WO2014003356A1 - Procédé de préparation d'un extrait de solution végétale de culture de mycélium à partir de vers contenant de la cordycépine en concentration élevée, et composition pour le traitement ou la prévention des maladies vasculaires, contenant ledit extrait - Google Patents

Procédé de préparation d'un extrait de solution végétale de culture de mycélium à partir de vers contenant de la cordycépine en concentration élevée, et composition pour le traitement ou la prévention des maladies vasculaires, contenant ledit extrait Download PDF

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WO2014003356A1
WO2014003356A1 PCT/KR2013/005413 KR2013005413W WO2014003356A1 WO 2014003356 A1 WO2014003356 A1 WO 2014003356A1 KR 2013005413 W KR2013005413 W KR 2013005413W WO 2014003356 A1 WO2014003356 A1 WO 2014003356A1
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extract
cordyceps
culture filtrate
mycelium
butanol
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PCT/KR2013/005413
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English (en)
Korean (ko)
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안병락
김종래
권호균
이종진
노준희
김원기
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환인제약 주식회사
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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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

Definitions

  • the present invention relates to a method for preparing an extract of Cordyceps mycelium culture filtrate containing a high concentration of cordycepin, and a composition for treating or preventing vascular diseases containing the extract.
  • Cerebrovascular diseases such as atherosclerosis, cerebral hemorrhage, stroke and cerebral infarction and vascular diseases such as heart disease are adult diseases that occupy the first and second leading causes of death. As the adult disease develops into a modern society, it is frequently occurring in middle-aged people due to changes in dietary habits and internal and external environmental stresses. The main cause of these diseases is known as thrombi, and thrombus is recognized as a pathology mediated by excess platelet aggregation. Platelets are activated by stimulation of various agonists such as collagen, thrombin, ADP, etc. upon blood vessel damage, causing adhesion, release and aggregation reactions. These reactions are involved in hemostasis, but play an important role in the development of vascular diseases, including blood clots.
  • Cordyceps sinensis is a mushroom that produces fruiting bodies based on insects. There are now hundreds of species worldwide (Mycologia, 50, 169 ⁇ 222, 1985), and there are about 80 species in Korea (Kyo). -Hak publishing co.Seoul, 13-18, 1996). Among them, Cordyceps sinensis and Cordyceps sp. Have been reported to be used as herbal medicines from tuberculosis, asthma, drug addiction, and nourishment tonic since ancient times (Sci. Rept. Tokyo Bunrika daikaku Sect). B., 5, 253-260, 1940; Icons of Medicinal Fungi from China Science Press China., P.575, 1989; USDA Agricultural Reserch Service, 1990).
  • Cordyceps sinensis Cordyceps sinensis
  • Cordyceps sinensis Cordyceps militaris
  • Cordyceps ophioglossoides Cordyceps sobolifera
  • Cordyps sobolifera Cordyceps beauveria
  • Cordyceps bassiana Representative Cordyceps sinensis , Cordyceps sinensis , Cordyceps militaris , Cordyceps ophioglossoides , Cordyceps sobolifera , Cordyps sobolifera Cordyceps beauveria , Cordyceps bassiana .
  • cordycepin which is attracting the most attention as a useful component of Cordyceps sinensis, has been researched about pharmacological activity since it was discovered by Cunningham et al. In 1950.
  • Cordycepin is known to inhibit the synthesis of m-RNA and have antibacterial, antifungal, immunopotentiating and anticancer effects. (Nature, 166, 949-954, 1950; Cancer Res., 21, 216-220, 1961 ; Biochem.Biophys.Acta., 80, 640-647, 1964).
  • Korean Patent No. 464876 discloses an antithrombotic composition containing cordycepin of Cordyceps sinensis.
  • Korean Patent Laid-Open No. 2001-0054264 discloses a cordycepin for anticancer drug extracted from Cordyceps sinensis and a manufacturing method thereof.
  • Korean Patent No. 509718 since the separation methods disclosed in Korean Patent Laid-Open No. 2001-0054264 are purification methods using HPLC, only a small amount of the active substance is separated through the method, which is not commercially suitable.
  • the Korean Patent No. 509718 discloses a method for separating and purifying a crude codycepine fraction comprising cordycepin and adenosine from the pupae cordyceps as an active ingredient, (1) a water-soluble extract from methanol, ethanol and butanol Step 1 to obtain a lower alcohol soluble fraction, such as (2) further purification with an eluate obtained at pH 6.0 or more by adsorption to a cation exchange resin, (3) a fraction containing cordycepin and adenosine via TLC Disclosed is a manufacturing process consisting of three steps of collecting and passing through the anion exchange resin to remove and elute the pigment and (4) four steps to remove impurities and finally purify the adsorbent.
  • cordycepin is secreted into the culture filtrate in suspension culture of Cordyceps mycelium, but there is no disclosure about a method for rapidly separating a large amount of cordycepin from the culture filtrate. Therefore, the present inventors, unlike the prior art that separates the composition containing cordycepin from the fruiting body or mycelium of Cordyceps sinensis, by adjusting the pH of the culture filtrate of Cordyceps sinensis mycelium, and extracting it with butanol solution, As high as possible, the present invention was completed by confirming that an extract containing high concentration of cordycepin could be prepared.
  • An object of the present invention is to provide a method for producing an extract of Cordyceps mycelium culture filtrate containing a high concentration of cordycepin, and to provide a composition for the treatment or prevention of vascular diseases containing the extract.
  • the present invention relates to a method for preparing an extract of Cordyceps mycelium culture filtrate containing a high concentration of cordycepin.
  • Step 1 after adjusting the culture filtrate of Cordyceps mycelia to pH 4.0 or more, and concentrating to 30 ⁇ 70 brix;
  • Step 2 adjusting the 30 ⁇ 70brix concentrate of the Cordyceps mycelium culture filtrate to pH 4.0 or more;
  • Step 4 separating the supernatant of the primary extract and the second residue by adding butanol solution to the remaining residue, and separating the supernatant of the secondary extract;
  • Step 5 After mixing the supernatant of the primary extract separated in step 3 and the supernatant of the secondary extract separated in step 4 and filtered, and drying the filtrate; and may be prepared.
  • Cordyceps mycelium used in the first step is Cordyceps sinensis , Cordyceps militaris , Cordyceps ophioglossoides , Cordyceps sobolifera , Cordyceps beauveria and Cordyceps bassiana may be one or more mycelium selected from the group consisting of.
  • butanol solution preferably 100% butanol can be used.
  • the butanol solution may be added 200 ⁇ 2000 parts by weight based on 100 parts by weight of the concentrated pH of the two steps.
  • butanol solution is added to the concentrated solution of the pH control of the second step, the primary extraction may be performed by stirring for 2 to 10 hours.
  • saturated butanol which is preferably a 75 to 85% butanol aqueous solution, may be used.
  • 200 to 2000 parts by weight of a butanol solution may be added to the residue remaining after separating the supernatant of the primary extract, based on 100 parts by weight of the concentrate having the pH adjusted in the second step.
  • butanol solution may be added to the remaining residue, followed by stirring for 2 to 10 hours to extract the secondary.
  • step 5 may be performed by concentration of the filtrate under reduced pressure, azeotropic concentration and lyophilization.
  • the present invention provides an extract of Cordyceps mycelium culture filtrate prepared by the above method, and preferably, provides a composition for preventing or treating vascular disease containing the extract of Cordyceps sinensis mycelium culture filtrate.
  • the vascular disease may be selected from the group consisting of atherosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications that occur after carotid angioplasty, cerebral infarction, cerebral hemorrhage, and stroke.
  • the present invention provides a health functional food for the prevention or improvement of vascular diseases containing the extract of Cordyceps sinensis mycelia culture filtrate prepared by the above method.
  • the culture filtrate of Cordyceps mycelium mycelium of step 1 can be obtained by filtering the culture solution obtained through the liquid culture of Cordyceps mycelium mycelium prepared by a conventional method, it is not limited to the culture method. However, preferably,
  • Solid medium a conventional medium for culturing microorganisms may be used, and preferably, PDA (Difco Potato Dextrose Agar) may be used.
  • PDA Denco Potato Dextrose Agar
  • the step of culturing the Cordyceps mycelium in the liquid medium does not limit the number of cultures, but it is suitable to enhance the culture activity of the mycelium by culturing in steps of 1 to 6 times.
  • a liquid medium for culturing microorganisms As a medium that can be used for the liquid medium, a liquid medium for culturing microorganisms can be used, and preferably, WIMG production medium (Glucose 20g / L, Yeast extract 10g / L, MgSO 4 2.5g / L, KH 2 PO 4 1 g / l, K 2 HPO 4 0.46 g / l, Glycine 1 g / l) can be used.
  • WIMG production medium Glucose 20g / L, Yeast extract 10g / L, MgSO 4 2.5g / L, KH 2 PO 4 1 g / l, K 2 HPO 4 0.46 g / l, Glycine 1 g / l
  • the solid culture may be incubated for 5 to 15 days at 20 ⁇ 30 °C, the liquid culture may be incubated for 5 to 15 days at 20 ⁇ 30 °C, agitation rate 50 ⁇ 500rpm, if necessary, aeration Liquid culture can be added by adding 0.1 ⁇ 2.0vvm condition.
  • the culture solution of the Cordyceps mycelium mycelium can be filtered through a conventional filtration method, preferably can be filtered using a filter paper, nonwoven fabric, cotton and the like.
  • the recovery rate of cordycepin may be lowered.
  • the pH of the culture filtrate immediately after the cultivation is usually within 3.0 ⁇ 7.0, at this time, if the concentration of the culture filtrate is less than pH 4.0, the recovery rate of cordycepin is lowered.
  • the pH control is 4.0 or more, the recovery rate of cordycepin is increased at all pH conditions, preferably pH 4.0-10.0, more preferably pH 4.0-7.0.
  • the step of concentrating the culture filtrate of Cordyceps sinensis mycelium can be concentrated using a reduced pressure concentration method.
  • the decompression concentration may be performed using a rotary vacuum concentrator.
  • the pH of the first step or the second step can be adjusted by using sodium hydroxide aqueous solution, potassium hydroxide aqueous solution or sulfuric acid aqueous solution, hydrochloric acid aqueous solution and the like.
  • the pH is adjusted again in the second step because the pH may be lowered below 4.0 during the concentration process.
  • the pH control is 4.0 or more, the recovery rate of cordycepin is increased at all pH conditions, preferably pH 4.0 to 10.0 is preferred, more preferably pH 4.0 to 7.0, and most preferably pH 4.0 to 6.0 This is the best.
  • the butanol solution may be added 200 to 2000 parts by weight based on 100 parts by weight of the pH-adjusted concentrate of the second step, preferably 200 to 600 parts by weight.
  • butanol solutions of any concentration may be used as the butanol solution, and most preferably 100% butanol may be used.
  • butanol solution of any concentration may be used as the butanol solution, but most preferably, saturated butanol which is 75-85% butanol aqueous solution.
  • the filtration process of step 5 may be repeated 1 to 3 times.
  • the filtration process of the five steps can be carried out through a conventional filtration method, preferably can be filtered using a filter paper, nonwoven fabric, cotton and the like.
  • the filtrate of step 5 may be concentrated using a reduced pressure concentration method, using a common drying method such as reduced pressure drying, spray drying, or freeze drying after azeotropic concentration with water to completely remove the remaining solvent. It can be dried, preferably lyophilized.
  • the reduced pressure concentration or reduced pressure drying may be performed using a rotary vacuum concentrator.
  • the extract of Cordyceps mycelium culture filtrate obtained through the method of the present invention if necessary, extraction with an organic solvent (alcohol, ether, acetone, etc.), distribution of the organic solvent (n-hexane, ethyl acetate, butanol) and water,
  • an organic solvent alcohol, ether, acetone, etc.
  • distribution of the organic solvent n-hexane, ethyl acetate, butanol
  • and water The method by column chromatography, the well-known method used for the isolation
  • Chromatography used in the present invention includes silica gel column chromatography, L-20 column chromatography, ion exchange resin chromatography, thin layer chromatography ( Thin layer chromatography (TLC), medium pressure liquid chromatography (MPLC), high performance liquid chromatography (High Performance Liquid Chromatography), and the like.
  • the present invention provides a pharmaceutical composition for the treatment or prevention of vascular diseases containing extracts of Cordyceps mycelium culture filtrate containing a high concentration of cordycepin prepared by the above method.
  • Pharmaceutical compositions for the prevention and treatment of vascular diseases containing extracts of Cordyceps sinensis mycelium culture filtrate containing high concentrations of cordycepin prepared by the method of the present invention respectively, powders, granules, tablets, capsules, suspensions Oral formulations such as emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions.
  • excipients and diluents that may be included in the composition containing the extract of Cordyceps mycelia mycelia culture filtrate containing high concentration of cordycepin prepared by the method of the present invention, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol , Erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc And magnesium stearate and mineral oil.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one or more extracts of Cordyceps mycelium culture filtrate containing high concentrations of cordycepin prepared by the method of the present invention.
  • Excipients For example, starch, calcium carbonate, sucrose or lactose, gelatin and the like are mixed and prepared.
  • lubricants such as magnesium stearate and talc are also used.
  • Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • the non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the dosage of the extract of Cordyceps mycelium culture filtrate containing a high concentration of cordycepin prepared by the method of the present invention is the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, administration It will depend on the route and judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 0.1 mg / kg / day to 500 mg / kg / day. Administration may be once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • the extract of Cordyceps sinensis mycelium culture filtrate containing a high concentration of cordycepin prepared by the method of the present invention can be administered to various mammals such as rats, livestock, humans. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.
  • the extract of Cordyceps sinensis mycelium culture filtrate containing a high concentration of cordycepin prepared by the method of the present invention is a composition derived from natural products, so there is almost no toxicity and side effects, so it is a drug that can be used safely for long-term use for prophylactic purposes.
  • the present invention provides a dietary supplement for the prevention or improvement of vascular diseases containing extracts of Cordyceps mycelium culture filtrate containing a high concentration of cordycepin prepared by the above method.
  • the health functional food may be added a food supplement acceptable food supplement.
  • the health functional food includes the form of tablets, capsules, pills or liquids, etc.
  • the health functional foods include dairy products, including soups, meat, sausages, bread, candy, snacks, noodles, ice cream, soups, Beverages containing ionic beverages, nutritional products including alcoholic beverages and vitamin complexes may be included.
  • the present invention relates to a method for preparing an extract of Cordyceps mycelium culture filtrate containing a high concentration of cordycepin, and a composition for treating or preventing vascular diseases containing the extract.
  • Cordyceps mycelium culture filtrate extract prepared by the method of the present invention is excellent in the recovery rate of cordycepin can be prepared extract containing a high content of cordycepin, Cordycepin is included from the culture filtrate of cordyceps mycelium previously discarded By extracting the effective ingredient can bring excellent cost reduction effect.
  • the extract of the culture filtrate of Cordyceps sinensis mycelia is excellent in the treatment effect of vascular diseases, such as atherosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications that occur after carotid angioplasty, cerebral infarction, cerebral hemorrhage, stroke, etc. It can also be used as a pharmaceutical composition for vascular disease.
  • vascular diseases such as atherosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications that occur after carotid angioplasty, cerebral infarction, cerebral hemorrhage, stroke, etc. It can also be used as a pharmaceutical composition for vascular disease.
  • FIG. 1 is a schematic diagram showing a method for preparing an extract of Cordyceps sinensis mycelium culture filtrate of the present invention.
  • Cordyceps mycelia isolated and identified in Example 1 were inoculated in a solid medium (Difco Potato Dextrose Agar) PDA and cultured so that the cells adhered to the whole for 24 days at 21 ° C. Thereafter, the primary cultivated Cordyceps sinensis mycelium was cut into pieces of about 5 mm in width and length using a sterile scalpel, and 10 pieces were cut into 100 ml of YM medium (yeast minimal media, Glucose 10 g / l, Malt extract 3 g / l).
  • YM medium yeast minimal media, Glucose 10 g / l, Malt extract 3 g / l.
  • the secondary seed culture was inoculated at 10% (v / v) in a 1 l baffled flask containing g / l, KH 2 PO 4 1 g / l, K 2 HPO 4 0.46 g / l) medium, Incubated for 4 days with stirring at 100 rpm in a 24 °C shaker.
  • the fourth species culture was inoculated with 10% (v / v) in 3.6 L of WIM medium, and then incubated for 1 day at a culture temperature of 24 ° C., aeration rate of 0.25vvm, and agitation speed of 100rpm.
  • the fifth seed culture was inoculated with 10% (v / v) of the fourth seed culture broth at 36 L of WIM medium, and then cultured for 1 day at a culture temperature of 24 ° C., aeration rate of 0.25vvm, and agitation speed of 100 rpm.
  • This culture is the fourth seed culture medium 360L WIMG production medium (Glucose 20g / L, Yeast extract 10g / L, MgSO 4 2.5g / L, KH 2 PO 4 1g / L, K 2 HPO 4 0.46g / L, Glycine 1g / l) inoculated at 10% (v / v) and then cultured for 10 days at a culture temperature 24 °C, aeration 0.05 ⁇ 0.5vvm, agitation speed 50 ⁇ 100rpm to finally obtain a culture of Cordyceps mycelia.
  • WIMG production medium Glucose 20g / L, Yeast extract 10g / L, MgSO 4 2.5g / L, KH 2 PO 4 1g / L, K 2 HPO 4 0.46g / L, Glycine 1g / l
  • Example 3-1 4.0 60 °C Example 3-2 5.0 60 °C Example 3-3 6.0 60 °C Example 3-4 7.0 60 °C Example 3-5 10.0 60 °C Example 3-6 4.0 80 °C Example 3-7 5.0 80 °C Example 3-8 6.0 80 °C Example 3-9 7.0 80 °C Example 3-10 10.0 80 °C Example 3-11 4.0 100 °C Example 3-12 5.0 100 °C Example 3-13 6.0 100 °C Example 3-14 7.0 100 °C Example 3-15 10.0 100 °C Example 3-16 4.0 40 °C Example 3-17 10.0 40 °C
  • Example 3 In the same manner as in Example 3 to prepare a concentrate of Cordyceps mycelia culture filtrate was prepared under the conditions of the concentration temperature and pH of Table 2.
  • 50brix concentrate of Cordyceps sinensis filtrate was prepared under the conditions of Example 3-2. Thereafter, 100 g of the 50brix concentrate was adjusted to the conditions shown in Table 3 below, and then the pH was adjusted (second pH adjustment), and then 300 g of the first extraction solvent was added, followed by stirring at 60 ° C. for 4 hours to extract the primary. After separating the first extracted supernatant, the second extractant 400g was further added to the remaining concentrate, followed by secondary extraction with stirring at 60 ° C. for 4 hours.
  • the second extracted supernatant was separated and mixed with the first extracted supernatant and filtered twice with filter paper.
  • the filtrate was concentrated under reduced pressure at 60 °C using a rotary vacuum concentrator, azeotropically concentrated with water to completely remove the remaining solvent and then lyophilized to prepare an extract of Cordyceps sinensis culture filtrate.
  • the extract of the Cordyceps mycelium culture filtrate of the present invention was prepared under the conditions of Table 4 (the conditions other than Table 4 were the same as in Example 5). In the following Table 4, there is no secondary extraction solvent, only the primary extraction is performed when the extract is filtered and dried).
  • Example 3-1 4.0 60 °C 98.2
  • Example 3-2 5.0 60 °C 100.0
  • Example 3-3 6.0 60 °C 98.5
  • Example 3-4 7.0 60 °C 99.4
  • Example 3-5 10.0 60 °C 98.9
  • Example 3-6 4.0 80 °C 96.8
  • Example 3-7 5.0 80 °C 98.2
  • Example 3-8 6.0 80 °C 99.0
  • Example 3-10 10.0 80 °C 99.5
  • Example 3-11 4.0 100 °C 92.2
  • Example 3-12 5.0 100 °C 100.0
  • Example 3-13 6.0 100 °C 99.9
  • Example 3-14 7.0 100 °C 98.6
  • Example 3-15 10.0 100 °C 98.6
  • Example 3-16 4.0 40 °C 95.3
  • Example 3-17 10.0 40 °C 96.5 Comparative Example 1-1 2.0 60 °C 71.9 Comparative Example 1-2 3.0 60
  • Example 3-2 pH Adjustment of Concentrate Primary Extraction Solvent Second extraction solvent Extraction temperature Cordycepin recovery (%)
  • Example 4-1 pH 4.0 100% Butanol Saturated Butanol 60 °C 97.6
  • Example 4-2 pH 4.0 100% Butanol Saturated Butanol 100 °C 96.7
  • Example 4-3 pH 5.0 100% Butanol Saturated Butanol 60 °C 96.5
  • Example 4-4 pH 5.0 100% Butanol Saturated Butanol 100 °C 96.8
  • Example 4-5 pH 6.0 100% Butanol Saturated Butanol 60 °C 98.9
  • Example 4-6 pH 6.0 100% Butanol Saturated Butanol 100 °C 97.6
  • Example 4-7 pH 7.0 100% Butanol Saturated Butanol 60 °C 95.2
  • Example 4-8 pH 6.0 Saturated Butanol 100% Butanol 60 °C 92.1 Comparative Example 2-1 pH 3.0 100% Butan
  • the animals were anesthetized with N 2 O and O 2 mixed gas and 3% isoflurane at a volume ratio of 7: 3, and then the common artery of the animals
  • the carotid artery and the external carotid artery are ligated and rounded by burning one end of a 19 mm probe (approximately 2.1 cm 4-0 surgical nylon thread) from the branch of the internal carotid artery.
  • the right sheath endovascular middlecerebral artery occlusion was used to induce local cerebral ischemia. After that, the blood flow of the middle cerebral artery was blocked for 90 minutes to maintain an ischemic state, and the surgical site was again exposed to remove the probe and reperfused.
  • Example 4 Samples of Example 4 and Comparative Example 2 were orally administered twice at 3 and 8 hours after ischemia induction (100 mg / kg, respectively). At this time, no sample was treated in the control group.
  • the brains of the experimental animals were extracted, and brain sections were made to a thickness of 2 mm using a brain frame, and then 2,3,5-triphenyltetrazolium chloride ( 2,3,5-triphenyltetrazonlium chloride, TTC) solution was deposited for 20-30 minutes at 37 °C.
  • TTC 2,3,5-triphenyltetrazonlium chloride
  • NMDA receptor blocker MK-801 ⁇ (+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d] cyclohepten-5,10-imine maleate ⁇ was used as a positive control. .
  • Example 7 As shown in Table 7, the sample of Example 4 at the cerebral infarction of the middle cerebral artery occlusion / reperfusion injury model was significantly reduced in the volume of infarct area caused by ischemia compared to the sample of Comparative Example 2, indicating that the brain tissue protection was excellent. Confirmed.
  • Example 4-1 20 g of the extract of the Cordyceps mycelia culture filtrate of Example 4-1 was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.
  • Example 4-1 2 g of the extract of Cordyceps sinensis mycelium culture filtrate of Example 4-1 was dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.
  • the extract of Cordyceps sinensis mycelium culture filtrate of Example 4-1 was prepared in an amount of 0.2 to 10% by weight to prepare a cooking seasoning for health promotion.
  • the extract of Cordyceps sinensis mycelium culture filtrate of Example 4-1 was added to the flour at 0.1 to 5.0% by weight, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.
  • the extract of Cordyceps sinensis mycelium culture filtrate of Example 4-1 was added to the soup and the juice to 0.1 ⁇ 1.0% by weight to prepare meat products for health promotion, soup and noodles of noodles.
  • the extract of Cordyceps mycelia mycelia culture filtrate of Example 4-1 was added to milk at 0.1 to 1.0% by weight, and various dairy products such as butter and ice cream were prepared using the milk.
  • Example 4-1 0.5 g of the extract of Cordyceps sinensis mycelium culture filtrate of Example 4-1 was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.
  • Example 3-2 Fruit juice manufacturing
  • Example 4-1 0.1 g of the extract of Cordyceps sinensis mycelium culture filtrate of Example 4-1 was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

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Abstract

La présente invention concerne un procédé de préparation d'un extrait de solution végétale de culture de mycélium à partir de vers contenant de la cordycépine en concentration élevée, et une composition destinée au traitement ou à la prévention des maladies vasculaires, contenant ledit extrait. Il est possible de : préparer un extrait contenant une proportion élevée de cordycépine étant donné que l'extrait d'une solution végétale de culture du mycélium à partir de vers préparée selon le procédé de la présente invention présente un excellent taux de récupération de la cordycépine ; et permet de réduire les coûts de façon notable, en extrayant un principe actif contenant la cordycépine d'une solution végétale de culture de mycélium à partir de vers, qui a été traditionnellement mise de côté. En outre, l'extrait de solution végétale de culture de mycélium à partir de vers est remarquablement efficace pour le traitement de maladies vasculaires, et peut ainsi également être utilisé en tant que composition pharmaceutique pour des maladies vasculaires telles que l'artériosclérose, l'hypertension, l'angine de poitrine, l'infarctus du myocarde, les maladies cardiaques ischémiques, l'insuffisance cardiaque, les complications pouvant résulter d'une angioplastie transluminale percutanée, l'infarctus cérébral, l'hémorragie cérébrale, l'apoplexie cérébrale et analogues.
PCT/KR2013/005413 2012-06-28 2013-06-19 Procédé de préparation d'un extrait de solution végétale de culture de mycélium à partir de vers contenant de la cordycépine en concentration élevée, et composition pour le traitement ou la prévention des maladies vasculaires, contenant ledit extrait WO2014003356A1 (fr)

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KR10-2012-0069718 2012-06-28

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