CN1616442A - Method for extracting 9-dihydrogen-13-acetyl baccatin III and taxol - Google Patents
Method for extracting 9-dihydrogen-13-acetyl baccatin III and taxol Download PDFInfo
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- CN1616442A CN1616442A CN 200410054174 CN200410054174A CN1616442A CN 1616442 A CN1616442 A CN 1616442A CN 200410054174 CN200410054174 CN 200410054174 CN 200410054174 A CN200410054174 A CN 200410054174A CN 1616442 A CN1616442 A CN 1616442A
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Abstract
The extraction process of 9-dihydrogen-13-acetyl baccatin III and taxol from taxad includes the following successive steps: leaching taxad branch and leaf with organic leaching liquid through multiple stage diacolation to obtain organic leached solution containing taxane compounds; chromatographic separation of the organic leached solution in silica gel column with C4-C8 alkane or petroleum ether, acetic acid and C1-C6 alkyl ester for elution to obtain coarse solution of 9-dihydrogen-13-acetyl baccatin III and taxol; and separation with C1-C4 alkyl halide, acetic acid and C1-C6 alkyl ester to obtain separated 9-dihydrogen-13-acetyl baccatin III and taxol.
Description
Technical field
The present invention relates to a kind of from Ramulus et folium taxi cuspidatae the method for high efficiency extraction 9-dihydro-13-acetyl baccatin III and taxol.
Background technology
Bearing taxanes is the compound that a class has the diterpene mother nucleus structure, and it mainly is present in the Ramulus et folium taxi cuspidatae at nature.
1971 (Wani Mc, et al, J.A.C.S.93,2325,1971.) such as Wani extract the taxol that obtains belonging to described bearing taxanes first from yewtree (Taxusbrevifolia) bark, its structural formula is as follows:
Known taxol shows extremely strong antitumour activity 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, its mechanism of action can be impelled tubulin polymerization for it and become 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, Phannacolther 25,83, and 1984].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 again as the advanced breast cancer curative in December, 1993, and this medicine is more than 20 state approval listings at present.
The method of extracting natural Japanese yew alcohol at present comprises the mixture that extracts bearing taxanes earlier from Ramulus et folium taxi cuspidatae, isolation of taxol from described mixture again.The method of described extraction bearing taxanes generally comprises following steps:
(1) pack in the container Ramulus et folium taxi cuspidatae of drying and pulverizing soaks for some time with vat liquor;
(2) after the liquid-solid separation, add vat liquor more isolating solid is soaked once more, separate obtaining vat liquor;
(3) repeating step (2).
The shortcoming of this multistage infusion method is that required time is long, and the vat liquor consumption is big, thereby makes the product concentration that obtains low, and follow-up to remove the workload desolvate big, limited extracting efficiency.
Therefore, need exploitation a kind of from Ramulus et folium taxi cuspidatae the method for high efficiency extraction bearing taxanes.
In addition, think that at present the active structure of taxol is the propylene oxide structure [Chen Weiming etc., Acta Pharmaceutica Sinica .1990,25 (3): 27] of C-13 position side chain in its molecular structure and C-4, C-5, C-20 position:
Because taxol is evident in efficacy, and it is in great demand.Taxol is mainly by separation and Extraction from Ramulus et folium taxi cuspidatae at present.Growth of taxol is very slow, and at the content of its taxol in bark generally only between 0.01～0.03 weight %, the content of taxol is lower in Ramulus et folium taxi cuspidatae.Therefore, the output of natural Japanese yew alcohol far can not meet the demands.Be necessary to seek new taxol source or its substitute.
9-dihydro-13-acetyl baccatin III is a kind of in the described bearing taxanes, and it also is to separate the natural organic-compound that obtains from Ramulus et folium taxi cuspidatae, is used for synthetic anti-cancer medicine paclitaxel and analogue thereof, and its structure is as follows:
For the research that obtains more taxol raw material, carried out utilizing tissue culture, method such as semi-synthetic and complete synthesis to obtain taxol both at home and abroad, in these methods, the output that tissue culture obtains is little, and that is that all right is ripe for the industrialization feasibility; Complete synthesis yield is low and cost is huge, also is difficult to carry out industrialization; And utilize 9-dihydro-13-acetyl baccatin III to carry out semi-synthetic taxol and analogue is simple and easy to do.For example, WO98/50378 discloses a kind of C-7 position hydroxyl with due care base protection 9-dihydro-1 3-ethanoyl baccatin III, oxidation C-9 position and add suitable side chain, the method for taxol biosynthesis and analogue thereof in the C-13 position.
The content of 9-dihydro-13-acetyl baccatin III is higher in Ramulus et folium taxi cuspidatae; generally between 0.01%～0.08 weight %; the content of 9-dihydro-13-acetyl baccatin III can reach 0.07%～0.08 weight % in the branches and leaves of T. canadensis; be more than the three-to-four-fold of content of taxol; branches and leaves are renewable resources in addition, therefore utilize 9-dihydro-13-acetyl baccatin III to have broad application prospect and commercial value for the method for semi-synthetic taxol of starting raw material and analogue thereof.
Therefore, except from Ramulus et folium taxi cuspidatae, extracting taxol compound, also need to develop a kind of mixture of the bearing taxanes that extracts from Ramulus et folium taxi cuspidatae the method for separation and purification 9-dihydro-13-acetyl baccatin III quickly and efficiently.
The content of invention
The purpose of this invention is to provide a kind of method of from Ramulus et folium taxi cuspidatae, extracting 9-dihydro-13-acetyl baccatin III and taxol.
Therefore, the invention provides a kind of method of extracting 9-dihydro-13-acetyl baccatin III and taxol from Ramulus et folium taxi cuspidatae, it comprises the steps:
(1) with organic vat liquor Ramulus et folium taxi cuspidatae is carried out lixiviate with multistage continuous diacolation extraction method, obtain containing organic extracting solution of bearing taxanes;
(2) remove organic solvent in organic extracting solution, obtain the water-based vat liquor;
(3) with polar organic solvent described water-based vat liquor is extracted, obtain organic extract liquid;
(4) remove organic solvent in the extraction liquid, obtain containing the medicinal extract of described bearing taxanes;
(5) described medicinal extract is carried out silica gel column chromatography, with 4: 1-1: 1 C
4-C
8Alkane-acetate C
1-C
6Alkane ester or with 4: 1-1: 1 sherwood oil-acetate C
1-C
6Alkane ester wash-out obtains containing the thick solution of 9-dihydro-13-acetyl baccatin III and taxol;
(6) remove C in the described thick solution
4-C
8Alkane-acetate C
1-C
6Alkane ester or sherwood oil-acetate C
1-C
6The alkane ester solvent obtains containing the dry extract of 9-dihydro-13-acetyl baccatin III and taxol.
The accompanying drawing summary
Fig. 1 is the synoptic diagram that the multistage continuous diacolation of the present invention extracts;
Fig. 2 is the separation and purification schema in better example of the present invention;
Fig. 3 is the synoptic diagram of the diacolation bucket of a better example of the present invention.
Embodiment
The present invention is described with reference to the accompanying drawings in more detail.
The extracting method of 9-dihydro-13-acetyl baccatin III of the present invention and taxol is made up of three parts:
1. extraction bearing taxanes
The extracting method of bearing taxanes comprises with multistage continuous diacolation extraction method Ramulus et folium taxi cuspidatae is carried out lixiviate.Described multistage continuous diacolation extraction method as shown in Figure 1, it comprises a plurality of placed in-line diacolation sections, the Ramulus et folium taxi cuspidatae that adds a certain amount of drying during use earlier in each diacolation section, add vat liquor lentamente to the first diacolation section subsequently, this vat liquor is flowed through and is flowed to the second diacolation section behind the Ramulus et folium taxi cuspidatae of the first diacolation section.Vat liquor flows to last diacolation section gradually under the driving of the vat liquor of follow-up adding.After vat liquor is flowed through last diacolation section, collect a certain amount of vat liquor, stop to add vat liquor and the second diacolation section that alters course adding vat liquor to the first diacolation section, promptly this moment, the second diacolation section was promoted as the first diacolation section.Add the Ramulus et folium taxi cuspidatae of new not lixiviate after the first original diacolation section discharging as last diacolation section.
Stop to add vat liquor and making to depend on the amount of the Ramulus et folium taxi cuspidatae that the diacolation section is contained and the flow of vat liquor into to the opportunity that the second diacolation section adds vat liquor to the first diacolation section.In general, collect 1-3 that the vat liquor volume (unit is milliliter) that obtains reaches the average contained Ramulus et folium taxi cuspidatae weight of each diacolation section (unit is gram) when flowing through last diacolation section doubly, better 1.5-2.5 doubly when being preferably 2 times, just can implement the reinforced conversion of described vat liquor.
In the inventive method, described vat liquor can add in a continuous manner, also can add in mode intermittently.
In a better example of the present invention, red bean branches and leaves raw material on average is placed on each diacolation section, when collecting the vat liquor volume (ml) that obtains when flowing through last step diacolation section and reaching 1-3 times of the contained Ramulus et folium taxi cuspidatae weight of each diacolation section (g), stop to add extracting solution to first step diacolation section, and in the end one-level increases a diacolation section again, adds extracting solution to second stage diacolation section subsequently.At this moment, the diacolation section that increases newly can be to form after adding new Ramulus et folium taxi cuspidatae again after the discharging of former first step diacolation section.
The diacolation device of implementing the multistage continuous diacolation extraction of the present invention is without particular limitation.In a better example of the present invention, use diacolation bucket as shown in Figure 3.
Diacolation bucket shown in Figure 3 comprises barrel 1, bottom screen cloth 2 and optional valve 3.Described barrel is made by materials such as stainless steel, glass, potteries usually, it can be straight-cylindrical, also can be tubaeform (for example side and bottom surface are 120-130 ℃, better 125 ℃ angle).The bottom surface of barrel 1 has one deck screen cloth 2, and the mesh size of screen cloth is without particular limitation, depends on the required vat liquor flow and the degree of grinding of Ramulus et folium taxi cuspidatae.In a better example of the present invention, described screen cloth is 20 purpose stainless steel meshs.
Diacolation bucket screen cloth below also can randomly comprise a valve, thereby but when this diacolation bucket withdraws from the diacolation flow process valve-off, in order to avoid inner residue outflow and polluting.
The progression of described multistage continuous diacolation extraction method does not have special restriction, but considers that from the angle of economy described multistage continuous diacolation extraction method is preferably the 4-15 level, and more preferably the 6-12 level is preferably 10 grades.
Before lixiviate, need Ramulus et folium taxi cuspidatae is carried out drying.The exsiccant method is well-known in the art.Its indefiniteness example comprises heat drying.The water content of dry back Ramulus et folium taxi cuspidatae is without particular limitation.But, if water content is too high, then can reduce the concentration of the bearing taxanes water-based vat liquor that makes, cause the aftertreatment difficulty; Water content is too low, then can increase dry required cost.Therefore, in general, water content is preferably 5%～15 weight %, is preferably 10 weight %.
The multistage continuous percolation of the present invention is with an organic solvent as vat liquor.Described organic solvent is without particular limitation, as long as the bearing taxanes in its energy lixiviate Ramulus et folium taxi cuspidatae.The indefiniteness example of described organic solvent comprises alcohol, better is C
1-6Fatty Alcohol(C12-C14 and C12-C18), for example aqueous solution of methyl alcohol, ethanol, propyl alcohol and butanols or their 50-100%; Ketone, for example acetone or its 50-100% aqueous solution, methylethylketone or its 50-100% aqueous solution, butanone, pimelinketone; Ether, for example ether or its 50-100% aqueous solution, methyl ethyl ether or its 50-100% aqueous solution; Ester, for example acetate C
1-4The alkane ester is as ethyl acetate, methyl acetate, propyl acetate, butylacetate.In a better example of the present invention, described organic solvent is that concentration is the aqueous solution of methyl alcohol, ethanol or the acetone of 50-100%.
In order to increase surface in contact to improve diacolation efficient, for example in advance Ramulus et folium taxi cuspidatae is ground into generally that particle diameter is 0.5-5mm, be preferably the powder of 1-2mm.
After obtaining containing organic extracting solution of bearing taxanes with the organic vat liquor of the present invention, remove the organic solvent in described organic extracting solution, owing to contain the cause that moisture or use contain water extract in the Ramulus et folium taxi cuspidatae, what therefore obtain is the water-based vat liquor (being also referred to as moisture crude extract) of bearing taxanes.Described method of removing organic solvent is the ordinary method of this area, and for example it can be the method for heated volatile or underpressure distillation.
The moisture crude extract of the bearing taxanes that obtains with aforesaid method contains many non-bearing taxanes impurity.In order to improve the purity of bearing taxanes medicinal extract, the inventive method also can comprise with polar organic solvent its step that extracts, when implementing described extraction step, contain to the bearing taxanes that obtains earlier and add a certain amount of water in the aqueous extract, extract subsequently.The add-on of described water is without particular limitation, as long as can reach the purpose of extraction.Those of ordinary skill in the art can easily determine the add-on of water as the case may be.In a better example of the present invention, the add-on of water be contain the aqueous extract volume 0.5-2 doubly, be preferably 1 times.The polar organic solvent that extraction step is used is without particular limitation, as long as this solvent can extract bearing taxanes as extraction agent from the aqueous solution.The example of described extraction agent comprises the lower halogenated hydrocarbon, for example methyl chloride, methylene dichloride, trichloromethane, monochloroethane, and 1,1-ethylene dichloride, 1,2-ethylene dichloride etc. better are trichloromethanes.
The temperature that the present invention is used for diacolation can be a normal temperature, also can carry out under heating up to improve the efficient of diacolation.But extraction temperature should be lower than the boiling point of organic solvent in the vat liquor, in order to avoid organism seethes with excitement and influences diacolation efficient.
Each diacolation section of the present invention also can be equipped with whipping appts, with the surface in contact of further raising vat liquor and Ramulus et folium taxi cuspidatae, improves extraction rate.
The extraction yield of bearing taxanes of the present invention is that benchmark is measured with 9-dihydro-13-acetyl baccatin III; its testing method is as follows: get 10g tree powder and extract with 95% ethanol repeated ultrasonic of 8 times of weight of tree powder; each 2 hours; exist with the peak that the HPLC method detects less than 9-dihydro-13-acetyl baccatin III until extracting solution; extracting solution is merged; reclaim; constant volume is in the 100mL volumetric flask; measure 9-dihydro-13-acetyl baccatin III content (external standard method with the HPLC method; referring to two appendix VD of Pharmacopoeia of the People's Republic of China version in 2000), obtain setting the content of 9-dihydro-13-acetyl baccatin III in the powder.Other gets the percolate that the multistage diacolation that is equivalent to 10g tree powder extracts gained, and constant volume is measured 9-dihydro-13-acetyl baccatin III content in the 100mL volumetric flask with the HPLC method as stated above, compares with the content in the tree powder and promptly gets extraction yield.
Extracting method of the present invention has extraction efficiency height (it can be extracted into the bearing taxanes more than 99% in the Ramulus et folium taxi cuspidatae in the medicinal extract); extraction time short (extraction time shortens more than the twice than traditional extraction); the advantage of solvent load few (this method solvent load is 1/10th～ten sixths of traditional extraction solvent load), thus reduce manufacturing cost and helped environment protection.
In a better example of the present invention, described extracting method comprises the steps:
1) is 10 weight % with Ramulus et folium taxi cuspidatae seasoning to water content, is ground into particle diameter and is 1～2 millimeter powder;
2) extracting the Duan Zhongyong concentration at 10 grades of continuous diacolations is that the aqueous solution of 50%～100% methyl alcohol, ethanol or acetone carries out lixiviate to Ramulus et folium taxi cuspidatae, obtains dense percolate;
3) described dense percolate reduction vaporization is not had to obtain moisture crude extract behind methyl alcohol, ethanol or the acetone substantially to solution;
4) in this moisture crude extract, add the dilution of equal-volume water, with this diluent of chloroform extraction;
5) reclaim chloroform and get medicinal extract, by the gross weight of red bean branches and leaves, the bearing taxanes extraction yield is 5～10 weight % greater than the yield of extract of 95 weight %.
Described yield of extract is that the weight of the dry extract (the bearing taxanes extraction yield is greater than 95 weight %) that will finally obtain is divided by the Ramulus et folium taxi cuspidatae gross weight meter that is used for the lixiviate drying.Because the present invention adopts continuous diacolation extraction method, therefore, the described Ramulus et folium taxi cuspidatae gross weight that is used for the drying of lixiviate is the summation of Ramulus et folium taxi cuspidatae loadings (m) of (n) at different levels drying of continuous diacolation lixiviate
2. from described bearing taxanes, extract mixing of 9-dihydro-13-acetyl baccatin III and taxol
Compound
The method that the present invention extracts the mixture of 9-dihydro-13-acetyl baccatin III and taxol from above in the medicinal extract comprises the following steps:
(1) described medicinal extract is carried out silica gel column chromatography, with 4: 1-1: 1 C
4-C
8Alkane-acetate C
1-C
6Alkane ester or 4: 1-1: 1 sherwood oil-acetate C
1-C
6Alkane ester wash-out obtains containing the thick solution of 9-dihydro-13-acetyl baccatin III and taxol;
(2) remove C in the described thick solution
4-C
8Alkane-acetate C
1-C
6Alkane ester or sherwood oil-acetate C
1-C
6The alkane ester solvent obtains containing the dry extract of 9-dihydro-13-acetyl baccatin III and taxol.
The silica gel column chromatography that uses in the inventive method is without particular limitation, and it can be this area silica gel column chromatography commonly used.In a better example of the present invention, the silica gel that described silica gel column chromatography uses is available from Shandong Zhifu silica gel factory.
It is without particular limitation to be used to dissolve the solvent that contains bearing taxanes medicinal extract before carrying out the silica gel column chromatography separation, as long as this solvent can dissolve described medicinal extract and 9-dihydro-13-acetyl baccatin III and taxol in the described solution are adsorbed on the silicagel column.The indefiniteness example of described solvent has ethyl acetate, methylene dichloride, chloroform or its mixture.
The elutriant that is used for wash-out 9-dihydro-13-acetyl baccatin III and taxol is 4: 1-1: the C of 1 (v/v)
4-C
8Alkane-acetate C
1-C
6The alkane ester is preferably 3: 1-1.5: the C of 1 (v/v)
4-C
8Alkane-acetate C
1-C
6The alkane ester perhaps can use 4: 1-1: the sherwood oil of 1 (v/v)-acetate C
1-C
6The alkane ester is preferably 3: 1-1.5: the sherwood oil of 1 (v/v)-acetate C
1-C
6The alkane ester.
In the present invention, term " C
4-C
8Alkane " be meant straight or branched alkane or naphthenic hydrocarbon with 4-8 carbon atom, for example normal butane, Trimethylmethane, uncle's butane, Skellysolve A, iso-pentane, normal hexane, hexanaphthene and normal heptane, octane etc.Term " acetate C
1-C
6The alkane ester " be meant acetate C
1-C
6Straight or branched alkane ester, for example methyl esters, ethyl ester, propyl ester, isopropyl ester, butyl ester, the tert-butyl ester, pentyl ester, isopentyl ester, own ester etc.Be preferably acetate C
1-C
4The alkane ester is as methyl acetate, ethyl acetate, n-propyl acetate and isopropyl acetate, more preferably ethyl acetate.
In a better example of the present invention, normal hexane-ethyl acetate of using 2: 1 (v/v) is as elutriant.
Then remove the 9-dihydro-13-acetyl baccatin III that obtains and the solvent in the thick solution of taxol, obtain containing the dry extract of 9-dihydro-13-acetyl baccatin III and taxol.Described without particular limitation except that the method for desolvating, can be any method of this area, as long as it has no adverse effect to 9-dihydro-13-acetyl baccatin III product.For example, it can adopt methods such as evaporation, spraying drying.
3. isolation of taxol and 9-dihydro-13-acetyl baccatin III
In order to obtain highly purified 9-dihydro-13-acetyl baccatin III and paclitaxel prodrugs, also need the 9-dihydro-13-acetyl baccatin III that obtains is separated with the taxol dry extract.Described separating step comprises: described dry extract is carried out silica gel column chromatography once more, with 4: 1-1: the C of 1 (v/v)
1-C
4Haloalkane-acetate C
1-C
6Alkane ester wash-out, the stream part of collecting 4-5 column volume obtains paclitaxel prodrugs; Collect stream part of 6-7 column volume, obtain 9-dihydro-13-acetyl baccatin III product.
The silica gel column chromatography that uses in the separating step of the present invention is without particular limitation, and it can be this area silica gel column chromatography commonly used.In a better example of the present invention, described silica gel column chromatography uses the silica gel available from Shandong Zhifu silica gel factory identical with above-mentioned separating step.
It is without particular limitation to be used to dissolve the solvent that contains 9-dihydro-13-acetyl baccatin III and taxol mixture dry extract before carrying out the silica gel column chromatography separation, as long as this solvent can dissolve described medicinal extract and 9-dihydro-13-acetyl baccatin III and taxol in the described solution are adsorbed on the silicagel column respectively.The indefiniteness example of described solvent has ethyl acetate, methylene dichloride, chloroform or its mixture.
The acetate C that is used for the elutriant of wash-out
1-C
6The alkane ester is meant acetate C
1-C
6Straight or branched alkane ester, for example methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butylacetate, tert.-butyl acetate, pentyl acetate, Isoamyl Acetate FCC, hexyl acetate etc.Be preferably acetate C
1-C
4The alkane ester is as methyl acetate, ethyl acetate, n-propyl acetate and isopropyl acetate, more preferably ethyl acetate.
C in the elutriant
1-C
4The indefiniteness example of haloalkane has methyl chloride, methylene dichloride, trichloromethane, 1,1-ethylene dichloride, 1,2-ethylene dichloride, 1, propylidene chloride 1,1,3-propylene dichloride, 1,1-dichlorobutane, trifluoromethane, 1,1 ,-C2H4F2 C2H4F2,1,3-C2H4F2 C2H4F2 etc.Better be methylene dichloride, trichloromethane; Methylene dichloride preferably.
In the elutriant, C
1-C
4Haloalkane and acetate C
1-C
6The volume ratio of alkane ester is generally 4: 1-1: 1, be preferably 3: 1-1.5: and 1, more preferably 2: 1.
During wash-out, collect stream part of 4-5 column volume, what obtain is paclitaxel prodrugs; Collect stream part of 6-7 column volume, what obtain is 9-dihydro-13-acetyl baccatin III product.
Sometimes in order to obtain more highly purified 9-dihydro-13-acetyl baccatin III, also can carry out recrystallization to the 9-dihydro-13-acetyl baccatin III that this separation obtains, the solvent that described recrystallization uses is without particular limitation.Those of ordinary skill in the art can easily learn suitable recrystallization solvent after having read content disclosed by the invention.In a better example of the present invention, the solvent that described recrystallization adopts is selected from 4: 1-1: ethyl acetate-normal hexane of 1,4: 1-1: methyl acetate-normal hexane of 1,4: 1-1: ethyl acetate-hexanaphthene of 1,4: 1-1: methyl acetate-hexanaphthene of 1,4: 1-1: propyl acetate-normal heptane of 1,4: 1-1: propyl acetate-normal hexane of 1,4: 1-1: propyl acetate-hexanaphthene of 1,4: 1-1: butylacetate-normal hexane of 1,4: 1-1: butylacetate-hexanaphthene of 1,4: 1-1: butylacetate-normal heptane of 1 etc. are preferably 4: 1-1: 1 ethyl acetate-normal hexane solvent as solvent.
Fig. 2 is the schema of 9-dihydro-13-acetyl baccatin III described in example of the present invention and taxol separation method.It comprises that the medicinal extract to the mixture that contains bearing taxanes carries out the silica gel column chromatography separation; obtain the mixture of 9-dihydro-13-acetyl baccatin III and taxol; subsequently the mixture that obtains is carried out silicagel column and separate, obtain 9-dihydro-13-acetyl baccatin III and paclitaxel prodrugs respectively.
Equally, also can carry out recrystallization purifying to the paclitaxel prodrugs that obtains.The solvent that recrystallization uses is without particular limitation, can be this area solvent commonly used.In a better example of the present invention, the recrystallization solvent of use is the aqueous solution of alcohol, and for example the aqueous solution of the 30-60% of methyl alcohol, ethanol, propyl alcohol, glycerol (v/v) better is the methyl alcohol or the alcoholic acid aqueous solution of 50% (v/v).
Further specify the present invention below in conjunction with embodiment.
Embodiment
Testing method
1. 9-dihydro-13-acetyl baccatin III extraction yield
The testing method of 9-dihydro-13-acetyl baccatin III extraction yield of the present invention is as follows: get 10g tree powder and extract with 95% ethanol repeated ultrasonic of 8 times of weight of tree powder; each 2 hours; exist with the peak that the HPLC method detects less than 9-dihydro-13-acetyl baccatin III until extracting solution; extracting solution is merged; reclaim; constant volume is in the 100mL volumetric flask; measure the amount A (external standard method of 9-dihydro-13-acetyl baccatin III with the HPLC method; referring to two appendix VD of Pharmacopoeia of the People's Republic of China version in 2000), obtain setting the content of 9-dihydro-13-acetyl baccatin III in the powder.
Other gets the percolate that the multistage diacolation that is equivalent to 10g tree powder extracts gained, and constant volume is measured 9-dihydro-13-acetyl baccatin III content in the 100mL volumetric flask with the HPLC method as stated above, compares with the content in the tree powder and promptly gets extraction yield.
2. the mensuration of 9-dihydro-13-acetyl baccatin III purity
Measure the purity (external standard method is referring to two appendix VD of Pharmacopoeia of the People's Republic of China version in 2000) of 9-dihydro-13-acetyl baccatin III with the HPLC method; Used high performance liquid chromatograph is day LC-10A pump and the SPD-10A UV-detector of island proper Tianjin company production among the embodiment.
3. the extraction yield of taxol and purity testing
Use the measuring method identical to measure described extraction yield and purity with 9-dihydro-13-acetyl baccatin III.
Embodiment 1
A. prepare medicinal extract
Getting 1Kg is the T. canadensis cauline leaf of 10 weight % through seasoning to water content, be ground into granularity and be 1～2 millimeter powder, other gets the diacolation bucket of ten 250ml, every diacolation barreled is gone into 50g tree powder, by shown in Figure 1, disclose the ethanolic soln of the middle 100ml 95% of adding toward first diacolation in mode intermittently, percolate is disclosed the back at first diacolation of flowing through and is flowed into second diacolation bucket, and the like, the continuous ethanolic soln that adds 100ml 95% in first diacolation bucket, finish until the tenth diacolation bucket diacolation, the percolate of collecting the tenth diacolation bucket is until reaching 100ml, stop in first diacolation bucket, adding percolate, make in second diacolation bucket, to add the ethanolic soln of 100ml95% into, add the dry tree of 50g powder after the discharging of first diacolation bucket as new last step diacolation section, the percolate second time that is about to the tenth diacolation bucket adds first diacolation bucket, regather the percolate first time of first diacolation bucket, after reaching 100ml, stop to add percolate to second diacolation bucket, make in the 3rd diacolation bucket, to add the ethanolic soln of 100ml 95% into, add the dry tree of 50g powder after the discharging of second diacolation bucket, and the like.The dense percolate that merges 500g tree powder amounts to 1000ml, and reclaim under reduced pressure is not to there being the alcohol flavor, and thin up is to about 500ml, and with 100ml chloroform extraction three times, water does not have the peak of taxol and 9-dihydro-13-acetyl baccatin III substantially with the HPLC inspection.Reclaim under reduced pressure chloroform extraction liquid gets medicinal extract 30g.
With the extraction yield of bearing taxanes in the aforesaid method mensuration medicinal extract, the extraction yield of bearing taxanes is 99% as a result.This medicinal extract leaching process about 8 hours consuming time altogether.
B. the mixture that separates 9-dihydro-13-acetyl baccatin III and taxol
30 gram steps A gained medicinal extract are dissolved in carry out silica gel column chromatography in the ethyl acetate solvent; the silica gel that uses is available from Shandong Zhifu silica gel factory; petroleum ether-ethyl acetate gradient elution with 4: 1; collection contains the part of 9-dihydro-13-acetyl baccatin III and taxol, reclaims the dry extract that solvent obtains containing 9-dihydro-13-acetyl baccatin III and taxol.
C. separate 9-dihydro-13-acetyl baccatin III and taxol
The dry extract that gained is contained 9-dihydro-13-acetyl baccatin III and taxol is dissolved in the mixed solvent of methylene dichloride-ethyl acetate; pass through silica gel column chromatography once more; the silica gel that uses is available from Shandong Zhifu silica gel factory; with 2: 1 methylene dichloride-ethyl acetate gradient elution, collect the stream part (6-7 column volume) that contains stream part (4-5 column volume) of taxol and contain 9-dihydro-13-acetyl baccatin III.
Recovery contains the solvent of 9-dihydro-13-acetyl baccatin III stream part; obtain purity and be 95% 9-dihydro-13-acetyl baccatin III; through ethyl acetate-normal hexane recrystallization; obtain purity greater than 98% 9-dihydro-13-acetyl baccatin III, 0.36 gram, product is confirmed as 9-dihydro-13-acetyl baccatin III through NMR:
1H-NMR(CDCl
3)δ：16～19CH3：1.62(3H，S)，1.43(3H，S)，2.37(3H，S)，2.06(3H，S)；OAc(CH
3)：2.02(3H，S)，2.05(3H，S)，2.06(3H，S)。
13C-NMR(CDCl
3)δ：(1～20)78.3，73.7，38.6，81.3，84.8，33.2，69.5，57.5，36.4，75.6，135.7，142.6，71.2，38.4，43.8，22.3，28.4，15.7，14.1，76.8，OBzC＝O：166.7，131.5，127.9，130.7，133.6，OAc(CH
3)：21.2，22.7，22.6；(C＝O)：170.1，171.1，170.5。
In addition, reclaim the solvent contain taxol stream part, obtain purity and be 96% taxol, through 50% methanol aqueous solution recrystallization, obtain purity greater than 99% taxol 0.12 gram, product is confirmed as taxol through NMR:
1H-NMR data (CDCl
3) δ: 16～19CH
3: 1.63 (3H, S), 1.25 (3H, S), 1.78 (3H, S), 1.81 (3H, S); OAc (CH
3): 2.10 (3H, S), 2.23 (3H, S)
13C-NMR data (CDCl
3) δ: (1～20) 78.2,73.1,46.2,82.8,83.7,36.8,69.8,56.7,209.7,76.1,135.1,138.4,76.5,35.3,42.9,22.1,27.9,14.3,12.5,76.4; OBzC=O:166.5,130.2,130.0,128.4,133.2,1 '～3 ': 170.5,73.2,54.1, Ph:138.3,126.9,128.2,127.5, NBzC=O:167.0,133.2,126.8,131.2; OAc (CH
3): 20.3,21.2; (C=O): 170.0,170.1.
Extraction yield with 9-dihydro-13-acetyl baccatin III and taxol in the Ramulus et folium taxi cuspidatae of aforesaid method mensuration is respectively 99% and 99.2%.
Comparative example 1
500g and the foregoing description 1 identical Ramulus et folium taxi cuspidatae powder is placed a container, with the ethanol lixiviates of 8 times of tree grain weight amounts, extract four times at every turn, each 5 hours, obtain 95% ethanolic soln 16000mL, extraction time is 20 hours.With the method identical with embodiment 1 with this ethanolic soln reclaim under reduced pressure to there not being the alcohol flavor, thin up is to about 500ml, with 100ml chloroform extraction three times, water does not have the peak of taxol and 9-dihydro-13-acetyl baccatin III substantially with the HPLC inspection.Reclaim under reduced pressure chloroform extraction liquid gets medicinal extract 30g.With the extraction yield of bearing taxanes in the aforesaid method mensuration medicinal extract, the extraction yield of bearing taxanes is 95% as a result.
A. prepare medicinal extract
Getting 10Kg is the T. canadensis cauline leaf of 5 weight % through seasoning to water content, be ground into the powder that granularity is the 2-5 millimeter, other gets the diacolation bucket of eight 2500ml, every diacolation barreled is gone into 500g tree powder, by shown in Figure 1, the methanol solution of adding 95% in disclosing toward first diacolation lentamente in a continuous manner, percolate adds second diacolation bucket, and the like, the volume of each percolate of collecting is 500ml, with the method identical, but be to use methyl chloride to replace chloroform to extract with embodiment 1, altogether medicinal extract 320g.
With the extraction yield of bearing taxanes in the aforesaid method mensuration medicinal extract, the extraction yield of bearing taxanes is 99% as a result.
B. the mixture that separates 9-dihydro-13-acetyl baccatin III and taxol
30 gram steps A gained medicinal extract are dissolved in carry out silica gel column chromatography in the ethyl acetate solvent; the silica gel that uses is available from Shandong Zhifu silica gel factory; with 1: 1 normal hexane-methyl acetate gradient elution; collection contains the part of 9-dihydro-13-acetyl baccatin III and taxol, reclaims the dry extract that solvent obtains containing 9-dihydro-13-acetyl baccatin III and taxol.
C. separate 9-dihydro-13-acetyl baccatin III and taxol
The dry extract that gained is contained 9-dihydro-13-acetyl baccatin III is dissolved in the mixed solvent of trichloromethane-methyl acetate; pass through silica gel column chromatography once more; the silica gel that uses is available from Shandong Zhifu silica gel factory; with 3: 1 trichloromethanes-methyl acetate gradient elution, collect the stream part (6-7 column volume) that contains stream part (4-5 column volume) of taxol and contain 9-dihydro-13-acetyl baccatin III.
Recovery contains the solvent of 9-dihydro-13-acetyl baccatin III stream part; obtain purity and be 95% 9-dihydro-13-acetyl baccatin III; through methyl acetate-normal hexane recrystallization; obtain purity greater than 98% 9-dihydro-13-acetyl baccatin III, 0.37 gram, product is confirmed as 9-dihydro-13-acetyl baccatin III through NMR:
1H-NMR(CDCl
3)δ：16～19CH3：1.62(3H，S)，1.43(3H，S)，2.37(3H，S)，2.06(3H，S)；OAc(CH
3)：2.02(3H，S)，2.05(3H，S)，2.06(3H，S)。
13C-NMR(CDCl
3)δ：(1～20)78.3，73.7，38.6，81.3，84.8，33.2，69.5，57.5，36.4，75.6，135.7，142.6，71.2，38.4，43.8，22.3，28.4，15.7，14.1，76.8，OBzC＝O：166.7，131.5，127.9，130.7，133.6，OAc(CH
3)：21.2，22.7，22.6；(C＝O)：170.1，171.1，170.5。
In addition, reclaim the solvent contain taxol stream part, obtain purity and be 96% taxol, through 50% aqueous ethanolic solution recrystallization, obtain purity greater than 99% taxol 0.13 gram, product is confirmed as taxol through NMR:
1H-NMR data (CDCl
3) δ: 16～19CH
3: 1.63 (3H, S), 1.25 (3H, S), 1.78 (3H, S), 1.81 (3H, S); OAc (CH
3): 2.10 (3H, S), 2.23 (3H, S)
13C-NMR data (CDCl
3) δ: (1～20) 78.2,73.1,46.2,82.8,83.7,36.8,69.8,56.7,209.7,76.1,135.1,138.4,76.5,35.3,42.9,22.1,27.9,14.3,12.5,76.4; OBzC=O:166.5,130.2,130.0,128.4,133.2,1 '～3 ': 170.5,73.2,54.1, Ph:138.3,126.9,128.2,127.5, NBzC=O:167.0,133.2,126.8,131.2; OAc (CH
3): 20.3,21.2:(C=O): 170.0,170.1.
Extraction yield with 9-dihydro-13-acetyl baccatin III and taxol in the Ramulus et folium taxi cuspidatae of aforesaid method mensuration is respectively 99.2% and 99.3%.
A. prepare medicinal extract
Get 2000g through seasoning to water content be 12 weight % the T. canadensis cauline leaf, be ground into granularity and be 0.5～2 millimeter powder, other gets the diacolation bucket of 12 250ml, every diacolation barreled is gone into 50g tree powder, by shown in Figure 1, disclose the acetone soln of the middle 150ml 70% of adding toward first diacolation in mode intermittently, percolate is disclosed the back at first diacolation of flowing through and is flowed into second diacolation bucket, and the like, the continuous acetone soln that adds 150ml 70% in first diacolation bucket, finish until the 12 diacolation bucket diacolation, the percolate of collecting the 12 diacolation bucket is until reaching 150ml, the acetone soln that adds 150ml 70% then in second diacolation bucket, add the dry tree of 50g powder after the discharging of first diacolation bucket, the percolate second time of the 12 diacolation bucket is added first diacolation bucket, regather the percolate first time of first diacolation bucket, after reaching 150ml, stop to add percolate to second diacolation bucket, the acetone soln that adds 150ml 70% in the 3rd the diacolation bucket, add the dry tree of 50g powder after the discharging of second diacolation bucket, and the like.The dense percolate that merges 600g tree powder amounts to 1800ml; reclaim under reduced pressure to gas-chromatography does not have the acetone peak, and thin up is to about 500ml, with 100ml 1; the peak that does not have taxol and 9-dihydro-13-acetyl baccatin III is substantially checked in 2-ethylene dichloride extraction three times, water with HPLC.The reclaim under reduced pressure extraction liquid gets medicinal extract 36g.
With the extraction yield of bearing taxanes in the aforesaid method mensuration medicinal extract, the extraction yield of bearing taxanes is 99% as a result.This medicinal extract leaching process about 8 hours consuming time altogether.
B. the mixture that separates 9-dihydro-13-acetyl baccatin III and taxol
30 gram steps A gained medicinal extract are dissolved in carry out silica gel column chromatography in the ethyl acetate solvent; the silica gel that uses is available from Shandong Zhifu silica gel factory; with 3: 1 Skellysolve As-butylacetate gradient elution; collection contains the part of 9-dihydro-13-acetyl baccatin III and taxol, reclaims the dry extract that solvent obtains containing 9-dihydro-13-acetyl baccatin III and taxol.
C. separate 9-dihydro-13-acetyl baccatin III and taxol
The dry extract that gained is contained 9-dihydro-13-acetyl baccatin III and taxol is dissolved in ethylene dichloride-butyl acetate solvent; pass through silica gel column chromatography once more; the silica gel that uses is available from Shandong Zhifu silica gel factory; with 1.5: 11; 1-ethylene dichloride-butylacetate gradient elution is collected the stream part (6-7 column volume) that contains stream part (4-5 column volume) of taxol and contain 9-dihydro-13-acetyl baccatin III.
Recovery contains the solvent of 9-dihydro-13-acetyl baccatin III stream part; obtain purity and be 95% 9-dihydro-13-acetyl baccatin III; through ethyl acetate-hexanaphthene recrystallization; obtain purity greater than 98% 9-dihydro-13-acetyl baccatin III, 0.344 gram, product is confirmed as 9-dihydro-13-acetyl baccatin III through NMR:
1H-NMR(CDCl
3)δ：16～19CH3：1.62(3H，S)，1.43(3H，S)，2.37(3H，S)，2.06(3H，S)；OAc(CH
3)：2.02(3H，S)，2.05(3H，S)，2.06(3H，S)。
13C-NMR(CDCl
3)δ：(1～20)78.3，73.7，38.6，81.3，84.8，33.2，69.5，57.5，36.4，75.6，135.7，142.6，71.2，38.4，43.8，22.3，28.4，15.7，14.1，76.8，OBzC＝O：166.7，131.5，127.9，130.7，133.6，OAc(CH
3)：21.2，22.7，22.6；(C＝O)：170.1，171.1，170.5。
In addition, reclaim the solvent contain taxol stream part, obtain purity and be 96% taxol, through 40% glycerin solution recrystallization, obtain purity greater than 99% taxol 0.115 gram, product is confirmed as taxol through NMR:
1H-NMR data (CDCl
3) δ: 16～19CH
3: 1.63 (3H, S), 1.25 (3H, S), 1.78 (3H, S), 1.81 (3H, S); OAc (CH
3): 2.10 (3H, S), 2.23 (3H, S)
13C-NMR data (CDCl
3) δ: (1～20) 78.2,73.1,46.2,82.8,83.7,36.8,69.8,56.7,209.7,76.1,135.1,138.4,76.5,35.3,42.9,22.1,27.9,14.3,12.5,76.4; OBzC=O:166.5,130.2,130.0,128.4,133.2,1 '～3 ': 170.5,73.2,54.1, Ph:138.3,126.9,128.2,127.5, NBzC=O:167.0,133.2,126.8,131.2; OAc (CH
3): 20.3,21.2; (C=O): 170.0,170.1.
Extraction yield with 9-dihydro-13-acetyl baccatin III and taxol in the Ramulus et folium taxi cuspidatae of aforesaid method mensuration is respectively 99.2% and 99.3%.
Embodiment 4
A. prepare medicinal extract
With the method continuous diacolation Ramulus et folium taxi cuspidatae powder identical with embodiment 1, but be to use 90% ether as vat liquor, use 1, the 1-ethylene dichloride is as extraction agent, and the extraction yield of bearing taxanes is 98.5% as a result.This medicinal extract leaching process about 8.5 hours consuming time altogether.
B. separate 9-dihydro-13-acetyl baccatin III and taxol
With separating 9-dihydro-13-acetyl baccatin III and taxol with embodiment 1 same procedure, still wash-out uses sherwood oil-butylacetate of 2: 1 for the first time, obtains containing the dry extract of 9-dihydro-13-acetyl baccatin III and taxol; For the second time wash-out use 2: 11, propylidene chloride 1-butylacetate is collected the stream part (6-7 column volume) that contains stream part (4-5 column volume) of taxol and contain 9-dihydro-13-acetyl baccatin III respectively.
Reclaim 9-dihydro-13-acetyl baccatin III with the method identical with embodiment 1, obtain the 0.345g product, extraction yield is 98.5%, and NMR confirms as 9-dihydro-13-acetyl baccatin III:
1H-NMR(CDCl
3)δ：16～19CH3：1.62(3H，S)，1.43(3H，S)，2.37(3H，S)，2.06(3H，S)；OAc(CH
3)：2.02(3H，S)，2.05(3H，S)，2.06(3H，S)。
13C-NMR(CDCl
3)δ：(1～20)78.3，73.7，38.6，81.3，84.8，33.2，69.5，57.5，36.4，75.6，135.7，142.6，71.2，38.4，43.8，22.3，28.4，15.7，14.1，76.8，OBzC＝O：166.7，131.5，127.9，130.7，133.6，OAc(CH
3)：21.2，22.7，22.6；(C＝O)：170.1，171.1，170.5。
In addition, use the method identical with embodiment 1 to reclaim taxol, obtain 0.112 gram product, extraction yield is 99.2%, and NMR confirms as taxol:
1H-NMR data (CDCl
3) δ: 16～19CH
3: 1.63 (3H, S), 1.25 (3H, S), 1.78 (3H, S), 1.81 (3H, S); OAc (CH
3): 2.10 (3H, S), 2.23 (3H, S)
13C-NMR data (CDCl
3) δ: (1～20) 78.2,73.1,46.2,82.8,83.7,36.8,69.8,56.7,209.7,76.1,135.1,138.4,76.5,35.3,42.9,22.1,27.9,14.3,12.5,76.4; OBzC=O:166.5,130.2,130.0,128.4,133.2,1 '～3 ': 170.5,73.2,54.1, Ph:138.3,126.9,128.2,127.5, NBzC=O:167.0,133.2,126.8,131.2; OAc (CH
3): 20.3,21.2; (C=O): 170.0,170.1.
Embodiment 5
A. prepare medicinal extract
With the method continuous diacolation Ramulus et folium taxi cuspidatae powder identical with embodiment 1, but be to use ethyl acetate as vat liquor, use methylene dichloride as extraction agent, the extraction yield of bearing taxanes is 98.0% as a result.This medicinal extract leaching process about 9 hours consuming time altogether.
B. separate 9-dihydro-13-acetyl baccatin III and taxol
With separating 9-dihydro-13-acetyl baccatin III and taxol with embodiment 2 same procedure, still wash-out uses normal butane-propyl acetate of 3.5: 1 for the first time, obtains containing the dry extract of 9-dihydro-13-acetyl baccatin III and taxol; For the second time wash-out use 2: 11,1 ,-C2H4F2 C2H4F2-methyl acetate is collected the stream part that contains taxol and the stream part that contains 9-dihydro-13-acetyl baccatin III respectively.After carrying out recrystallization purifying respectively, use NMR to confirm that it is respectively 9-dihydro-13-acetyl baccatin III and taxol, extraction yield is respectively 98% and 99%.
Taxol:
1H-NMR data (CDCl
3) δ: 16～19CH
3: 1.63 (3H, S), 1.25 (3H, S), 1.78 (3H, S), 1.81 (3H, S); OAc (CH
3): 2.10 (3H, S), 2.23 (3H, S)
13C-NMR data (CDCl
3) δ: (1～20) 78.2,73.1,46.2,82.8,83.7,36.8,69.8,56.7,209.7,76.1,135.1,138.4,76.5,35.3,42.9,22.1,27.9,14.3,12.5,76.4; OBzC=O:166.5,130.2,130.0,128.4,133.2,1 '～3 ': 170.5,73.2,54.1, Ph:138.3,126.9,128.2,127.5, NBzC=O:167.0,133.2,126.8,131.2; OAc (CH
3): 20.3,21.2; (C=O): 170.0,170.1.
9-dihydro-13-acetyl baccatin III:
1H-NMR(CDCl
3)δ：16～19CH3：1.62(3H，S)，1.43(3H，S)，2.37(3H，S)，2.06(3H，S)；OAc(CH
3)：2.02(3H，S)，2.05(3H，S)，2.06(3H，S)。
13C-NMR(CDCl
3)δ：(1～20)78.3，73.7，38.6，81.3，84.8，33.2，69.5，57.5，36.4，75.6，135.7，142.6，71.2，38.4，43.8，22.3，28.4，15.7，14.1，76.8，OBzC＝O：166.7，131.5，127.9，130.7，133.6，OAc(CH
3)：21.2，22.7，22.6；(C＝O)：170.1，171.1，170.5。
Embodiment 6
A. prepare medicinal extract
With the method continuous diacolation Ramulus et folium taxi cuspidatae powder identical with embodiment 1, but be to use the 98% methyl ethyl ether aqueous solution as vat liquor, use monochloroethane as extraction agent, the extraction yield of bearing taxanes is 98.0% as a result.This medicinal extract leaching process about 9 hours consuming time altogether.
B. separate 9-dihydro-13-acetyl baccatin III and taxol
With separating 9-dihydro-13-acetyl baccatin III and taxol with embodiment 4 same procedure, still wash-out uses normal hexane-ethyl acetate of 3.1: 1 for the first time, obtains containing the dry extract of 9-dihydro-13-acetyl baccatin III and taxol; For the second time wash-out use 2.5: 11; 2-methylene dichloride-ethyl acetate; obtain taxol and 9-dihydro-13-acetyl baccatin III respectively; use confirms that with NMR it is respectively 9-dihydro-13-acetyl baccatin III; the extraction yield of taxol is 98.8%, and the extraction yield of 9-dihydro-13-acetyl baccatin III is 99%.
Taxol:
1H-NMR data (CDCl
3) δ: 16～19CH
3: 1.63 (3H, S), 1.25 (3H, S), 1.78 (3H, S), 1.81 (3H, S); OAc (CH
3): 2.10 (3H, S), 2.23 (3H, S)
13C-NMR data (CDCl
3) δ: (1～20) 78.2,73.1,46.2,82.8,83.7,36.8,69.8,56.7,209.7,76.1,135.1,138.4,76.5,35.3,42.9,22.1,27.9,14.3,12.5,76.4; OBzC=O:166.5,130.2,130.0,128.4,133.2,1 '～3 ': 170.5,73.2,54.1, Ph:138.3,126.9,128.2,127.5, NBzC=O:167.0,133.2,126.8,131.2; OAc (CH
3): 20.3,21.2; (C=O): 170.0,170.1.
9-dihydro-13-acetyl baccatin III:
1H-NMR(CDCl
3)δ：16～19CH3：1.62(3H，S)，1.43(3H，S)，2.37(3H，S)，2.06(3H，S)；OAc(CH
3)：2.02(3H，S)，2.05(3H，S)，2.06(3H，S)。
13C-NMR(CDCl
3)δ：(1～20)78.3，73.7，38.6，81.3，84.8，33.2，69.5，57.5，36.4，75.6，135.7，142.6，71.2，38.4，43.8，22.3，28.4，15.7，14.1，76.8，OBzC＝O：166.7，131.5，127.9，130.7，133.6，OAc(CH
3)：21.2，22.7，22.6；(C＝O)：170.1，171.1，170.5。
Embodiment 7
A. prepare medicinal extract
With the method continuous diacolation Ramulus et folium taxi cuspidatae powder identical with embodiment 1, but be to use 98% methylethylketone as vat liquor, use 1, the 1-ethylene dichloride is as extraction agent, and the extraction yield of bearing taxanes is 98.5% as a result.This medicinal extract leaching process about 8.5 hours consuming time altogether.
B. separate 9-dihydro-13-acetyl baccatin III and taxol
With separating 9-dihydro-13-acetyl baccatin III and taxol with embodiment 2 same procedure, still wash-out uses Skellysolve A-pentyl acetate of 3.2: 1 for the first time, obtains containing the dry extract of taxol and 9-dihydro-13-acetyl baccatin III; Wash-out uses trifluoromethane-methyl acetate of 2: 1 for the second time, obtains 9-dihydro-13-acetyl baccatin III and taxol respectively.Use NMR to confirm as 9-dihydro-13-acetyl baccatin III and taxol, 9-dihydro-13-acetyl baccatin III extraction yield is 99%, and the extraction yield of taxol is 99.3%.
Taxol:
1H-NMR data (CDCl
3) δ: 16～19CH
3: 1.63 (3H, S), 1.25 (3H, S), 1.78 (3H, S), 1.81 (3H, S); OAc (CH
3): 2.10 (3H, S), 2.23 (3H, S)
13C-NMR data (CDCl
3) δ: (1～20) 78.2,73.1,46.2,82.8,83.7,36.8,69.8,56.7,209.7,76.1,135.1,138.4,76.5,35.3,42.9,22.1,27.9,14.3,12.5,76.4; OBzC=O:166.5,130.2,130.0,128.4,133.2,1 '～3 ': 170.5,73.2,54.1, Ph:138.3,126.9,128.2,127.5, NBzC=O:167.0,133.2,126.8,131.2; OAc (CH
3): 20.3,21.2; (C=O): 170.0,170.1.
9-dihydro-13-acetyl baccatin III:
1H-NMR(CDCl
3)δ：16～19CH3：1.62(3H，S)，1.43(3H，S)，2.37(3H，S)，2.06(3H，S)；OAc(CH
3)：2.02(3H，S)，2.05(3H，S)，2.06(3H，S)。
13C-NMR(CDCl
3)δ：(1～20)78.3，73.7，38.6，81.3，84.8，33.2，69.5，57.5，36.4，75.6，135.7，142.6，71.2，38.4，43.8，22.3，28.4，15.7，14.1，76.8，OBzC＝O：166.7，131.5，127.9，130.7，133.6，OAc(CH
3)：21.2，22.7，22.6；(C＝O)：170.1，171.1，170.5。
By top description as seen, be example with embodiment 1 and comparative example 1, from 500g tree powder, extract the ethanol that bearing taxanes needs 1000mL 95% approximately with the inventive method, extraction time needs 8 hours approximately; If by traditional extraction operation, then need 95% ethanol 16000mL, extraction time is 20 hours.Be that its solvent load is 16 times of the inventive method.Therefore, compare with existing method, the inventive method can significantly improve extracting efficiency, reduces solvent load, helps to reduce cost and alleviates pressure to environment.
In addition, use the inventive method, the extraction efficiency of 9-dihydro-13-acetyl baccatin III and taxol compound reaches as high as 99.3%, and this is that prior art is not mentioned.Therefore, use extracting method of the present invention to obtain good effect.
Claims (10)
1. method of from Ramulus et folium taxi cuspidatae, extracting 9-dihydro-13-acetyl baccatin III, it in turn includes the following steps:
With organic vat liquor Ramulus et folium taxi cuspidatae is carried out lixiviate with multistage continuous diacolation extraction method, obtain containing organic extracting solution of bearing taxanes;
The organic liquor that obtains is carried out silica gel column chromatography, with 4: 1-1: 1 C
4-C
8Alkane-acetate C
1-C
6Alkane ester or with 4: 1-1: 1 sherwood oil-acetate C
1-C
6Alkane ester wash-out obtains containing the thick solution of 9-dihydro-13-acetyl baccatin III;
Remove the solvent in the described thick solution, obtain containing the dry extract of 9-dihydro-13-acetyl baccatin III;
Carrying out silica gel column chromatography once more, is 4 with volume ratio: 1-1: 1 C
1-C
4Haloalkane-acetate C
1-C
6Alkane ester wash-out, the stream part of collecting 6-7 column volume obtains 9-dihydro-13-acetyl baccatin III.
2. method of from Ramulus et folium taxi cuspidatae, extracting taxol, it in turn includes the following steps:
With organic vat liquor Ramulus et folium taxi cuspidatae is carried out lixiviate with multistage continuous diacolation extraction method, obtain containing organic extracting solution of bearing taxanes;
The organic liquor that obtains is carried out silica gel column chromatography, with 4: 1-1: 1 C
4-C
8Alkane-acetate C
1-C
6Alkane ester or with 4: 1-1: 1 sherwood oil-acetate C
1-C
6Alkane ester wash-out obtains containing the thick solution of taxol;
Remove the solvent in the described thick solution, obtain containing the dry extract of taxol;
Carrying out silica gel column chromatography once more, is 4 with volume ratio: 1-1: 1 C
1-C
4Haloalkane-acetate C
1-C
6Alkane ester wash-out, the stream part of collecting 4-5 column volume obtains taxol.
3. method as claimed in claim 1 or 2 is characterized in that after obtaining containing organic extracting solution of bearing taxanes, but carries out before the silica gel column chromatography, and it also in turn includes the following steps:
Remove the organic solvent in organic extracting solution, obtain the water-based vat liquor;
With polar organic solvent described water-based vat liquor is extracted, obtain organic extract liquid;
Remove the organic solvent in the extraction liquid, obtain containing the medicinal extract of described bearing taxanes;
Form organic liquor with this medicinal extract of organic solvent dissolution.
4. method as claimed in claim 1 or 2 is characterized in that described organic vat liquor is selected from C
1-4Fatty Alcohol(C12-C14 and C12-C18) or its 50-100% aqueous solution; Acetone or its 50-100% aqueous solution, methylethylketone or its 50-100% aqueous solution, ether or its 50-100% aqueous solution, methyl ethyl ether or its 50-100% aqueous solution, acetate C
1-4The alkane ester.
5. method as claimed in claim 3 is characterized in that the organic polar solvent that is used to extract is selected from methyl chloride, methylene dichloride, trichloromethane, monochloroethane, 1, and 1-ethylene dichloride or 1,2-ethylene dichloride.
6. method as claimed in claim 3 is characterized in that described multistage continuous diacolation extraction method is the 4-15 level, and described organic vat liquor is a 50-100% ethanol, and the described organic polar solvent that is used to extract is a trichloromethane.
7. method as claimed in claim 1 or 2, it is characterized in that red bean branches and leaves raw material on average is placed on each diacolation section, the 1-3 of the weight that it is unit with the gram that the volume of collecting the extracting solution that obtains when flowing through last step diacolation section and with the milliliter be unit reaches the average contained Ramulus et folium taxi cuspidatae of each diacolation section doubly, when being preferably 2 times, stop to add vat liquor to first step diacolation section, and in the end one-level increases a diacolation section again, adds vat liquor to second stage diacolation section subsequently.
8. method as claimed in claim 1 or 2, to be selected from volume ratio be 4 to the described elutriant that is used for wash-out 9-dihydro-13-acetyl baccatin III or taxol when it is characterized in that for the first time silica gel column chromatography separates: 1-1: 1 C
4-C
8Alkane-acetate C
1-C
6Alkane ester or 4: 1-1: the sherwood oil of 1 (v/v)-acetate C
1-C
6The alkane ester.
9. method as claimed in claim 1 or 2, it is 4 that the elutriant that it is characterized in that being used for when for the second time silica gel column chromatography separates wash-out 9-dihydro-13-acetyl baccatin III or taxol is selected from volume ratio: 1-1: 1 C
1-C
4Haloalkane-acetate C
1-C
6The alkane ester.
10. method as claimed in claim 1 or 2 is characterized in that it also comprises the step of the product that obtains being carried out recrystallization purifying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410054174 CN1616442A (en) | 2004-09-01 | 2004-09-01 | Method for extracting 9-dihydrogen-13-acetyl baccatin III and taxol |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410054174 CN1616442A (en) | 2004-09-01 | 2004-09-01 | Method for extracting 9-dihydrogen-13-acetyl baccatin III and taxol |
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| Publication Number | Publication Date |
|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103588736A (en) * | 2013-12-03 | 2014-02-19 | 云南汉德生物技术有限公司 | 13-acetyl-9-dihydrobaccatin III extraction separation method |
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2004
- 2004-09-01 CN CN 200410054174 patent/CN1616442A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103588736A (en) * | 2013-12-03 | 2014-02-19 | 云南汉德生物技术有限公司 | 13-acetyl-9-dihydrobaccatin III extraction separation method |
| CN103588736B (en) * | 2013-12-03 | 2015-06-03 | 云南汉德生物技术有限公司 | 13-acetyl-9-dihydrobaccatin III extraction separation method |
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