黄绿密环菌生物转化合成天麻素的研究
|
摘要
天麻素(gastrodin)是一种酚型糖苷类化合物,它具有显著的生物学活性和药理活性。本论文主要研究黄绿密环菌生物转化合成天麻素的转化条件优化以及产物的分离纯化,对分离得到的产物进行结构鉴定,并对合成的天麻素进行动物试验评价,研究其抗炎以及免疫调节功能,并初步研究生物转化体系中葡萄糖基转移酶的酶学性质,为后续深入研究和调控天麻素生物合成提供试验依据。
首先研究建立转化体系中天麻素和对羟基苯甲醇的检测方法,确定转化体系中天麻素和对羟基苯甲醇的反相高效液相色谱法(RP-HPLC),色谱柱为Waterssymmetry C18反相柱(4.6 mm×150 mm,5μm),流动相:0.02%磷酸溶液-甲醇(体积比为5:95),检测波长:220 nm,流速:0.5 mL/min,柱温35:℃。
通过单因素实验和响应面实验设计对生物转化中的条件进行优化,确定相应的转化条件为初始pH 4.5,在30 mL转化液中底物浓度为3 mg/mL,加入静息细胞量为6.5 g,添加质量分数为3%的葡萄糖和1.5%的吐温80,并对转化动力学进行相应的研究,初步构建了转化合成动力学模型。
初步研究转化体系中糖基转移酶的酶活测定方法和酶学性质,测得糖基转移酶的最适反应温度为50℃,在30℃稳定性最好;糖基转移酶的最适作用pH为6,在pH为6-7范围内稳定性较好。
采用硅胶柱色谱、葡聚糖凝胶Sephadex LH-20等色谱分离方法相结合对转化产物进行分离纯化,分离得到的两个组分经RP-HPLC、核磁共振分析等分析证实是天麻素和对羟基苯甲醇。
对合成的天麻素进行动物实验,研究其抗炎及对免疫功能的影响。研究证实合成的天麻素具有抗炎的作用,并且对正常小鼠的非特异性免疫和细胞免疫功能具有增强作用。
Gastrodin is the important ingredients of Gastrodia elata Blume owing to its important pharmacological properties. In this thesis, the major research is focused on the optimization of biotransformation conditions for gastrodin from HBA, separation and purification of the products, and structural identification, and then carrying out the animal experiments to study its anti-inflammatory effection and immune function using the synthetic gastrodin, and finally investigating the characterisitcs of glucosyltransferase from the biotransformation system. This study will provide the basis for understanding the biotransformation mechanism and regulating the gastrodin production.
The HPLC system used throughout this study consisted of two Waters 510 pumps, a sample injector (Rheodyne, Cotati, CA, USA) with a 20μL loop, and a Waters 996 photodiode array detector. The column used was a reversed-phase Symmetry C18 column (150 mm×3.9 mm i.d., 5 urn; Waters). The mobile phase was acetonitrile (solvent A)-0.02% phosphoric acid solution (solvent B) in the aso-gradient mode as follows: 95% A and 5% B. The flow rate was 0.5 mL/min, and the effluent was monitored at 220 nm. Routine sample calculations were made by comparison of the peak area with that of the standard curve.
Single factor experiments and response surface methodology were applied to optimize the bioconversion conditions for gastrodin production. The optimized results showed that the optimum transformation conditions composed of 3 mg/mL HBA, 6.5 g/30 mL inoculums level, 1.5% Tween 80, pH 4.5, and transformation temperature at 28℃. The bioconversion kinetics model was simulated and constructed in this study.
The glucosyltransferase enzyme for biosynthesis of gastrodin from HBA was preliminary studied. The optimum temperature of the enzyme reaction was 50℃, and the pH was 6.0. The enzyme was stable at less than 50℃, and pH 6.0～7.0. The transformed products were obtained and identified by extraction, chromatography and its analysis from ~1H, ~(13)C-NMR. The bioactive functions of the synthetic gastrodin were examined in vivo design, which include anti-inflammatory and immune function. It was confirmed that the synthetic gastrodin had anti-inflammatory effection and immune stimulatory function.
首先研究建立转化体系中天麻素和对羟基苯甲醇的检测方法,确定转化体系中天麻素和对羟基苯甲醇的反相高效液相色谱法(RP-HPLC),色谱柱为Waterssymmetry C18反相柱(4.6 mm×150 mm,5μm),流动相:0.02%磷酸溶液-甲醇(体积比为5:95),检测波长:220 nm,流速:0.5 mL/min,柱温35:℃。
通过单因素实验和响应面实验设计对生物转化中的条件进行优化,确定相应的转化条件为初始pH 4.5,在30 mL转化液中底物浓度为3 mg/mL,加入静息细胞量为6.5 g,添加质量分数为3%的葡萄糖和1.5%的吐温80,并对转化动力学进行相应的研究,初步构建了转化合成动力学模型。
初步研究转化体系中糖基转移酶的酶活测定方法和酶学性质,测得糖基转移酶的最适反应温度为50℃,在30℃稳定性最好;糖基转移酶的最适作用pH为6,在pH为6-7范围内稳定性较好。
采用硅胶柱色谱、葡聚糖凝胶Sephadex LH-20等色谱分离方法相结合对转化产物进行分离纯化,分离得到的两个组分经RP-HPLC、核磁共振分析等分析证实是天麻素和对羟基苯甲醇。
对合成的天麻素进行动物实验,研究其抗炎及对免疫功能的影响。研究证实合成的天麻素具有抗炎的作用,并且对正常小鼠的非特异性免疫和细胞免疫功能具有增强作用。
Gastrodin is the important ingredients of Gastrodia elata Blume owing to its important pharmacological properties. In this thesis, the major research is focused on the optimization of biotransformation conditions for gastrodin from HBA, separation and purification of the products, and structural identification, and then carrying out the animal experiments to study its anti-inflammatory effection and immune function using the synthetic gastrodin, and finally investigating the characterisitcs of glucosyltransferase from the biotransformation system. This study will provide the basis for understanding the biotransformation mechanism and regulating the gastrodin production.
The HPLC system used throughout this study consisted of two Waters 510 pumps, a sample injector (Rheodyne, Cotati, CA, USA) with a 20μL loop, and a Waters 996 photodiode array detector. The column used was a reversed-phase Symmetry C18 column (150 mm×3.9 mm i.d., 5 urn; Waters). The mobile phase was acetonitrile (solvent A)-0.02% phosphoric acid solution (solvent B) in the aso-gradient mode as follows: 95% A and 5% B. The flow rate was 0.5 mL/min, and the effluent was monitored at 220 nm. Routine sample calculations were made by comparison of the peak area with that of the standard curve.
Single factor experiments and response surface methodology were applied to optimize the bioconversion conditions for gastrodin production. The optimized results showed that the optimum transformation conditions composed of 3 mg/mL HBA, 6.5 g/30 mL inoculums level, 1.5% Tween 80, pH 4.5, and transformation temperature at 28℃. The bioconversion kinetics model was simulated and constructed in this study.
The glucosyltransferase enzyme for biosynthesis of gastrodin from HBA was preliminary studied. The optimum temperature of the enzyme reaction was 50℃, and the pH was 6.0. The enzyme was stable at less than 50℃, and pH 6.0～7.0. The transformed products were obtained and identified by extraction, chromatography and its analysis from ~1H, ~(13)C-NMR. The bioactive functions of the synthetic gastrodin were examined in vivo design, which include anti-inflammatory and immune function. It was confirmed that the synthetic gastrodin had anti-inflammatory effection and immune stimulatory function.
引文
[1]Dordick JS,Khmelnitsky YL,Sergeeva MV.The evolution of biotransformation technologies.Current Opinion in Microbiology,1998,1:311-318.
[2]Suga T,Hirata T.Biotransformation of exogenous substrates by plant cell cultures.Phytochemistry,1990,29(8):2393-2406.
[3]Whitesides GM,Wong CH.Enzymes as Catalysts in Synthetic Organic Chemistry.Angew.Chem.Int.Ed.Engl.,1985,24(8):617-718.
[4]徐仁生.大然药物化学.北京:科学出版社,1993,12:894-911.
[5]宋欣.微生物酶转化技术.北京:化学业出版社,2004,5:11.
[6]Ooi Y,Hashimoto T,Mitsuo N,Satoh T.Enzymic synthesis of chemically unstable cardiac glycosides by β-galactosidase from Aspergillus oryzae.Tetrahedron Lett,1984,25(21):2241-2244.
[7]Ogawa J,Shimizu S.Microbial enzymes:new industrial applicatiofis from traditional screening methods.Tibtech,1999,17:13-20.
[8]Stewart JD.Organic transformations catalyzed by engineered yeast cells and related systems.Current Opinion in Biotechnology,2000,11:363-368.
[9]Davies GJ,Gloster TM,Henrissat B.Recent structural insights into the expanding world of carbohydrate-active enzymes.Current Opinion in structural Biology,2005,15:637-645.
[10]Blanchard S,Thorson JS.Enzymatic tools for engineering natural product glycosylation.Current Opionion in Chemical Biology,2006,10:263-271.
[11]张玉彬.生物催化的手性合成.北京:化学工业出版社,2001,8:119.
[12]戈梅,陈代杰.关注微生物来源的新药开发.生命科学,2005,17(1):15-18.
[13]Hanson RL,Wasylyk JM,Nanduri VB.Site-specific enzymatic hydrolysis of taxanes at C-10and C-13.J Biol Chem,1994,269:22145-22149.
[14]Patel R N.Tour de paclitaxel:biocatalysis for semisynthesis.Annu.Rev.Microbiol,1998,98:361-395.
[15]Patel R N,Banerjee A,Nanduri V.Enzymatic acetylation of 10-deacetylbaccatin Ⅲ to baccatin Ⅲ by C-10 deacetylase from Nocardioides luteus SC13913.Enzyme Microb.Technol.,2000,27:371-375.
[16]褚志义.生物合成药物学.北京:化学工业出版社,2000,259-262.
[17]占纪勋.青篙素等五种大然活性成分的微生物转化研究.华东理工大学博士论文,2002.
[18]Staebler A,Cruz A,van der Goot W,Pinheiro HM,Cabral JMS,Fernandes P.Optimization of androstenedione production in an organic-aqueous two-liquid phase system.Journal of Molecular Catalysis B:Enzymatic,2004,29:19-23.
[19]Mian L,Kim J-W,Peeples T L.Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.Journal of Biotechnology,2002,93:15-26.
[20]Bie ST,Du LX,Zhang LM,Lu FP.Bioconversion of methyl-testosterone in a biphasic system.Process Biochemistry,2005,40:3309-3313.
[21]Cull SG,Holbrey JD,Vargas-Mora V,et al.Room-temperature ionic liquids as replacements for organic solvents in multiphase bioprocess operations.Biotechnol Bioeng,2000,69:227-233.
[22]Howarth J,James P,Dai J.Immobilized baker's yeast reduction of ketones in an ionic liquid,[bmim]PF_6 and water mix.Tetrahedron Letters,2001,42:7517-7519.
[23]刘幽燕,李青云,覃益民等.聚氨酯泡沫固定化产碱杆菌细胞生物转化氰化物.环境科学,2006,27(3):586-589.
[24]李胜华,伍贤进,刘惠君.大孔树脂分离纯化翻白草中总黄酮优化工艺研究,时珍国医国药,2008,19(6):1301-1303.
[25]张莉华,许新德,孙晓霞等.硅胶柱层析法纯化天然维生素E.中国食品添加剂,2008,3:105-109.
[26]袁华,吴莉,吴元欣等.硅胶柱层析法提纯茶多酚的研究,华中师范大学学报:自然科学版,2007,41(4):553-556.
[27]袁文鹏,丛日山,杨秀霞等.水溶性海参皂苷的分离纯化及其抗真菌活性研究.山东大学学报:理学版,2007,42(5):69-73.
[28]Changjun Yang,Daxiang Li,Xiaochun Wan.Combination of HSCCC and Sephadex LH-20methods:An approach to isolation and purification of the main individual theaflavins from black tea.Journal of Chromatography B,2008,861(1):140-144.
[29]Gerold J,Tanja S,Kathrin F,et al.Separation of betalains from berries of Phytolacca americana by ion-pair high-speed counter-current chromatography.Journal of Chromatography A,2008,1190(1):63-73.
[30]Li Hua-Bin,Chen Feng.Preparative isolation and purification of gastrodin from the Chinese medicinal plant Gastrodia elata by high-speed counter-current chromatography.Journal of Chromatography A,2004,1052(2):229-232.
[31]Sutherland IA.,Fisher D.Role of counter-current chromatography in the modernisation of Chinese herbal medicines.Journal of Chromatography A,2009,1216(4):740-753.
[32]Marston A,Hostettmann K.Developments in the application of counter-current chromatography to plant analysis.Journal of Chromatography A,2006,1112(1-2):181-194.
[33]Mong Liang,Vivin Y.T.Chen,Hsiu-Ling Chen.et al.A simple and direct isolation of whey components from raw milk by gel filtration chromatography and structural characterization by Fourier transform Raman spectroscopy.Talanta,2006,69(5):1269-1277.
[34]Anna C,Fisichella S,Foti S,et al.Isolation by gel-permeation chromatography of a non-covalent complex of Cibacron Blue F3G-A with human serum albumin.Journal of Chromatography A,1996,736(1-2):115-123.
[35]赵金芬.复方天麻密环糖肽片治疗心脑血管疾病的药理与临床应用进展.临床和实验医学杂志,2009,8(8):146-147.
[36]塞冬.黄芪天麻对老年心脑血管疾病患者发中微量元素影响的研究.锦卅医学院学报,1994,15(4):12-13.
[37]任惠锋.天麻钩藤饮加味治疗脑血管疾病126例.中国中医药杂志,2008,6(3):49-51.
[38]杨进林,兰进,徐锦堂.天麻的研究进展.中草药,2000,31(1):66-69.
[39]Ojemann LM,Nelson WL,Shin,DS et al.Tian ma,an ancient Chinese herb,offers new options for the treatment of epilesy and other conditions.Epilepsy and Behavior,2006,8:376-383.
[40]Gong XD,Sucher,NJ.Stroke therapy in traditional Chinese medicine(TCM):prospects for drug discovery and development.TIPS,1999,20:191-196.
[41]王惠国,冯宝民.密环菌多糖免疫调节活性的实验研究.陕西科技大学学报:自然科学版,2009,27(2):62-64.
[42]闫良.复方天麻密环菌片联合尼莫地平治疗颈性眩晕30例疗效观察.世界中西医结合杂志,2009,4(1):34-35.
[43]林志彬,章达敏,夏冬.密环菌多糖小鼠免疫功能的影响.中国药理学通报,1988,4(2):92-96.
[44]杨峻山,苏亚伦,王玉兰.密环菌菌丝体化学成分的研究Ⅳ:密环菌癸素的分离与结构 鉴定.药学学报,1990,25(5):353-356.
[45]沈业寿,洪毅.密环菌多糖分离纯化及其部分理化性质.中国食用菌,1999,18(1):38-40.
[46]郭顺星,徐锦堂,肖培根.密环菌的化学成分及应用研究.微生物学通报,1996,23(4):239-240.
[47]张庆林.密环菌多糖的分离纯化及组成分析.中国药学杂志,1995,30(7):401-403.
[48]张赫名.天麻的研究进展.中药研究与信息,2005,7(11):19-22.
[49]史清文,杨章群,顾吉顺.中药天麻的研究概况.中医药信息,1992,1:36.
[50]王兴,周明眉.天麻中有效成分的提取工艺.华西药学杂志,2003,18(4):269.
[51]杨世林,兰进,徐锦堂.天麻的研究进展.中草药,2000,31(1):66-69.
[52]郭增军,苏纪兰,王开.天麻提取工艺的研究.中药材,2002,25(5):348-349.
[53]刘明,李更生.天麻提取工艺的优化选择.基层中药杂志,2000,14(2):28-29.
[54]刘金龙,陈根洪,郑子江.提高天麻素提取纯度研究.食品科学,2005,26(9):329-333.
[55]张赫名,年四辉.水提天麻工艺的初步研究.云南中医学院学报,2006,29(2):17-19.
[56]周俊,杨雁宾,杨崇仁.天麻的化学研究Ⅱ一天麻素及其类似物的合成.化学学报,1980,32(2):162-166.
[57]戴晓畅,彭啸,吴松福,杨万松,毛宇.天麻素及其类似酚性糖普的化学合成工艺研究.云南民族大学学报(自然科学版),2004,13(2):83.
[58]Lin Yong-Da,Liang Yu-Da,Liang Shi-Gin,et al.Synthesis of fluorine-containing natural gastrodin and its analogues.Journal of Fluorine Chemistry,1990,46(3):367-374.
[59]朱宏利.微生物转化法合成天麻素等天然活性成分的研究.西北大学.
[60]蔡洁,丁家宜,华亚男.人参毛状根生物合成天麻素转化体系的建立.植物资源与环境学报,2005,14(2):29-31.
[61]Zhan JX,Guo H Zh,Dai J Cx.Biotransformation of gastrodin by Mucor spinosus.Journal of Chinese Pharmaceutical Sciences,2001,10(4):187-189.
[61]Dai JQ,Gong ZH,Zhu DM,et al.Biotransfonmation of gastro din by cell suspension cultures of Catharanthus roseus.Acta Botanica Sinica,2002,44(3):377-378.
[62]戴均贵,鲁丹丹,崔亚君等.桔梗悬浮培养对细胞天麻素的生物转化.药学学报,2001,36(12):942-943.
[63]李婧.HPLC测定天麻素的含量和有关物质.西南民族大学学报:自然科学版.2009,35(4):781-784.
[64]Zhang W,Sheng YX,Zhang JL.Determination and pharmacokinetics of gastrodin and p-hydroxybenzylalcohol after oral administration of Gastrodia elata Bl.extract in rats by high-performance liquid chromatography-electrospray ionization mass spectrometric method.Phytomedicine,2008,15(10):844-850.
[65]Huang Xiaodong,Kong Liang,Li Xin,et al.Strategy for analysis and screening of bioactive compounds in traditional Chinese medicines.Journal of Chromatography B,2004,812(1-2):71-84.
[66]田野,李秀芳,杨莲等.HPLC法对三产地天麻中天麻素的含量测定.云南中医学院学报,2008,31(3):6-8.
[67]沈卫华,顾雪中.天麻素紫外分光光度法定量探讨.时珍国医国药,1999,10(9):666-666.
[68]申玉华,董文慧.双波长薄层扫描法测定天芎头痛宁胶囊中天麻素的含量.中药新药与临床药理,1995,6(3):33-35.
[69]熊慧林,薛捷.薄层扫描法测定天麻杜仲胶囊中天麻素的含量.中国民族民间医药杂志,1998.3:38-40.
[70]何丹鸿,陈鼎雄.二阶导数光谱法测定复方天麻头痛口服液中天麻素含量.海峡药学,1998,10(4):47-48.
[71]Zhao Yunkun,Cao Qiu-e,Xiang Yanqiao,et al.Identification and determination of active components in Gastrodia elata Bl.by capillary electrophoresis.Journal of Chromatography A,1999,849(1):277-283.
[72]王德先,杨更亮,刘海光等.天麻及其伪品的毛细管电泳鉴别及天麻素的测定.中草药,2001,32(4):352.
[73]陈启和.弹性蛋白酶发酵工艺及其发酵动力学研究[D].浙江大学.2003.
[74]张永勤,薛长湖,汤浩源等.还原糖的可见分光光度法研究进展.食品与发酵工业,2007,33(5):97-99.
[75]Chen QH,He GQ,Ali Mokhtar AM.Optimization of medium composition for the production of elastase by Bacillus sp.EL31410 with response surface methodology.Enzyme and Microbial Technology,2002,30(5):667-672.
[76]Hirotani M,Kuroda R,Suzuki H,Yoshikava T.Cloning and expression of UDP-glucose:flavonoid 7-O-glucosyl-transferase from hairy root cultures of Scutellaria baicalensis.Planta.2000,210:1006-1013.
[77]Hancock SM,Vaughan MD,Withers SG.Engineering of glycosidases and glycosyltransferases.Current Opinion in Chemical Biology,2006,10:509-519.
[78]Kikuchi N,Narimatsu H.Bioinformatics for comprehensive finding and analysis of glycosyltransferases.Biochimica et Biophysica Acta,2006,1760:578-583.
[79]Isayenkova J,Wray V,Nimtz M,Strack D,et al.Cloning and functional characterization of two regioselective flavonoid glucosyltransferases from Beta vulgaris.Phytochemistry,2006,67:1598-1612.
[80]Kaminaga Y.,Sahin F.P.,Mizukami H..Molecular cloning and characterization of a glucosyltransferase catalyzing glucosylation of curcumin in cultured Catharanthus roseus cells.FEBS Letters,2004,567:197-202.
[81]Taguchi G,Ubukata T,Hayashida N,et al.Cloning and characterization of a glucosyltransferase that reacts on 7-hydroxyl group of flavanol and 3-hydroxyl group of coumarin from tobacco cells.Archives of Biochemistry and Biophysics,2003,420:95-102.
[82]Kim JH,Kim BG,Ko JH,Lee YS,Hur HG,et al.Molecular cloning,expression and characterization of a flavonoid glycosyltransferase from Arabidopsis thaliana.Plant Science,2006,170:897-903.
[83]Kita M,Hirata Y,Moriguchi T,Tomoko El,Mastumoto R,et al..Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus.FEBS Letters,2000,469:172-178.
[84]Charles PM,et al.Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato.The Plant Journal,1997,11:227.
[85]Luque T,Okano K,Reilly DDO.Characterization of a novel silkworm phenol UDP-glucosyltransferase.Eur J Biochem,2002,269:819-825.
[86]Stasyk OV,Nazarko TY,Stasyk OG,Krasovska OS,et al.Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica.Cell Biology International,2003,27:947-952.
[87]Lee Shiow-Ling,Chen Wen-Chang.Optimization of medium composition for the production of glucosyltransferase by Aspergillus niger with response surface methodology.Enzyme and Microbial Technology,1997,21:436-440.
[88]李永兆,王骏,梁秀琴.柱色谱硅胶的应用.生物技术产业,2006,2:66-69.
[89]洪燕龙,徐德生,冯怡等.天麻提取物的提取、纯化工艺研究.中成药,2008,30(2):204-207.
[90]杨屹,唐睿,丁明玉.大孔吸附树脂对天麻素的吸附与分离特性的研究.分析试验室,2004,23(11):1-4.
[91]张慧丽,孙印石,郑毅男.葡聚糖凝胶LH-20柱层析法分离人参皂苷Re的研究.吉林农业 大学学报,2005,27(3):293-295.
[92]司传领,邓小娟,王丹等.三球悬铃木树皮的酚类化学成分研究.林产化学与工业,2008,28(4):31-33.
[93]黄占波,宋冬梅,陈发奎.天麻化学成分的研究.中国药物化学杂志,2005,15(4):227-229.
[94]鞠桂春.天麻及其制剂的药理作用和临床应用研究进展.中国药业,2008,17(1):64-66.
[95]杨杰,赵朝华,宗长宏等.短暂性MCAO大鼠注射天麻素具有神经保护作用.第四军医大学学报,2008,29(4):295-297.
[96]胡京红,司银楚,洪庆涛等.天麻素对体外模拟脑缺血损伤大鼠脑微血管内皮细胞的保护作用.中华中医药杂志(原中国医药学报),2007,22(2):62-64.
[97]Zeng X,Zhang Y,Zhang S,et al.A microdialysis study of effects of gastrodin on neurochemical changes in the ischemic/reperfused rat cerebral hippocampus.Biol Pham Bull,2007,30(4):801-804.
[98]王昭君,习杨彦彬,刘佳等.天麻素对快速衰老小鼠大脑组织衰老相关基因表达的影响.解剖科学进展,2007,13(4):353-357.
[99]Ji Wook Jung,Byung Hoon Yoon,Hye Rim Oh,et al.Anxiolytic-Like Effects of Gastrodia elata and Its Phenolic Constituents in Mice.Biol Pharm Bull,2006,29(2):261-265.
[100]黄秀凤,唐红,肖颐等.合成天麻素对心肌细胞中毒性损伤的保护作用.成都中医学院学报,1990,13(2):27-28.
[2]Suga T,Hirata T.Biotransformation of exogenous substrates by plant cell cultures.Phytochemistry,1990,29(8):2393-2406.
[3]Whitesides GM,Wong CH.Enzymes as Catalysts in Synthetic Organic Chemistry.Angew.Chem.Int.Ed.Engl.,1985,24(8):617-718.
[4]徐仁生.大然药物化学.北京:科学出版社,1993,12:894-911.
[5]宋欣.微生物酶转化技术.北京:化学业出版社,2004,5:11.
[6]Ooi Y,Hashimoto T,Mitsuo N,Satoh T.Enzymic synthesis of chemically unstable cardiac glycosides by β-galactosidase from Aspergillus oryzae.Tetrahedron Lett,1984,25(21):2241-2244.
[7]Ogawa J,Shimizu S.Microbial enzymes:new industrial applicatiofis from traditional screening methods.Tibtech,1999,17:13-20.
[8]Stewart JD.Organic transformations catalyzed by engineered yeast cells and related systems.Current Opinion in Biotechnology,2000,11:363-368.
[9]Davies GJ,Gloster TM,Henrissat B.Recent structural insights into the expanding world of carbohydrate-active enzymes.Current Opinion in structural Biology,2005,15:637-645.
[10]Blanchard S,Thorson JS.Enzymatic tools for engineering natural product glycosylation.Current Opionion in Chemical Biology,2006,10:263-271.
[11]张玉彬.生物催化的手性合成.北京:化学工业出版社,2001,8:119.
[12]戈梅,陈代杰.关注微生物来源的新药开发.生命科学,2005,17(1):15-18.
[13]Hanson RL,Wasylyk JM,Nanduri VB.Site-specific enzymatic hydrolysis of taxanes at C-10and C-13.J Biol Chem,1994,269:22145-22149.
[14]Patel R N.Tour de paclitaxel:biocatalysis for semisynthesis.Annu.Rev.Microbiol,1998,98:361-395.
[15]Patel R N,Banerjee A,Nanduri V.Enzymatic acetylation of 10-deacetylbaccatin Ⅲ to baccatin Ⅲ by C-10 deacetylase from Nocardioides luteus SC13913.Enzyme Microb.Technol.,2000,27:371-375.
[16]褚志义.生物合成药物学.北京:化学工业出版社,2000,259-262.
[17]占纪勋.青篙素等五种大然活性成分的微生物转化研究.华东理工大学博士论文,2002.
[18]Staebler A,Cruz A,van der Goot W,Pinheiro HM,Cabral JMS,Fernandes P.Optimization of androstenedione production in an organic-aqueous two-liquid phase system.Journal of Molecular Catalysis B:Enzymatic,2004,29:19-23.
[19]Mian L,Kim J-W,Peeples T L.Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.Journal of Biotechnology,2002,93:15-26.
[20]Bie ST,Du LX,Zhang LM,Lu FP.Bioconversion of methyl-testosterone in a biphasic system.Process Biochemistry,2005,40:3309-3313.
[21]Cull SG,Holbrey JD,Vargas-Mora V,et al.Room-temperature ionic liquids as replacements for organic solvents in multiphase bioprocess operations.Biotechnol Bioeng,2000,69:227-233.
[22]Howarth J,James P,Dai J.Immobilized baker's yeast reduction of ketones in an ionic liquid,[bmim]PF_6 and water mix.Tetrahedron Letters,2001,42:7517-7519.
[23]刘幽燕,李青云,覃益民等.聚氨酯泡沫固定化产碱杆菌细胞生物转化氰化物.环境科学,2006,27(3):586-589.
[24]李胜华,伍贤进,刘惠君.大孔树脂分离纯化翻白草中总黄酮优化工艺研究,时珍国医国药,2008,19(6):1301-1303.
[25]张莉华,许新德,孙晓霞等.硅胶柱层析法纯化天然维生素E.中国食品添加剂,2008,3:105-109.
[26]袁华,吴莉,吴元欣等.硅胶柱层析法提纯茶多酚的研究,华中师范大学学报:自然科学版,2007,41(4):553-556.
[27]袁文鹏,丛日山,杨秀霞等.水溶性海参皂苷的分离纯化及其抗真菌活性研究.山东大学学报:理学版,2007,42(5):69-73.
[28]Changjun Yang,Daxiang Li,Xiaochun Wan.Combination of HSCCC and Sephadex LH-20methods:An approach to isolation and purification of the main individual theaflavins from black tea.Journal of Chromatography B,2008,861(1):140-144.
[29]Gerold J,Tanja S,Kathrin F,et al.Separation of betalains from berries of Phytolacca americana by ion-pair high-speed counter-current chromatography.Journal of Chromatography A,2008,1190(1):63-73.
[30]Li Hua-Bin,Chen Feng.Preparative isolation and purification of gastrodin from the Chinese medicinal plant Gastrodia elata by high-speed counter-current chromatography.Journal of Chromatography A,2004,1052(2):229-232.
[31]Sutherland IA.,Fisher D.Role of counter-current chromatography in the modernisation of Chinese herbal medicines.Journal of Chromatography A,2009,1216(4):740-753.
[32]Marston A,Hostettmann K.Developments in the application of counter-current chromatography to plant analysis.Journal of Chromatography A,2006,1112(1-2):181-194.
[33]Mong Liang,Vivin Y.T.Chen,Hsiu-Ling Chen.et al.A simple and direct isolation of whey components from raw milk by gel filtration chromatography and structural characterization by Fourier transform Raman spectroscopy.Talanta,2006,69(5):1269-1277.
[34]Anna C,Fisichella S,Foti S,et al.Isolation by gel-permeation chromatography of a non-covalent complex of Cibacron Blue F3G-A with human serum albumin.Journal of Chromatography A,1996,736(1-2):115-123.
[35]赵金芬.复方天麻密环糖肽片治疗心脑血管疾病的药理与临床应用进展.临床和实验医学杂志,2009,8(8):146-147.
[36]塞冬.黄芪天麻对老年心脑血管疾病患者发中微量元素影响的研究.锦卅医学院学报,1994,15(4):12-13.
[37]任惠锋.天麻钩藤饮加味治疗脑血管疾病126例.中国中医药杂志,2008,6(3):49-51.
[38]杨进林,兰进,徐锦堂.天麻的研究进展.中草药,2000,31(1):66-69.
[39]Ojemann LM,Nelson WL,Shin,DS et al.Tian ma,an ancient Chinese herb,offers new options for the treatment of epilesy and other conditions.Epilepsy and Behavior,2006,8:376-383.
[40]Gong XD,Sucher,NJ.Stroke therapy in traditional Chinese medicine(TCM):prospects for drug discovery and development.TIPS,1999,20:191-196.
[41]王惠国,冯宝民.密环菌多糖免疫调节活性的实验研究.陕西科技大学学报:自然科学版,2009,27(2):62-64.
[42]闫良.复方天麻密环菌片联合尼莫地平治疗颈性眩晕30例疗效观察.世界中西医结合杂志,2009,4(1):34-35.
[43]林志彬,章达敏,夏冬.密环菌多糖小鼠免疫功能的影响.中国药理学通报,1988,4(2):92-96.
[44]杨峻山,苏亚伦,王玉兰.密环菌菌丝体化学成分的研究Ⅳ:密环菌癸素的分离与结构 鉴定.药学学报,1990,25(5):353-356.
[45]沈业寿,洪毅.密环菌多糖分离纯化及其部分理化性质.中国食用菌,1999,18(1):38-40.
[46]郭顺星,徐锦堂,肖培根.密环菌的化学成分及应用研究.微生物学通报,1996,23(4):239-240.
[47]张庆林.密环菌多糖的分离纯化及组成分析.中国药学杂志,1995,30(7):401-403.
[48]张赫名.天麻的研究进展.中药研究与信息,2005,7(11):19-22.
[49]史清文,杨章群,顾吉顺.中药天麻的研究概况.中医药信息,1992,1:36.
[50]王兴,周明眉.天麻中有效成分的提取工艺.华西药学杂志,2003,18(4):269.
[51]杨世林,兰进,徐锦堂.天麻的研究进展.中草药,2000,31(1):66-69.
[52]郭增军,苏纪兰,王开.天麻提取工艺的研究.中药材,2002,25(5):348-349.
[53]刘明,李更生.天麻提取工艺的优化选择.基层中药杂志,2000,14(2):28-29.
[54]刘金龙,陈根洪,郑子江.提高天麻素提取纯度研究.食品科学,2005,26(9):329-333.
[55]张赫名,年四辉.水提天麻工艺的初步研究.云南中医学院学报,2006,29(2):17-19.
[56]周俊,杨雁宾,杨崇仁.天麻的化学研究Ⅱ一天麻素及其类似物的合成.化学学报,1980,32(2):162-166.
[57]戴晓畅,彭啸,吴松福,杨万松,毛宇.天麻素及其类似酚性糖普的化学合成工艺研究.云南民族大学学报(自然科学版),2004,13(2):83.
[58]Lin Yong-Da,Liang Yu-Da,Liang Shi-Gin,et al.Synthesis of fluorine-containing natural gastrodin and its analogues.Journal of Fluorine Chemistry,1990,46(3):367-374.
[59]朱宏利.微生物转化法合成天麻素等天然活性成分的研究.西北大学.
[60]蔡洁,丁家宜,华亚男.人参毛状根生物合成天麻素转化体系的建立.植物资源与环境学报,2005,14(2):29-31.
[61]Zhan JX,Guo H Zh,Dai J Cx.Biotransformation of gastrodin by Mucor spinosus.Journal of Chinese Pharmaceutical Sciences,2001,10(4):187-189.
[61]Dai JQ,Gong ZH,Zhu DM,et al.Biotransfonmation of gastro din by cell suspension cultures of Catharanthus roseus.Acta Botanica Sinica,2002,44(3):377-378.
[62]戴均贵,鲁丹丹,崔亚君等.桔梗悬浮培养对细胞天麻素的生物转化.药学学报,2001,36(12):942-943.
[63]李婧.HPLC测定天麻素的含量和有关物质.西南民族大学学报:自然科学版.2009,35(4):781-784.
[64]Zhang W,Sheng YX,Zhang JL.Determination and pharmacokinetics of gastrodin and p-hydroxybenzylalcohol after oral administration of Gastrodia elata Bl.extract in rats by high-performance liquid chromatography-electrospray ionization mass spectrometric method.Phytomedicine,2008,15(10):844-850.
[65]Huang Xiaodong,Kong Liang,Li Xin,et al.Strategy for analysis and screening of bioactive compounds in traditional Chinese medicines.Journal of Chromatography B,2004,812(1-2):71-84.
[66]田野,李秀芳,杨莲等.HPLC法对三产地天麻中天麻素的含量测定.云南中医学院学报,2008,31(3):6-8.
[67]沈卫华,顾雪中.天麻素紫外分光光度法定量探讨.时珍国医国药,1999,10(9):666-666.
[68]申玉华,董文慧.双波长薄层扫描法测定天芎头痛宁胶囊中天麻素的含量.中药新药与临床药理,1995,6(3):33-35.
[69]熊慧林,薛捷.薄层扫描法测定天麻杜仲胶囊中天麻素的含量.中国民族民间医药杂志,1998.3:38-40.
[70]何丹鸿,陈鼎雄.二阶导数光谱法测定复方天麻头痛口服液中天麻素含量.海峡药学,1998,10(4):47-48.
[71]Zhao Yunkun,Cao Qiu-e,Xiang Yanqiao,et al.Identification and determination of active components in Gastrodia elata Bl.by capillary electrophoresis.Journal of Chromatography A,1999,849(1):277-283.
[72]王德先,杨更亮,刘海光等.天麻及其伪品的毛细管电泳鉴别及天麻素的测定.中草药,2001,32(4):352.
[73]陈启和.弹性蛋白酶发酵工艺及其发酵动力学研究[D].浙江大学.2003.
[74]张永勤,薛长湖,汤浩源等.还原糖的可见分光光度法研究进展.食品与发酵工业,2007,33(5):97-99.
[75]Chen QH,He GQ,Ali Mokhtar AM.Optimization of medium composition for the production of elastase by Bacillus sp.EL31410 with response surface methodology.Enzyme and Microbial Technology,2002,30(5):667-672.
[76]Hirotani M,Kuroda R,Suzuki H,Yoshikava T.Cloning and expression of UDP-glucose:flavonoid 7-O-glucosyl-transferase from hairy root cultures of Scutellaria baicalensis.Planta.2000,210:1006-1013.
[77]Hancock SM,Vaughan MD,Withers SG.Engineering of glycosidases and glycosyltransferases.Current Opinion in Chemical Biology,2006,10:509-519.
[78]Kikuchi N,Narimatsu H.Bioinformatics for comprehensive finding and analysis of glycosyltransferases.Biochimica et Biophysica Acta,2006,1760:578-583.
[79]Isayenkova J,Wray V,Nimtz M,Strack D,et al.Cloning and functional characterization of two regioselective flavonoid glucosyltransferases from Beta vulgaris.Phytochemistry,2006,67:1598-1612.
[80]Kaminaga Y.,Sahin F.P.,Mizukami H..Molecular cloning and characterization of a glucosyltransferase catalyzing glucosylation of curcumin in cultured Catharanthus roseus cells.FEBS Letters,2004,567:197-202.
[81]Taguchi G,Ubukata T,Hayashida N,et al.Cloning and characterization of a glucosyltransferase that reacts on 7-hydroxyl group of flavanol and 3-hydroxyl group of coumarin from tobacco cells.Archives of Biochemistry and Biophysics,2003,420:95-102.
[82]Kim JH,Kim BG,Ko JH,Lee YS,Hur HG,et al.Molecular cloning,expression and characterization of a flavonoid glycosyltransferase from Arabidopsis thaliana.Plant Science,2006,170:897-903.
[83]Kita M,Hirata Y,Moriguchi T,Tomoko El,Mastumoto R,et al..Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in Citrus.FEBS Letters,2000,469:172-178.
[84]Charles PM,et al.Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato.The Plant Journal,1997,11:227.
[85]Luque T,Okano K,Reilly DDO.Characterization of a novel silkworm phenol UDP-glucosyltransferase.Eur J Biochem,2002,269:819-825.
[86]Stasyk OV,Nazarko TY,Stasyk OG,Krasovska OS,et al.Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica.Cell Biology International,2003,27:947-952.
[87]Lee Shiow-Ling,Chen Wen-Chang.Optimization of medium composition for the production of glucosyltransferase by Aspergillus niger with response surface methodology.Enzyme and Microbial Technology,1997,21:436-440.
[88]李永兆,王骏,梁秀琴.柱色谱硅胶的应用.生物技术产业,2006,2:66-69.
[89]洪燕龙,徐德生,冯怡等.天麻提取物的提取、纯化工艺研究.中成药,2008,30(2):204-207.
[90]杨屹,唐睿,丁明玉.大孔吸附树脂对天麻素的吸附与分离特性的研究.分析试验室,2004,23(11):1-4.
[91]张慧丽,孙印石,郑毅男.葡聚糖凝胶LH-20柱层析法分离人参皂苷Re的研究.吉林农业 大学学报,2005,27(3):293-295.
[92]司传领,邓小娟,王丹等.三球悬铃木树皮的酚类化学成分研究.林产化学与工业,2008,28(4):31-33.
[93]黄占波,宋冬梅,陈发奎.天麻化学成分的研究.中国药物化学杂志,2005,15(4):227-229.
[94]鞠桂春.天麻及其制剂的药理作用和临床应用研究进展.中国药业,2008,17(1):64-66.
[95]杨杰,赵朝华,宗长宏等.短暂性MCAO大鼠注射天麻素具有神经保护作用.第四军医大学学报,2008,29(4):295-297.
[96]胡京红,司银楚,洪庆涛等.天麻素对体外模拟脑缺血损伤大鼠脑微血管内皮细胞的保护作用.中华中医药杂志(原中国医药学报),2007,22(2):62-64.
[97]Zeng X,Zhang Y,Zhang S,et al.A microdialysis study of effects of gastrodin on neurochemical changes in the ischemic/reperfused rat cerebral hippocampus.Biol Pham Bull,2007,30(4):801-804.
[98]王昭君,习杨彦彬,刘佳等.天麻素对快速衰老小鼠大脑组织衰老相关基因表达的影响.解剖科学进展,2007,13(4):353-357.
[99]Ji Wook Jung,Byung Hoon Yoon,Hye Rim Oh,et al.Anxiolytic-Like Effects of Gastrodia elata and Its Phenolic Constituents in Mice.Biol Pharm Bull,2006,29(2):261-265.
[100]黄秀凤,唐红,肖颐等.合成天麻素对心肌细胞中毒性损伤的保护作用.成都中医学院学报,1990,13(2):27-28.