CN101914002A - Method for extracting glaucocalyxin A - Google Patents
Method for extracting glaucocalyxin A Download PDFInfo
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- CN101914002A CN101914002A CN2010102646585A CN201010264658A CN101914002A CN 101914002 A CN101914002 A CN 101914002A CN 2010102646585 A CN2010102646585 A CN 2010102646585A CN 201010264658 A CN201010264658 A CN 201010264658A CN 101914002 A CN101914002 A CN 101914002A
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Abstract
The invention belongs to the field of extraction of traditional Chinese medicines, and in particular relates to a method for preparing high-purity glaucocalyxin A from rabdosia japonica var. galaucocalyx serving as a medicinal material. The method comprises the following steps of: (1) collecting and crushing over-ground part of the rabdosia japonica var. galaucocalyx serving as the raw medicinal material, refluxing and extracting for 2 to 3 times with 75 to 80 percent aqueous solution of ethanol under normal pressure, 2 to 3 hours every time, combining extract, and recovering a solvent under reduced pressure to obtain concentrated extract; (2) adding silica gel into the concentrated extract obtained by the step (1), uniformly stirring the mixture, drying the mixture under reduced pressure and performing silica gel column chromatography; and (3) recrystallizing anhydrous alcohol to obtain the glaucocalyxin A. The glaucocalyxin A obtained by the method has high purity of 95 to 100 percent, is suitable to be prepared into various formulations and has high yield of over 10 percent; and the method has the advantages of high repeatability, no organic solvent residue measured by an organic solvent residue measurement method in pharmacopoeia appendix 2010, no toxicity of suitable organic solvent and conformance to the requirements of the pharmacopoeia 2010.
Description
Technical field
The invention belongs to the traditional Chinese medicine extraction field, being specifically related to a kind of is the method for feedstock production high purity glaucocalyxin A with the rabdosia japonica medicinal material.
Background technology
Glaucocalyxin A is the activeconstituents of rabdosia japonica, glaucocalyxin A has significant pharmacologically active, the rabbit platelet that glaucocalyxin A can suppress Calcimycin and stimulate generates platelet activation factor, the rabbit platelet thromboxane A2 that can suppress arachidonic acid-induction generates, and the prostaglandin E2 that raises simultaneously, can significantly suppress ADP, AA, inductive rabbit platelets such as PAF are assembled, cAMP level in the remarkable increased platelets counts (referring to: Zhang B, Long K.Effects of glaucocalyxin A on aggtegation and cAMP levels of rabbit platelets in vitro.Acta Pharmacologica Sinica 1993,14 (4): 347.) glaucocalyxin A has cytotoxicity, ehrlich carcinoma there is certain restraining effect, in the body S180 solid tumor inhibiting rate is reached 30.4% (10mg/kg, 7 days), glaucocalyxin A is to DNA, RNA and protein synthesis have reversible inhibition, and be dosage correlation (referring to Zhang Shuxiang etc., blue honor first element external to DNA, the biosynthetic pharmacy Intelligence Newsletter 1990,8 (3) that influences of RNA and protein: 238.).Glaucocalyxin A has stronger cytotoxic activity to A549 (human lung carcinoma cell), LOVO (people's colon-cancer cell), 6T-CEM (human T cell leukemia cell), HL-60 (human leukemia cell), can be used for preparing cancer therapy drug (referring to Chen Haisheng etc., the application CN101455652-3320283 of diterpene-kind compound in the preparation cancer therapy drug in the rabdosia japonica).
In the prior art, the method for extracting glaucocalyxin A from the medicinal material rabdosia japonica is complex steps not only, and yield is on the low side, generally be lower than 10%, and because employing has toxic solvent to organism, residual noxious solvent does not meet the pharmacopeia requirement, specifically may further comprise the steps:
(1) gathers medicinal material rabdosia japonica over-ground part and pulverizing, under normal pressure, use percent by volume 85%～95% aqueous ethanolic solution refluxing extraction 2～3 times, each refluxing extraction 2 hours, united extraction liquid, decompression and solvent recovery, extremely every milliliter of solvent contains crude drug 3～10g;
(2) with isopyknic petroleum ether extraction three times, petroleum ether layer discards, and water layer is used the equal-volume chloroform extraction three times again, and the combined chloroform layer also concentrates, and obtains chloroform extraction liquid;
(3) add silica gel in step (2) gained chloroform extraction liquid and stir, drying under reduced pressure carries out silica gel column chromatography; With chloroform: methyl alcohol 200: 1 or chloroform: 100: 1 wash-outs of methyl alcohol, with the thin-layer chromatography contrast, collect the elutriant that contains glaucocalyxin A, elutriant is reclaimed solvent;
(4) with dehydrated alcohol or methyl alcohol periodic crystallisation, get glaucocalyxin A, test purity is calculated yield.
The purity that adopts technique scheme gained glaucocalyxin A is generally more than 90%, but yield is generally 7%～9%, and has used deleterious solvent in the process, chloroform, methyl alcohol, and residual noxious solvent can influence the use of glaucocalyxin A as medicine, does not meet the pharmacopeia requirement.
Summary of the invention
The object of the invention provides a kind of extracting method of glaucocalyxin A, when improving purity and yield, avoids using deleterious solvent, makes the glaucocalyxin A of extraction meet the pharmacopeia requirement.
For achieving the above object, the technical solution used in the present invention is: a kind of extracting method of glaucocalyxin A, and extraction, silica gel column chromatography wash-out pigment and impurity, purification step specifically may further comprise the steps:
(1) gather crude drug rabdosia japonica over-ground part and also pulverize, under normal pressure, use percent by volume 75～80% aqueous ethanolic solution refluxing extraction 2～3 times, each refluxing extraction 2～3 hours, united extraction liquid, partial solvent is removed in decompression, gets concentrated extracting solution;
(2) in step (1) gained concentrated extracting solution, add silica gel and stirring, drying under reduced pressure, carry out silica gel column chromatography: with the silicagel column volume is 1 parts by volume, at first with the elutriant A wash-out fat-soluble pigment of 2～3 parts of parts by volume, again with the elutriant B wash-out impurity of 4～6 parts of parts by volume, at last with the elutriant C wash-out of 10～15 parts of parts by volume; Collect elutriant, concentrate the back and place, separate out crystal;
Wherein, described elutriant A is the mixture of sherwood oil and ethyl acetate, and by volume, sherwood oil: ethyl acetate=8: 1; Described elutriant B is the mixture of sherwood oil and ethyl acetate, and by volume, sherwood oil: ethyl acetate=4: 1; Described elutriant C is the mixture of sherwood oil and ethyl acetate, and by volume, sherwood oil: ethyl acetate=2: 1;
(3) dehydrated alcohol recrystallization gets the glaucocalyxin A crystal.
In the technique scheme, in the step (1), the partial solvent that decompression is removed can reclaim recirculation and use; The amount of removing partial solvent depends on the concentration of concentrated extracting solution, and the concentration of concentrated extracting solution is: the volume of concentrated extracting solution: the quality=1ml of crude drug: 3～4g; For example, the quality of crude drug is 1200g, and then the volume of concentrated extracting solution corresponds to 300～400ml.
Because the technique scheme utilization, the present invention compared with prior art has following advantage:
The purity of the glaucocalyxin A that rabdosia japonica medicinal material (recording medicinal material medium blue calyx first cellulose content 0.1% through high-efficient liquid phase technique) obtains through present method is 95～100%, the purity height, be suitable for preparing various formulations, yield is more than 10%, the yield height, and the circulation ratio of method is strong, measure according to organic solvent residual measuring method in 2010 editions pharmacopeia appendix, organic solvent-free is residual, and the organic solvent nontoxicity that is suitable for, and meets 2010 editions pharmacopeia requirements.
Embodiment
Below in conjunction with embodiment the present invention is further described:
Embodiment one:
Get rabdosia japonica over-ground part 10kg, through pulverizing, in the atmospheric pressure reflux extraction element, with 75% alcohol reflux 2 times, each refluxing extraction 2 hours, united extraction liquid, the decompression and solvent recovery concentrated extracting solution, the volume after concentrating is 3300 milliliters, adds 1650 gram silica gel and stirs, drying under reduced pressure carries out silica gel column chromatography; The silicagel column volume is 17600 milliliters, sherwood oil with 52800 milliliters: 8: 1 wash-out fat-soluble pigments of ethyl acetate, again with 105600 milliliters sherwood oil: 4: 1 wash-out impurity of ethyl acetate, at last with 264000 milliliters sherwood oil: 2: 1 wash-outs of ethyl acetate, collect elutriant, be concentrated into 20 milliliters, place, crystallization at last with 20 milliliters of recrystallizations of dehydrated alcohol, gets glaucocalyxin A 1.5 grams, its purity is 96.0%, yield 15%, and according to organic solvent residual measuring method mensuration in 2010 editions pharmacopeia appendix, organic solvent-free is residual.
Embodiment two:
Get rabdosia japonica over-ground part 10kg, through pulverizing, in the atmospheric pressure reflux extraction element, with 80% alcohol reflux 3 times, each refluxing extraction 3 hours, united extraction liquid, the decompression and solvent recovery concentrated extracting solution, the volume after concentrating is 3250 milliliters, adds 1600 gram silica gel and stirs, drying under reduced pressure carries out silica gel column chromatography; The silicagel column volume is 18000 milliliters, sherwood oil with 54000 milliliters: 8: 1 wash-out fat-soluble pigments of ethyl acetate, again with 108000 milliliters sherwood oil: 4: 1 wash-out impurity of ethyl acetate, at last with 270000 milliliters sherwood oil: 2: 1 wash-outs of ethyl acetate, collect elutriant, be concentrated into 20 milliliters, place, crystallization at last with 20 milliliters of recrystallizations of dehydrated alcohol, gets glaucocalyxin A 1.4 grams, its purity is 95.6%, yield 14%, and according to organic solvent residual measuring method mensuration in 2010 editions pharmacopeia appendix, organic solvent-free is residual.
Following examples are for adopting the comparative example of prior art:
Embodiment three:
Get rabdosia japonica over-ground part 1kg, through pulverizing, in the atmospheric pressure reflux extraction element, with 85% alcohol reflux 2 times, each refluxing extraction 2 hours, united extraction liquid, the decompression and solvent recovery concentrated extracting solution, the volume after concentrating is 250 milliliters, earlier with isopyknic petroleum ether extraction three times, petroleum ether layer discards, water layer is used the equal-volume chloroform extraction three times again, and the combined chloroform layer also is concentrated into 10 milliliters, adds 20 gram silica gel and stirs, drying under reduced pressure carries out silica gel column chromatography; Chloroform: 200: 1 wash-outs of methyl alcohol, with the thin-layer chromatography contrast, collect the elutriant that contains glaucocalyxin A, elutriant is reclaimed solvent, with the dehydrated alcohol periodic crystallisation, get glaucocalyxin A 0.07 gram, its purity is 90%, yield 7%.
Embodiment four:
Rabdosia japonica over-ground part 1kg is through pulverizing, in the atmospheric pressure reflux extraction element, with 95% alcohol reflux 3 times, each refluxing extraction 2 hours, united extraction liquid, the decompression and solvent recovery concentrated extracting solution, the volume after concentrating is 100 milliliters, earlier with isopyknic petroleum ether extraction three times, petroleum ether layer discards, water layer is used the equal-volume chloroform extraction three times again, and the combined chloroform layer also is concentrated into 10 milliliters, adds 20 gram silica gel and stirs, drying under reduced pressure carries out silica gel column chromatography; Chloroform: 100: 1 wash-outs of methyl alcohol, with the thin-layer chromatography contrast, collect the elutriant that contains glaucocalyxin A, elutriant is reclaimed solvent, with the methyl alcohol periodic crystallisation, get glaucocalyxin A 0.09 gram, its purity is 92%, yield 9%.
Embodiment five:
Rabdosia japonica over-ground part 1kg is through pulverizing, in the atmospheric pressure reflux extraction element, with 95% alcohol reflux 3 times, each refluxing extraction 2 hours, united extraction liquid, the decompression and solvent recovery concentrated extracting solution, the volume after concentrating is 100 milliliters, earlier with isopyknic petroleum ether extraction three times, petroleum ether layer discards, water layer is used the equal-volume chloroform extraction three times again, and the combined chloroform layer also is concentrated into 15 milliliters, adds 30 gram silica gel and stirs, drying under reduced pressure carries out silica gel column chromatography; With chloroform wash-out pigment, again with chloroform: 200: 1 wash-outs of methyl alcohol, with the thin-layer chromatography contrast, collect the elutriant that contains glaucocalyxin A, elutriant is concentrated, add the methyl alcohol of ten times of amounts, place, filter, with recrystallizing methanol, get glaucocalyxin A 0.07 gram, its purity is 96%, yield 7%.
Claims (2)
1. the extracting method of a glaucocalyxin A extracts, silica gel column chromatography wash-out pigment and impurity, purification step, it is characterized in that, specifically may further comprise the steps:
(1) gather crude drug rabdosia japonica over-ground part and also pulverize, under normal pressure, use percent by volume 75～80% aqueous ethanolic solution refluxing extraction 2～3 times, each refluxing extraction 2～3 hours, united extraction liquid, partial solvent is removed in decompression, gets concentrated extracting solution;
(2) in step (1) gained concentrated extracting solution, add silica gel and stirring, drying under reduced pressure, carry out silica gel column chromatography: with the silicagel column volume is 1 parts by volume, at first with the elutriant A wash-out fat-soluble pigment of 2～3 parts of parts by volume, again with the elutriant B wash-out impurity of 4～6 parts of parts by volume, at last with the elutriant C wash-out of 10～15 parts of parts by volume; Collect elutriant, concentrate the back and place, separate out crystal;
Wherein, described elutriant A is the mixture of sherwood oil and ethyl acetate, and by volume, sherwood oil: ethyl acetate=8: 1; Described elutriant B is the mixture of sherwood oil and ethyl acetate, and by volume, sherwood oil: ethyl acetate=4: 1; Described elutriant C is the mixture of sherwood oil and ethyl acetate, and by volume, sherwood oil: ethyl acetate=2: 1;
(3) dehydrated alcohol recrystallization gets the glaucocalyxin A crystal.
2. according to the extracting method of the described glaucocalyxin A of claim 1, it is characterized in that: in the step (1), the amount of the partial solvent that decompression is removed depends on the concentration of concentrated extracting solution, and the concentration of concentrated extracting solution is: the volume of concentrated extracting solution: the quality=1ml of crude drug: 3～4g.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102389456A (en) * | 2011-11-29 | 2012-03-28 | 苏州大学 | Method for extracting isodon japonica var.galaucocalyx total diterpenoids or Glaucocalyxin A |
| CN102911025A (en) * | 2011-08-02 | 2013-02-06 | 苏州宝泽堂医药科技有限公司 | Method for extracting 4-hydroxyderricin from angelica keiskei |
| CN103399120A (en) * | 2013-07-30 | 2013-11-20 | 广州白云山和记黄埔中药有限公司 | Thin-layer identification method of medicine linearstripe rabdosia herb |
| KR101540310B1 (en) * | 2013-08-07 | 2015-07-30 | 건국대학교 산학협력단 | Composition for Preventing or Treating Neuroinflammation by Rabdosia japonica Extracts, and Extracting Method of Rabdosia japonica Extracts |
| WO2016150208A1 (en) * | 2015-03-26 | 2016-09-29 | 苏州沪云肿瘤研究中心股份有限公司 | Pharmaceutical composition and dosage form containing glaucocalyxin a derivative or salt thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101455652A (en) * | 2009-01-05 | 2009-06-17 | 中国人民解放军第二军医大学 | Use of diterpenoids from Isodon japonica var.galaucocalyx in preparing anti-cancer medicine |
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2010
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101455652A (en) * | 2009-01-05 | 2009-06-17 | 中国人民解放军第二军医大学 | Use of diterpenoids from Isodon japonica var.galaucocalyx in preparing anti-cancer medicine |
Non-Patent Citations (3)
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| ISAO KUBO ETAL: "Antibacterial Activity of ent-Kaurene Diterpenoids from Rabdosia rosthornii", 《PHYTOTHERAPY RESEARCH》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911025A (en) * | 2011-08-02 | 2013-02-06 | 苏州宝泽堂医药科技有限公司 | Method for extracting 4-hydroxyderricin from angelica keiskei |
| CN102389456A (en) * | 2011-11-29 | 2012-03-28 | 苏州大学 | Method for extracting isodon japonica var.galaucocalyx total diterpenoids or Glaucocalyxin A |
| CN103399120A (en) * | 2013-07-30 | 2013-11-20 | 广州白云山和记黄埔中药有限公司 | Thin-layer identification method of medicine linearstripe rabdosia herb |
| KR101540310B1 (en) * | 2013-08-07 | 2015-07-30 | 건국대학교 산학협력단 | Composition for Preventing or Treating Neuroinflammation by Rabdosia japonica Extracts, and Extracting Method of Rabdosia japonica Extracts |
| WO2016150208A1 (en) * | 2015-03-26 | 2016-09-29 | 苏州沪云肿瘤研究中心股份有限公司 | Pharmaceutical composition and dosage form containing glaucocalyxin a derivative or salt thereof |
| US10196344B2 (en) | 2015-03-26 | 2019-02-05 | Suzhou Pharmavan Cancer Research Center Co., Ltd. | Glaucocalyxin a derivative and preparation method and application thereof |
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