基于相似结构特征的黄连混合生物碱转化为单体黄连碱的研究
|
摘要
本文以具有共同的异喹啉环结构的小檗碱、黄连碱、巴马汀和药根碱所组成的黄连混合生物碱为起始原料,经过总碱的还原加氢、甲氧基、亚甲二氧基的羟基化、邻位羟基的环合和氧化脱氢四步反应合成黄连碱,总收率可达16%。通过单因素实验设计,详细研究了由黄连混合生物碱制备单体黄连碱的工艺条件,比较了由混合生物碱制备黄连碱的两种工艺条件的优劣,确定了最佳工艺条件；并以白色念珠菌、金黄色葡萄球菌、克柔念珠菌及隐球菌为实验菌株,测定了制备黄连碱和天然黄连碱的最低抑菌浓度MIC值。
实验得到的最佳工艺条件：黄连混合生物碱先加氢再脱甲氧基、亚甲二氧基,然后邻位的羟基环合最后脱氢得到单体黄连碱。四氢黄连混合生物碱的合成条件：还原剂硼氢化钠与原料的在室温条件下的反应摩尔比为4：1,反应时间4 h,甲醇的浓度为80%。四氢四羟基黄连碱的合成条件：三溴化硼与中间体的反应摩尔比为6：1,反应时间为14 h。四氢黄连碱的合成条件：反应温度120℃,反应时间为30 h,溴氯甲烷与原料的反应摩尔比为6：1,氟化铯与原料的反应摩尔比为10：1。黄连碱的合成条件：催化剂用醋酸钠,碘与原料的反应摩尔比为3：1,反应时间10 h。
抗菌结果表明：制备黄连碱保持甚至提高了抗菌性能。其中,制备的单体黄连碱对金黄色葡萄球菌具有较高的抗菌活性,MIC值为8μg/mL。其抗菌机理可能是由于制备产物具有更高的亲脂性,增强了药物对细胞膜的渗透性。
本文制备黄连碱的方法与传统的黄连碱的制取方法相比,省去了药物合成中原料的纯化步骤,且将药力分散不明、纯度低的粗黄连混合碱转化为了抗菌专属性更高、活性更强的单体黄连碱；该方法操作简便,成本低廉,适合黄连碱的批量生产,具有广阔的工业应用前景,并可为其它中草药的研究提供借鉴,对于提高中草药的利用价值和发展创制新药具有很好的指导意义,为其实际应用提供基础。
Coptisine in overall yield of 16% was synthesized from the alkaloids with the same main structure of isoquinoline included berberine, berberurine, palmatine and jatrorrhizine by a four-step reaction of hydro genati on, demethylation or demethylene-dioxy, cyclization, dehydrogenation and so on.Through the single-factor experimental design, the synthetic conditions of Coptisine were studied. The conditions were compared with the two process. The optimum process conditions were obtianed. Candida albicans, Staphylococcus aureus, Candida krusei and Cryptococcus were for the experimental bacterium.The value of minimum inhibitory concentration is tested.
The optimal conditions for the experiment are as follows:alkaloids first were hydrogenated and then demethoxy and demethylenedioxy to obtain the monomer coptisine after the final dehydrogenation of the hydroxyl ring. The conditions for synthesis tetrahydroberberine alkaloids:the most appropriate molar ratio of the sodium borohydride and raw materials is 4:1 and the reaction time is 4 h, using anhydrous potassium carbonate as catalyst and the appropriate methanol solution concentration was 80%.. The conditions for the synthesis of tetrahydrotetrahydroxy coptisine is that the most appropriate molar ratio of borontribromide and the intermediates is 6:1, reaction time is 14 h. The temperature of the tetrahydrocoptisine synthesis is 120℃and the reaction time is 30 h; the most appropriate molar ratio of bromochloromethane and the intermediates is 6:1. The molar ratio of catalys and the intermediates is 10:1.And the reaction time of coptisine synthesis is 10 h. the most appropriate catalys is sodium acetate.
The results of the antibacterial show that:the prepared coptisine monomer maintain or even improve the antibacterial properties. The prepared monomer coptisine has, a high antibacterial activity for Staphylococcus aureus, the value of MIC is 8μ/mL. The mechanism of their antibacterial may due to that the prepared product has higher lipophilic and enhance the permeability of the drug on the cell membrane.
The method which used in this article compared with the conventional method of prepareing coptisine eliminated the purification of the raw materials used for the drugs synthesis, and at the same time it contraved the mixture of bases into monomer coptisine which has higher antimicrobial specificity and activity.This method was simple and easy to operate.The cost was low and suitable for coptisine's mass production.The transformation of the main components to one component would provide useful ideas for the Chinese herbal medicine's value and good guide for the development of new medicine.
实验得到的最佳工艺条件：黄连混合生物碱先加氢再脱甲氧基、亚甲二氧基,然后邻位的羟基环合最后脱氢得到单体黄连碱。四氢黄连混合生物碱的合成条件：还原剂硼氢化钠与原料的在室温条件下的反应摩尔比为4：1,反应时间4 h,甲醇的浓度为80%。四氢四羟基黄连碱的合成条件：三溴化硼与中间体的反应摩尔比为6：1,反应时间为14 h。四氢黄连碱的合成条件：反应温度120℃,反应时间为30 h,溴氯甲烷与原料的反应摩尔比为6：1,氟化铯与原料的反应摩尔比为10：1。黄连碱的合成条件：催化剂用醋酸钠,碘与原料的反应摩尔比为3：1,反应时间10 h。
抗菌结果表明：制备黄连碱保持甚至提高了抗菌性能。其中,制备的单体黄连碱对金黄色葡萄球菌具有较高的抗菌活性,MIC值为8μg/mL。其抗菌机理可能是由于制备产物具有更高的亲脂性,增强了药物对细胞膜的渗透性。
本文制备黄连碱的方法与传统的黄连碱的制取方法相比,省去了药物合成中原料的纯化步骤,且将药力分散不明、纯度低的粗黄连混合碱转化为了抗菌专属性更高、活性更强的单体黄连碱；该方法操作简便,成本低廉,适合黄连碱的批量生产,具有广阔的工业应用前景,并可为其它中草药的研究提供借鉴,对于提高中草药的利用价值和发展创制新药具有很好的指导意义,为其实际应用提供基础。
Coptisine in overall yield of 16% was synthesized from the alkaloids with the same main structure of isoquinoline included berberine, berberurine, palmatine and jatrorrhizine by a four-step reaction of hydro genati on, demethylation or demethylene-dioxy, cyclization, dehydrogenation and so on.Through the single-factor experimental design, the synthetic conditions of Coptisine were studied. The conditions were compared with the two process. The optimum process conditions were obtianed. Candida albicans, Staphylococcus aureus, Candida krusei and Cryptococcus were for the experimental bacterium.The value of minimum inhibitory concentration is tested.
The optimal conditions for the experiment are as follows:alkaloids first were hydrogenated and then demethoxy and demethylenedioxy to obtain the monomer coptisine after the final dehydrogenation of the hydroxyl ring. The conditions for synthesis tetrahydroberberine alkaloids:the most appropriate molar ratio of the sodium borohydride and raw materials is 4:1 and the reaction time is 4 h, using anhydrous potassium carbonate as catalyst and the appropriate methanol solution concentration was 80%.. The conditions for the synthesis of tetrahydrotetrahydroxy coptisine is that the most appropriate molar ratio of borontribromide and the intermediates is 6:1, reaction time is 14 h. The temperature of the tetrahydrocoptisine synthesis is 120℃and the reaction time is 30 h; the most appropriate molar ratio of bromochloromethane and the intermediates is 6:1. The molar ratio of catalys and the intermediates is 10:1.And the reaction time of coptisine synthesis is 10 h. the most appropriate catalys is sodium acetate.
The results of the antibacterial show that:the prepared coptisine monomer maintain or even improve the antibacterial properties. The prepared monomer coptisine has, a high antibacterial activity for Staphylococcus aureus, the value of MIC is 8μ/mL. The mechanism of their antibacterial may due to that the prepared product has higher lipophilic and enhance the permeability of the drug on the cell membrane.
The method which used in this article compared with the conventional method of prepareing coptisine eliminated the purification of the raw materials used for the drugs synthesis, and at the same time it contraved the mixture of bases into monomer coptisine which has higher antimicrobial specificity and activity.This method was simple and easy to operate.The cost was low and suitable for coptisine's mass production.The transformation of the main components to one component would provide useful ideas for the Chinese herbal medicine's value and good guide for the development of new medicine.
引文
[1]尤启冬.药物化学[M].北京：化学工业出版社,2009：217-227.
[2]李彩虹,周克元,等.黄连活性成分的作用及机制研究进展[J].时珍国医国药,2010,21(2)：466-468.
[3]杨勇,叶小利,李学刚.4种黄连生物碱的抑菌作用[J].时珍国医国药,2007,18(12)：3013-3014.
[4]Youn MJ, So HS, Cho HJ, etal. Berbefine a natural product combined with cisplatin enhanced apoptosis through a mitochondria/caspase-mediated pathway in HeLa cells[J]. Biol Pharm Bull,2008,31(5):789.
[5]Wen-Hsin Hsu, Yih-Shou Hsieh, Hsing-Chun Kuo etal. Berberine induces apoptosis in SW620 human colonic carcinoma cells through generation of reactive oxygen species and activation of JNK/p38 MAPK and Fals[J]. Arch Toxicol,2007,81(10):719-728.
[6]吴柯,周岐新.黄连生物碱抗结肠癌作用实验及其分子机制研究[D].重庆：重庆医科大学,2007.
[7]骆叶,郝玉.小檗碱抑制肿瘤细胞Cyclin D1相关信号通路的研究[D].北京：北京中医药大学,2007.
[8]崔国辉,黄秀兰,周克元.黄连及其主要成分小檗碱对人鼻咽癌CNE -2Z生长的抑制作用[J].广东医学,2008,29(5)：737.
[9]王邦茂,翟春颖,方维丽,等.黄连素对脱氧胆酸诱导的HT-29人结肠癌细胞增殖的抑制作用及机制研究[J].中国消化杂志,2006,26(11)：749.
[10]Dove-Edwin I, Thomas HJW. The prevention of colorectal cancer[J]. Alimentary Pharmacology and Therapeutics,2001,15(3):323-336.
[11]孙焕,陆付耳,王增四,等.小檗碱改善高糖高脂诱导的胰岛NIT21细胞凋亡的分子机制[J].中国药理学通报,2008,24(6)：762.
[12]陶大昌,郑凌云,彭艳,等.黄连素对缺氧复氧心肌细胞Bcl-2和Bax基因表达变化的免疫组织化学研究[J].四川医学,2008,29(3)：266.
[13]Yung-Tsuan Ho, Jai-Sing Yang, Tsai-Chung Li etal. Berberine suppresses in vitro migration and invasion of human SCC-4 tongue squamous cancer cells through the inhibitions of FAK, IKK, NF-KB, u-PA and MMP-2 and-9[J]. Cancer Lett,2009,279(2):155.
[14]J.X.Kang, Jing Liu, Jingdong Wang etal. The extract of huang lian, a medicinal herb, induces cell growth arrest and apoptosis by upregulation of interferon-O and TNF-cdn human breast cancer cells[J]. Carcinogenesis vol,2005,26(6): 1934.
[15]Leng SH, Lu FE, Xu LJ. Therapeutic effects of berberine in impaireducose tolerance rats and its influence on insulin secretion[J]. ActaPhannacol Sin, 2004,25(4):496.
[16]Tang LQ, Wei W, etal. Effects of herberine on diabetes induced by alloxan and a high-fat/high-cholesterol diet in rats[J]. Ethnopharmacol,2006,108(1): 109-115.
[17]Yin J, Gao Z, Liu D, Liu Z. Berberine improves glucose metab-olism through induction of glyeolysis[J]. Endocrinol Metab,2008,294(1):148-156.
[18]应懿.黄连生物碱的提取及其对实验性糖尿病周围神经病变和糖尿病肌病的保护作用及机制研究[D].重庆：第三军医大学,2007,5：10.
[19]JiYin Zhou, Shi Wen Zhou, KeBin Zhang etal. Chronic Effects of Berberine on Blood, Liver, Glucolipid metabolism and Liver PPARs Expression in Diabetic Hyderlipidemie Rats[J]. Biol.Pharm.Bull,2008,31(6):1169.
[20]Dong Y etal. Absorption of extractive Rhizoma Coptidis in rat ever'ted gut scas[J]. Zhongguo Zhong Yao Za Zhi,2008,33(9):1056.
[21]Weihua Liu, Yanhui Deng etal. Berberine reduces fibronectin and collagen accumulation in rat glomerular mesangial cells cultured under high glucose condition[J]. Molecular and Cellular Biochemistry,2009,325(1-2):99-105.
[22]Wang Y, Huang Y, etal. Berberine prevents hyperglycemia-induced endothelial injury and enhances vasodilatation via adenosinemonophosphate-activated protein kinase and endothelial nitric oxidesynthase[J]. Cardiovasc Res,2009, 82(3):484.
[23]Eun kyung Kim, Kang-Beom Kwon, Mi-Jeong Han etal. Coptidisrhizoma extract protects against cytokine-induced death of panereatiel3-ells through suppression of NF-KB activation[J]. Exp.Mol.Med.Vol,2007,39(2):149.
[24]周吉银,周世文.小檗碱降糖调脂作用与PPARs/P-TEFb信号转导通路的关系[D].重庆：第三军医大学,2008.
[25]孙强,朱冰芝,陈亚娟.黄连灌肠治疗慢性溃疡性结肠炎173例[J].现代中西医结合杂志,2003,12(10)：1024.
[26]尹立子,欧阳萍,徐雪,周丽光.西藏胡黄连化学成分的分离和鉴定[J].高等学校化学学报,2010,31(1)：84-87.
[27]张红梅,程雪梅,王长虹,王峥涛.黄连中生物碱提取工艺的正交试验研 究[J].中国医药工业杂志,2008,39(8)：588-590.
[28]柳明,叶小利,梁艳婷,王亮.黄连须根总生物碱及单体的联产工艺研究[J].时珍国医国药,2010,21(7)：1644-1646.
[29]董新荣,曾建国.北美黄连主要生物碱的提取与分离[J].精细化工中间体,2001.31(3),33-35.
[30]马桔云,赵晶岩,姜颖,等.纤维素酶在黄连提取工艺中的应用[J].中草药,2003,1(2)：103-104.
[31]李群林,顾军,李灵.齐墩果酸衍生物的合成[J].合成化学,2010,18(3)：290-296.
[32]姜效军,李井会.pH校正最小二乘紫外分光光度法同时测定喹啉异喹啉和萘[J].环境工程,1995,10,13(5)：43-46.
[33]杜莹,薛赛凤等.紫外吸收光谱法测定瓜环与喹啉衍生物包结物包结比[J].贵州大学学报,2005,5,22(2)：166-180.
[34]武彦文,肖小河,孙素琴,刘红霞.黄连不同提取物的红外光谱研究[J].光谱学与光谱分析,2009,29(1)：93-96.
[35]李立军,再帕尔-不力孜,项贇,王爱国,冯孝章.黄连总碱的MS/MS及LC-MS/MS研究[J].质谱学报,21(3,4)：81-82.
[36]秦海林,尚玉俊,赵伟,等.核磁共振氢谱法鉴别黄连的研究[J].中草药,2000,31(1)：48-50.
[37]应懿,何志红,周世文,汤建林,杨雪,王璐.测定黄连中5种生物碱含量的高效液相色谱法研究[J].第三军医大学学报,2007,29(9)：843-845.
[38]贺福元,罗杰英,阳长明,等.大黄蒽醌和黄连小檗碱衍生物含量变化及中药成分谱的研究[J].湖南中医学院学报,2000,20(4)：27-29.
[39]利健文,蔡沛祥.毛细管电泳安培法检测黄连中的生物碱[J].化学研究与应用,2009,21(10)：1381-1385.
[40]陈帅,童元峰,吴松.盐酸黄连碱的全合成[J].合成化学,2009,17(4)：512-513.
[41]Xiang Juan Cui, Ying Ying Lu, Min Zhao, Shan Qianand Yong Wu. The new shortcut synthesis of 8-oxocoptisine[J]. Chinese Chemical Letters,2010,21(11): 1281-1282.
[42]Maria Laura Colombo, etal. Cytotoxicity evaluation of natural coptisine and synthesis of coptisine from berberine[J]. Fameco,2001,56(5-7):403-409.
[43]张灿,黄文龙.原小檗碱季铵类化合物的合成及抗心律失常活性研究[J].中国药科大学学报,2003,34(1)：7-12.
[2]李彩虹,周克元,等.黄连活性成分的作用及机制研究进展[J].时珍国医国药,2010,21(2)：466-468.
[3]杨勇,叶小利,李学刚.4种黄连生物碱的抑菌作用[J].时珍国医国药,2007,18(12)：3013-3014.
[4]Youn MJ, So HS, Cho HJ, etal. Berbefine a natural product combined with cisplatin enhanced apoptosis through a mitochondria/caspase-mediated pathway in HeLa cells[J]. Biol Pharm Bull,2008,31(5):789.
[5]Wen-Hsin Hsu, Yih-Shou Hsieh, Hsing-Chun Kuo etal. Berberine induces apoptosis in SW620 human colonic carcinoma cells through generation of reactive oxygen species and activation of JNK/p38 MAPK and Fals[J]. Arch Toxicol,2007,81(10):719-728.
[6]吴柯,周岐新.黄连生物碱抗结肠癌作用实验及其分子机制研究[D].重庆：重庆医科大学,2007.
[7]骆叶,郝玉.小檗碱抑制肿瘤细胞Cyclin D1相关信号通路的研究[D].北京：北京中医药大学,2007.
[8]崔国辉,黄秀兰,周克元.黄连及其主要成分小檗碱对人鼻咽癌CNE -2Z生长的抑制作用[J].广东医学,2008,29(5)：737.
[9]王邦茂,翟春颖,方维丽,等.黄连素对脱氧胆酸诱导的HT-29人结肠癌细胞增殖的抑制作用及机制研究[J].中国消化杂志,2006,26(11)：749.
[10]Dove-Edwin I, Thomas HJW. The prevention of colorectal cancer[J]. Alimentary Pharmacology and Therapeutics,2001,15(3):323-336.
[11]孙焕,陆付耳,王增四,等.小檗碱改善高糖高脂诱导的胰岛NIT21细胞凋亡的分子机制[J].中国药理学通报,2008,24(6)：762.
[12]陶大昌,郑凌云,彭艳,等.黄连素对缺氧复氧心肌细胞Bcl-2和Bax基因表达变化的免疫组织化学研究[J].四川医学,2008,29(3)：266.
[13]Yung-Tsuan Ho, Jai-Sing Yang, Tsai-Chung Li etal. Berberine suppresses in vitro migration and invasion of human SCC-4 tongue squamous cancer cells through the inhibitions of FAK, IKK, NF-KB, u-PA and MMP-2 and-9[J]. Cancer Lett,2009,279(2):155.
[14]J.X.Kang, Jing Liu, Jingdong Wang etal. The extract of huang lian, a medicinal herb, induces cell growth arrest and apoptosis by upregulation of interferon-O and TNF-cdn human breast cancer cells[J]. Carcinogenesis vol,2005,26(6): 1934.
[15]Leng SH, Lu FE, Xu LJ. Therapeutic effects of berberine in impaireducose tolerance rats and its influence on insulin secretion[J]. ActaPhannacol Sin, 2004,25(4):496.
[16]Tang LQ, Wei W, etal. Effects of herberine on diabetes induced by alloxan and a high-fat/high-cholesterol diet in rats[J]. Ethnopharmacol,2006,108(1): 109-115.
[17]Yin J, Gao Z, Liu D, Liu Z. Berberine improves glucose metab-olism through induction of glyeolysis[J]. Endocrinol Metab,2008,294(1):148-156.
[18]应懿.黄连生物碱的提取及其对实验性糖尿病周围神经病变和糖尿病肌病的保护作用及机制研究[D].重庆：第三军医大学,2007,5：10.
[19]JiYin Zhou, Shi Wen Zhou, KeBin Zhang etal. Chronic Effects of Berberine on Blood, Liver, Glucolipid metabolism and Liver PPARs Expression in Diabetic Hyderlipidemie Rats[J]. Biol.Pharm.Bull,2008,31(6):1169.
[20]Dong Y etal. Absorption of extractive Rhizoma Coptidis in rat ever'ted gut scas[J]. Zhongguo Zhong Yao Za Zhi,2008,33(9):1056.
[21]Weihua Liu, Yanhui Deng etal. Berberine reduces fibronectin and collagen accumulation in rat glomerular mesangial cells cultured under high glucose condition[J]. Molecular and Cellular Biochemistry,2009,325(1-2):99-105.
[22]Wang Y, Huang Y, etal. Berberine prevents hyperglycemia-induced endothelial injury and enhances vasodilatation via adenosinemonophosphate-activated protein kinase and endothelial nitric oxidesynthase[J]. Cardiovasc Res,2009, 82(3):484.
[23]Eun kyung Kim, Kang-Beom Kwon, Mi-Jeong Han etal. Coptidisrhizoma extract protects against cytokine-induced death of panereatiel3-ells through suppression of NF-KB activation[J]. Exp.Mol.Med.Vol,2007,39(2):149.
[24]周吉银,周世文.小檗碱降糖调脂作用与PPARs/P-TEFb信号转导通路的关系[D].重庆：第三军医大学,2008.
[25]孙强,朱冰芝,陈亚娟.黄连灌肠治疗慢性溃疡性结肠炎173例[J].现代中西医结合杂志,2003,12(10)：1024.
[26]尹立子,欧阳萍,徐雪,周丽光.西藏胡黄连化学成分的分离和鉴定[J].高等学校化学学报,2010,31(1)：84-87.
[27]张红梅,程雪梅,王长虹,王峥涛.黄连中生物碱提取工艺的正交试验研 究[J].中国医药工业杂志,2008,39(8)：588-590.
[28]柳明,叶小利,梁艳婷,王亮.黄连须根总生物碱及单体的联产工艺研究[J].时珍国医国药,2010,21(7)：1644-1646.
[29]董新荣,曾建国.北美黄连主要生物碱的提取与分离[J].精细化工中间体,2001.31(3),33-35.
[30]马桔云,赵晶岩,姜颖,等.纤维素酶在黄连提取工艺中的应用[J].中草药,2003,1(2)：103-104.
[31]李群林,顾军,李灵.齐墩果酸衍生物的合成[J].合成化学,2010,18(3)：290-296.
[32]姜效军,李井会.pH校正最小二乘紫外分光光度法同时测定喹啉异喹啉和萘[J].环境工程,1995,10,13(5)：43-46.
[33]杜莹,薛赛凤等.紫外吸收光谱法测定瓜环与喹啉衍生物包结物包结比[J].贵州大学学报,2005,5,22(2)：166-180.
[34]武彦文,肖小河,孙素琴,刘红霞.黄连不同提取物的红外光谱研究[J].光谱学与光谱分析,2009,29(1)：93-96.
[35]李立军,再帕尔-不力孜,项贇,王爱国,冯孝章.黄连总碱的MS/MS及LC-MS/MS研究[J].质谱学报,21(3,4)：81-82.
[36]秦海林,尚玉俊,赵伟,等.核磁共振氢谱法鉴别黄连的研究[J].中草药,2000,31(1)：48-50.
[37]应懿,何志红,周世文,汤建林,杨雪,王璐.测定黄连中5种生物碱含量的高效液相色谱法研究[J].第三军医大学学报,2007,29(9)：843-845.
[38]贺福元,罗杰英,阳长明,等.大黄蒽醌和黄连小檗碱衍生物含量变化及中药成分谱的研究[J].湖南中医学院学报,2000,20(4)：27-29.
[39]利健文,蔡沛祥.毛细管电泳安培法检测黄连中的生物碱[J].化学研究与应用,2009,21(10)：1381-1385.
[40]陈帅,童元峰,吴松.盐酸黄连碱的全合成[J].合成化学,2009,17(4)：512-513.
[41]Xiang Juan Cui, Ying Ying Lu, Min Zhao, Shan Qianand Yong Wu. The new shortcut synthesis of 8-oxocoptisine[J]. Chinese Chemical Letters,2010,21(11): 1281-1282.
[42]Maria Laura Colombo, etal. Cytotoxicity evaluation of natural coptisine and synthesis of coptisine from berberine[J]. Fameco,2001,56(5-7):403-409.
[43]张灿,黄文龙.原小檗碱季铵类化合物的合成及抗心律失常活性研究[J].中国药科大学学报,2003,34(1)：7-12.