CN100488978C - A continuous extraction method of effective ingredients from fruitbodies of Cordyceps militaris - Google Patents

A continuous extraction method of effective ingredients from fruitbodies of Cordyceps militaris Download PDF

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Publication number
CN100488978C
CN100488978C CNB2006100235784A CN200610023578A CN100488978C CN 100488978 C CN100488978 C CN 100488978C CN B2006100235784 A CNB2006100235784 A CN B2006100235784A CN 200610023578 A CN200610023578 A CN 200610023578A CN 100488978 C CN100488978 C CN 100488978C
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cordycepin
extract
residue
cordyceps
solution
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CN101007025A (en
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陈伟
高新华
唐乐珊
沈明
吴畏
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Shanghai CORT agricultural (Group) Co., Ltd.
Shanghai Academy of Agricultural Sciences
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Shanghai Academy of Agricultural Sciences
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention disclosed a way to successively extract active ingredients from carpohole of north aweto. The invention adopts super critical CO2 to extract sterin substances, precipitating with alcohol to get nucleoside substances with cordycepin and Cordyceps polyose, separating with normal phase low pressure gel silica chromatography to get crude cordycipin, preparative reversed-phase high-performance liquid chromatography to get cordycepin crystal with high purity. The invention can not only successively extract sterin substances and Cordyceps polyose (CM-1) from the carpohole of north aweto, it can also get the cordycepin metamer; it is not applicable to small experiments but also applicable to industrial production. Since the producers can extract many substances by one treatment of carpohole, the invention can not only decrease the industrial cost but also make the biggest economic returns.

Description

A kind of method of continuous extraction effective ingredients from fruitbodies of Cordyceps militaris
Technical field
The present invention relates to the extracting method of biologically active substance, specifically a kind of method of continuous extraction effective ingredients from fruitbodies of Cordyceps militaris.
Background technology
Effective constituent in the cordyceps militaris fruit body comprises phytosterin compound, Cordyceps polysaccharide, cordycepin etc., the extraction of domestic enterprise or R﹠D institution's effective constituent at present, the method that adopts single component to extract to cordyceps militaris fruit body more, this kind method is to make the maximization of target component yield, other composition is not paid close attention to damaed cordition as impurity.In addition, when single active ingredient was extracted, employed raw material all was the primary cordyceps militaris fruit body, and technology is fairly simple, grasped easily.The extraction of single active ingredient makes that a large amount of other effective constituents are lost in vain in the cordyceps militaris fruit body, the biologically active substance that makes the multiple physiologically active ingredient of cordyceps militaris fruit body or have a various clinical purposes is difficult to develop, and the product cost that causes single product to make is too high.
Summary of the invention
The method that a kind of continuous extraction effective ingredients from fruitbodies of Cordyceps militaris that provides at the deficiencies in the prior art is provided, it is when extracting a certain effective constituent, can not only make this effective component yield maximization, but also guarantee not lose other effective constituent; Can the effective constituent with in the cordyceps militaris fruit body many, fast, good, that economize extract in turn, separate, prepare, and help fully utilizing and saving expensive sporophore raw material.
The concrete technical scheme that realizes the object of the invention is:
A kind of method of continuous extraction effective ingredients from fruitbodies of Cordyceps militaris, it is through CO 2Supercritical fluid extraction gets the sterols material, must contain the nucleosides material of cordycepin and Cordyceps polysaccharide, separate and enrichment gets the cordycepin crude product, utilizes that the reverse high performance liquid chromatography of preparation type separates, purifying gets the high-purity cordycepin crystal again through positive low pressure silica gel chromatography through alcohol precipitation; It specifically may further comprise the steps:
(1), CO 2Supercritical fluid extraction
Get 700-1000 gram cordyceps militaris fruit body, pulverize, temperature remains on 40-80 ℃ in the extraction kettle of packing into, and pressure is 300-500bar, feeds CO 2, flow velocity is 50-8g/min, adds CO 295% ethanol of the 5-15% of content is made solubility promoter, balance 10-25min, and the extraction time is 0.5-3h, obtains faint yellow oily extract and residue, faint yellow oily extract is the sterols material;
(2), alcohol precipitation
With the residue that above-mentioned steps obtains, put into extractor and extract, add 500-2000ml distilled water, in 50-90 ℃ of insulation 1-2.5h, take out centrifugally, again residue is reentered in the distilled water, repeat to extract three times, merge No. three times extracting solution, abandon or adopt residue; Extracting solution is concentrated with rotatory evaporator, and temperature is 40-80 ℃, and vacuum tightness is 0.5-1Mpa, is concentrated into 500-2000ml, and is centrifugal, removes residue, adds 95% ethanol of three times of extracting liquid volumes, and placement is spent the night; Utilize whizzer centrifugal, obtain supernatant liquor and throw out, supernatant liquor is the nucleosides material that contains cordycepin, and throw out is with Cordyceps polysaccharide;
(3), positive low pressure silica gel chromatography separation, enrichment
With supernatant liquor in the step 2 is material, utilize positive low pressure silica gel chromatography to separate and enrichment, chromatographic condition: chromatographic column is the sealing glass post of 50mm * 1000mm, filler is the GF254 chromatographic silica gel, and with trichloromethane: ethyl acetate: Virahol: water, mass ratio are: 400: 100: 300: 24 eluent carries out gradient elution; Get an amount of supernatant liquor and add silicagel column, go up sample, evenly add eluent with 1-5ml/min under the room temperature, the solution behind the collection wash-out merges the elutriant that contains cordycepin, and lyophilize promptly gets the cordycepin crude product.
(4), preparation type RPLC separates, purifying
Preparative chromatography condition: chromatographic column: Sino ODS C1820 * 250mm, flow velocity 6-20ml/min; Sample size: 2ml or chromatographic column: Sino ODSC18 50 * 250mm, flow velocity 50-200ml/min; Sample size: 10-15ml, with dissolved in distilled water cordycepin crude product and be settled to proper volume, the moving phase of above chromatographic condition is methyl alcohol: water=15: 85 gradient elutions, detect wavelength: 260nm, collect solution, said process reaches more than 99% until solution purity repeatedly, freeze-drying solution can obtain the high-purity cordycepin crystal again.
The present invention not only can extract phytosterin compound, the Cordyceps polysaccharide (CM-1) in the cordyceps militaris fruit body continuously, and can prepare highly purified cordycepin monomer; Its both suitable small test, also can be used for industrialization production, because disposable processing sporophore, can extract multiple material, not only having reduced the industrialization cost, and easily created maximum economic benefit, is a kind of method that the recycling economy direction is extended and met to industrial chain that is suitable for, and in implementation process, no any material and other factors that causes environmental pollution.
Embodiment
Embodiment
1, CO 2Supercritical fluid extraction
Get 700 gram cordyceps militaris fruit bodies, pulverize, remain on 60 ℃ in the extraction kettle of packing into, pressure is 400bar, feeds CO 2, flow velocity is 30g/min, adds CO 210% 95% ethanol of content is made solubility promoter, balance 20min, and the extraction time is 2h, obtains faint yellow oily extract and residue, uses reversed phase high efficiency chromatography (chromatographic column: Kromasil ODS C respectively 184.6 * 250mm, 5um; Moving phase: methyl alcohol: water=15: 85; Flow velocity: 0.8ml/min; Detect wavelength: 260nm; Detected temperatures: 30 ℃; Sample size is 2ul) cordycepin content changing conditions in complete monitoring sporophore and the intermediate product.
2, alcohol precipitation
With the residue that above-mentioned steps obtains, put into extractor and extract, add 1200ml distilled water, in 70 ℃ of insulation 2h, take out centrifugally, again residue is reentered in the distilled water, repeat to extract three times, merge No. three times extracting solution, abandon or adopt residue; Extracting solution is concentrated with rotatory evaporator, and temperature is 60 ℃, and vacuum tightness is 0.8Mpa, is concentrated into 1200ml, and is centrifugal, removes residue, adds 95% ethanol of three times of extracting liquid volumes, and placement is spent the night; Utilize whizzer centrifugal, obtain supernatant liquor and throw out, supernatant liquor is the nucleosides material that contains cordycepin, and throw out is Cordyceps polysaccharide (CM-1).
3, positive low pressure silica gel chromatography separation, enrichment
With supernatant liquor in the step 2 is material, utilize positive low pressure silica gel chromatography to separate and enrichment, chromatographic condition: chromatographic column is the sealing glass post of 50mm * 1000mm, filler is the GF254 chromatographic silica gel, and with trichloromethane: ethyl acetate: Virahol: water, mass ratio are: 400: 100: 300: 24 eluent carries out gradient elution; Get an amount of supernatant liquor and add silicagel column, go up sample, evenly add eluent with 3ml/min under the room temperature, the solution behind the collection wash-out merges the elutriant that contains cordycepin, and lyophilize promptly gets the cordycepin crude product.
4, preparation type RPLC separates, purifying
Preparative chromatography condition: chromatographic column: Sino ODS C1820 * 250mm, flow velocity 6-20ml/min; Sample size: 2ml or chromatographic column: Sino ODSC18 50 * 250mm, flow velocity 150ml/min; Sample size: 13ml, with dissolved in distilled water cordycepin crude product and be settled to proper volume, the moving phase of above chromatographic condition is methyl alcohol: water=15: 85 gradient elutions, detect wavelength: 260nm, collect solution, said process reaches more than 99% until solution purity repeatedly, freeze-drying solution can obtain 1.5 gram high-purity cordycepin crystal again.

Claims (1)

1, a kind of method of continuous extraction effective ingredients from fruitbodies of Cordyceps militaris is characterized in that it is through CO 2Supercritical fluid extraction gets the sterols material, must contain the nucleosides material of cordycepin and Cordyceps polysaccharide, separate and enrichment gets the cordycepin crude product, utilizes that the reverse high performance liquid chromatography of preparation type separates, purifying gets the high-purity cordycepin crystal again through positive low pressure silica gel chromatography through alcohol precipitation: it comprises following concrete steps:
(1), CO 2Supercritical fluid extraction
Get 700-1000 gram cordyceps militaris fruit body, pulverize, temperature remains on 40-80 ℃ in the extraction kettle of packing into, and pressure is 300-500bar, feeds CO 2, flow velocity is 50-8g/min, adds CO 295% ethanol of the 5-15% of content is made solubility promoter, balance 10-25min, and the extraction time is 0.5-3h, obtains faint yellow oily extract and residue, faint yellow oily extract is the sterols material;
(2), alcohol precipitation
With the residue that above-mentioned steps obtains, put into extractor and extract, add 500-2000ml distilled water, in 50-90 ℃ of insulation 1-2.5h, take out centrifugally, again residue is reentered in the distilled water, repeat to extract three times, merge No. three times extracting solution, abandon or adopt residue; Extracting solution is concentrated with rotatory evaporator, and temperature is 40-80 ℃, and vacuum tightness is 0.5-1Mpa, is concentrated into 500-2000ml, and is centrifugal, removes residue, adds 95% ethanol of three times of extracting liquid volumes, and placement is spent the night; Utilize whizzer centrifugal, obtain supernatant liquor and throw out, supernatant liquor is the nucleosides material that contains cordycepin, and throw out is a Cordyceps polysaccharide;
(3), positive low pressure silica gel chromatography separation, enrichment
With supernatant liquor in the step 2 is material, utilize positive low pressure silica gel chromatography to separate and enrichment, chromatographic condition: chromatographic column is the sealing glass post of 50mm * 1000mm, filler is the GF254 chromatographic silica gel, and with trichloromethane: ethyl acetate: Virahol: water, mass ratio are: 400: 100: 300: 24 eluent carries out wash-out; Get an amount of supernatant liquor and add silicagel column, go up sample, evenly add eluent with 1-5ml/min under the room temperature, the solution behind the collection wash-out merges the elutriant that contains cordycepin, and lyophilize promptly gets the cordycepin crude product;
(4), preparation type RPLC separates, purifying
Preparative chromatography condition: chromatographic column: Sino ODSC1820 * 250mm, flow velocity 6-20ml/min; Sample size: 2ml or chromatographic column: Sino ODSC1850 * 250mm, flow velocity 50-200ml/min; Sample size: 10-15ml, with dissolved in distilled water cordycepin crude product and be settled to proper volume, the moving phase of above chromatographic condition is methyl alcohol: water=15: 85 wash-outs, detect wavelength: 260nm, collect solution, said process reaches more than 99% until solution purity repeatedly, freeze-drying solution can obtain the high-purity cordycepin crystal again.
CNB2006100235784A 2006-01-24 2006-01-24 A continuous extraction method of effective ingredients from fruitbodies of Cordyceps militaris Expired - Fee Related CN100488978C (en)

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Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102643318B (en) * 2012-03-28 2014-10-22 辽宁大学 Method for extracting refined cordycepin from Cordyceps militaris fruit body
CN103520222A (en) * 2012-07-02 2014-01-22 上海国宝企业发展中心 Cordyceps militaris extractive and application thereof in preparation of medicines for treating tumors
CN103446189A (en) * 2013-07-03 2013-12-18 广州市浩立生物科技有限公司 Medicinal liquor of cordyceps sinensis fungi powder and preparation method of medicinal liquor
CN103445138B (en) * 2013-07-03 2016-01-06 广州市浩立生物科技有限公司 A kind of Chinese caterpillar fungus bacterium powder tablet or capsule and preparation method thereof
CN103784481B (en) * 2014-01-27 2016-06-01 正源堂（天津）生物科技有限公司 A kind of method and application thereof extracting antitumor component from Cordyceps militaris (L.) Link.
CN103751224B (en) * 2014-01-27 2015-12-02 正源堂（天津）生物科技有限公司 Method and the application thereof of antitumor component is extracted from Cordyceps militaris (L.) Link.
CN103908476B (en) * 2014-04-29 2017-01-25 洛阳奥达特食用菌技术开发有限公司 Cordyceps militaris extract as well as preparation method and preparation thereof
CN103936806B (en) * 2014-05-13 2015-10-21 邓华 The processing method of cordycepin is extracted from Cordyceps militaris (L.) Link.
CN107576736B (en) * 2017-08-07 2019-12-10 广东东阳光药业有限公司 Method for simultaneously measuring contents of 4 sterols in fresh cordyceps sinensis by HPLC-ELSD and application
CN109553647A (en) * 2018-12-26 2019-04-02 中国科学院西北高原生物研究所 The extracting method of metabolite, additive
CN113504330B (en) * 2021-06-18 2023-06-02 汇毓安莱博(苏州)医药技术有限公司 Purification process and device for oligosilyl oil
CN113907169A (en) * 2021-11-05 2022-01-11 安吉三叶青生物科技有限公司 Radix astragali buccal tablet and preparation method and application thereof

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CN1291989A (en) * 1998-02-27 2001-04-18 久光制药株式会社 Substance having steroid-like structure, process for production thereof and antitumor agents containing same
CN1263103A (en) * 1999-11-19 2000-08-16 上海交通大学 Supercritical fluid extraction method of high-purity sterol

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