CN101575277A - Method for preparing high purity irisquinone, pallason A - Google Patents
Method for preparing high purity irisquinone, pallason A Download PDFInfo
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- CN101575277A CN101575277A CNA2009100159820A CN200910015982A CN101575277A CN 101575277 A CN101575277 A CN 101575277A CN A2009100159820 A CNA2009100159820 A CN A2009100159820A CN 200910015982 A CN200910015982 A CN 200910015982A CN 101575277 A CN101575277 A CN 101575277A
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- irisquinone
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Abstract
The invention provides a method for preparing high purity irisquinone, pallason A, which is characterized in that: the method adopts a reverse phase preparative chromatography, and comprises elution in a liquid chromatography column and a post treatment process of eluted component parts, wherein the elution conditions are that: a sample is irisquinone solution for the preparation, and prepared from Chinese iris seed extract through dissolution by ethanol and acetonitrile, crystallization and filtration respectively; a mobile phase is water-containing acetonitrile solution; the temperature is between 20 and 30 DEG C; and the post treatment process is to crystallize, filter, wash and dry the eluted component parts containing the irisquinone, pallason A after the elution to prepare the high purity irisquinone, pallason A. The method has the characteristics of high purity of prepared products and simple and easy operation.
Description
Technical field
The present invention relates to pharmacy field, be specifically related to use reverse phase preparative chromatography to prepare high purity irisquinone, pallason A.
Background technology
Irisquinonum is by the main anticancer active constituent that extracts in the irides Semen Iridis, mainly contains Irisquinone A and Pallasone B.
The structure of Irisquinone A (I) is:
The structure of Pallasone B (II) is:
Irisquinonum is Western medicine two kind new medicines that develop jointly Shandong XinHua Pharmacy stock Co., Ltd and Tianjin Institute of Medicine Science, and listing in 1996 is clinically share as radiotherapeutic sensitizer and radiotherapy.
The two structural similitude of Irisquinone A and Pallasone B, not easily separated and mensuration.The HPLC method that Irisquinonum component separating measuring method has been reported: pharmaceutical analysis magazine, 10 (4), 1990, P230-232.The method that provides is that (65: 30: 20: 20) be moving phase, but this moving phase ether highly volatile, the component change was bigger, was not suitable for preparation and used with methanol-water-acetonitrile-ether.Herbal medicine, 16 (3), 1985, P5.The method that provides is to add Silver Nitrate (5 * 10 with anhydrous methanol
-2M) be moving phase, but this moving phase has infringement to instrument and chromatographic column, and Silver Nitrate is difficult for removing from sample, equally also is not suitable for preparation and uses.
Irisquinone A also can prepare with complete synthesis method: chemical journal, 47 (9), 1989, P896-900.Complete synthesis route is long, and reagent is relatively more expensive, and difficulty is bigger.
Using reverse phase preparative chromatography to prepare high purity irisquinone, pallason A does not appear in the newspapers so far.
Summary of the invention
The purpose of this invention is to provide a kind of method for preparing high purity irisquinone, pallason A, the product purity height is operated simple and easy.
The present invention prepares the method for high purity irisquinone, pallason A, it is characterized in that adopting reverse phase preparative chromatography, is included in the last handling process of wash-out and wash-out stream in the liquid-phase chromatographic column part,
Wherein the condition of wash-out is:
Sample: Irisquinonum solution is used in preparation, is to be dissolved through ethanol, acetonitrile respectively by the Semen Iridis extract, and crystallization, filtration obtain,
Moving phase: aqueous acetonitrile solution
Temperature: 20 ℃～30 ℃
Last handling process: contain the wash-out stream part of Irisquinone A behind the wash-out, through crystallization, filtration, wash, be drying to obtain.
The performance liquid chromatographic column that uses is a C-18 preparative chromatography post, and diameter is 10mm, 25mm; Length is 250mm.There are a lot of suppliers can produce the preparative chromatography post of this character, as Phenomenex company etc.
The preparation preparation of Irisquinonum solution: with the Semen Iridis kind skin of buying, through pulverizing, extracting, filter with ethyl acetate backflow, the filtrate temperature control is below 60 ℃, pressure-0.07～-0.09MPa is evaporated to dried, with the dissolving of ethanol room temperature, alumina column chromatography, ethanol elution.The elutriant temperature control is below 60 ℃, pressure-0.07～-0.09MPa is evaporated to 1/2nd ,-20 ℃ of freezing and crystallizings of original volume, filter crude product.Crude product is by sherwood oil, ethanol recrystallization (50 ℃ of dissolvings ,-20 ℃ of crystallizations) repeatedly, the Semen Iridis extract, Irisquinone A 83%～86% (W/W) wherein, Pallasone B 9%～12% (W/W).
The Semen Iridis extract must prepare through pre-treatment uses Irisquinonum solution.Concrete grammar is: the Semen Iridis extract is cooled to 35 ± 5 ℃ with the dissolving down of dehydrated alcohol reflux temperature, is incubated 2 hours, filter, filtrate is cooled to subzero 15 ± 5 ℃, crystallization, filter, behind the filter cake drying under reduced pressure, with the acetonitrile dissolving, solvent temperature is 50 ± 5 ℃, be cooled to 25 ± 5 ℃, be incubated 2 hours, filter, must prepare and use Irisquinonum solution.
Each composition of Irisquinonum adsorbs and contained the acetonitrile moving phase wash-out of water on stationary phase, wherein the volume percent of water is 1% to 10%, and is preferred 2% to 6%, and optimum is 3% to 5%.
Reversed-phase preparative chromatography method of the present invention is carried out under 20 ℃ to 30 ℃.Comparatively high temps is stable influential to Irisquinone A, and lesser temps is separated out crystallization easily.
Contain wash-out stream part of Irisquinone A behind the purifying,, filter in subzero 20 ± 5 ℃ of crystallizations, subzero 20 ± 5 ℃ acetonitrile washing, Vanadium Pentoxide in FLAKES is a siccative room temperature drying under reduced pressure, gets Irisquinone A, the HPLC method is measured content＞99% (area normalization method).
Advantage of the present invention:
Use the inventive method to prepare Irisquinone A, the product purity height is operated simple and easy.Method provided by the invention is compared with the method that document provides and had following characteristics: 1) the moving phase component is reasonable.Irisquinone A and Pallasone B structural similitude, difference are that the tenth of Irisquinone A side chain has a two key, and the Pallasone B side chain is a saturated carbon chains, and rest part is identical.According to its structural difference, both have obtained good separation as the mobile mutual-assistance to have selected acetonitrile/water.Sample solubleness in this moving phase is bigger, is easy to enlarge sample size.This moving phase is harmless to instrument and chromatographic column, and is stable, is fit to preparation and uses.2) wash-out that contains Irisquinone A flows part, and subzero 20 ± 5 ℃ of crystallizations are filtered, subzero 20 ± 5 ℃ acetonitrile washing, and Vanadium Pentoxide in FLAKES is a siccative room temperature drying under reduced pressure.Do not need concentration, avoided the temperature height, easily the phenomenon of decomposing.3) preparation cycle is short.
Embodiment
By following examples foregoing of the present invention is described in further detail.For a person skilled in the art, this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment; All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
The preparation process of Semen Iridis extract (known technology):
With the Semen Iridis kind skin of buying, through pulverizing, extract, filter with ethyl acetate backflow, the filtrate temperature control is below 60 ℃, pressure-0.07～-0.09MPa be evaporated to dried, with the dissolving of ethanol room temperature, alumina column chromatography, ethanol elution.The elutriant temperature control is below 60 ℃, pressure-0.07～-0.09MPa is evaporated to 1/2nd ,-20 ℃ of freezing and crystallizings of original volume, filter crude product.Crude product is by sherwood oil, ethanol recrystallization (50 ℃ of dissolvings ,-20 ℃ of crystallizations) repeatedly, the Semen Iridis extract, Irisquinone A 83%～86% (W/W) wherein, Pallasone B 9%～12% (W/W).
The preparation preparation of Irisquinonum solution:
The Semen Iridis extract refluxes with dehydrated alcohol and dissolves.The concentration of Semen Iridis extract is 0.1g/mL, is cooled to 35 ± 5 ℃, is incubated 2 hours, filters.Filtrate is cooled to subzero 15 ± 5 ℃, and crystallization 8 hours is filtered.
Behind the filter cake drying under reduced pressure, with the acetonitrile dissolving, the concentration of Semen Iridis extract is 0.06g/mL, and solvent temperature is 50 ± 5 ℃, is cooled to 25 ± 5 ℃, is incubated 2 hours, filters, and must prepare and use Irisquinonum solution.
Content assaying method is according to promulgation " national drug standards " WS of State Food and Drug Administration
1-(X-234)-measuring method that 2003Z announces.
Embodiment 1
Last sample component: prepare with Irisquinonum solution (containing Irisquinone A 83%, Pallasone B 12%)
Stationary phase: the silica gel of octadecyl (C18) part
Pillar: the Luna of Phenomenex company chromatographic column, 250 * 10mm
Flow velocity: 5mL/min
Temperature: 20 ℃-30 ℃
Detect wavelength: 270nm
Moving phase: 95% (v/v) acetonitrile solution
Collect the wash-out stream part at the plain peak of first, subzero 20 ± 5 ℃ of crystallizations 24 hours are filtered, subzero 20 ± 5 ℃ acetonitrile washing three times.Vanadium Pentoxide in FLAKES is that siccative room temperature drying under reduced pressure is to constant weight.Get Irisquinone A, the HPLC method is measured content 99% (area normalization method).
Embodiment 2
Last sample component: prepare with Irisquinonum solution (containing Irisquinone A 85%, Pallasone B 9%)
Stationary phase: the silica gel of octadecyl (C18) part
Pillar: the Jupiter of Phenomenex company chromatographic column, 250 * 25mm
Flow velocity: 10mL/min
Temperature: 20 ℃-30 ℃
Detect wavelength: 270nm
Moving phase: 97% (v/v) acetonitrile solution
Collect the wash-out stream part at the plain peak of first, subzero 20 ℃～subzero 25 ℃ of crystallizations 24 hours are filtered.Cold acetonitrile washing three times.Vanadium Pentoxide in FLAKES is that siccative room temperature drying under reduced pressure is to constant weight.Get Irisquinone A, the HPLC method is measured content 99% (area normalization method).
Claims (5)
1, a kind of method for preparing high purity irisquinone, pallason A is characterized in that adopting reverse phase preparative chromatography, is included in the last handling process of wash-out and wash-out stream in the chromatographic column part,
Wherein the condition of wash-out is:
Sample: Irisquinonum solution is used in preparation, is to be dissolved through ethanol, acetonitrile respectively by the Semen Iridis extract, and crystallization, filtration obtain,
Moving phase: aqueous acetonitrile solution
Temperature: 20 ℃～30 ℃
Last handling process: contain the wash-out stream part of Irisquinone A behind the wash-out, through crystallization, filtration, wash, be drying to obtain.
2, method according to claim 1 is characterized in that the method for preparing with Irisquinonum solution is: the Semen Iridis extract is cooled to 35 ± 5 ℃ with the dissolving down of dehydrated alcohol reflux temperature, be incubated 2 hours, filter, filtrate is cooled to subzero 15 ± 5 ℃, crystallization is filtered, behind the filter cake drying under reduced pressure, dissolve with acetonitrile, solvent temperature is 50 ± 5 ℃, is cooled to 25 ± 5 ℃, is incubated 2 hours, filter, must prepare and use Irisquinonum solution.
3, method according to claim 1 is characterized in that moving phase is aqueous acetonitrile solution, and wherein the volume percent of water is 1 to 10.
4, method according to claim 1 is characterized in that last handling process is to contain wash-out stream part of Irisquinone A, in subzero 20 ± 5 ℃ of crystallizations, to filter, subzero 20 ± 5 ℃ acetonitrile washing, and Vanadium Pentoxide in FLAKES is a siccative room temperature drying under reduced pressure.
5, method according to claim 1 is characterized in that containing Irisquinone A 83%～86% in the described Semen Iridis extract, and Pallasone B 9%～12% is all in mass percent.
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| CN 200910015982 CN101575277B (en) | 2009-06-08 | 2009-06-08 | Method for preparing high purity irisquinone, pallason A |
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| CN 200910015982 CN101575277B (en) | 2009-06-08 | 2009-06-08 | Method for preparing high purity irisquinone, pallason A |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102649722A (en) * | 2011-02-25 | 2012-08-29 | 苏州宝泽堂医药科技有限公司 | Irisquinone purification method |
| CN102649723A (en) * | 2011-02-25 | 2012-08-29 | 苏州宝泽堂医药科技有限公司 | Method for extracting irisquinone from Chinese iris seed coat |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100502848C (en) * | 2004-06-23 | 2009-06-24 | 和记黄埔医药企业有限公司 | Use of Chinese small iris seed extract A and its derivative and analog in preparation of chemical therapeutical sensitizing agent |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102649722A (en) * | 2011-02-25 | 2012-08-29 | 苏州宝泽堂医药科技有限公司 | Irisquinone purification method |
| CN102649723A (en) * | 2011-02-25 | 2012-08-29 | 苏州宝泽堂医药科技有限公司 | Method for extracting irisquinone from Chinese iris seed coat |
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| CN101575277B (en) | 2012-12-12 |
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