WO2003028748A1 - Procede pour extraire des composants desoxyadenosines du cordyceps sinensis - Google Patents

Procede pour extraire des composants desoxyadenosines du cordyceps sinensis Download PDF

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Publication number
WO2003028748A1
WO2003028748A1 PCT/CN2002/000683 CN0200683W WO03028748A1 WO 2003028748 A1 WO2003028748 A1 WO 2003028748A1 CN 0200683 W CN0200683 W CN 0200683W WO 03028748 A1 WO03028748 A1 WO 03028748A1
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WIPO (PCT)
Prior art keywords
raw material
components
cordyceps
pressure
extraction
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PCT/CN2002/000683
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English (en)
Chinese (zh)
Inventor
Shunzhi Chen
Jianping Gao
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Qian, Kangnan
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Publication of WO2003028748A1 publication Critical patent/WO2003028748A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/066—Clavicipitaceae
    • A61K36/068—Cordyceps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the invention belongs to the field of deep processing of edible medicinal fungi. Specifically, the present invention relates to a method for extracting deoxyadenosine components from Cordyceps. Background technique
  • Cordyceps fungi are mostly precious edible medicinal fungi. Advances in artificial culture technology in recent years have made rare wild fungi economically valuable.
  • Cordyceps a model species in the genus Cordyceps, can be cultivated in large quantities. The active ingredients and pharmacological properties of its artificial products are comparable to natural products. Therefore, many artificial culture products in the genus Cordyceps have valuable edible medicinal value.
  • the active ingredient in Cordyceps fungi is deoxyadenosine, which includes, for example, 3'-deoxyadenosine (commonly known as cordycepin and its CA registration number is [73-03-0], adenosine-3'deoxy), 3 ' -Amino-3'-deoxyadenosine and other compounds, and the main active ingredient is 3'-deoxyadenosine (ie cordycepin).
  • the active ingredients in Cordyceps fungi are usually extracted by techniques such as water boiling, alcohol extraction, cooking and concentration. These technologies consume large amounts of energy, are cumbersome to operate, and there are many wastes that cause environmental pollution.
  • the invention relates to a method for extracting deoxyadenosine components in Cordyceps sinensis.
  • the method comprises: using a fermented product of a Cordyceps strain as a raw material, performing supercritical carbon dioxide extraction, and using a material selected from the group consisting of ethanol, water or propidium.
  • the reagent was used as an entrainer and separated under reduced pressure.
  • the supercritical carbon dioxide extraction method of the present invention has the advantages of low-temperature inert environment, does not affect the activity of active ingredients, is non-toxic and non-flammable, is safe to use, does not pollute the environment, has no solvent residues, and has no heavy metal ions. It overcomes boiling and alcohol extraction
  • the traditional techniques such as concentration, concentration, etc. have the disadvantages of large energy consumption, long time and tediousness, can quickly obtain the natural active ingredients in Cordyceps strains, and can also comprehensively utilize raw materials.
  • the above components prepared by the present invention have antitumor, antiviral and immunomodulatory effects, and are also important reagents in the field of genetic engineering. They may also be related to renal failure and become adjuvants for the treatment of renal failure. Therefore, the present invention has broad application prospects. Brief description of the drawings
  • Figure 1 shows a high-pressure liquid chromatogram with a cordycepin content of 54.65% prepared by the present invention.
  • Fig. 2 is a high-pressure liquid chromatogram of the deoxyadenosine component prepared by the present invention when the content of cordycepin is 75.18%.
  • Fig. 3 is the ultraviolet spectrum characteristic of the composition shown in Fig. 2.
  • Figure 4 shows the liquid chromatogram of the components obtained when 10% ethanol was used as the entrainer.
  • Figure 5 shows the UV spectrum characteristics of the main peak in Figure 4.
  • the invention relates to a method for extracting deoxyadenosine components in Cordyceps sinensis.
  • the method comprises: using a fermented product of a Cordyceps strain as a raw material, performing supercritical carbon dioxide extraction, and using a reagent selected from ethanol, water or propane As an entrainer, it was then separated under reduced pressure.
  • Cordyceps fungi such as Cordyceps Sinensis, Cordyceps militaris, Cordyceps Hawkesii, Paecitmyces militarae, and so on.
  • the present invention is not limited to these specific strains.
  • strains were successfully cultured and inoculated on more than 50 kinds of media such as rice medium, corn, sorghum, wheat, millet, silkworm pupa, tussah pupa, bat larvae, and egg white.
  • media such as rice medium, corn, sorghum, wheat, millet, silkworm pupa, tussah pupa, bat larvae, and egg white.
  • Cordyceps is more difficult to form fruiting bodies
  • Cordyceps militaris is more likely to form fruiting bodies.
  • the products of these strains after artificial culture can be divided into two categories: one is fruit body; the other is fermented material (medium, fermented bacterial powder).
  • Fruiting bodies can be directly made into products and sold on the market; and fermented materials, such as Cordyceps fermented fungus powder, Cordyceps militaris, etc., can be used as health food or medicine.
  • large-yield medium containing bacterial base and bacterial residue such as y
  • the raw material in the present invention refers to the product of the above-mentioned Cordyceps strain after artificial culture or the fungus residue after picking the constellation. Specifically, it mainly refers to solid or semi-solid substances containing cordycepin, such as solid fermentation medium, semi-solid fermented corn porridge medium, dried fungal powder after liquid fermentation, and Remaining raw materials, etc. These products are easy to store and transport at a moisture content of 6-10%.
  • Cordyceps fermented fungus powder Cordyceps militaris powder, Humulus japonica rice culture medium, Paecilomyces cerevisiae fermentation fungus powder and rice culture medium, sub-fragrant stick fermentation fungus powder, etc.
  • the remaining raw material after extracting Cordyceps oil has a moisture content of about 1-5%, which is much lower than the commercially available rice moisture content and does not affect the extraction of deoxyadenosine.
  • the fungal culture needs to be pulverized and added to the extraction kettle.
  • the particle size of the solid raw material should be 20-60 mesh, which is too fine and easily clogged, too coarse, the extraction time is long, it is easy to remain, and the yield is low.
  • the artificial culture of Cordyceps strains used in the method of the present invention should have a cordycepin (3'-deoxyadenosine) content of 0.5 mg / g or more, more preferably 0.8 mg / g or more and 1.8 mg / g or less. Because cordycepin is the main component of deoxyadenosine, it is its signature component and active component.
  • the content of cordycepin in natural Cordyceps and Cordyceps militaris is less than 0.05-0.08%, and the content of cordycepin in artificial culture is higher.
  • the cordycepin content in the yarrow fungus powder is as high as 1.0-5.0%, or higher. Therefore, the selection of the raw materials according to the content of cordycepin is beneficial to the production and implementation, and is beneficial to reducing the cost, which is consistent with the process of this process.
  • Supercritical carbon dioxide extraction refers to making carbon dioxide into a liquid under high pressure, and as a solvent, the components of the extracted Chinese medicinal materials are dissolved in the kettle. In the process of carbon dioxide flow, the pressure and temperature in the separation kettle are different, so that the required component can be analyzed. Combined with the I, II, III separation kettle or fractionation column, a certain component can be analyzed. Then, carbon dioxide is volatilized under reduced pressure to obtain a substance containing the deoxyadenosine component according to the present invention as a main component.
  • the mixed components (crude extract) can be re-extracted once in the kettle, that is, 50% or 75% of the crystals are re-extracted; a single component can also be obtained by ordinary crystal separation methods.
  • the pressure of the extraction kettle is 15-50Mpa; the pressure of the separation kettle is preferably 5-25Mpa, but the pressure of the separation kettle is always lower than the pressure of the extraction kettle in the same process.
  • the carbon dioxide flow rate in the extraction kettle is 15-25 liters / h, and the temperature of the extraction kettle is about 50-60 ° C.
  • the pressure and temperature of the separation kettle are changed according to the required conditions. Experiments have shown that extraction temperatures up to 100 ° C have not significantly damaged structures such as Cordyceps.
  • another feature is the use of an agent selected from the group consisting of ethanol, water and propidium as an entrainer.
  • Ethanol should be used.
  • the concentration of ethanol should be at least 5%.
  • Commercially available anhydrous ethanol can also be used as an entrainer.
  • the optimal concentration of ethanol is 10-30%.
  • Propylamidine also works well as an entrainer. When water is used as an entrainer, it takes a long time. It is separated by the principle of solvent distribution, so the yield of extraction needs to be improved. In general, the initial experimental yield is 10-20%. Improving the experimental conditions (time, flow rate, temperature, pressure, and carrier of the fractionation column) can improve the product yield, so that it can reach 30-50% or more. The principle is combined within the scope of the patent right. Such as the use of extended separation time, increased pressure difference, continuous multiple extraction or use of a distillation column.
  • FIG. 1 shows a high-pressure liquid chromatogram with a cordycepin content of 54.65% prepared by the present invention.
  • FIG. 2 is a high-pressure liquid chromatogram of the deoxyadenosine component prepared by the present invention when the content of cordycepin is 75.18%.
  • the peak washing time is 6.10-6.30 minutes, the second peak is 4.20-4.60 minutes, accounting for 6.49%; the three peaks are 10.48 minutes, accounting for 4.28%; the four peaks are 7.78 minutes, accounting for 4.23%.
  • Figure 3 is the ultraviolet spectrum characteristics of the components shown in Figure 2, 217nm is 4.027; there is a trough at 245nm; the absorption peak is at 259nm.
  • FIG. 4 shows the liquid chromatogram of the components obtained when 10% ethanol is used as the entrainer. There is only one main peak, washing out time 6.11 Points, accounting for 75.05%.
  • Fig. 5 is an ultraviolet characteristic of the main peak of Fig. 4. Its 219nm is 4.074, and the 241nm trough absorbance is 1.68; the 259nm peak is 2.045; the wavelength at half the peak absorbance is about 280nm.
  • Cordycepin monomer obtained by super-reproducibility technology has only 2-3 peaks in liquid chromatography, the main peak is> 98.6%, and the sum of the two and three peaks is 1.4%.
  • the UV characteristics of the single product of this technology are: the trough is at 227nm ; the peak is at 259nm and the half height is at 274nm. Because more than 98% is close to pure substance, when the purity of monomer is> 99.0%, liquid chromatography can show a main peak labeled as 100%, and the secondary components only show waveforms without scores. Compared with traditional products, there are many similar Processing, but the waveforms are different; if necessary, use infrared spectroscopy to distinguish them.
  • the deoxyadenosine components obtained in the method of the present invention are still dissolved in water, in the ethanol mixture or in the propane liquid, and conventional methods such as activated carbon adsorption, impurity removal, organic solvent removal, recrystallization, or ionic resin need to be used.
  • the powdery raw material is obtained by processing and removing impurities, and then freeze-drying or vacuum drying, so as to obtain a powdery raw material having a cordycepin content of 50-99.9% by weight, and then adding an adjuvant to produce tablets or capsules.
  • Each tablet or capsule prepared may contain 5 to 200 mg of 3'-deoxyadenosine, preferably between 25 to 100 mg.
  • the deoxyadenosine component powder obtained by the method of the present invention is processed into a lyophilized injection, and the content of cordycepin in each injection is 12.5 mg-200 mg, and the optimum is 25-100 mg / piece.
  • the extract is about 540ml, combined with activated carbon to remove impurities, and concentrated in vacuo to obtain 235mg of pale yellow pellets with a content of 75.05%.
  • the yield is about 16.79%. If you want to increase the yield, you can use entrainer extraction repeatedly.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

Cette invention se rapporte à un procédé servant à extraire des composants désoxyadénosines du cordyceps sinensis et consistant à cet effet : à cultiver artificiellement des souches de cordyceps sinensis, puis à extraire les micro-organismes fermentés ainsi obtenus, utilisés comme matière brute, par procédé au CO2 hypercritique, dans lequel les agents entraînants sont choisis parmi des réactifs tels que l'alcool, l'eau ou le propane, et séparés ensuite sous vide. Ce procédé n'affecte pas les activités de la composition active, il est non toxique et ininflammable, il ne contamine pas l'environnement, il ne produit pas de solvant résiduaire ni d'ions de métaux lourds, notamment, et il permet d'obtenir rapidement des composants naturellement actifs de souches de cordyceps sinensis, et il utilise également la matière brute intégralement.
PCT/CN2002/000683 2001-09-28 2002-09-27 Procede pour extraire des composants desoxyadenosines du cordyceps sinensis WO2003028748A1 (fr)

Applications Claiming Priority (2)

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CNB011269103A CN1240706C (zh) 2001-09-28 2001-09-28 一种超临界萃取虫草脱氧核苷的生产方法
CN01126910.3 2001-09-28

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AU2003271037A1 (en) * 2003-09-28 2005-04-14 Ailing Li The extractive of aweto and process for its preparation and uses
CN101124988B (zh) * 2007-09-25 2010-10-06 江苏瑞迪生科技有限公司 从蛹虫草中提取精制虫草素和虫草多糖的方法
CN101406491B (zh) * 2008-11-26 2011-04-06 吉林大学 一种水包油型虫草乳剂及其制备工艺
CN102349934B (zh) * 2011-10-24 2013-05-01 周伯扬 一种虫草菌丝体有效成分的提取工艺

Citations (2)

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Publication number Priority date Publication date Assignee Title
CN1086732A (zh) * 1993-07-03 1994-05-18 李春燕 北冬虫夏草提取液及其制备方法
US5582828A (en) * 1995-03-15 1996-12-10 Ching-Yuang Lin Active fractions of Cordyceps sinensis and method of isolation thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1086732A (zh) * 1993-07-03 1994-05-18 李春燕 北冬虫夏草提取液及其制备方法
US5582828A (en) * 1995-03-15 1996-12-10 Ching-Yuang Lin Active fractions of Cordyceps sinensis and method of isolation thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JIANG LIXIA ET AL.: "Determinate main nucleoside and nucleobase of cordyceps sinensis by HPLC", CHINESE PATENT MEDICINE, vol. 15, no. 5, 1993 *
LI XIAOMING ET AL.: "Extract adenosine from cordyceps sinensis mycelium", JOURNAL OF CHINESE MATERIA MEDICA, vol. 15, no. 1, 1990 *
LIU JINGMING ET AL.: "Research of chemical components in cordyceps miliaris", JOURNAL OF CHINESE MATERIA MEDICA, vol. 14, no. 10, 1989 *
MAO LIZHEN ET AL.: "Determinate adenosine content of cordyceps sinensis---filifonn spores", CHINESE MIDICNAL HERB., vol. 24, no. 9, 1993 *
WU CHUNMIN ET AL.: "Determinate adenosine content of cordyceps sinensis", CHINESE MEDICINAL HERB., vol. 30, no. 9, 1999 *

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CN1240706C (zh) 2006-02-08

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