长春碱高效提取纯化的工艺研究
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摘要
长春花是夹竹桃科长春花属植物，在我国广为种植。长春花中含有大量的生物碱，其中长春碱具有广谱抗肿瘤活性，能治疗多种癌症。本文建立了长春碱的RP-HPLC分析方法和样品制备方法，研究了从鲜长春花中高效提取、纯化长春碱的方法，确定了全新的生产工艺，并进行了中试放大实验。主要结论如下：
1．确立了长春花中长春碱、长春新碱、长春质碱和文多灵等四种生物碱的检测方法：色谱柱：Diamonsil~(TM)C_(18)ODS (250×4.6mm i．d．，5μm)；流动相；甲醇：乙腈=4：1(溶液A)；水：二乙胺=986：14，用磷酸调节pH=7.2(溶液B)；380ml A和620ml B混合作为流动相；流速：2ml／min；进样量：10μl；柱温：23℃；检测波长：220nm。
2．确定了长春碱提取、纯化的工艺路线，其工艺流程如下：
具体工艺参数如下：
(1)鲜磨匀浆提取工艺条件为：萃取溶剂为75％乙醇溶液，料液比为1：3，匀浆时间为6min。负压空化强化提取工艺条件为：提取溶剂为75％的乙醇溶液，料液比1：5，萃取时间25min，通气量为2m~3／h。萃取工艺中长春碱的提取率为97.3％，经萃取、浓缩后得到纯度为10％的长春碱粗提粉。
(2)长春碱纯化工艺条件为：长春花粗提粉：水：沸腾乙醇=100：200：4000，经结晶后，得到纯度为40％的长春碱粗品。取纯度为40％的长春碱，硅胶为300-500目，上样量3g，洗脱剂：氯仿：甲醇=100：2，洗脱液流速：3BV／h。此工艺条件下收集到纯度在95％以上的组分1.13g。将纯度为95％以上的硫酸长春碱在60℃下溶解于90％的乙醇溶液中，冷却至0℃，得到硫酸长春碱晶体，纯度为98.2％。
利用这一工艺提取长春碱，长春碱最终得率为十万分之六，产品纯度可达98.2％。在此基础上制定了长春碱的质量标准，并进行了年产9kg硫酸长春碱的可行性研究。
Catharanthus roseus (L.) G Don is one of the Catharanthus in Apocynaceae. It widely grows in China. There are lots of alkaloids in it, of which vinblastine has a broad-spectrum anticancer activity, it can treat much kinds of cancer. In this paper we established RP-HPLC determination method and sample preparation method, and studied on the technology of vinblastine high effective extraction and purification, and finished the middle experiment from fresh catharanthus roseus. And finally we established the new vinblastine produce process. Here are the main conclusions:
1. Established the HPLC detection method of four vinca alkaloids: vinblastine, vincristine, catharanthine, vindoline.
These 4 ingredients were separated on a Diamonsil (C18 column 250x4.6mm i.d., 5 )with methanol: acetonitrile =4:1 as solvent A, and water: diethylamine =986:14 , H3PO4 adjust pH to 7.2, as solvent B, and the mobile phase was composed of 380ml A and 620ml B. The detection was performed at a flow rate of 2.0ml/min, and detected at 220nm within 30min.
2. Established extraction and purification process of vinblastine. The process is:
Extracted Extracted
Fresh catharanthus roseus Extracted liquid Crude extraction
Concentrated and dryed
Acidifyed Column chromatography
Crude vinblastine Purified vinblastine
Extracted Acidifyed
VLB-sulfate
Recrystalized
The detailed parameters are:
(1) Fresh material grinding extraction process: using 75% alcohol as extract solvent, sample and extraction solvent rate is 1:3, in 2min. Aggrandizement extraction process is: using 75% alcohol as extract solvent, sample and extraction solvent rate is 1:5, air flew rate is 2m3/h, and extraction time is 25min. In this process, vinblastine extraction rate is 97.3%. And it can get to 10% purity after extraction and
concentration process.
(2) vinblastine purification process is: crude vinblastine: water: boiling alcohol= 100:200:4000, from this process we got crude vinblastine(40%). And then using silicon gel(300-500mu) to purify process, the sample quality is 3g, elution solvent is CHCl3:MeOH=100:2 i n 3 BV/h. Yield 1 .13g v inblastine(95%). I n 6 0C, using 90% alcohol as crystal solvent, then cooled slowly to 0癈, yield vinblastine crystalloid(98.2%).
In this process, vinblastine utmost yield is 0.006% with purity of 98.2%. We established vinblastine quality criteria and also studied on the feasibility of produce 9kg vinblastine per year base on this process.
1．确立了长春花中长春碱、长春新碱、长春质碱和文多灵等四种生物碱的检测方法：色谱柱：Diamonsil~(TM)C_(18)ODS (250×4.6mm i．d．，5μm)；流动相；甲醇：乙腈=4：1(溶液A)；水：二乙胺=986：14，用磷酸调节pH=7.2(溶液B)；380ml A和620ml B混合作为流动相；流速：2ml／min；进样量：10μl；柱温：23℃；检测波长：220nm。
2．确定了长春碱提取、纯化的工艺路线，其工艺流程如下：
具体工艺参数如下：
(1)鲜磨匀浆提取工艺条件为：萃取溶剂为75％乙醇溶液，料液比为1：3，匀浆时间为6min。负压空化强化提取工艺条件为：提取溶剂为75％的乙醇溶液，料液比1：5，萃取时间25min，通气量为2m~3／h。萃取工艺中长春碱的提取率为97.3％，经萃取、浓缩后得到纯度为10％的长春碱粗提粉。
(2)长春碱纯化工艺条件为：长春花粗提粉：水：沸腾乙醇=100：200：4000，经结晶后，得到纯度为40％的长春碱粗品。取纯度为40％的长春碱，硅胶为300-500目，上样量3g，洗脱剂：氯仿：甲醇=100：2，洗脱液流速：3BV／h。此工艺条件下收集到纯度在95％以上的组分1.13g。将纯度为95％以上的硫酸长春碱在60℃下溶解于90％的乙醇溶液中，冷却至0℃，得到硫酸长春碱晶体，纯度为98.2％。
利用这一工艺提取长春碱，长春碱最终得率为十万分之六，产品纯度可达98.2％。在此基础上制定了长春碱的质量标准，并进行了年产9kg硫酸长春碱的可行性研究。
Catharanthus roseus (L.) G Don is one of the Catharanthus in Apocynaceae. It widely grows in China. There are lots of alkaloids in it, of which vinblastine has a broad-spectrum anticancer activity, it can treat much kinds of cancer. In this paper we established RP-HPLC determination method and sample preparation method, and studied on the technology of vinblastine high effective extraction and purification, and finished the middle experiment from fresh catharanthus roseus. And finally we established the new vinblastine produce process. Here are the main conclusions:
1. Established the HPLC detection method of four vinca alkaloids: vinblastine, vincristine, catharanthine, vindoline.
These 4 ingredients were separated on a Diamonsil (C18 column 250x4.6mm i.d., 5 )with methanol: acetonitrile =4:1 as solvent A, and water: diethylamine =986:14 , H3PO4 adjust pH to 7.2, as solvent B, and the mobile phase was composed of 380ml A and 620ml B. The detection was performed at a flow rate of 2.0ml/min, and detected at 220nm within 30min.
2. Established extraction and purification process of vinblastine. The process is:
Extracted Extracted
Fresh catharanthus roseus Extracted liquid Crude extraction
Concentrated and dryed
Acidifyed Column chromatography
Crude vinblastine Purified vinblastine
Extracted Acidifyed
VLB-sulfate
Recrystalized
The detailed parameters are:
(1) Fresh material grinding extraction process: using 75% alcohol as extract solvent, sample and extraction solvent rate is 1:3, in 2min. Aggrandizement extraction process is: using 75% alcohol as extract solvent, sample and extraction solvent rate is 1:5, air flew rate is 2m3/h, and extraction time is 25min. In this process, vinblastine extraction rate is 97.3%. And it can get to 10% purity after extraction and
concentration process.
(2) vinblastine purification process is: crude vinblastine: water: boiling alcohol= 100:200:4000, from this process we got crude vinblastine(40%). And then using silicon gel(300-500mu) to purify process, the sample quality is 3g, elution solvent is CHCl3:MeOH=100:2 i n 3 BV/h. Yield 1 .13g v inblastine(95%). I n 6 0C, using 90% alcohol as crystal solvent, then cooled slowly to 0癈, yield vinblastine crystalloid(98.2%).
In this process, vinblastine utmost yield is 0.006% with purity of 98.2%. We established vinblastine quality criteria and also studied on the feasibility of produce 9kg vinblastine per year base on this process.
引文
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2. Bensch KG, Malawista SE. Microtubule crystals: a new biophysical phenomenon induced by Vinca alkaloids[J]. Nature, 1968, 218:1176～1177.
3. Bhadra R, Shanks JV. Transient Stydies of Nutrient Uptake,Growth,and Indole Alkaloid Accumulation in Heterotrophic Cultures of Hairy Roots of Catharanthus roseus[J]. Biotechnology and Bioengineering. 1997, 55(3): 528～534.
4. Brade W, Nagel GA, Seeber S, et al. Critical review of pharmacology, toxicology, pharmacokinetics of vincristine, vindesine, vinblastine. International Vinca Alkaloid Symposium- Vindesine. 1981, 95～123.
5. Chabner BA,Calabresi P. Goodman and Gilman's The Pharmacological Basis of Therapeutics[M]. New York:McGraw-Hill Publishing Co., 1996:1225～1289.
6. Chatterjee S K, Laffray J, Patel P. Interaction of Tubulin with a New Fluorescent Analogue of Vinblastine[J]. Biochemistry, 2002, 41 (47): 14010～14018.
7. CN 1,365,978 A. 2002(从长春花中提取抗癌活性成分的新工艺).
8. Conrad RA, Cullinan GJ, Gerzon K, et al. Structure-Activity Relationships of Dimeric Catharanthus Alkaloids. 2. Experimental Antitumor Activities of N-Substituted Deacetyl- vinblastine Amide( Vindesine ) Sulfates[J]. J Am Chem Soc, 1979, 22(4): 391～400.
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