CN103588736B - 13-acetyl-9-dihydrobaccatin III extraction separation method - Google Patents
13-acetyl-9-dihydrobaccatin III extraction separation method Download PDFInfo
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- 238000000605 extraction Methods 0.000 title claims abstract description 17
- 238000000926 separation method Methods 0.000 title claims abstract description 12
- TUSIZTVSUSBSQI-UHFFFAOYSA-N Dihydrocarveol acetate Chemical compound CC1CCC(C(C)=C)CC1OC(C)=O TUSIZTVSUSBSQI-UHFFFAOYSA-N 0.000 title abstract description 8
- 239000011347 resin Substances 0.000 claims abstract description 19
- 229920005989 resin Polymers 0.000 claims abstract description 19
- 241000015728 Taxus canadensis Species 0.000 claims abstract description 5
- 238000007654 immersion Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 108
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 92
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- OVMSOCFBDVBLFW-VHLOTGQHSA-N (-)-Baccatin III Natural products O([C@@H]1[C@@]2(C[C@H](O)C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)O)C(=O)C1=CC=CC=C1 OVMSOCFBDVBLFW-VHLOTGQHSA-N 0.000 claims description 15
- 229930014667 baccatin III Natural products 0.000 claims description 15
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 14
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 12
- 238000002425 crystallisation Methods 0.000 claims description 11
- 230000008025 crystallization Effects 0.000 claims description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000000465 moulding Methods 0.000 claims description 7
- 230000002378 acidificating effect Effects 0.000 claims description 6
- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 0.000 claims description 6
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- 238000010521 absorption reaction Methods 0.000 claims description 4
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- 239000007788 liquid Substances 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims 1
- 229930012538 Paclitaxel Natural products 0.000 abstract description 16
- 229960001592 paclitaxel Drugs 0.000 abstract description 16
- 238000000034 method Methods 0.000 abstract description 14
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 9
- 239000002994 raw material Substances 0.000 abstract description 6
- YWLXLRUDGLRYDR-UHFFFAOYSA-N 10-deacetylbaccatin Chemical compound CC(=O)OC12COC1CC(O)C(C(C(O)C1=C(C)C(O)CC3(O)C1(C)C)=O)(C)C2C3OC(=O)C1=CC=CC=C1 YWLXLRUDGLRYDR-UHFFFAOYSA-N 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 2
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 abstract description 2
- 238000003809 water extraction Methods 0.000 abstract description 2
- -1 9-DHB Chemical class 0.000 abstract 1
- 239000002253 acid Substances 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 26
- TYLVGQKNNUHXIP-MHHARFCSSA-N 10-deacetyltaxol Chemical group O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)C=4C=CC=CC=4)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 TYLVGQKNNUHXIP-MHHARFCSSA-N 0.000 description 12
- 238000001914 filtration Methods 0.000 description 11
- 239000000463 material Substances 0.000 description 10
- 238000011084 recovery Methods 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 229930182986 10-Deacetyltaxol Natural products 0.000 description 6
- DBXFAPJCZABTDR-KUEXGRMWSA-N Cephalomannine Natural products O=C(O[C@@H]1C(C)=C2[C@@H](OC(=O)C)C(=O)[C@]3(C)[C@@H](O)C[C@@H]4[C@](OC(=O)C)([C@H]3[C@H](OC(=O)c3ccccc3)[C@@](O)(C2(C)C)C1)CO4)[C@@H](O)[C@H](NC(=O)/C(=C\C)/C)c1ccccc1 DBXFAPJCZABTDR-KUEXGRMWSA-N 0.000 description 6
- DBXFAPJCZABTDR-WBYYIXQISA-N cephalomannine Chemical compound O([C@@H]1[C@]2(O)C[C@@H](C(=C([C@@H](OC(C)=O)C(=O)[C@]3(C)[C@@H](O)C[C@H]4OC[C@]4([C@H]31)OC(C)=O)C2(C)C)C)OC(=O)[C@H](O)[C@@H](NC(=O)C(/C)=C/C)C=1C=CC=CC=1)C(=O)C1=CC=CC=C1 DBXFAPJCZABTDR-WBYYIXQISA-N 0.000 description 6
- 125000002456 taxol group Chemical group 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 229940123237 Taxane Drugs 0.000 description 3
- 241001149649 Taxus wallichiana var. chinensis Species 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960003668 docetaxel Drugs 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
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- 238000002955 isolation Methods 0.000 description 2
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- 230000000737 periodic effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 208000019901 Anxiety disease Diseases 0.000 description 1
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- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 241001116500 Taxus Species 0.000 description 1
- 241001330459 Taxus wallichiana var. wallichiana Species 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
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- 208000029078 coronary artery disease Diseases 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
- C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a 13-acetyl-9-dihydrobaccatin III extraction separation method. The 13-acetyl-9-dihydrobaccatin III extraction separation method is characterized in that branches and leaves of Taxus canadensis Marsh are used as raw materials, 13-acetyl-9-dihydrobaccatin III having the content more than 40% is prepared by immersion, acid water extraction and resin column separation and separation effects are obvious; and the 13-acetyl-9-dihydrobaccatin III is re-crystallized to form a 13-acetyl-9-dihydrobaccatin III product having the content more than 99% and simultaneously, active taxane compounds such as 9-DHB, taxol and 10-DAB are effectively separated. The 13-acetyl-9-dihydrobaccatin III extraction separation method has simple processes and a high yield and is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of method extracting active compound in T. canadensis plant, particularly the extraction and separation method of 13-ethanoyl-9-hydroxyl baccatin III.
Background technology
The taxol extracted in Ramulus et folium taxi cuspidatae has the effect of cancer preventing and treating, and without obvious untoward reaction, be the cancer therapy drug that the plant of generally acknowledging in the world is purified, clinical application has good effect to kinds cancer.The clinical study of the U.S. proves there is outstanding curative effect to ovary and mammary cancer.In addition, also certain effect is had to hypertension, diabetes, coronary heart disease.The taxol contained in Ramulus et folium taxi cuspidatae, have unique anticancer mechanism and higher antitumour activity, can stop the breeding of cancer cells, the migration of inhibition tumor cell, being recognized is most important anti-cancer active matter in current natural medicine field.
Current China has the Ramulus et folium taxi cuspidatae of four kinds and a mutation, and namely the taxol of taxusyunnanensis, Xizang Taxus chinensis, taxus chinensis in northeast, Chinese Ramulus et folium taxi cuspidatae and southerm yew (mutation) natural Chinese yew is mainly distributed in bark, and whole strain utilization ratio is low.And T. canadensis aboundresources, the content of bearing taxanes is higher, and in branches and leaves, 13-ethanoyl-9-hydroxyl baccatin III (9-DHB) content can reach 5-8.This compound and taxol have identical female ring structure, have good utility value at semi-synthetic taxol and Docetaxel field.The traditional extraction technique of Ramulus et folium taxi cuspidatae directly adopts alcohol steep, concentrate and obtain medicinal extract, medicinal extract is through the silica gel column chromatography of more than 3 times, can output content reach 40% bearing taxanes, and be separated containing a large amount of pigments and oily matter in the enriched material that obtains, have a strong impact on effect and the yield of recrystallization, traditional taxane compounds treatment process route is loaded down with trivial details, one time yield can only reach 30%, and yield is lower;
13-ethanoyl-9-hydroxyl baccatin III is white powder material, is called for short 9-DHB, English name: 13-Acetyl-9-dihydrobaccatin III, No. CAS: 142203-65-4, molecular formula: C
33h
42o
12molecular weight: 630.67938; Structural formula is such as formula I:
The main raw material of current semi-synthetic taxol and Docetaxel is 10-DAB, and along with the increase of the market requirement, the price of 10-DAB also rises steadily, and brings huge financial pressure to the manufacturer of taxol biosynthesis.Effective Isolation and purification of 9-DHB, can increase the raw material of taxol biosynthesis, greatly alleviates the situation of raw material anxiety, improves the utilization ratio of Ramulus et folium taxi cuspidatae raw material.
Summary of the invention
The object of the invention is the extraction and separation method providing a kind of 13-ethanoyl-9-hydroxyl baccatin III, the method with T. canadensis branches and leaves for raw material, after dried and crushed, the ratio of 4-7L methanol-water solution is added in every 1 kilogram of Ramulus et folium taxi cuspidatae powder, add methanol-water solution at Ramulus et folium taxi cuspidatae powder and soak 8-24h, filter, filter residue soaks with 3-4L methanol-water solution again, repeat 2-3 time, merging filtrate, concentrating filter liquor adds hydrochloric acid soln and regulates filtrate pH value to be 5-6 after reclaiming methyl alcohol, then chloroform extraction is added 3-4 time, collect combined chloroform layer, concentrate and obtain chloroform medicinal extract, the absorption of R type resin column is adopted after chloroform medicinal extract dissolves, enrichment 13-ethanoyl-9-hydroxyl baccatin III, with acetone-water, methanol-water, one in alcohol-water is as elutriant, elutriant pressurizes, wash-out is carried out according to the flow velocity of 2 column volumes per hour, collect 13-ethanoyl-9-hydroxyl baccatin III section elutriant, concentrated, crystallization, be drying to obtain 13-ethanoyl-9-hydroxyl baccatin III, wherein moulding pressure is less than 3 kilograms.
The methanol aqueous solution that the methanol-water solution used when soaking described in the present invention is mass percent concentration is 40-90%.
The type of R described in the present invention resin is Chroma-R204.
Described in the present invention before adsorbing with R type resin column, chloroform medicinal extract first adopts acidic alumina column chromatography, concrete operations are chloroform medicinal extract mass percent concentration is 60-80% dissolve with methanol solution, centrifugal segregation precipitates, the methanol solution quality of wherein adding is 10-15 times of medicinal extract quality, and then centrifugal rear solution is with the flow velocity of 2 times of column volumes per hour, peracidity alumina column, collect effluent liquid, concentrated obtain medicinal extract, medicinal extract adopts after dissolving that R type resin column adsorbs, wash-out again.
Described acidic alumina is 100-250 object neutral alumina mass percent concentration is that after the glacial acetic acid solution immersion 2-3h of 30-50%, 200-300 DEG C of oven dry is obtained.
The advantage of the inventive method and technique effect: this technique selects sour water extraction treatment on the basis of traditional extract drugs technique, a large amount of pigments and impurity can be removed, effective reduction paste volume, decrease the difficulty of subsequent purification process, then R type resin isolation is adopted, chromatography just can output content reach more than 40% 13-ethanoyl-9-hydroxyl baccatin III, separating effect is remarkable, the product of content more than 99% just can be being obtained by simple recrystallization, simultaneously can by 9-DHB, taxol, 10-DAB isoreactivity taxanes compounds is effectively separated, and present method technique is simple, the rate of recovery is high, be applicable to suitability for industrialized production.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail; but protection scope of the present invention is not limited to described content, in the inventive method if no special instructions, all adopt ordinary method; use reagent if no special instructions, all adopt the reagent of ordinary method preparation or commercially available conventional reagent.
Embodiment 1: get dry Canadian branches and leaves 1000g, being crushed to granularity is 10-20 order, add 5L methanol-water solution (mass percent concentration is 70%) immersion and steep filtration after 12 hours, filtrate is to be concentrated, filter residue again adds 3L methanol aqueous solution and soaks 20 hours, filtrate is to be concentrated, filter residue adds 3L methanol aqueous solution again and soaks filtration after 10 hours, merge three times and filter gained filtrate, add after filtrate concentration and recovery methyl alcohol mass percent concentration be 10% hydrochloric acid soln regulate filtrate pH value to be 5, then add 1L chloroform to extract, re-extract 3 times, collect combined chloroform layer, chloroform layer is concentrated into and dryly obtains 50g chloroform medicinal extract, after chloroform medicinal extract acetone solution, join in Chroma-R204 resin (400 order) post, with pressurization mass percent concentration be 40%, 50%, the acetone-water solution of 90% carries out wash-out (moulding pressure is 2 kilograms) successively according to the flow velocity of 2 column volumes per hour, elutant adopts TLC plate figure to detect, what 40% elutriant washed out is 9-DHB section, what 50% elutriant washed out is 10-deacetyl taxol section, what 90% elutriant washed out is taxol and Cephalomannine, collect 9-DHB section elutriant, enriched material 1.8g is obtained after concentrated, it is 42% that HPLC detects 9-DHB content, add 100ml acetone solution post crystallization, obtain white crystalline powder 0.5g, content 99.1%.
Embodiment 2: get dry Canadian branches and leaves 1000g, be crushed to 10 orders, add 7L methyl alcohol (mass percent concentration is 40%) and soak filtration after 24 hours, filtrate is to be concentrated, filter residue continues to add 4L methyl alcohol and soaks 24 hours, filtrate is to be concentrated, filter residue adds 4L methyl alcohol and soaks filtration after 24 hours, merge three times and filter gained filtrate, add after filtrate concentration and recovery methyl alcohol mass percent concentration be 10% hydrochloric acid soln regulate filtrate pH value to be 6, then add 1L chloroform and carry out extraction 4 times, collect combined chloroform layer, chloroform is concentrated into mutually and dryly obtains 60g chloroform medicinal extract, after chloroform medicinal extract acetone solution, join in Chroma-R204 resin (500 order) post, with pressurization mass percent concentration be 40%, 50%, the methanol-water solution of 90% carries out wash-out (moulding pressure is 1 kilogram) successively according to the flow velocity of 2 column volumes per hour, elutant adopts TLC plate figure to detect, what 40% elutriant washed out is 9-DHB section, what 50% elutriant washed out is 10-deacetyl taxol section, what 90% elutriant washed out is taxol and Cephalomannine, collect 9-DHB section, enriched material 1.6g is obtained after concentrated, it is 40% that HPLC detects 9-DHB content, add 50ml acetone solution post crystallization, obtain white crystalline powder 0.4g, content 99.3%.
Embodiment 3: get dry Canadian branches and leaves 1000g, be crushed to 20 orders, add 4L methyl alcohol (mass percent concentration is 90%) and soak filtration after 8 hours, filtrate is to be concentrated, filter residue continues to add 3.5L methyl alcohol and soaks 10 hours, repeat 3 times, merge four times and filter gained filtrate, add after filtrate concentration and recovery methyl alcohol mass percent concentration be 10% hydrochloric acid soln regulate filtrate pH value to be 5.5, then add 2L chloroform and carry out extraction 3 times, collect combined chloroform layer, chloroform is concentrated into mutually and dryly obtains 57g chloroform medicinal extract, after chloroform medicinal extract acetone solution, join in Chroma-R204 resin (400 order) post, with pressurization mass percent concentration be 40%, 50%, the ethanol-water solution of 90% carries out wash-out (moulding pressure is 2 kilograms) successively according to the flow velocity of 2 column volumes per hour, elutant adopts TLC plate figure to detect, what 40% elutriant washed out is 9-DHB section, what 50% elutriant washed out is 10-deacetyl taxol section, what 90% elutriant washed out is taxol and Cephalomannine, collect 9-DHB section, obtain enriched material 1.7g after concentrated, it is 41% that HPLC detects 9-DHB content, adds 70ml acetone solution post crystallization, obtains white crystalline powder 0.6g, content 99.5%.
Embodiment 4: get dry Canadian branches and leaves 1000g, be crushed to 15 orders, add 6L methyl alcohol (mass percent concentration is 60%) and soak filtration after 20 hours, filtrate is to be concentrated, filter residue continues to add 3L methyl alcohol and soaks 15 hours, repeat 3 times, merge four times and filter gained filtrate, add after filtrate concentration and recovery methyl alcohol mass percent concentration be 10% hydrochloric acid soln regulate filtrate pH value to be 5.5, then add 1.5L chloroform and carry out extraction 3 times, collect combined chloroform layer, chloroform is concentrated into mutually and dryly obtains 61g chloroform medicinal extract, chloroform medicinal extract mass percent concentration is 70% dissolve with methanol solution, centrifugal, the methanol solution quality of wherein adding is 15 times of medicinal extract quality, then by centrifugal rear solution with the flow velocity peracidity alumina column of 2 times of column volumes per hour (acidic alumina to be 100 order neutral alumina mass percent concentrations be 50% glacial acetic acid solution soak 2h after 200 DEG C dry obtained), collect effluent liquid, concentrate and obtain medicinal extract, after medicinal extract acetone solution, join in Chroma-R204 resin (300 order) post, with pressurization mass percent concentration be 40%, 50%, the acetone-water solution of 90% carries out wash-out (moulding pressure is 2 kilograms) successively according to the flow velocity of 2 column volumes per hour, elutant adopts TLC plate figure to detect, what 40% elutriant washed out is 9-DHB section, what 50% elutriant washed out is 10-deacetyl taxol section, what 90% elutriant washed out is taxol and Cephalomannine, collect 9-DHB section, enriched material 2.1g is obtained after concentrated, it is 45% that HPLC detects 9-DHB content, add 50ml acetone solution post crystallization, obtain white crystalline powder 0.8g, content 99.8%.
Embodiment 5: get dry Canadian branches and leaves 1000g, be crushed to 10 orders, add 5L methyl alcohol (mass percent concentration is 80%) and soak filtration after 15 hours, filtrate is to be concentrated, filter residue continues to add 3L methyl alcohol and soaks 20 hours, repeat 3 times, merge four times and filter gained filtrate, add after filtrate concentration and recovery methyl alcohol mass percent concentration be 10% hydrochloric acid soln regulate filtrate pH value to be 6, then add 1L chloroform and carry out extraction 3 times, collect combined chloroform layer, chloroform is concentrated into mutually and dryly obtains 59g chloroform medicinal extract, chloroform medicinal extract mass percent concentration is 60% dissolve with methanol solution, centrifugal, the methanol solution quality of wherein adding is 10 times of medicinal extract quality, then by centrifugal rear solution with the flow velocity peracidity alumina column of 2 times of column volumes per hour (acidic alumina to be 150 order neutral alumina mass percent concentrations be 40% glacial acetic acid solution soak 3h after 300 DEG C dry obtained), collect effluent liquid, concentrate and obtain medicinal extract, after medicinal extract acetone solution, join in Chroma-R204 resin column (400 order), with pressurization mass percent concentration be 40%, 50%, the acetone-water solution of 90% carries out wash-out (moulding pressure is 2 kilograms) successively according to the flow velocity of 2 column volumes per hour, elutant adopts TLC plate figure to detect, what 40% elutriant washed out is 9-DHB section, what 50% elutriant washed out is 10-deacetyl taxol section, what 90% elutriant washed out is taxol and Cephalomannine, collect 9-DHB section, enriched material 1.9g is obtained after concentrated, it is 46% that HPLC detects 9-DHB content, add 50ml acetone solution post crystallization, obtain white crystalline powder 0.9g, content 99.7%.
Embodiment 6: adopt traditional D101 type resin column to test in contrast, particular content is as follows:
Get dry Canadian branches and leaves 1000g, be crushed to 5-10 order, add 4L methyl alcohol and soak filtration after 12 hours, filtrate is to be concentrated, filter residue continues to add 3L methyl alcohol and soaks 12 hours, filtrate is to be concentrated, filter residue adds 3L methyl alcohol and soaks filtration after 12 hours, merge three times and filter gained filtrate, concentration and recovery methyl alcohol, after adding the dissolving of 500ml chloroform, add 300ml water to extract, aqueous phase discards, chloroform is concentrated into mutually and dryly obtains 100g chloroform medicinal extract, after chloroform medicinal extract acetone solution, join in D101 type resin column, adding mass percent concentration by the mode of normal pressure is 30%, the acetone-water eluant solution of 70%, elutant adopts TLC plate figure to detect, what 30% elutriant washed out is a large amount of pigments and impurity, that 70% elutriant washes out is 9-DHB, 10-deacetyl taxol, taxol and Cephalomannine, each compound intersects mutually, could not effectively separate, enriched material 12.3g is obtained after concentrated, it is 5% that HPLC detects 9-DHB content, continue to add 50ml chloroform to dissolve, with silica gel column chromatography, TLC plate figure detects, collect 9-DHB section, obtain enriched material 2.3g after concentrated and add 80ml acetone solution post crystallization, periodic crystallisation repeatedly obtains white crystalline powder 0.4g, content 97.5%.
Embodiment 7: adopt traditional HPD045 type resin column to test in contrast, particular content is as follows:
Get dry Canadian branches and leaves 1000g, be crushed to 20 orders, add 5L methanol aqueous solution (percent mass hundred concentration is 90%) and soak filtration after 24 hours, filtrate is to be concentrated, filter residue continues to add 3L methanol aqueous solution and soaks 24 hours, filtrate is to be concentrated, filter residue adds 3L methanol aqueous solution and soaks filtration after 12 hours, merge three times and filter gained filtrate, concentration and recovery methyl alcohol, add 1L chloroform to extract, aqueous phase discards, chloroform is concentrated into mutually and dryly obtains 60g chloroform medicinal extract, after chloroform medicinal extract acetone solution, join in HPD045 type resin column, adding mass percent concentration by the mode of normal pressure is 30%, the methanol-water solution wash-out of 90%, elutant adopts TLC plate figure to detect, collect 9-DHB section, enriched material 4.8g is obtained after concentrated, it is 13% that HPLC detects 9-DHB content, add 40ml methylene dichloride and dissolve post crystallization, periodic crystallisation repeatedly obtains white crystalline powder 0.3g, content 98.3%.
Claims (3)
1. the extraction and separation method of a 13-ethanoyl-9-hydroxyl baccatin III, it is characterized in that: T. canadensis branches and leaves are dried, after pulverizing, the ratio of 4-7L methanol-water solution is added in every 1 kilogram of Ramulus et folium taxi cuspidatae powder, add methanol-water solution at Ramulus et folium taxi cuspidatae powder and soak 8-24h, filter, filter residue soaks with 3-4L methanol-water solution again, repeat 2-3 time, merging filtrate, concentrating filter liquor adds hydrochloric acid soln and regulates filtrate pH value to be 5-6 after reclaiming methyl alcohol, then chloroform extraction is added 3-4 time, collect combined chloroform layer, concentrate and obtain chloroform medicinal extract, the absorption of R type resin column is adopted after chloroform medicinal extract dissolves, enrichment 13-ethanoyl-9-hydroxyl baccatin III, with acetone-water, methanol-water, one in alcohol-water is as elutriant, elutriant pressurizes, wash-out is carried out according to the flow velocity of 2 column volumes per hour, collect 13-ethanoyl-9-hydroxyl baccatin III section elutriant, concentrated, crystallization, be drying to obtain 13-ethanoyl-9-hydroxyl baccatin III, wherein moulding pressure is less than 3 kilograms,
The methanol aqueous solution that the methanol-water solution used during described immersion is mass percent concentration is 40-90%;
Described R type resin is Chroma-R204.
2. the extraction and separation method of 13-ethanoyl-9-hydroxyl baccatin III according to claim 1; it is characterized in that: before with the absorption of R type resin column; chloroform medicinal extract first adopts acidic alumina column chromatography; concrete steps are chloroform medicinal extract mass percent concentration is the dissolve with methanol solution of 60-80%; centrifugal; the methanol solution quality of wherein adding is 10-15 times of medicinal extract quality; then by centrifugal rear solution with the flow velocity peracidity alumina column of 2 times of column volumes per hour; collect effluent liquid, concentrated obtain medicinal extract, after medicinal extract dissolves, adopt the absorption of R type resin column.
3. the extraction and separation method of 13-ethanoyl-9-hydroxyl baccatin III according to claim 2, is characterized in that: the glacial acetic acid solution of acidic alumina to be neutral alumina mass percent concentration be 30-50% soaks 2-3h post-drying and obtains.
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| CN1266059A (en) * | 1999-01-07 | 2000-09-13 | 刘健 | Low pressure color spectrum separating and purifying paclitaxel and other relative taxusane compounds by industrial preparation on polyresin column |
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| CN1266059A (en) * | 1999-01-07 | 2000-09-13 | 刘健 | Low pressure color spectrum separating and purifying paclitaxel and other relative taxusane compounds by industrial preparation on polyresin column |
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