阶梯生物催化协同提取薯蓣皂苷元及其洁净工艺研究
|
摘要
天然药物是现代新药发现的重要源泉,对天然药物进行化学与生物学活性研究,发现具有开发前景的结构新类型作为先导化合物,利用适当的分离纯化手段,将其提取出来,进行结构修饰和优化以发现新药,成为目前制备合成药物和中药一类新药的重要发展方向。
利用生物酶催化技术实现天然药物先导化合物的提取和分离是一项新兴的技术。植物的细胞壁以及黏液质、果胶、淀粉等杂质是提取有效成分的主要屏障,这些成分影响植物细胞中活性成分的浸出,如果选用恰当的酶,通过酶催化反应使这些杂质成分水解或降解,则可加速有效成分的释放提取。
本文选用根茎类植物的典型代表——盾叶薯蓣作为研究对象,考察在提取盾叶薯蓣有效成分—薯蓣皂苷元的过程中,多种生物酶阶梯分步加入的工艺条件及其对产品收率和质量的影响,同时对薯蓣皂苷元生产废液以及废粗纤维渣的综合利用进行了研究,对阶梯生物催化协同提取工艺和其它四种提取工艺作了简要的对比。本文还对酶的催化机制作了初步探讨。
利用阶梯生物催化协同提取薯蓣皂苷元技术时,依次加入纤维素酶和果胶酶复合酶制剂、淀粉酶、糖化酶,各种酶的最佳作用条件依次为——纤维素酶和果胶酶复合酶制剂:液用量为1.0‰(1g原料加0.001mL的酶液),作用时间为2h,反应温度为50℃,反应液pH值为6.0;淀粉酶:液用量为1.5‰,反应温度为75℃,反应液pH值为5.5,其催化终点根据碘试剂的显色反应来确定,当碘试剂遇反应液不再显蓝色时视为终点;糖化酶:液用量为1.0‰,作用时间为2h,反应温度为60℃,反应液pH值为5.5。
薯蓣皂苷元的生产废液经过中和、活性炭脱色、浓缩、结晶等工序后,可以提取出葡萄糖;薯蓣粗纤维渣可以采用乙醇-水的有机溶媒体系,以氢氧化钠为碱化剂,一氯乙酸为醚化剂来制取CMC。
与其它四种工艺相比,阶梯生物催化协同提取工艺产品收率最高,达到2.79%;该工艺能够将薯蓣植物中98%的薯蓣皂苷元提取出来,所得皂苷元产品与标准品的红外谱图像吻合,经HPLC测定纯度达95%以上,熔点为202℃-204℃,符合陕西省优级品标准。
本文说明生物酶技术在天然药物有效成分的提取中具有广阔的发展前景。
Natural products are the significant source of new medicine nowadays. The chemical and biological properties of natural products are studied in order to find lead compounds with great prospect, which structure can be modified after separation and purification. It is an important means of developing new medicine.
It is a new technology to extract and separate active constituents from natural plants with enzyme catalysis. Cell walls,pectin,starch and others are common impurities in plants, which do harm to the extraction of effective constituents. If proper enzymes are employed to act on natural plants, the impurities above can be decomposed, which is of great advantage to the extraction of effective ones.
Dioscorea zingiberensis C. H. Wright, a typical medical plant, was chosen to be studied in the paper. The process conditions of stepwise enzyme catalysis as well as the influence of it on the yield and melting point of diosgenin were studied. Meanwhile, the reuse of waste liquor and residue in production process was discussed. In the paper, comparison of this method and others was also made according to the yield and melting point of product. The mechanism of enzyme catalysis was done some research on here.
Cellulase and pectinase compound, amylase and diastatic enzyme were added in order in the method of stepwise enzyme catalysis, and their optimum conditions respectively were: dose 1‰, 1.5‰, 1‰, temperature 50℃, 75℃, 60℃, pH6.0, 5.5, 5.5. The reaction time of cellulase and pectinase compound and diastatic enzyme was 2h and 6h. But that of amylase was determined by color reaction of iodine, which meant end point when iodine didn’t turn blue.
Glucose could be extracted from waste liquor after treatment of neutralization, decolorization of activated carbon, condensation and crystallization. Coarse fabric of the plant could be made into CMC-an important chemical with wide use, after being alkalified and etherifed.
Compared with other methods, it can improve the yield and quality with less energy consumption. 98% diosgenin from plants is extracted. IR spectrum of the product is in accordance with that of the standard, the purity of it is more than 95% according to its HPLC chromatogram, and its melting point is 202~204℃, which reaches the first - grade standard.
利用生物酶催化技术实现天然药物先导化合物的提取和分离是一项新兴的技术。植物的细胞壁以及黏液质、果胶、淀粉等杂质是提取有效成分的主要屏障,这些成分影响植物细胞中活性成分的浸出,如果选用恰当的酶,通过酶催化反应使这些杂质成分水解或降解,则可加速有效成分的释放提取。
本文选用根茎类植物的典型代表——盾叶薯蓣作为研究对象,考察在提取盾叶薯蓣有效成分—薯蓣皂苷元的过程中,多种生物酶阶梯分步加入的工艺条件及其对产品收率和质量的影响,同时对薯蓣皂苷元生产废液以及废粗纤维渣的综合利用进行了研究,对阶梯生物催化协同提取工艺和其它四种提取工艺作了简要的对比。本文还对酶的催化机制作了初步探讨。
利用阶梯生物催化协同提取薯蓣皂苷元技术时,依次加入纤维素酶和果胶酶复合酶制剂、淀粉酶、糖化酶,各种酶的最佳作用条件依次为——纤维素酶和果胶酶复合酶制剂:液用量为1.0‰(1g原料加0.001mL的酶液),作用时间为2h,反应温度为50℃,反应液pH值为6.0;淀粉酶:液用量为1.5‰,反应温度为75℃,反应液pH值为5.5,其催化终点根据碘试剂的显色反应来确定,当碘试剂遇反应液不再显蓝色时视为终点;糖化酶:液用量为1.0‰,作用时间为2h,反应温度为60℃,反应液pH值为5.5。
薯蓣皂苷元的生产废液经过中和、活性炭脱色、浓缩、结晶等工序后,可以提取出葡萄糖;薯蓣粗纤维渣可以采用乙醇-水的有机溶媒体系,以氢氧化钠为碱化剂,一氯乙酸为醚化剂来制取CMC。
与其它四种工艺相比,阶梯生物催化协同提取工艺产品收率最高,达到2.79%;该工艺能够将薯蓣植物中98%的薯蓣皂苷元提取出来,所得皂苷元产品与标准品的红外谱图像吻合,经HPLC测定纯度达95%以上,熔点为202℃-204℃,符合陕西省优级品标准。
本文说明生物酶技术在天然药物有效成分的提取中具有广阔的发展前景。
Natural products are the significant source of new medicine nowadays. The chemical and biological properties of natural products are studied in order to find lead compounds with great prospect, which structure can be modified after separation and purification. It is an important means of developing new medicine.
It is a new technology to extract and separate active constituents from natural plants with enzyme catalysis. Cell walls,pectin,starch and others are common impurities in plants, which do harm to the extraction of effective constituents. If proper enzymes are employed to act on natural plants, the impurities above can be decomposed, which is of great advantage to the extraction of effective ones.
Dioscorea zingiberensis C. H. Wright, a typical medical plant, was chosen to be studied in the paper. The process conditions of stepwise enzyme catalysis as well as the influence of it on the yield and melting point of diosgenin were studied. Meanwhile, the reuse of waste liquor and residue in production process was discussed. In the paper, comparison of this method and others was also made according to the yield and melting point of product. The mechanism of enzyme catalysis was done some research on here.
Cellulase and pectinase compound, amylase and diastatic enzyme were added in order in the method of stepwise enzyme catalysis, and their optimum conditions respectively were: dose 1‰, 1.5‰, 1‰, temperature 50℃, 75℃, 60℃, pH6.0, 5.5, 5.5. The reaction time of cellulase and pectinase compound and diastatic enzyme was 2h and 6h. But that of amylase was determined by color reaction of iodine, which meant end point when iodine didn’t turn blue.
Glucose could be extracted from waste liquor after treatment of neutralization, decolorization of activated carbon, condensation and crystallization. Coarse fabric of the plant could be made into CMC-an important chemical with wide use, after being alkalified and etherifed.
Compared with other methods, it can improve the yield and quality with less energy consumption. 98% diosgenin from plants is extracted. IR spectrum of the product is in accordance with that of the standard, the purity of it is more than 95% according to its HPLC chromatogram, and its melting point is 202~204℃, which reaches the first - grade standard.
引文
[1] 黄宇芳,天然药物的研究与开发,河南科技,2003,8:25~27
[2] 凌良元,新药开发现状及趋势,时珍国医国药,2005,16(4):362~363
[3] 邱家学,郭小斌,邵蓉,加入 WTO 与医药知识产权保护,中国药业,2000,9(10):8~11
[4] 彭军,李元建,创新:中国“入世”后新药研制的必由之路,湖南医科大学学报,2000,1:21~25
[5] 于德泉,展望从天然产物创新药物研究,中国医学科学院学报,2002,24(4):335~338
[6] 孙曼霁,周廷冲,我国的新药研究与开发,化学通报,1994:93~97
[7] 于德泉,从天然产物创制新药的发展趋势,中国工程科学,1999,1(1):87-90
[8] Shu YZ, Recent natural products based drug development: a pharmaceutical industry perspective, J Nat Prod, 1998,61(8):1053~1071
[9] 刘惠莲,抓住机遇求发展,迎接挑战勇向前一试论入世后我国中药新药研究的机遇与挑战,第六次全国中西医结合实验医学学术研讨会:357-360
[10] 石建功,于德泉,展望天然产物化学发展方向,21 世纪有机化学发展战略,北京:化工出版社,2002,171~198
[11] Baker JT, Borris RP, Ccragg GM, et al, Natural product drug discovery and development: new perspectives on intermational collaboration, J Nat Prod, 1995, 58(8):1325~1357
[12] Fahy JW,Zalcmann AT, Talalay P, The chemical diversity and distribution of glucosinalates and isothiocyanates among plants, Phytochem, 2001, 56(1):5~51
[13] Vogler B, Klaiber I, Kraus W, et al, Combination of LC-MS and LC-NMR as a tool for the structuredetermination of natural products, J Nat Prod, 1998, 61(2):175-178
[14] Gordon EM, Barett RW, Dower WJ, et al, Application of combinatorial technologies Ⅱ Combinatorial organic synthesis, library screen strategies and future direction, J Med Chem, 1994, 37:1385
[15] Andrew FA, Junsden, Barrie Wilkinson, et al, Engineering Brosder Specificity into an Antibiotic-Polyketide Synthase, Science, 1998, 279:199~202
[16] John R Jacobsen, Richard Hutchinson, David E Cane, et al, Precursor-Directed Biosynthesis of Erythromycin Analogs by an Engineered Polyketide Synthase, Science, 1997, 277:363~369
[17] David E Cane, Christopher T Walsh, Chaitan Khosla, et al, Harnessing the Biosynthetic Code: Combinations Permutations, and Mutations, Science, 1998, 282:63~68
[18] 杨晓春,吴镭,天然药物化学研究在我国新药创制中的作用,中国新药杂志,2000,9(6):361~363
[19] 郝小江,植物化学与天然新药研究之管见,贵州科学,2000,18(1):27~30
[20] 陈尚妍,刘诚,范国荣,天然产物中活性成分提取分离及分析技术,江西林业科技,2005,3:32~36
[21] 姜彬慧,胡筱敏,左小红,酶技术与中药现代化,世界科学技术,2004,6(2):46~49
[22] 侯嵘峤,孟宪纾,徐育新,以酶解法从中药及药渣中制备β—葡萄糖的研究,沈阳药学院学报,1994,11(4):289~293
[23] 林桂云,生物技术与中药现代化,成都大学学报,2001,20(2):34~36
[24] 胡之璧,刘涤,生物技术在中药现代化中的地位和作用,世界科学技术——中药现代化,2000,2(5):23~26
[25] 赵立亚,金凤燮,鱼红闪,酶法生产稀有人参皂苷极其产物成分的分析,大连轻工业学院学报,2002,21(2):112~115
[26] 戴均贵,果德安,现代中药生物技术研究综述及展望,世界科学技术——中药现代化,2000,2(5):27~30
[27] 金凤燮,鱼红闪,中草药活性物质转化与其酶学,中国天然药物研究与发展论,2003:108~109
[28] 江苏植物研究所. 新华本草纲要(第一册),上海: 上海科学技术出版社, 1988,195~196
[29] 丁怡,天然甾体皂苷研究进展,中国新药杂志,2000,9(8):521~524
[30] 裴鉴, 丁志遵, 中国植物志(第十六卷), 北京: 科学出版社, 1985,60~12l.
[31] 丁志遵,唐世蓉,秦慧贞,甾体激素药源植物,北京:科学出版社,1983,14~111
[32] 宋发军,甾体药物源植物薯蓣属植物中薯蓣皂苷元的研究及生长状况,中成药. 2003, 25(3):232-234.
[33] 徐成基,中国薯蓣资源,成都:四川科学技术出版社,2000,1~349.
[34] 王泽斌,湖南省薯蓣资源研究初报,中国野生植物资源,2002,21(1):31~32
[35] 聂凌鸿, 林淑英, 宁正祥,薯蓣属植物中薯蓣皂苷元的研究进展,中国生化药物杂志,2004,25(5):318~320
[36] Liu CHL, Chen YY, Tang YF, et al, Isolation and identification of steroidal saponins from Dioscorea zingiberensis Wright, Acta Botanica Sinica, 1984, 26(3):283~289
[37] 徐任生,天然产物化学,北京:科学出版社,2004,437~444
[38] 康阿龙,孙文基,汤迎爽,薄层扫描法测定盾叶薯蓣中盾叶新苷和薯蓣皂苷的含量,药物分析杂志,2003,1:59~60
[39] 胡湘春,鱼红闪,金凤燮,穿龙薯蓣皂苷的分离提纯,大连轻工业学院学报,2004,23(1):25~29
[40] 巨勇,彭海若,贾忠建,黑叶菝葜甾体皂苷成分研究,兰州大学学报,1994,30(1):64~67
[41] Fukuhara K, Kubo I, Isolation of steroidal glycoalkaloids from Solamum Incanum by two countercurrent chromatographic methods, Phytochem, 1991, 30:685-687
[42] Rothrock JW, Hammes PA, Mcaleer WJ, Isolation of Diosgenin by Acid Hydrolysis of Saponin, Ind Eng Chem Res, 1957,49:186-189
[43] Martin Weissenberg, Isolation of solasodine and other steroidal alkaloids and sapogenins by direct hydrolysis-extraction of Solanum plants or glycosides therefrom, Phytochem, 2001, 58:501-508
[44] John C, Analysis of Fenugreek sapogenins by gas and liquid chromatography, J Chromat, 1997, 133:222~225.
[45] 周振起,封玉贤,薯蓣皂苷元分离工艺的研究及其综合利用,西北植物学报, 1995,15(3):254~260.
[46] 葛发欢, 史庆龙, 林香仙, 等,超临界CO2 从黄山药中萃取薯蓣皂素的工艺研究,中草药,2000 , 31(3):181~183
[47] Liu B, Lockwood GB, Gifford LA,Supercritical fluid extraction of diosgenin from tubers of Dioscorea nipponical,J of Chromat, 1995, 690(2):250-253
[48] Hardman R, Wood CN,The effect of ripening and aqueous incubation on the yield of diosgenin and yamogenin from the fruit of Balanites pedicellaris,Planta Med, 1971, 20 (4):350-355
[49] Hardman R, Wood CN,The effect of incubation with chlorinated phenoxy acids on the yield of steroidal sapogenin from the fruit parts of Balanites ofbicularis,Phytochem, 1971, 10:757-763
[50] Paseshnichenko VA,Fermentation of Dioscorea deltoidea rhizomes,Prikl Biokhim Mikrobiol, 1973,9(4):583~587
[51] 北京医学院药学系,预发酵提高薯蓣皂苷元收率的研究,中草药通讯,1977,(4):17~20
[52] 四川省生物研究所薯蓣综合利用组,预发酵提高薯蓣皂素产率及淀粉综合利用的研究,植物学报,1975,17(3):242~246
[53] Fonin VS, Shain SS, Vandyshev VV, et al, Enzymatic hydrolysis of steroid glycosides in plant raw material for medicinal use, Prikl Biokhim Mikrobiol, 1996, 32:514-518
[54] 佟玲, 张胜科,李锦,酶解法提取薯蓣皂素的工艺研究,陕西师范大学学报(自然科学版),2003,31(2):81
[55] 张黎明,张露亿,杜连祥,酶解法提取葫芦巴种子中薯蓣皂苷元的工艺研究,农业工程学报,2005,21(2):161~164
[56] Sanjay PM, Vilas GG, Recovery of Diosgenin From Dioscorea Rhizomes Using Aqueous Hydrotropic Solutions of Sodium Cumeme Sulfonate, Ind Eng Chem Res, 2004,43:5339~5346
[57] Brain KR, Fazli RY, Hardman R, Rapid determination of C25 ipimeric steroidal sapogenin in plants, Phytochem, 1968, 7:1810-1817
[58] 江天生,旋光法测定薯蓣皂苷元含量, 吉林大学学报, 1997 ,18 (2):63~64.
[59] Csizer E. Microdeterminationg of △5-steroids after thin-layer chromatographicseparation, J Chromat, 1973, 76:502-505
[60] 都述虎. RP-HPLC法测定穿龙薯蓣总皂苷中薯蓣皂苷元的含量. 中国药科大学学报, 2001, 32 (1):37~40
[61] Rozanski A, A simplified method of extraction of diosgenin from Dioscorea Tubers and its determination by using gas-liquid chromatography, Analyst, 1972, 97(11):968-972
[62] 金川嘉,单文腆,王翔燕,薯蓣皂苷元的棒状薄层色谱-火焰离子检测扫描测定法,药物分析杂志,1985,5(2):117-118
[63] 泰铁英,李晓雄,比色法用于薯蓣皂素提取的终点控制,中草药,1993,24(11):613
[64] 徐礼焱,刘爱茹,皂苷元分析方法研究Ⅲ:薯蓣皂苷元库仑滴定法,药学学报,1981,16(1):39-44
[65] 汤兴利,徐增来,夏冰,等,用盾叶薯蓣生产薯蓣皂苷元预发酵与水解条件、优化,植物资源与环境学报,2004,13(3):35~37
[66] Mei Dong, Xi-Zhi Feng, Ben-xiang Wang, et al, Two Novel stanol Saponins from the Rhizomes of Dioscorea Panthaica Prain et Burkill Their Cytotoxic Activity, Tetrahedro, 2001, 57:498-506
[67] 刘春,皂素生产废水污染特点及治理对策探讨,环境保护科学, 2001, 27 (6):22- 24
[68] 单丽伟, 冯贵颖, 朱丹, 等,上流式厌氧污泥床(UASB) 处理皂素废水的研究,西北农林科技大学学报,2003,10(31):109~112
[69] 宋凤敏,呼世斌,酵母菌处理皂素生产废水的研究,环境污染治理技术与设备,2004,5(4):66~69
[70] 彭志英,食品酶学导论,北京:中国轻工业出版社,2003,113~121
[71] 周云龙,植物生物学,北京:高教出版社,2004,17~37
[72] Lenting HBM, Warmoeskerken MCG, Mechanism of interaction between cellulose action and applied shear force, an hypothesis, J Biotech, 2001, 89(2):217-226
[73] Sebastien Violot, Nushin Aghajari, Mirjam Czjzek, et al, Structure of a Full Length Psychrophilic Cellulase from Pseudoalteromonas haloplanktis revealed by X-ray Diffraction and Small Angle X-ray Scattering, J Mol Biol, 2005(348):1211–1224
[74] Jin-Won Park, Kwinam Park, Hocheol Song, et al, Saccharification and adsorption characteristics of modified cellulases with hydrophilic/hydrophobic copolymers, J Biotech, 2002, 93:203–208
[75] 诸葛斌,姚惠源,诸葛健,生淀粉糖化酶催化位点氨基酸酶合成调控的初步研究,工业微生物,2002,32(3):24~27
[2] 凌良元,新药开发现状及趋势,时珍国医国药,2005,16(4):362~363
[3] 邱家学,郭小斌,邵蓉,加入 WTO 与医药知识产权保护,中国药业,2000,9(10):8~11
[4] 彭军,李元建,创新:中国“入世”后新药研制的必由之路,湖南医科大学学报,2000,1:21~25
[5] 于德泉,展望从天然产物创新药物研究,中国医学科学院学报,2002,24(4):335~338
[6] 孙曼霁,周廷冲,我国的新药研究与开发,化学通报,1994:93~97
[7] 于德泉,从天然产物创制新药的发展趋势,中国工程科学,1999,1(1):87-90
[8] Shu YZ, Recent natural products based drug development: a pharmaceutical industry perspective, J Nat Prod, 1998,61(8):1053~1071
[9] 刘惠莲,抓住机遇求发展,迎接挑战勇向前一试论入世后我国中药新药研究的机遇与挑战,第六次全国中西医结合实验医学学术研讨会:357-360
[10] 石建功,于德泉,展望天然产物化学发展方向,21 世纪有机化学发展战略,北京:化工出版社,2002,171~198
[11] Baker JT, Borris RP, Ccragg GM, et al, Natural product drug discovery and development: new perspectives on intermational collaboration, J Nat Prod, 1995, 58(8):1325~1357
[12] Fahy JW,Zalcmann AT, Talalay P, The chemical diversity and distribution of glucosinalates and isothiocyanates among plants, Phytochem, 2001, 56(1):5~51
[13] Vogler B, Klaiber I, Kraus W, et al, Combination of LC-MS and LC-NMR as a tool for the structuredetermination of natural products, J Nat Prod, 1998, 61(2):175-178
[14] Gordon EM, Barett RW, Dower WJ, et al, Application of combinatorial technologies Ⅱ Combinatorial organic synthesis, library screen strategies and future direction, J Med Chem, 1994, 37:1385
[15] Andrew FA, Junsden, Barrie Wilkinson, et al, Engineering Brosder Specificity into an Antibiotic-Polyketide Synthase, Science, 1998, 279:199~202
[16] John R Jacobsen, Richard Hutchinson, David E Cane, et al, Precursor-Directed Biosynthesis of Erythromycin Analogs by an Engineered Polyketide Synthase, Science, 1997, 277:363~369
[17] David E Cane, Christopher T Walsh, Chaitan Khosla, et al, Harnessing the Biosynthetic Code: Combinations Permutations, and Mutations, Science, 1998, 282:63~68
[18] 杨晓春,吴镭,天然药物化学研究在我国新药创制中的作用,中国新药杂志,2000,9(6):361~363
[19] 郝小江,植物化学与天然新药研究之管见,贵州科学,2000,18(1):27~30
[20] 陈尚妍,刘诚,范国荣,天然产物中活性成分提取分离及分析技术,江西林业科技,2005,3:32~36
[21] 姜彬慧,胡筱敏,左小红,酶技术与中药现代化,世界科学技术,2004,6(2):46~49
[22] 侯嵘峤,孟宪纾,徐育新,以酶解法从中药及药渣中制备β—葡萄糖的研究,沈阳药学院学报,1994,11(4):289~293
[23] 林桂云,生物技术与中药现代化,成都大学学报,2001,20(2):34~36
[24] 胡之璧,刘涤,生物技术在中药现代化中的地位和作用,世界科学技术——中药现代化,2000,2(5):23~26
[25] 赵立亚,金凤燮,鱼红闪,酶法生产稀有人参皂苷极其产物成分的分析,大连轻工业学院学报,2002,21(2):112~115
[26] 戴均贵,果德安,现代中药生物技术研究综述及展望,世界科学技术——中药现代化,2000,2(5):27~30
[27] 金凤燮,鱼红闪,中草药活性物质转化与其酶学,中国天然药物研究与发展论,2003:108~109
[28] 江苏植物研究所. 新华本草纲要(第一册),上海: 上海科学技术出版社, 1988,195~196
[29] 丁怡,天然甾体皂苷研究进展,中国新药杂志,2000,9(8):521~524
[30] 裴鉴, 丁志遵, 中国植物志(第十六卷), 北京: 科学出版社, 1985,60~12l.
[31] 丁志遵,唐世蓉,秦慧贞,甾体激素药源植物,北京:科学出版社,1983,14~111
[32] 宋发军,甾体药物源植物薯蓣属植物中薯蓣皂苷元的研究及生长状况,中成药. 2003, 25(3):232-234.
[33] 徐成基,中国薯蓣资源,成都:四川科学技术出版社,2000,1~349.
[34] 王泽斌,湖南省薯蓣资源研究初报,中国野生植物资源,2002,21(1):31~32
[35] 聂凌鸿, 林淑英, 宁正祥,薯蓣属植物中薯蓣皂苷元的研究进展,中国生化药物杂志,2004,25(5):318~320
[36] Liu CHL, Chen YY, Tang YF, et al, Isolation and identification of steroidal saponins from Dioscorea zingiberensis Wright, Acta Botanica Sinica, 1984, 26(3):283~289
[37] 徐任生,天然产物化学,北京:科学出版社,2004,437~444
[38] 康阿龙,孙文基,汤迎爽,薄层扫描法测定盾叶薯蓣中盾叶新苷和薯蓣皂苷的含量,药物分析杂志,2003,1:59~60
[39] 胡湘春,鱼红闪,金凤燮,穿龙薯蓣皂苷的分离提纯,大连轻工业学院学报,2004,23(1):25~29
[40] 巨勇,彭海若,贾忠建,黑叶菝葜甾体皂苷成分研究,兰州大学学报,1994,30(1):64~67
[41] Fukuhara K, Kubo I, Isolation of steroidal glycoalkaloids from Solamum Incanum by two countercurrent chromatographic methods, Phytochem, 1991, 30:685-687
[42] Rothrock JW, Hammes PA, Mcaleer WJ, Isolation of Diosgenin by Acid Hydrolysis of Saponin, Ind Eng Chem Res, 1957,49:186-189
[43] Martin Weissenberg, Isolation of solasodine and other steroidal alkaloids and sapogenins by direct hydrolysis-extraction of Solanum plants or glycosides therefrom, Phytochem, 2001, 58:501-508
[44] John C, Analysis of Fenugreek sapogenins by gas and liquid chromatography, J Chromat, 1997, 133:222~225.
[45] 周振起,封玉贤,薯蓣皂苷元分离工艺的研究及其综合利用,西北植物学报, 1995,15(3):254~260.
[46] 葛发欢, 史庆龙, 林香仙, 等,超临界CO2 从黄山药中萃取薯蓣皂素的工艺研究,中草药,2000 , 31(3):181~183
[47] Liu B, Lockwood GB, Gifford LA,Supercritical fluid extraction of diosgenin from tubers of Dioscorea nipponical,J of Chromat, 1995, 690(2):250-253
[48] Hardman R, Wood CN,The effect of ripening and aqueous incubation on the yield of diosgenin and yamogenin from the fruit of Balanites pedicellaris,Planta Med, 1971, 20 (4):350-355
[49] Hardman R, Wood CN,The effect of incubation with chlorinated phenoxy acids on the yield of steroidal sapogenin from the fruit parts of Balanites ofbicularis,Phytochem, 1971, 10:757-763
[50] Paseshnichenko VA,Fermentation of Dioscorea deltoidea rhizomes,Prikl Biokhim Mikrobiol, 1973,9(4):583~587
[51] 北京医学院药学系,预发酵提高薯蓣皂苷元收率的研究,中草药通讯,1977,(4):17~20
[52] 四川省生物研究所薯蓣综合利用组,预发酵提高薯蓣皂素产率及淀粉综合利用的研究,植物学报,1975,17(3):242~246
[53] Fonin VS, Shain SS, Vandyshev VV, et al, Enzymatic hydrolysis of steroid glycosides in plant raw material for medicinal use, Prikl Biokhim Mikrobiol, 1996, 32:514-518
[54] 佟玲, 张胜科,李锦,酶解法提取薯蓣皂素的工艺研究,陕西师范大学学报(自然科学版),2003,31(2):81
[55] 张黎明,张露亿,杜连祥,酶解法提取葫芦巴种子中薯蓣皂苷元的工艺研究,农业工程学报,2005,21(2):161~164
[56] Sanjay PM, Vilas GG, Recovery of Diosgenin From Dioscorea Rhizomes Using Aqueous Hydrotropic Solutions of Sodium Cumeme Sulfonate, Ind Eng Chem Res, 2004,43:5339~5346
[57] Brain KR, Fazli RY, Hardman R, Rapid determination of C25 ipimeric steroidal sapogenin in plants, Phytochem, 1968, 7:1810-1817
[58] 江天生,旋光法测定薯蓣皂苷元含量, 吉林大学学报, 1997 ,18 (2):63~64.
[59] Csizer E. Microdeterminationg of △5-steroids after thin-layer chromatographicseparation, J Chromat, 1973, 76:502-505
[60] 都述虎. RP-HPLC法测定穿龙薯蓣总皂苷中薯蓣皂苷元的含量. 中国药科大学学报, 2001, 32 (1):37~40
[61] Rozanski A, A simplified method of extraction of diosgenin from Dioscorea Tubers and its determination by using gas-liquid chromatography, Analyst, 1972, 97(11):968-972
[62] 金川嘉,单文腆,王翔燕,薯蓣皂苷元的棒状薄层色谱-火焰离子检测扫描测定法,药物分析杂志,1985,5(2):117-118
[63] 泰铁英,李晓雄,比色法用于薯蓣皂素提取的终点控制,中草药,1993,24(11):613
[64] 徐礼焱,刘爱茹,皂苷元分析方法研究Ⅲ:薯蓣皂苷元库仑滴定法,药学学报,1981,16(1):39-44
[65] 汤兴利,徐增来,夏冰,等,用盾叶薯蓣生产薯蓣皂苷元预发酵与水解条件、优化,植物资源与环境学报,2004,13(3):35~37
[66] Mei Dong, Xi-Zhi Feng, Ben-xiang Wang, et al, Two Novel stanol Saponins from the Rhizomes of Dioscorea Panthaica Prain et Burkill Their Cytotoxic Activity, Tetrahedro, 2001, 57:498-506
[67] 刘春,皂素生产废水污染特点及治理对策探讨,环境保护科学, 2001, 27 (6):22- 24
[68] 单丽伟, 冯贵颖, 朱丹, 等,上流式厌氧污泥床(UASB) 处理皂素废水的研究,西北农林科技大学学报,2003,10(31):109~112
[69] 宋凤敏,呼世斌,酵母菌处理皂素生产废水的研究,环境污染治理技术与设备,2004,5(4):66~69
[70] 彭志英,食品酶学导论,北京:中国轻工业出版社,2003,113~121
[71] 周云龙,植物生物学,北京:高教出版社,2004,17~37
[72] Lenting HBM, Warmoeskerken MCG, Mechanism of interaction between cellulose action and applied shear force, an hypothesis, J Biotech, 2001, 89(2):217-226
[73] Sebastien Violot, Nushin Aghajari, Mirjam Czjzek, et al, Structure of a Full Length Psychrophilic Cellulase from Pseudoalteromonas haloplanktis revealed by X-ray Diffraction and Small Angle X-ray Scattering, J Mol Biol, 2005(348):1211–1224
[74] Jin-Won Park, Kwinam Park, Hocheol Song, et al, Saccharification and adsorption characteristics of modified cellulases with hydrophilic/hydrophobic copolymers, J Biotech, 2002, 93:203–208
[75] 诸葛斌,姚惠源,诸葛健,生淀粉糖化酶催化位点氨基酸酶合成调控的初步研究,工业微生物,2002,32(3):24~27