紫杉醇分离纯化工艺的研究
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摘要
本文以吸附层析为主,探索研究了适合于从红豆杉粗提物及内生真菌发酵液中分离纯化紫杉醇的技术。
传统的液液萃取由于溶剂用量大、溶液易乳化等缺点限制了其在真菌发酵液中分离紫杉醇的应用。本文以树脂层析取代传统的液液萃取步骤,解决了上述不足。从六种树脂中筛选出了吸附性能最好的HZ818大孔吸附树脂,通过单因素及正交试验对工艺进行了优化。通过一步树脂层析可使原料中的紫杉醇同时达到脱色和纯化的双重目的。此步平均回收率在98%以上。
以正相层析作为整体工艺的第二步。研究了硅胶及碱性氧化铝两种介质柱的层析效果。通过考察洗脱液组成、洗脱液强度、洗脱速率、高径比等因素对两种柱进行了对比,其中样品经氧化铝层析后,紫杉醇含量有所增加,这主要是由于氧化铝的催化作用,使其它某些紫杉烷类物质转化成了紫杉醇。树脂处理后的样品再经氧化铝层析后纯度达到30%,平均回收率在125%左右。
最后一步采用C18反相层析对前两步纯化得到的产品进行精制,通过优化层析洗脱剂组成、洗脱剂强度、洗脱方式以及进料量等操作条件,采用双柱串联的方法,得到纯度在93-94%的紫杉醇,再继以结晶重结晶步骤,除去少量杂质得到纯度在98%左右的紫杉醇晶体。经本工艺分离纯化后的紫杉醇总回收率约为88%。采用波长扫描、高压液相及质谱对制得产品进行鉴定,结果与文献上报道得紫杉醇性质一致。
Separation and purification of taxol from Taxus Yunnanensis extract and fermentation liquor of yew endophyte were mainly carried out by adsorption chromatography.
Because of large usage of organic solvent and emulsification , traditional liquor-liquor extraction was limited on some degree,especially on separation from fermentation liquor of yew endophyte. This thesis used resin chromatography insteadly as the first step,and HZ818 macroreticular resin was screened from six kind resins,it showed excellent effects of adsorption and separation. Meanwhile, single-factor and orthogonal experiments were carried out to optimize the technological parameters. Through this chromatography process,decoloration and purification were achieved,the even recovery of taxol was more than 98%.
Normal-phase chromatography process was established as the second step. Silica gel and Al2O3 were chosen to study. Elutent composition、strength and rate of elution、rate of height and diameter were optimized. Results showed that the content of taxol was greatly increased after Al2O3 chromatography. It might be that some taxol-like taxanes converted to taxol under the catalyzing of Al2O3. Through resin and Al2O3 chromatography,the purity of taxol reached 30%, and the recovery reached about 125%.
C18 reversed-phase chromatography was selected as the final step to further purify taxol. By optimization of eluent composition, strength of elution, elution mode and capacity of loading, with two column in series, the purity of taxol can be reached nearly 93-94%, followed with crystallization and recrystallization, the purity can increase to about 98%. Through this whole process, the total recovery of taxol was about 88%.
The structure of taxol was identified with IR、HPLC and MS, which was identical with the results that reported on documents.
传统的液液萃取由于溶剂用量大、溶液易乳化等缺点限制了其在真菌发酵液中分离紫杉醇的应用。本文以树脂层析取代传统的液液萃取步骤,解决了上述不足。从六种树脂中筛选出了吸附性能最好的HZ818大孔吸附树脂,通过单因素及正交试验对工艺进行了优化。通过一步树脂层析可使原料中的紫杉醇同时达到脱色和纯化的双重目的。此步平均回收率在98%以上。
以正相层析作为整体工艺的第二步。研究了硅胶及碱性氧化铝两种介质柱的层析效果。通过考察洗脱液组成、洗脱液强度、洗脱速率、高径比等因素对两种柱进行了对比,其中样品经氧化铝层析后,紫杉醇含量有所增加,这主要是由于氧化铝的催化作用,使其它某些紫杉烷类物质转化成了紫杉醇。树脂处理后的样品再经氧化铝层析后纯度达到30%,平均回收率在125%左右。
最后一步采用C18反相层析对前两步纯化得到的产品进行精制,通过优化层析洗脱剂组成、洗脱剂强度、洗脱方式以及进料量等操作条件,采用双柱串联的方法,得到纯度在93-94%的紫杉醇,再继以结晶重结晶步骤,除去少量杂质得到纯度在98%左右的紫杉醇晶体。经本工艺分离纯化后的紫杉醇总回收率约为88%。采用波长扫描、高压液相及质谱对制得产品进行鉴定,结果与文献上报道得紫杉醇性质一致。
Separation and purification of taxol from Taxus Yunnanensis extract and fermentation liquor of yew endophyte were mainly carried out by adsorption chromatography.
Because of large usage of organic solvent and emulsification , traditional liquor-liquor extraction was limited on some degree,especially on separation from fermentation liquor of yew endophyte. This thesis used resin chromatography insteadly as the first step,and HZ818 macroreticular resin was screened from six kind resins,it showed excellent effects of adsorption and separation. Meanwhile, single-factor and orthogonal experiments were carried out to optimize the technological parameters. Through this chromatography process,decoloration and purification were achieved,the even recovery of taxol was more than 98%.
Normal-phase chromatography process was established as the second step. Silica gel and Al2O3 were chosen to study. Elutent composition、strength and rate of elution、rate of height and diameter were optimized. Results showed that the content of taxol was greatly increased after Al2O3 chromatography. It might be that some taxol-like taxanes converted to taxol under the catalyzing of Al2O3. Through resin and Al2O3 chromatography,the purity of taxol reached 30%, and the recovery reached about 125%.
C18 reversed-phase chromatography was selected as the final step to further purify taxol. By optimization of eluent composition, strength of elution, elution mode and capacity of loading, with two column in series, the purity of taxol can be reached nearly 93-94%, followed with crystallization and recrystallization, the purity can increase to about 98%. Through this whole process, the total recovery of taxol was about 88%.
The structure of taxol was identified with IR、HPLC and MS, which was identical with the results that reported on documents.
引文
[1] Wani M C, Taylor H L, Wall M E, et al. Plant antitumor agents VI. The Isolation and Structure of Taxol, a Novel Antileukemic and Antitumor Agent from Taxus brevifolia[J]. J Am Chem Soc, 1971, 93:2325-2327
[2] Schiff P B, Fant J, Horwitz S B. Promotion of microtubule assembly in vitro by taxol[J]. Nature, 1979, 277:665-667
[3] Hoke S H, Wood J M, Cooks R G, et al. Rapid screening for taxanes by tandem mass spectrometry[J]. Anal Chem,1992, 64:2313-2317
[4] Powell R G, Miller R W, Smith C R, et al. Taxol content and extraction from Taxus wallichiana[J]. J Chem Soc Communm, 1979:102-105
[5] Witherup K M, Look S A, Stasko M W, et al. Taxus spp. Needles contain amounts of taxol comparable to the bark of Taxus brevifolia: Analysis and Isolation[J]. J Nat Prod. 1990, 53(5):1249-1255
[6] Senilh V, Blechert S, Colin M, et al. New analogs of taxol extracts from Taxus baccata[J]. J Nat Prod. 1984, 47:131-137
[7] Nair. Process for the isolation and purification of taxol and taxanes from Taxus spp[P]. US patent, 5279949, 1994
[8] Rao K V, Gainesville F. Isolating and purification of taxol and its natural analogue[P]. US patent, 5380916, 1995
[9] Xu L X, Liu A R. Determination of taxol in the extract of Taxes chinensis by HPLC method[J]. Acts Pharm, 1991, 26(7):537-540
[10]锅田惠助. JP patent, 6157329[P], 1994
[11] Ferman G R, Gore T P. Isolation and purification of precursors of taxol and its analogues[P]. EP patent, 553780A, 1993
[12] Elsohly H N, Croom E M, Elsohly M A. Isolating taxanes from ornamental Taxus cultivates[P]. WO 9218492, 1992
[13] Strobel G, Yang X S, Sears J, et al. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana[J]. Microbiology, 1996(142):435-440
[14] 庞欣,章光明,张瑞萍. 固相萃取-高效液相色谱法测定真菌产紫杉醇[J]. 色谱,2004, 22(2):185
[15]Mattina M J I. Extraction, purification by solid-phase extraction and high -performance liquid chromatographic analysis of taxanes from ornamental Taxus needles[J]. Journal of Chromatography A, 1994, 679:269-275
[16]张志强,王云山,田桂莲. 固相萃取及反相层析分离提纯紫杉醇[J]. 药物生物技术,2000,7(3):157-160
[17]Jennings D W, Deutsch H M, Zal kow L H, et al. Supercritical Extraction of Taxol from the Bark of Taxes Brevifolia[J]. J Supercriti Flu., 1992, 5(1):1-5
[18] Vandana V, Amyn S.T, Leon H.Z. Supercritical Extraction and HPLC Analysis of Taxol from Taxus brevifolia Using Nitrous Oxide and Nitrous Oxide+Ethanol Mixtures[J]. Fluid Phase Equilibria. 1996, 116:162-169
[19]Nair J B. Supercritical extraction of taxanes. European patent[P]. EP patent, 0521675, 1993
[20]李华,李明,郑志坚等. 超临界 CO2 流体萃取法提取紫杉醇的研究[J]. 复旦学报,2003,43(2):453-456
[21]Carver D R, Prout T R, Workman C T, et al. Processing taxane solutes usingmembranes[P]. WO 9412268, 1994
[22]Ketchum R E B, Gibson D M. A Novel Method of Isolating Taxanes from Cell Suspension Cultures of Yew (Taxes Spp.)[J]. J.Liq.Chromatogr, 1995, 18(6):1093-1111
[23]元英进,那平,胡宗定. 东北红豆杉提取物吸附法脱色的初步探讨[J].离于交换与吸附.1994,10(6),543-545
[24]张志强,许建峰,苏志国. 树脂层析法脱色和浓缩紫杉醇[J]. 高校化学工程学报. 1999,13(2):160-164
[25]赵凌云,杨军,王健刚. 离子交换树脂固定床催化解禽紫杉醇键合物的研究[J]. 中草药,1997,28(增刊):21-23
[26]Cardelina J C. Taxol extraction from several Taxas species[J]. J.Liq.Chromatogr, 1991, 659:14-20
[27]Harvey S D, Campbel J A, Kelsey R G, et al. Separation of taxol from related taxanes in Taws brevijolia extracts by isocmtic elution reversed-phase microcolumn high-performance liquid chromatography[J]. Journal of Chromatography, 1991, 587:300-305
[28]Rao K V. Taxol and Related Taxanes.Ⅰ.Taxanes of Taxus brevifolia Bark[J]. Pharmaceutical Research, 1993, 10(4):521-524
[29]Wu D R. Preparative separation of taxol in normal and reversed-phase operation[J]. J.Chromatogr A, 1995, 702 (1/2) 233-236
[30] Hassan Y, Aboul-Enein, Vince Serignene. Liquid chromatographic determination of taxol and derivatives using a new polyfluorinated reversed-phase column[J]. Analytica Chimica Acta, 1996, 319:187-190
[31] Yang X F, Liu K L, Xie M. Purification of taxol by industrial preparative liquid chromatography[J]. Journal of Chromatography A, 1998, 813:201-204
[32]戴惠芳,梅忠,须媚. 紫杉醇、三尖杉宁碱和 7-表-10-去乙酰基紫杉醇的分离及 7-表-紫杉醇的转化研究[J]. 中国药物化学杂志,2003,13(5)288-290
[33]张志强,苏志国. 正相和反相柱层析组合分离纯化紫杉醇[J]. 生物工程学报,2000,16(1):69-73
[34]Stasko M W, Witherup K M, Ghiorzi T J, et al. Multimodal thin layer chromatographic separation of taxol and related compounds from Taxus brevifolia[J]. J.Liq.Chromatogr, 1989, 12 (11):2133-2143
[35]饶畅,刘欣,张佩玲,等.高速逆流色谱在天然产物分离中的应用-紫杉烷类二萜及二萜生物碱的制备分离[J].药学学报,1991,26(7):510-514
[36]Kingston D G O. The chemistry of taxol[J]. J.Nat.Prod, 1992, 55(2): 295-261
[37]Murray. Oxidation of cephalomannine with ozone in the presence of taxol[P]. US patent, 5334732, 1994
[38]陈建民,薛军,张黎,等. 紫杉醇臭氧处理纯化方法[P]. CN 1240789A,2000
[39]肖剑. 药理作用靶点法分离纯化紫杉醇的研究[D]. 天津:天津大学生物化工学院,1999
[40]Witherup K M, Look S A, Stasko M W, et al. High performance liquid chromatographic separation of taxol and related compounds from Taxus brevifolia[J]. J.Lid.Chromatogr, 1989, 12(11):2117-2132
[41]Harvey S D, Campbell J Am, Kelsey R G, et al. Separation of taxol from related taxanes in Taxus brevifolia extracts by isocratic elution reverse-phase microcolumn high-performance liquid chromatography[J]. J chromatogr, 1991, 587:300
[42]Richeimer S L, Tinnermeier D M, Timmons DW. High performance liquid chromatographic assay of taxol[J]. Anal Chem, 1992, 64:2316-2322
[43]吴锦霞,俞培忠.高效液相色谱法对紫杉醇及其有关物质的测定[J].药物分研杂志,1995,15(增刊):437-438
[44]Castor T P, Tyler T A. Determination of taxol in Taxus Media needles in the presence of interfering compounds[J]. J Liq Chromatogr, 1993, 16(3):723-731
[45]史作清,施荣富,范云鸽,等. 树脂吸附法在中草药有效成分提取中的应用[J]. 中草药,2001,32(7):660-662
[46]杨军. 紫杉醇分离纯化工艺及理论的研究[D]. 天津:天津大学生物化工,2001
[47]张志强. 吸附层析分离提纯紫杉醇[D]. 大连:大连理工大学生物化工学院,1999
[2] Schiff P B, Fant J, Horwitz S B. Promotion of microtubule assembly in vitro by taxol[J]. Nature, 1979, 277:665-667
[3] Hoke S H, Wood J M, Cooks R G, et al. Rapid screening for taxanes by tandem mass spectrometry[J]. Anal Chem,1992, 64:2313-2317
[4] Powell R G, Miller R W, Smith C R, et al. Taxol content and extraction from Taxus wallichiana[J]. J Chem Soc Communm, 1979:102-105
[5] Witherup K M, Look S A, Stasko M W, et al. Taxus spp. Needles contain amounts of taxol comparable to the bark of Taxus brevifolia: Analysis and Isolation[J]. J Nat Prod. 1990, 53(5):1249-1255
[6] Senilh V, Blechert S, Colin M, et al. New analogs of taxol extracts from Taxus baccata[J]. J Nat Prod. 1984, 47:131-137
[7] Nair. Process for the isolation and purification of taxol and taxanes from Taxus spp[P]. US patent, 5279949, 1994
[8] Rao K V, Gainesville F. Isolating and purification of taxol and its natural analogue[P]. US patent, 5380916, 1995
[9] Xu L X, Liu A R. Determination of taxol in the extract of Taxes chinensis by HPLC method[J]. Acts Pharm, 1991, 26(7):537-540
[10]锅田惠助. JP patent, 6157329[P], 1994
[11] Ferman G R, Gore T P. Isolation and purification of precursors of taxol and its analogues[P]. EP patent, 553780A, 1993
[12] Elsohly H N, Croom E M, Elsohly M A. Isolating taxanes from ornamental Taxus cultivates[P]. WO 9218492, 1992
[13] Strobel G, Yang X S, Sears J, et al. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana[J]. Microbiology, 1996(142):435-440
[14] 庞欣,章光明,张瑞萍. 固相萃取-高效液相色谱法测定真菌产紫杉醇[J]. 色谱,2004, 22(2):185
[15]Mattina M J I. Extraction, purification by solid-phase extraction and high -performance liquid chromatographic analysis of taxanes from ornamental Taxus needles[J]. Journal of Chromatography A, 1994, 679:269-275
[16]张志强,王云山,田桂莲. 固相萃取及反相层析分离提纯紫杉醇[J]. 药物生物技术,2000,7(3):157-160
[17]Jennings D W, Deutsch H M, Zal kow L H, et al. Supercritical Extraction of Taxol from the Bark of Taxes Brevifolia[J]. J Supercriti Flu., 1992, 5(1):1-5
[18] Vandana V, Amyn S.T, Leon H.Z. Supercritical Extraction and HPLC Analysis of Taxol from Taxus brevifolia Using Nitrous Oxide and Nitrous Oxide+Ethanol Mixtures[J]. Fluid Phase Equilibria. 1996, 116:162-169
[19]Nair J B. Supercritical extraction of taxanes. European patent[P]. EP patent, 0521675, 1993
[20]李华,李明,郑志坚等. 超临界 CO2 流体萃取法提取紫杉醇的研究[J]. 复旦学报,2003,43(2):453-456
[21]Carver D R, Prout T R, Workman C T, et al. Processing taxane solutes usingmembranes[P]. WO 9412268, 1994
[22]Ketchum R E B, Gibson D M. A Novel Method of Isolating Taxanes from Cell Suspension Cultures of Yew (Taxes Spp.)[J]. J.Liq.Chromatogr, 1995, 18(6):1093-1111
[23]元英进,那平,胡宗定. 东北红豆杉提取物吸附法脱色的初步探讨[J].离于交换与吸附.1994,10(6),543-545
[24]张志强,许建峰,苏志国. 树脂层析法脱色和浓缩紫杉醇[J]. 高校化学工程学报. 1999,13(2):160-164
[25]赵凌云,杨军,王健刚. 离子交换树脂固定床催化解禽紫杉醇键合物的研究[J]. 中草药,1997,28(增刊):21-23
[26]Cardelina J C. Taxol extraction from several Taxas species[J]. J.Liq.Chromatogr, 1991, 659:14-20
[27]Harvey S D, Campbel J A, Kelsey R G, et al. Separation of taxol from related taxanes in Taws brevijolia extracts by isocmtic elution reversed-phase microcolumn high-performance liquid chromatography[J]. Journal of Chromatography, 1991, 587:300-305
[28]Rao K V. Taxol and Related Taxanes.Ⅰ.Taxanes of Taxus brevifolia Bark[J]. Pharmaceutical Research, 1993, 10(4):521-524
[29]Wu D R. Preparative separation of taxol in normal and reversed-phase operation[J]. J.Chromatogr A, 1995, 702 (1/2) 233-236
[30] Hassan Y, Aboul-Enein, Vince Serignene. Liquid chromatographic determination of taxol and derivatives using a new polyfluorinated reversed-phase column[J]. Analytica Chimica Acta, 1996, 319:187-190
[31] Yang X F, Liu K L, Xie M. Purification of taxol by industrial preparative liquid chromatography[J]. Journal of Chromatography A, 1998, 813:201-204
[32]戴惠芳,梅忠,须媚. 紫杉醇、三尖杉宁碱和 7-表-10-去乙酰基紫杉醇的分离及 7-表-紫杉醇的转化研究[J]. 中国药物化学杂志,2003,13(5)288-290
[33]张志强,苏志国. 正相和反相柱层析组合分离纯化紫杉醇[J]. 生物工程学报,2000,16(1):69-73
[34]Stasko M W, Witherup K M, Ghiorzi T J, et al. Multimodal thin layer chromatographic separation of taxol and related compounds from Taxus brevifolia[J]. J.Liq.Chromatogr, 1989, 12 (11):2133-2143
[35]饶畅,刘欣,张佩玲,等.高速逆流色谱在天然产物分离中的应用-紫杉烷类二萜及二萜生物碱的制备分离[J].药学学报,1991,26(7):510-514
[36]Kingston D G O. The chemistry of taxol[J]. J.Nat.Prod, 1992, 55(2): 295-261
[37]Murray. Oxidation of cephalomannine with ozone in the presence of taxol[P]. US patent, 5334732, 1994
[38]陈建民,薛军,张黎,等. 紫杉醇臭氧处理纯化方法[P]. CN 1240789A,2000
[39]肖剑. 药理作用靶点法分离纯化紫杉醇的研究[D]. 天津:天津大学生物化工学院,1999
[40]Witherup K M, Look S A, Stasko M W, et al. High performance liquid chromatographic separation of taxol and related compounds from Taxus brevifolia[J]. J.Lid.Chromatogr, 1989, 12(11):2117-2132
[41]Harvey S D, Campbell J Am, Kelsey R G, et al. Separation of taxol from related taxanes in Taxus brevifolia extracts by isocratic elution reverse-phase microcolumn high-performance liquid chromatography[J]. J chromatogr, 1991, 587:300
[42]Richeimer S L, Tinnermeier D M, Timmons DW. High performance liquid chromatographic assay of taxol[J]. Anal Chem, 1992, 64:2316-2322
[43]吴锦霞,俞培忠.高效液相色谱法对紫杉醇及其有关物质的测定[J].药物分研杂志,1995,15(增刊):437-438
[44]Castor T P, Tyler T A. Determination of taxol in Taxus Media needles in the presence of interfering compounds[J]. J Liq Chromatogr, 1993, 16(3):723-731
[45]史作清,施荣富,范云鸽,等. 树脂吸附法在中草药有效成分提取中的应用[J]. 中草药,2001,32(7):660-662
[46]杨军. 紫杉醇分离纯化工艺及理论的研究[D]. 天津:天津大学生物化工,2001
[47]张志强. 吸附层析分离提纯紫杉醇[D]. 大连:大连理工大学生物化工学院,1999