摘要
目的:利用三尖杉和转基因西洋参冠瘿组织培养体系对青蒿素的生物合成中间体青蒿酸及二氢青蒿酸进行生物转化研究,获得三个青蒿酸及二氢青蒿酸衍生物,同时得到一个次级代谢产物。
方法:通过TLC和HPLC方法检测转化体系中的转化产物;利用硅胶柱层析、制备薄层、Sephadex LH20、ODS柱等方法分离纯化转化产物;检验产物的理化性质;用1H-NMR,13C-NMR和MS方法鉴定转化产物结构;用HPLC方法考察转化体系对底物进行生物转化的最佳共培养条件。
结果:国内首次使用三尖杉悬浮培养细胞进行生物转化研究,以青蒿素的生物合成途径中两个重要中间体青蒿酸和二氢青蒿酸为底物。青蒿酸作为底物与三尖杉悬浮培养细胞共培养两天,从三尖杉体系分离并鉴定一个产物3α-羟基青蒿酸(SQJ-2),最佳共培养时间为2d,总的摩尔转化率最高为8.42%。共培养时间也为两天,从体系中分离并鉴定两个转化产物分别为:15-羟基二氢青蒿酸(SQQJ-3)、15-羟基二氢青蒿酸-β-D-葡萄糖酯(SQQJ-5);培养体系中SQQJ-3的转化率在共培养的第2d达到最大值(6.02%),SQQJ-5在共培养的第3d达到最大值(4.95%)。使用转基因西洋参冠瘿组织对青蒿酸进行转化得到一个次级代谢产物:12,13-环氧-11-羟基-9-十八碳烯酸。
Abstract Objective:To investigate the biotransformation of artemisinic acid and dihydroartemisinic acid by cell suspension culture of Cephalotaxus fortunei Hook. f. and transgenic crown gall of Panax quinquefolium L, generated three transformed Products and a secondary metabolite.
Methods:Plant tissue culture technology was employed. Transformed products were detected by TLC and HPLC, isolated and purified by the column chromatography, recrystallization, Sephadex LH20, ODS etc. Their physico-chemical properties were checked up. Their structures were elucidated by 1H-NMR, 13C-NMR and MS. The optimal co-culture times of the products were determined by HPLC.
Results:The cell suspension culture of Cephalotaxus fortunei Hook. f. transformed the artemisinic acid and dihydroartemisinic acid to three Products, transformed artemisinic acid to one Product:3a-hydroxyartemisinic acid. After co-cultured for 2 days, the mole conversion ratio of artemisinic acid reached the highest (8.42%) in cell suspension culture of Cephalotaxus fortunei Hook. f. The cell suspension culture of Cephalotaxus fortunei Hook. f. transformed dihydroartemisinic acid to two Products: 15-hydroxy dihydroartemisinic acid and 15-hydroxy dihydroartemisinic acidβ-D-glucopyranosyl ester. The conversion rate of SQQJ-3 in the cultures reached the maximum (6.02%) in 2nd day, and conversion rate of SQQJ-5 in the cultures reached the maximum (4.95%) in 3rd day. The transgenic crown gall of Panax quinquefolium L transformed artemisinic acid, generated a secondary metabolite:12,13-epoxy-11-hydroxy-9-octadecenoic acid.
引文
[1]褚志义.生物合成药物学[M].北京:化学工业出版社,2000:259-262.
[2]Loughlin W.A. Bio transformations in organic synthesis [J]. Bioresource Technology, 2000,74(1):49-62.
[3]Rozzell J D. Commercial scale biocatalysis: Myths and realities [J]. Bioorg Med Chem,1999,7(10):2253-2261.
[4]占纪勋,魏秋芬,单启成,等.四个青蒿素生物转化产物抗体外培养恶性疟原虫活性研究[J].中国热带医学,2003,3(1):17-18.
[5]谢秀祯,郭勇.植物培养物生物转化系统在药物合成上的研究与应用[J].药物生物技术,2003,10(6):405-408.
[6]张礼和.化学学科进展[M].北京:化学工业出版社,2005:149-150.
[7]Li Lixin, SuYanfang, Liu Xiaofeng and Guo Dean. Biotransformation of Artemisinin by Hairy Root Cultures of Rheum Palmatum [J]. Journal of don-NA-utical Sciences,2002.11 (4):122-123.
[8]冯冰,马百平.天然产物的生物转化研究进展[J].中草药,2005,36(6):941-945.
[9]Adachi T, Saito M, Sasaki J, et al. Microbial hydroxylation of (-)-eburnamonine by Mucor circinelloides and Streptomyces violens [J]. Chem Pham Bull,1993,41(3): 611-613.
[10]Tapia A.A., Vallejo M.D., Gouiric S.C., et al. Hydroxylation of dehydroabietic acid by Fusarium species [J]. Phytochemistry,1997,46(1):131-133.
[11]Li W, Koike K., Asada Y., et al. Biotransformation of umbelliferone by Panax ginseng root cultures [J]. Tetrabedron Letters,2002,43(32):5633-5635.
[12]于荣敏.天然药物活性成分的生物合成与生物转化[J].中草药,2006,37(9):1281-1288.
[13]Kamel S., Brazier M., Desmet G., et al. Glucosylation of butyric acid by cell suspension culture of Nicotiana plumbaginifolia [J]. Phytochemistry,1992,31(5): 1581-1583.
[14]Fournier D., Hawari J., Streger S.H., et al.Biotransformation of N-nitrosodimethylamine by Pseudomonas mendocina KR1 [J]. Appl Environ Microbiol,2006,72(10):6693-6698.
[15]Chai W., Hamada H., Suhara J., et al. Biotransformation of (+)-and (-)-camphorquinones by plant cultured cells [J]. Phytochemistry,2001,57(5):669-673.
[16]Shimoda K., Hirata T., Biotransformation of enones with biocatalysts-two enone reductases from Astasia longa [J]. Journal of Molecular Catalysis B:Enzymatic, 2000,8(426):255-264.
[17]Lucero M.E., Mueller W., Hubsten berger J., et al. Tolerance to nitrogenous explosives and metabolism of TNT by cell suspensions of Datura innoxia [J]. In Vitro Cell Dev Biol (Plant),1999,35(6):480-486.
[18]Hughes J.B., Shanks J., Vanderford M., et al. Transformation of TNT by aquatic plants and plant tissue cultures [J]. Environ Sci Technol,1997,31(1):266-271.
[19]Sakui N., Kuroyanagi M., Ishitobi Y., et al. Biotransformation of sesquiterpenes by cultured cells of Curcuma zedoaria [J]. Phytochemistry,1992,31(1):143-147.
[20]Chai W., Hayashida Y., Sakamaki H., et al. The biocatalytic oxidation of thujopsene by plant cultured-cells [J]. Journal of Molecular Catalysis B: Enzymatic, 2004,27(223):55-60.
[21]Dai J., Gong Z., Zhu D., et al. Biotransformation of gastrodin by cell suspension cultures of Catharanthus roseus [J]. Zhiwu Xuebao,2002,44(3):377-378.
[22]严春艳,于荣敏.植物生物转化技术与中药活性化合物研究[J].食品与药品,2005,7(10A):428.
[23]王旭,徐威,游松.微生物转化在药学中的应用[J].沈阳药科大学学报,2006,23(7):477-482.
[24]谢春锋,娄红祥.天然产物的生物转化[J].天然产物研究与开发,2005,17(5):658-664.
[25]张春燕,白宝星,王明蓉.甾体药物生物转化体系的研究进展[J].国外医药:抗生素分册,2007,28(5):210-214.
[26]余伯阳.中药与天然药物生物技术研究进展与展望[J].中国药科大学学报,2002,33(5):359-363.
[27]卢定强,李衍亮,凌岫泉,等.手性药物拆分技术的研究进展[J].时珍国医国 药,2009,20(7):1731-1734.
[28]胡文浩,周静.手性药物及手性合成[J].纪学教学,2009,(5):123.
[29]Santaniello E., Ferraboschi P., Grisenti P, et al. The biocatalytic approach to the preparation of enantiomerically pure chiral building blocks [J]. Chem Rev,1992,92 (5): 1071-1140.
[30]宫丽,卞俊.手性药物的制备[J].中国药业,2008,17(23):64-66.
[31]吴再坤,钟宏,王微宏,等.手性药物的活性分析与生物转化[J].化工技术与开发,2006,35(9):24-27.
[32]张宪锋,郑裕国.酶法拆分手性化合物HPBE [J].生物加工过程,2003,1(2):34-38.
[33]Kapoor M., Anand N., Koul S., et al. Kinetic resolution of 1-chloro-3-(1-napht hyloxy)-2-propanol, an intermediate in t he synthesis of β-adrenergic receptor blockers [J]. Bioorganic Chem,2003,31(3):259-269.
[34]刘正威.酶法拆分技术研究[J].管理观察,2009,(5):58.
[35]蒋育澄,李淑妮,翟全国.手性药物的酶催化不对称定向合成[J].化学教育,2008,29(12):9-12.
[36]汤亚杰,李艳,徐小玲,等.天然活性先导化合物生物转化[J].中国生物工程杂志,2007,27(9):110-115.
[37]于德泉,吴毓林.天然产物化学进展[M].北京:化学工业出版社,2005:368-392.
[38]Sangwan R.S., Agarwal K., Luthra R., Thakur R.S., Neelam S.S. Biotrans-formation of arteannuic acid into arteannuin-B and artemisinin in Artemisia annua [J]. Phytochemistry,1993,34(5):1301-1302.
[39]许杏详,朱杰,黄大中,周维善.青蒿素及其一类物结构和合成的研究-从青蒿酸立体控制合成青蒿素和脱氧青蒿素[J].化学学报,1983,41(6):574-577.
[40]Haynes, R.K.青蒿酸催化氧化制备去氢青蒿素和青蒿素[J].国外医药-植物药分册,1991,6(1):29.
[41]周维善,张联,许杏详.青蒿素及其一类物的结构和合成-青蒿戊素的合成和α-环氧青蒿酸甲酯环氧的开环反应[J].化学学报,1985,43:845-851.
[42]Hiroshi Kawamoto, Yoshihisa Asada, Hiroshi Sekine and Tsutomu Furuya, Biotransformation of artemisinic acid by cultured cells of Artemisia annua, Phytochemistry,1999,48(8) 1329-1333.
[43]Zeng Q.P., Qiu F., Ling, Y. Production of artemisinin by genetically-modified microbes [J]. Biotechnology Letter,2008,48:581-592.
[44]Covello P.S., Keat H.T., Polichuk D.R., Reed D.W., Nowak G. Functional genomics and the biosynthesis of artemisinin [J]. Phytochemistry,2007,68: 1864-1871.
[45]Olsson M.E., Olofsson L.M., Lindahl A.L., Lundgren A., Brodelius M., Brodelius P.E. Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L [J]. Phytochemistry,2009,90: 1123-1128.
[46]Zhang Y.H., Keat H.T., Reed D.W., Maes L., Goossens A., Douglas J.H., Andrew R.S., Patrick S.C. The molecular cloning of artemisinic aldehyde △11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua [J]. The Journol of Biological Chemistry,2008,283(31):21501-1508.
[47]周生伟,王莎莉,王亚平.青蒿酸对人红白血病K562细胞增殖抑制的体外研究[J].重庆医科大学学报,2006,31(2):159-162.
[48]邓定安,蔡俊超.具有抗肿瘤活性的青蒿酸衍生物[J].有机化学,1991,11:540-543.
[49]Dhingra V., Pakki S.R., Narasu M.L. Antimicrobial activity of artemisinin and its precursors [J]. Current Science,2000,78:708-710.
[50]Huang L, Liu J.F., Liu L.X. Studies on the antipyretic and anti-inflammatory effects of Artemisia annua L [J]. China Journal Chinese Materia Medica,1993,18: 44-48.
[51]蔡月琴,林镇斌,陈礼光,刘学琴,等.三尖杉愈伤组织诱导及其分化的初步研究[J].福建林学院学报,2008,28(1):31-35.
[52]白雪芳,王靖楣,卜宗式,等.三尖杉悬浮细胞的培养及抗癌生物碱的产生[J].中国生化药物杂志,1999,20(3):139-142.
[53]惠月明,胡月红.三尖杉组织培养的初步研究[J].中草药,1989,20(1):31-35.
[54]符文英,杜道林,符木均.海南粗榧愈伤组织的诱导和培养[J].植物生理学通讯,2004,40(1):33-36.
[55]荣俊冬,蔡月琴,林镇斌,等.三尖杉无菌增殖培养技术的研究[J].福建林学院学报,2007,27(3):208-212.
[56]唐魏,杨映根,桂耀林,等.松柏类植物体细胞胚胎发生的研究与应用[J].植物通讯报,1996,13(1):25-31.
[57]姜新兵.香榧离体再生系统建立的研究[J].浙江:浙江大学农业与生物技术学院,2004.
[58]Becwar M.R., Nagmani R., Wann S.R. Initiation of embryogenc cultures and somatic embryo development in loblollypine (Pinus taeda L.) [J]. Can J For Res, 1990,20(6):810-817.
[59]Laine E, Bade P, David A. Recovery of plants from cryop resserved embryogeneic cell suspensions of Pinus caribaea [J]. Plant Cell Rep,1992,11(5-6):295-298.
[60]成静,郭勇.植物细胞工程药物生产的研究进展[J].江西科学,2000,18(1):60-62.
[61]张荫麟,宋经元,赵保华.丹参的冠瘿组织培养和丹参酮的产生[J].生物工程学报,1995,11(2):150-152.
[62]王宁宁,王淑芳,田俊英,李霞,朱亮基.土壤农杆菌转化的长春花冠瘿细胞培养[J].生物工程学报,1994,10(3):244-249.
[63]贾秀山,廖宇静等.黄花蒿组织培养研究[J].生物技术通讯,2008,19(2):259-262.
[64]Acton, N. Semisythesis of 3-β-hydroxyartermisinin [J]. Journal of Natural products,1999,62:790-793.
[65]Geoffrey D. Brown, Lai-King Sy, In vivo transformations of artemisinic acid in Artemisia annua plants [J]. Tetrahedron,2007,63:9548-9566.
[66]Geoffrey D. Brown, Lai-King Sy, In vivo transformations of dihydro-artemisinic acid in Artemisia annua plants [J]. Tetrahedron,2004,60: 1139-1159.
[67]于荣敏,宋永波,张辉,叶文才,张荫麟,姚新生.西洋参冠瘿组织培养及其人 参皂苷Re和人参皂苷Rg-1的产生[J].生物工程学报,2003,19:372-375.
[68]Ernst H. Oliw, Ulrike Garscha, Tomas Nilsson, Mirela Cristea, Payne rearrangement during analysis of epoxyalcohols of linoleic and a-linolenic acids by normal phase liquid chromatography with tandem mass spectrometry [J]. Analytical Biochemistry,2006,354:111-126.
[69]Markus O. Lederer, Axel Schuler, and Marc O., Reactivity of lysine moieties toward an epoxyhydroxylinoleic acid derivative:aminolysis versus hydrolysis [J]. J. Agric. Food Chem.,1999,47:4611-4620.
[70]Gerrit J.G., Gerrit A.V., Johannes F.G., et al. The formation of threo-11-hydroxy-trans-12:13-epoxy-9-cis-octadecenoic acid by enzymic isomerisation of 13-L-hydroperoxy-9-cis,-trans-octadecadienoic acid by soybean lipoxygenase-1 [J]. Eur. J. Biochem.,1976,62:33-36.