洗建中,洗超.苦参碱注射液治疗黄疽型病毒性肝炎疗效观察[[J].中国药业,1997,4: 45-46.
[34]杨志伟,周娅,曹秀琴.苦豆子生物碱体外抗柯萨奇B3病毒的作用[J].四川中医,2003,21 (3):16.
[35]李阳,周娅,等.宁夏贝母总生物碱影响小鼠免疫功能的初步研究[[J].宁夏医学院学报, 1997,19(3):1.
[36]李凡,石艳春,黄红兰,等.苦豆碱对小鼠免疫细胞功能的影响[J].白求恩医科大学学报,1997, 23(6):603-605.
[37]钱信忠.有毒中草药大辞典[M].天津:天津科技翻译出版公司,1992:146.
[38]衰黎明,吴平,夏滔,等.高速逆流色谱制备分离中药黄柏中的生物碱[J].色谱,2002, 20(2):185-186.
[39]颜继忠.高速逆流色谱分离中药中的生物碱[D].浙江工业大学硕士学位论文,2001.
[40]袁黎明,湛学先,刘国祥,等.高速逆流色谱对芦荟有效成分的制备性分离研究[J].分析化学, 2003,31(2):251.
[41]张存莉,马惠玲,张宏利,等.苦参生物碱工业生产新方法的探究[J].西北林学院学报2004, 11(1):113-114
[42]蔡保仁,汤苏阳.苦参生物碱的研究进展和临床应用[J].医学进展,2001,14(4):237-238.
[43]邓志国.醇沉法影响有效成分的含量要述[J].川药校刊,1991,(3):48.
[44] Fuquan Yang and Yoichiro Ito[J]. Chromatogr. A, 2002, 943(2): 219-225.
[45]黄健光,李总成.影响pH区带逆流色谱分离效果的土要因素[M].第一届制药工程科技与教 育研讨会会议论文,2003:1 9-23.
[46]王震平.现代中药提取分离法[J].内蒙古石油化工,2004,30:14-15.
[47]戴德舜,王义明,罗国安.高速逆流色谱研究进展[J].分析化学,2001,29(5):586-591.
[48]田桂莲,张天佑,杨福全.黄柏的化学成分及药理学研究进展[J].天然产物研究与开发,2000, 12(6):52-55.
[49]马朝阳.苦豆子生物碱提取分离纯化研究[D].江南大学硕士研究生学位论文,2004.
[50] Ku YR, Chang LY, Lin JH, et al. Determination of matrine and oxymatrine in Sophorasubprostata by CE. J Pharm Biomed Anal. 2002 Jun 1 ;28(5):05-10
[51] Garcia-Lora A, Algarra I, Collado A,et al. Tumour immunology, vaccination and escapestrategies.Eur J Immunogenet. 2003 Jun;30(3):177-183.
[52] Kaltenegger E, Brem B, Mereiter K, Kalchhauser H, Kahlig H, Hofer O,Vajrodaya S, GregerH. Photochemistry, 2003,63:803-816.
[53] Stevigny D, Jiwan J L H, Rozenberg R,Rapid . Commun, Mass Spectrom. 2004, 18: 523-528.
[54]潘五九,肖小河,苑海龙.中药生产关键性新技术研究进展,中草药,2004,35(4):361-367.
[55]焦霞,沈其峋.苦参生物碱的临床及药理研究进展[J].中药新药与临床药理,2002, 13(3):192.
[56]崔建国,陈小军,孙志良.白毛藤生物碱的提取及体外抗菌活性研究[J].中兽医医药杂志, 2004,5:41-42.
[57]王英豪.大孔吸附树脂分离提取中药碱类成分的探析[J].福建中医学院学报,2006,16 (2):51-53.
[58]秦学功,元英进.用大孔吸附树脂分离苦豆子生物碱[J].中国中药杂志,2002,??27(6):428-429.
[59] A.Lorence, F. Medina-Bolivar. Camptothecin and 10-hydroxycamptothecin from Camptothecaacuminata hairy roots[J]. Plant Cell Reports, 2004, 22(6): 437-441.
[60] Hua Lu.Elizabeth Gorman,Thomas D. McKnight. Molecular characterization of two anthranilatesynthase alpha subunit genes in Camptotheca acuminata[J]. Planta, 2003,14(3):234-238.
[61]姜平平,吕晓玲,姚秀玲,等.紫心甘薯花色苷抗氧化活性体外试验研究[J].中国食品添加 剂,2002(6):8-11.
[62]何耀松,张继东.抗菌中草药饲料添加剂的研究与应用[J].广东饲料,2007,16(4):35-36.
[63]杨小林,陈关平.兽用抗菌抗病毒中草药的研究进展[J].长江大学学报,2006,3(2): 151-154.
[64]孟浩,胡博路,张建生.火炬树树叶清除自由基作用的研究[J].食品科学,2002,23(8): 258-260.
[65]杨洋,韦小英,阮征.国内外天然食品抗氧化剂的研究进展[J].食品工业科技,2002,23 (10):137-140.
[66]姚雷,张少艾,王霞平.三种芳香植物的抗氧化性研究[J].香料香精化妆品,2002,(4): 4-8.
[67]金鸣,蔡亚欣,李金荣,等.邻二氮菲-亚铁氧化法检测双氧水/亚铁产生的羟自由基[J].生 物化学与生物物理进展,1996,23(6):54-55.
[68]朱启忠.五种中草药抗羟自由基作用的研究[J].滨州医学院学报,1999,22(5):435-436.
[69]张永忠,余龙江,金文闻等.玛咖多糖抗氧化保健作用研究[J].食品科技,2005(8):97-99.
[70] WuY J. Li WG Zhang ZM. et al. Antioxidative activity of 4-oxy-and-hydroxy-nitroxides in tissues and erythrocytes from rats[J]. Acta Pharmacol Sin. 1997, 18:150.
[71]宋俊梅,丁霄霖.白首乌对超氧阴离子自由基清除作用的研究[J].食品科学,1997,9(18): 61-64.
[72]万影,陈玉石.黄芪对超氧阴离子自由基的清除作用[J].数理医药杂志,1998,11(1):61-62.
[73]马长雨,杨悦武.微波萃取在中药提取和分析中的应用[J].中草药.2004,35(11),12-16.
[74]龚国清,刘同征,李立文,等.西红花酸的体外抗氧化作用的研究[J].中国药科大学学报, 2001,32(4):306-309.
[75]王欣.博落回中生物碱成分的研究Ⅱ[D].西北农林科技大学硕士研究生学位论文,2005: 30-36.
[76]肖观秀.超临界萃取技术提取石蒜中加兰他敏的工艺研究[D].天津大学硕士学位论 文,2005:42
[77] Li Fa-rong, Yang-xiong, Sheng xiao-ting, Li Shu-juan. Antioxidative activities of aucubin in vitro. Journal of Shaanxi Normal University (Natural Science Edition), 2004,32(3):98-101.
[78]凌关庭主编.抗氧化食品与健康[M].北京:化学工业出版社,2004,4.