CN1124735A - Process for extracting and separating taxol from yew trunk and leaves - Google Patents
Process for extracting and separating taxol from yew trunk and leaves Download PDFInfo
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- CN1124735A CN1124735A CN94114041A CN94114041A CN1124735A CN 1124735 A CN1124735 A CN 1124735A CN 94114041 A CN94114041 A CN 94114041A CN 94114041 A CN94114041 A CN 94114041A CN 1124735 A CN1124735 A CN 1124735A
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- folium taxi
- taxi cuspidatae
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Abstract
The process for extracting taxusol from Enqlish yew's stem and leaf includes such technological steps as extraction in mixed solvent, concentration, recovering solvent, silica gel column laminar eduction to obtain coarse product, recrystallizing and separation by column chromatographic analysis to obtain over 98% taxusol with yield about 0.007%, and the abundant source of Zhejiang yew stem and leaf is used as raw material.
Description
The present invention relates to a kind of from natural phant the method for extraction separation effective constituent, specifically a kind of from beautiful Ramulus et folium taxi cuspidatae (Taxus mairei) cauline leaf the method for extraction separation taxol (Taxol).
As everyone knows, taxol is a kind of diterpenoids compound.Separated first from yewtree (Taxus brevifolia) bark by (Wani Mc, et al, J.A.C.S.93,2325,1971.) such as Wani in 1971 and obtain.It shows the antitumour activity that is thoroughly satisfied to many tumor models such as B-16 melanoma, L-1210 and P-388 leukemia, MX-1 mammary cancer and heteroplastic CX-1 colorectal carcinoma, because its mechanism of action uniqueness, can impel tubulin polymerization to form microtubule, and microtubule no longer disintegrates in the presence of it, cancer cells is limited in the framework and dead [Dentsch-HM et al, J.M.C.32 (4), 788,1989; Manfredi JJ, et al, Phannacol ther 25,83,1984.].Therefore, it has become the focus of domestic and international scientist in antitumor drug research, and progress is very rapid.In December, 1992, the taxol of drugs approved by FDA BMS company is as the second line treatment medicine of transitivity ovarian cancer; Ratify this medicine in December, 1993 again as breast cancer treatment medicine in late period.In more than 20 state approval listing such as American and Britain, moral, method, its annual turnover is above 300,000,000 dollars for this medicine at present.Along with the satisfactory effect of II phase and III clinical trial phase is carried out to human body melanoma, leukemia, small cell lung cancer and colorectal carcinoma in the listing of this medicine and it, supply falls short of demand to have made taxol.U.S. relevant expert in 1992 prediction is difficult to find the natural drug higher than taxol curative effect again in recent two decades.And appeal that the whole world all is concerned about and improves the supply of taxol, with clinical needs [Chemical and Engineering, 69 (35), 11-18,1991 of alleviating it; Kingston DGI, et al, J.N.P.53 (1), 1,1990.].China Medicine University and the beautiful Ramulus et folium taxi cuspidatae stem of Gifu, Japan pharmaceutical university joint study skin fail to obtain taxol; U.S. Purdue university and the Taiwan Hinchu Industrial Technology Research Institute beautiful Ramulus et folium taxi cuspidatae needle of use research are only finished this plant of HPLC quantitative assay and are contained taxol 0.006-0.008%; Taxus chinensis in northeast (Taxus cuspidata) branches and leaves have been studied by China institute of materia medica, medical courses in general institute Beijing, and the yield of its report taxol is below 0.0007%; External more extensive at present to the research of the compounds such as 10 1 deacetylate Baccatine IIIs in the Ramulus et folium taxi cuspidatae class plant stem-leaf, but the percentage composition of taxol in this plant stem-leaf of HPLC quantitative assay that only appears in the newspapers does not prepare taxol and be reported from cauline leaf.French scientist in 1988 report separates from the needle of european yew (Taxus baccata) and obtains.Can be converted into taxol or other relevant active medicine by fairly simple semi-synthesis method.The whole world can have nine kinds and four mutation for the Ramulus et folium taxi cuspidatae of research, and China occupies four kinds and a mutation, aboundresources.In recent years, some R﹠D institutions on ground such as Beijing, Shanghai and Nanjing, Guangzhou, Kunming and universities and colleges are in succession to taxus chinensis in northeast (Taxus cuspidata), beautiful Ramulus et folium taxi cuspidatae [Liang Jingyu etc., chemical journal, 46,1053,1988; 46,210,1988; J.-y.Zhou et al, LaternationalResearch eongress on Natural Products Halifax, N.S., Canada July31-August4,1994, P:59; Tong Xiaojie etc., Acta Pharmaceutica Sinica, 29 (1), 55,1994.] and taxusyunnanensis (Taxus yusanenensis) carried out research [Chen Weiming etc., Acta Pharmaceutica Sinica, 26 (10), 747,1991; Mingky, el al, Phytochem 27,1534, and 1988.], but not only yield is low and extracting method is not perfect.From the needs of long-term production, extracting taxol from cauline leaf is a preferable approach.Gather needle and spray and can not damage plant, constantly regeneration will be played and protect national resource and the ecosystem, makes and benefits the mankind.Therefore, efficient, the easy method of extracting taxol from natural phant is still visited plain problem for people.
The purpose of this invention is to provide a kind of from the beautiful Ramulus et folium taxi cuspidatae of natural phant the method for extraction separation taxol.
Method of the present invention is to make raw material with the cauline leaf of beautiful Ramulus et folium taxi cuspidatae, can through pulverizing, solvent extraction, concentrate the deep green paste, add the suitable quantity of water dilution, use solvent extraction again, extracting solution concentrates the back to be separated with column chromatography method, desolventize the taxol crude product.This crude product through the second time column chromatography for separation can obtain purity and be 〉=98% taxol, yield is greater than 7/100000ths.
Used leach liquor is one of key that improves yield among the present invention.Leach liquor is two or more mixed solvent.Specifically the mixture of ethanol and the water miscible polar solvent except that ethanol also can contain a spot of water.Their weight ratio was followed successively by 1: 0.01-0.30: 0-0.20.The weight ratio of recommending is 1: 0.01 1: 0-0.10.Described water miscible polar solvent can be other the water miscible polar solvent except that ethanol.As the ester of low carbon chain, the alcohol of low carbon chain, acetone, acetonitrile etc.The ester of low carbon chain and alcohol can be methyl-formiate, ethyl formate, propyl formate, methyl acetate, ethyl acetate, propyl acetate, methyl propionate, ethyl propionate, methyl alcohol, propyl alcohol etc.
When leaching beautiful Ramulus et folium taxi cuspidatae cauline leaf with above-mentioned mixed solvent, at least will soak into beautiful Ramulus et folium taxi cuspidatae cauline leaf, preferably mixed solvent is more excessive, so that leach fully, also help leaching when leaching, but will increase workload and the energy consumption that concentrates or reclaim solvent with very a large amount of mixed solvents.The weight ratio of common beautiful Ramulus et folium taxi cuspidatae and mixed solvent is 1: 30.It also is favourable adopting more mixed solvent.The weight ratio of recommending to adopt is 1: 1-5.
When adopting mixed solvent to leach, preferably repeatedly leach with new mixed solvent, as leach 3-6 times, to improve leaching yield with above-mentioned method.Leach more frequently, not remarkable to improving the leaching yield effect.
Adopt in the method for the present invention, can adopt the many alkyl chloride of low carbon chain or mix many alkyl chloride extractions, as the mixed solution of methylene dichloride and trichloromethane.Column chromatography for separation can adopt silica gel column chromatography, with the hydrochloric ether of low carbon chain and the pure mixed solvent wash-out of low carbon chain, all washes out until taxol.Obtain thick product after concentrating.
Thick product can be used the alcohol and water recrystallization purifying, uses chromatographic separation again, separates as positive or anti-phase silica gel column chromatography, makes eluent with polar solvent or with water, the taxol of concentrated obtaining 〉=98% purity, and yield is greater than 7/100000ths.
Adopt method of the present invention, not only can adopt the beautiful Ramulus et folium taxi cuspidatae cauline leaf of growing on the high mountain, as the beautiful Ramulus et folium taxi cuspidatae in northeast Japanese yew (claiming Ramulus et folium taxi cuspidatae again) and Yunnan.And can adopt on hills and the level land, the beautiful faster Ramulus et folium taxi cuspidatae cauline leaf of growth makes raw material, as the beautiful Ramulus et folium taxi cuspidatae cauline leaf in Zhejiang.This also is an another important content of the present invention.
The main raw material of producing taxol at present is the bark of Chinese yew, but bark can cause the death of trees, finally can influence the eubiosis.And the easy artificial growth of the beautiful Ramulus et folium taxi cuspidatae in Zhejiang, the speed of growth is very fast, the experiment proved that the content ideal of taxol in this plant stem-leaf, is a kind of plant resources of rising, reproducible taxol.So we adopt and propose taxol from cauline leaf, can not destroy plant resources, remarkable in economical benefits is made and is benefited society.
As mentioned above, adopt method of the present invention, do not need from the bark of Ramulus et folium taxi cuspidatae or root, to extract taxol, adopt beautiful Ramulus et folium taxi cuspidatae cauline leaf to make raw material, help conservation of resources and raw material constantly provides.And method of the present invention is easy, and abundant raw material, yield height are suitable for scale production.
To help the understanding of the present invention by following embodiment, and not limit content of the present invention.
Fusing point is measured with the Bortius melting point detector among the embodiment, does not proofread and correct; Specific rotation is measured with the Perkin-Elmer241 polarimeter; Nuclear magnetic resonance spectrum Bruker AMX-600 type and AM-300 nmr determination; Mass spectrum is measured with JMS-DX300 and Quattro GC/ms/ms mass spectrograph; Column chromatography silica gel is Haiyang Chemical Plant, Qingdao's product, and HPLC measures with day island proper Tianjin HPLC instrument.Developer is 5%H
2SO
4Ethanolic soln.
Embodiment 1
Get 10Kg through the beautiful Ramulus et folium taxi cuspidatae cauline leaf in the Zhejiang of natural drying at room temperature, after crushed, with 1 of 95% ethanol and ethyl acetate, acetone, acetonitrile, propyl alcohol or methyl alcohol: the mixed solvent lixiviate of 0.01-0.3 weight ratio 3-6 times, each cauline leaf is 1: 5 with the weight of solvent ratio.The leach liquor concentrating under reduced pressure gets the deep green paste, adds an amount of H then
2CH is used in the O dilution
2Cl
2And CHCl
3The mixed solution extraction.Merge CH
2Cl
2Extracting solution, concentrating under reduced pressure get extract 400-450g.Then, with CH
2Cl
2Extract carries out silica gel column chromatography to be separated, and adopts CH
2Cl
2-MeOH mixed solvent gradient elution is collected 80-90 stream parts, every part of 300ml-400ml altogether.Check all stream parts with plate layer chromatography, merge stream part of containing taxol, boil off solvent, get 809.The gained crude product carries out silica gel column chromatography once more, and can obtain containing taxol 1.95g with the methanol crystallization purifying.Adopt then on rotating thin layer chromato-graphy (purification on normal-phase silica gel or reverse phase silica gel) or the reverse phase silica gel post high performance liquid phase instrument and separate at last, as eluent, get 〉=98% taxol 0.72g with methanol, yield is 7/100000ths. two.
Embodiment 2
With embodiment 1, adopt mixed solvent to leach 1 time.Obtain 98% taxol, yield 0.0061%.
Embodiment 3
Make raw material with the cauline leaf of Yunnan and taxus chinensis in northeast respectively, all the other are with embodiment 1.Experiment repeats secondary respectively, purity be the yield of 98% taxol all with above similar, be respectively 0.0075% and 0.0073%.
Structure is identified:
Taxol, molecular formula are C
47H
51NO
14White needles or white powdery crystallization (H
2O-MeOH or H
2O-CH
3CN), mp217-218 ℃, [α]
D 20-48.5 (CO.20, MeOH), thin layer shows purple, IR (KBr) CM
-13460 (OH, NH), 1730,1720,1710 (C=0), 1650 (NHC=0).FAB—MS?m/z：854(MH
+)，794(MH
+—HOAC)，569(MH
+—CH
16H
15O
4N)。490,430,404,324,307,105 (base peak).
1HNMR (CD
3Cl, 600MHz) δ (ppm): 1.16 (3H, s, 17-CH
3), 1.25 (3H, s, 16-Me), 1.69 (3H, s, 19-Me), 1.81 (3H, s, J=1.1,18-CH
3), 1.87 (1H, m, 6 β-H), 2.24 (3H, s, OAc), 2.30 (2H, m, 14-H
2), 2.39 (3H, s, OAc), 2.55 (1H, ddd, J=6.6,9.7,14,9Hz, 6 α-H), 3.81 (1H, d, J=7.0,3H), 4.20 and 4.31 (each 1H, d, J=8.5,20-H
2), 4.40 (1H, dd, J=6.6,10.8,7-H), 4.79 (1H, brs, Z '-H), 4.94 (1H, dd, J=2.1,9.5,5-H), 5.68 (1H, d, J=7,2-H), 5.78 (1H, d, J=8.8Hz, 3 '-H), 6.23 (1H, brt, J=8.3Hz, 13-H), 6.28 (1H, s, 10-H), 7.00 (1H, s, J=8.8Hz, NH), 7.40 (5H, m, Ar-H), 7.50 (4H, m, Ar-H), 7.61 (2H, t, J=7Hz, Ar-H), 7.76 (2H, d, J=7.2Hz, Ar-H), 8.14 (2H, d, J=7.2Hz, Ar-H), above-mentioned data are consistent with the document record.
Embodiment 4 compares both at home and abroad
Result of the present invention and the results are shown in the table of comparing both at home and abroad
| Japanese and middle traditional Chinese medicines University of Science and Technology | The U.S. and Taiwan | The present invention | Beijing medicine institute | |
| Material | T.Marei branch skin | The T.Marei needle | The T.Marei cauline leaf | The T.Cuspidada branches and leaves |
| Method | Lab scale research | HPLC measures content in the needle | Lab scale research | Lab scale research |
| Charging capacity | ??8.5kg | ??10kg | ??13.5kg | |
| The taxol yield | ??0％ | ????0.006- ????0.008％ | ??0.006- ?0.0075％ | ??0.0006- ?0.0007％ |
Claims (7)
1. method of from beautiful Ramulus et folium taxi cuspidatae (Tarus mairei) cauline leaf, extracting taxol, comprise solvent extraction, concentrate, add the suitable quantity of water dilution, solvent extraction, extracting solution uses column chromatography and concentrates, it is characterized in that adopting the mixed solvent of ethanol, the water soluble polar solvent except that ethanol and water to leach, weight ratio was followed successively by 1: 0.01-0.3: 0-0.20.
2. a method of extracting taxol from beautiful Ramulus et folium taxi cuspidatae cauline leaf as claimed in claim 1 is characterized in that the mixed solvent weight ratio of described ethanol, the water soluble polar solvent except that ethanol and water was followed successively by 1: 0.01-0.10: 0-0.10.
3. a method of extracting taxol from beautiful Ramulus et folium taxi cuspidatae cauline leaf as claimed in claim 1 is characterized in that described water soluble polar solvent is the ester of acetone, acetonitrile, low carbon chain or the alcohol except that ethanol.
4. a method of extracting taxol from beautiful Ramulus et folium taxi cuspidatae as claimed in claim 1 is characterized in that extracting 3-6 times with described mixed solvent.
5. method of extracting taxol from beautiful Ramulus et folium taxi cuspidatae as claimed in claim 1 is characterized in that with many alkyl chloride of low carbon chain or mixes many alkyl chloride extractions, through silica gel column chromatography.
6. a method of extracting taxol from beautiful Ramulus et folium taxi cuspidatae as claimed in claim 1 is characterized in that again through recrystallization and chromatographic separation.
7. one kind as claim 1,2, the 3 or 4 described methods of extracting taxol from beautiful Ramulus et folium taxi cuspidatae, it is characterized in that described beautiful Ramulus et folium taxi cuspidatae cauline leaf is the cauline leaf of the beautiful Ramulus et folium taxi cuspidatae that produces of Zhejiang.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94114041A CN1124735A (en) | 1994-12-17 | 1994-12-17 | Process for extracting and separating taxol from yew trunk and leaves |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94114041A CN1124735A (en) | 1994-12-17 | 1994-12-17 | Process for extracting and separating taxol from yew trunk and leaves |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1124735A true CN1124735A (en) | 1996-06-19 |
Family
ID=5036996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN94114041A Pending CN1124735A (en) | 1994-12-17 | 1994-12-17 | Process for extracting and separating taxol from yew trunk and leaves |
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| Country | Link |
|---|---|
| CN (1) | CN1124735A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1055688C (en) * | 1996-11-19 | 2000-08-23 | 西藏自治区卫生厅 | Method for extracting taxol from Xizang Taxus chinensis |
| US6136989A (en) * | 1998-12-30 | 2000-10-24 | Phytogen Life Sciences, Incorporated | Method for high yield and large scale extraction of paclitaxel from paclitaxel-containing material |
| CN1060476C (en) * | 1998-05-07 | 2001-01-10 | 中国人民解放军第二军医大学 | Preparation process of taxad alcohol |
| CN1091105C (en) * | 1997-08-01 | 2002-09-18 | 中国科学院武汉植物研究所 | Method for separation and purification of taxol and taxane |
| CN101671319B (en) * | 2009-09-18 | 2012-02-29 | 应成杰 | Taxus nontoxic extract process |
| CN103172598A (en) * | 2013-03-26 | 2013-06-26 | 四川祥光农业科技开发有限公司 | Method for extracting paclitaxel from taxus chinensis branch and leaf |
-
1994
- 1994-12-17 CN CN94114041A patent/CN1124735A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1055688C (en) * | 1996-11-19 | 2000-08-23 | 西藏自治区卫生厅 | Method for extracting taxol from Xizang Taxus chinensis |
| CN1091105C (en) * | 1997-08-01 | 2002-09-18 | 中国科学院武汉植物研究所 | Method for separation and purification of taxol and taxane |
| CN1060476C (en) * | 1998-05-07 | 2001-01-10 | 中国人民解放军第二军医大学 | Preparation process of taxad alcohol |
| US6136989A (en) * | 1998-12-30 | 2000-10-24 | Phytogen Life Sciences, Incorporated | Method for high yield and large scale extraction of paclitaxel from paclitaxel-containing material |
| CN101671319B (en) * | 2009-09-18 | 2012-02-29 | 应成杰 | Taxus nontoxic extract process |
| CN103172598A (en) * | 2013-03-26 | 2013-06-26 | 四川祥光农业科技开发有限公司 | Method for extracting paclitaxel from taxus chinensis branch and leaf |
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