WO2009102083A1 - Nouveaux dérivés de clitocybine, leur procédé de préparation et composition contenant l'extrait de clitocybe aurantiaca kctc 11143bp ou les nouveaux dérivés de clitocybine comme ingrédient actif pour la prévention du vieillissement - Google Patents

Nouveaux dérivés de clitocybine, leur procédé de préparation et composition contenant l'extrait de clitocybe aurantiaca kctc 11143bp ou les nouveaux dérivés de clitocybine comme ingrédient actif pour la prévention du vieillissement Download PDF

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WO2009102083A1
WO2009102083A1 PCT/KR2008/000804 KR2008000804W WO2009102083A1 WO 2009102083 A1 WO2009102083 A1 WO 2009102083A1 KR 2008000804 W KR2008000804 W KR 2008000804W WO 2009102083 A1 WO2009102083 A1 WO 2009102083A1
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Prior art keywords
extract
clitocybin
ethyl acetate
clitocybe
aurantiaca
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PCT/KR2008/000804
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English (en)
Inventor
Ick-Dong Yoo
In-Ja Ryoo
Young-Hee Kim
Soo-Jin Choo
Sangku Lee
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Korea Research Institute Of Bioscience And Biotechnology
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Priority to PCT/KR2008/000804 priority Critical patent/WO2009102083A1/fr
Publication of WO2009102083A1 publication Critical patent/WO2009102083A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4906—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom
    • A61K8/4913—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having five membered rings, e.g. pyrrolidone carboxylic acid
    • A61K8/492—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with one nitrogen as the only hetero atom having five membered rings, e.g. pyrrolidone carboxylic acid having condensed rings, e.g. indol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/46—Iso-indoles; Hydrogenated iso-indoles with an oxygen atom in position 1

Definitions

  • the present invention relates to novel clitocybin derivatives, preparation methods thereof, and anti-aging compositions comprising Clitocybe aurantiaca extracts or novel clitocybin derivatives as an active ingredients .
  • Reactive oxygen species including oxygen ions, free radicals, and peroxides, such as superoxide (0 2 ⁇ ) , hydroxyl radical (-0H) and hydrogen peroxide (H 2 O 2 ) , which are generated by various oxidative reactions, chemicals, foods, diseases, radiation, and other factors, oxidize the lipids in cell membranes, which are abundant in unsaturated fatty acid, with the concomitant production of lipid peroxides .
  • Lipid peroxides accumulated in cell membranes lower the fluidity and functionality of cell membrane, suppressing overall cellular functionality and causing changes in cell structure. Thus, tissues are regionally injured, resulting in various diseases.
  • reactive oxygen and free radicals modify or destroy cellular constituents, such as nucleic acids, carbohydrates, etc., causing various diseases including cancer, liver diseases such as alcoholic hepatitis, cerebral apoplexy, myocardial infarction, diabetic vascular diseases, hyperlipidemia, acute inflammation, and rheumatoid diseases [Arthritis. Res. Ther. 6(6): 265-78 (2004), J. Neuropathol. Exp. Neurol. 65(7): 631-41 (2006), Trends. MoI. Med. 12(11): 521-8 (2006), J ⁇ t. J. Biochem. Cell. Biol. 39(1); 44-84 (2007)].
  • liver diseases such as alcoholic hepatitis, cerebral apoplexy, myocardial infarction, diabetic vascular diseases, hyperlipidemia, acute inflammation, and rheumatoid diseases
  • reactive oxygen is a cause of food rancidity because it is likely to react with unsaturated fatty acids in food, thus compromising the safety and quality of food.
  • problems caused by reactive oxygen species are found in various fields, including oxidative stress on animals and plants and a decrease in yield of microorganism fermentation .
  • non-toxic materials with potent antioxidative activity are very usefully applied to the development of therapeutics or as anti-aging cosmetic additives and food additives .
  • BHT butylated hydroxy toluene
  • BHA butylated hydroxy anisol
  • carbazole-type anti-oxidant materials Typical of the carbazole-type anti-oxidant materials is carquinostatin, which is an acetone extract from Streptomyces exfoliates.
  • carquinostatin an acetone extract from Streptomyces exfoliates.
  • antiostatin, carazostatin, neocarozostatin, and carbazomycin have been reported.
  • phenazine-type anti-oxidant materials have been reported, which include benthocyanin, benthophoenin and phenazoviridin.
  • thiazolestatin?, naphterpin, and pyrrolostatin have been reported as natural thiazole-type anti-oxidant agents.
  • KRIBB Korean Research Institute of Bioscience and biotechnology
  • KRIBB has studied anti-oxidant materials from Korean wild mushrooms, with the achievement of excellent research results, including "New Indole Derivatives with Free Radical Scavenging Activity from Agrocybe cylindracea” (J. of Natural Products, 60 : 721- 723, 1997), "Novel antioxidative compounds from Daldinia concentrica and antioxidative composition comprising the same as an active material" (Korean Patent No.
  • an antioxidant agent comprising an extract from Clitocybe aurantiaca (KCTC 11143BP) or a novel clitocybin derivative as an active ingredient.
  • a UV screening agent comprising an extract from Clitocybe aurantiaca (KCTC 11143BP) or a novel clitocybin derivative as an active ingredient.
  • a pharmaceutical composition for the prophylaxis and treatment of diseases attributable to the accumulation of excess active oxygen comprising an extract from Clitocybe aurantiaca (KCTC 11143BP) or a novel clitocybin derivative as an active ingredient.
  • a pharmaceutical composition for the prevention of aging comprising an extract from Clitocybe aurantiaca (KCTC 11143BP) or a novel clitocybin derivative as an active ingredient .
  • a cosmetic composition for the prevention of aging comprising an extract from Clitocybe aurantiaca (KCTC 11143BP) or a novel clitocybin derivative as an active ingredient .
  • a method for the treatment of diseases attributable to the accumulation of active oxygen comprising the administration of the novel clitocybin derivative to patients suffering therefrom.
  • a method for the treatment of diseases attributable to the accumulation of active oxygen comprising the administration of an extract from Clitocybe aurantiaca in water, acetone or a mixture thereof to a patient in need thereof.
  • a method for the treatment of diseases attributable to the accumulation of active oxygen comprising the administration of an ethyl acetate fraction from the extract from Clitocybe aurantiaca in water, acetone or a mixture thereof to a patient in need thereof.
  • an ethyl acetate extract from Clitocybe aurantiaca in the prevention and treatment of a disease attributable to accumulation of excess active oxygen, the ethyl acetate extract being prepared by fractioning an extract from Clitocybe aurantiaca in water, acetone or a mixture thereof into ethyl acetate.
  • the extract from Clitocybe aurantiaca (KCTC 11143BP) or the novel clitocybin derivative in accordance with the present invention can be useful in the prophylaxis and treatment of diseases attributable to the accumulation of excess active oxygen and can be applied to the preparation of pharmaceutical or cosmetic compositions for the prevention of aging.
  • FIG. 1 is a 13 C NMR spectrum of the novel Clitocybin A compound according to the present invention.
  • FIG. 2 is a 1 H NMR spectrum of the novel Clitocybin A compound according to the present invention.
  • FIG. 3 is an HMBC spectrum of the novel Clitocybin A compound according to the present invention.
  • FIG. 4 is a 13 C NMR spectrum of the novel Clitocybin B compound according to the present invention.
  • FIG. 5 is a 1 H NMR spectrum of the novel Clitocybin B compound according to the present invention
  • FIG. 6 is a 13 C NMR spectrum of the novel Clitocybin C compound according to the present invention.
  • FIG. 7 is a 1 H NMR spectrum of the novel Clitocybin C compound according to the present invention.
  • FIG. 8 provides fluorescence microphotographs displaying images of lymphocytes without or with damaged DNA after treatment with PBS only (a, control), novel clitocybin A (b) ; novel clitocybin B (c) ; novel clitocybin C (d) ; hydrogen peroxide (e) ; novel clitocybin A + hydrogen peroxide (f) ; novel clitocybin B + hydrogen peroxide (g) and novel clitocybin C + hydrogen peroxide (h) ; and
  • FIG. 9 is a graph in which erythema reduction is plotted for animals treated with novel clitocybin A (a) , novel clitocybin B (b) , novel clitocybin C (c) and control (d).
  • the present invention provides a novel clitocybin derivative represented by the following Chemical Formula 1.
  • R 1 and R 2 are independently OCH 3 or OH
  • the clitocybin derivative represented by Chemical Formula 1 can be further illustrated by the following Chemical Formulas 11 ⁇ 13.
  • the compound of Chemical Formula 11 according to the present invention is prepared as a pale brown powder having the molecular formula C 14 H 11 O 4 N and named clitocybin A.
  • the compound of Chemical Formula 12 according to the present invention is prepared as a pale yellow powder having the molecular formula C 15 H 13 O 4 N and named clitocybin B.
  • the compound of Chemical Formula 13 according to the present invention is prepared as a white powder having the molecular formula C 16 H 15 O 4 N and named clitocybin C.
  • the compound of Chemical Formula 1 according to the present invention may be obtained preferably from Clitocybe aurantiaca using extraction and purification processes and may be synthesized chemically.
  • the derivative represented by Chemical Formula 1 in accordance with the present invention may be in the form of pharmaceutically acceptable salts.
  • acid addition salts formed with pharmaceutically acceptable free acids.
  • Useful as the free acids are inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, and phosphorous acid, and non-toxic organic acids such as aliphatic mono- and dicarboxylate, phenyl-substituted alkanoate, hydroxy alkanoate and alkanedioate, aromatic acids, aliphatic and aromatic sulfonic acids.
  • non-toxic bases examples include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprilate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne- 1,4-dioate, hexyne-1, ⁇ -dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluenesulfonate
  • the acid addition salts of the compound according to the present invention may be prepared using a conventional method, for example, by dissolving the compound of Chemical Formula 1 in excess acid in water and precipitating the resulting salt in a water-miscible organic solvent, e.g., methanol, ethanol, acetone or acetonitrile.
  • a water-miscible organic solvent e.g., methanol, ethanol, acetone or acetonitrile.
  • the compound of Chemical formula 1 may be heated along with the same amount of acid or alcohol in water, followed by evaporating the mixture and drying or filtering the precipitate to prepare acid addition salts thereof .
  • metal salts formed with bases may fall within the range of pharmaceutically acceptable salts of the compound of the present invention.
  • the metal salts useful in the present invention include alkali metal salts and alkaline earth metal salts.
  • the compound of the present invention may be dissolved in excess alkali metal hydroxide or alkaline earth metal hydroxide in water, and, after the filtration of the solution to remove non-dissolved compound salts, the filtrate may be dried to afford the pharmaceutically acceptable salts of the compound of the present invention.
  • Suitable for use in pharmaceutics are sodium, potassium or calcium salts.
  • Corresponding silver salts may be obtained by reacting the alkali metal or alkaline earth metal salts with suitable silver salt (e.g., silver nitrate).
  • the present invention provides a method for the preparation of the novel clitocybin derivative of the present invention.
  • the novel clitocybin derivative according to the present invention may be prepared by extraction and purification from Clitocybe aurantiaca or through organic synthesis .
  • the method for the preparation of the novel clitocybin derivative comprises :
  • Step 2 Forming an extract from the culture of Step 1 with acetone and ethyl acetate (Step 2); Separating an active fraction from the extract of Step 2 through silica gel column chromatography eluting with a gradient of a mixture of chloroform and methanol (Step 3) ; and Purifying the active fraction of Step 3 through Sephadex LH-20 column chromatography eluting with 100% methanol (Step 4) .
  • Step 1 Clitocybe aurantiaca (KCTC 11143BP) is cultured in a medium.
  • the medium for culturing Clitocybe aurantiaca is preferably a yeast extract broth (YPS) .
  • Step 2 is to extract active ingredients from the culture of Step 1 with acetone and ethyl acetate.
  • the culture obtained in Step 1 is treated with acetone, followed by re-extraction with ethyl acetone.
  • a typical extraction process may be applied to this.
  • the ethyl acetate layer after the re-extraction is used for the next step.
  • Step 2 the extract of Step 2 is subjected to silica gel column chromatography eluting with a gradient of a mixture of chloroform and methanol in Step 4.
  • the mixture of chloroform and methanol had a concentration gradient from 50:1 to 1:1, and elution at a rate of 10 ⁇ 15 ml/min for 150 min affords an active fraction.
  • Step 3 the active fraction of Step 3 is purified by Sephadex LH-20 column chromatography eluting with 100% ethanol to afford the clitocybin derivative of Chemical Formula 1 in Step 4.
  • the method for the preparation of the novel clitocybin derivative comprises reacting the compound of Chemical Formula 2 with the compound of Chemical Formula 3 in an organic solvent to produce the compound of Chemical Formula 13 (Step a) .
  • the method for the preparation of the novel clitocybin derivative as illustrated in the following Reaction Scheme 2, comprises:
  • Step a the compound of Chemical Formula 2 is reacted with the compound of Chemical Formula 3 in an organic solvent to produce the compound of Chemical Formula 13.
  • Suitable for use as an organic solvent in this reaction is methanol.
  • the compounds of Chemical Formulas 2 and 3 are preferably mixed at an equivalent ratio of 1:1.
  • step b the compound of Chemical Formula 13 is reacted with hydrobromic acid and acetic acid, followed by extraction with ethyl acetate to afford the compound of Chemical Formula 1 (Chemical Formulas 11 and 12) .
  • the compound of Chemical Formula 13, obtained in Step a is reacted with hydrobromic aid and acetic acid with heating reflux for 15 ⁇ 20 hrs.
  • hydrobromic aid and acetic acid with heating reflux for 15 ⁇ 20 hrs.
  • To this reaction is added ice water, followed by extraction with ethyl acetate.
  • the organic layer thus formed is washed with water, dried over anhydrous magnesium sulfate, and concentrated in a vacuum. Purification through silica gel column chromatography eluting with a mixture of 10:1 dichloromethane : methanol gives the clitocybin derivatives of Chemical Formulas 11 and 12 at a yield of 80% or higher.
  • the present invention provides an antioxidative agent comprising an extract from Clitocybe aurantiaca or the novel clitocybin derivative as an active ingredient.
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivative in accordance with the present invention was found to have superior radical-scavenging activity over trolox, BHA, ferulic acid and catechin, as measured using superoxide, DPPH and ABTS + (FIG. 3) .
  • the increased oxidative DNA damage by hydrogen peroxide expressed by % tail DNA was found to be significantly inhibited by administering the novel clitocybin derivative of the present invention (FIG. 8 and Table 4 ) .
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivative in accordance with the present invention can be useful as an antioxidant agent .
  • the present invention provides a sun block agent comprising the extract from Clitocybe aurantiaca or the novel clitocybin derivative as an active ingredient.
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivative according to the present invention was found to have a sun block effect comparable to that of a control, as determined by measuring erythema (FIG. 9) . These results indicate that the extract from Clitocybe aurantiaca or the novel clitocybin derivative can be used as a sun block agent.
  • the present invention provides a pharmaceutical composition for the protection and treatment of diseases attributable to the accumulation of active oxygen, comprising the extract from Clitocybe aurantiaca or the novel clitocybin derivative as an active ingredient.
  • the present invention provides an anti-aging pharmaceutical composition
  • an anti-aging pharmaceutical composition comprising the extract from Clitocybe aurantiaca or the novel clitocybin derivative as an active ingredient .
  • the present invention provides a method for the treatment of diseases attributable to the accumulation of active oxygen, comprising administering the novel clitocybin derivative to the patient.
  • the present invention provides a method for the treatment of diseases attributable to the accumulation of active oxygen, comprising administering the patient with an extract from Clitocybe aurantiaca in water, acetone, or a mixture thereof.
  • the present invention provides a method for the treatment of diseases attributable to the accumulation of active oxygen, comprising administering the patient with an ethyl acetate fraction of an extract from Clitocybe aurantiaca in water, acetone, or a mixture thereof.
  • the present invention provides the use of the novel clitocybin derivative in the prevention and treatment of diseases attributable to the accumulation of excess active oxygen.
  • the present invention provides the use of an extract from Clitocybe aurantiaca in water, acetone or a mixture thereof in the prevention and treatment of diseases attributable to the accumulation of excess active oxygen.
  • the present invention provides the use of an acetone fraction of an extract from Clitocybe aurantiaca in water, acetone or a mixture thereof in the prevention and treatment of diseases attributable to the accumulation of excess active oxygen.
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivative in accordance with the present invention showed higher activity than did controls, such as trolox, BHA, ferulic acid and catechin (Table 3) .
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivative according to the present invention was found to have an effect of protecting DNA from active oxygen, as the increased oxidative DNA damage by hydrogen peroxide, expressed by % tail DNA, was significantly inhibited by administering the extract from Clitocybe aurantiaca or the novel clitocybin derivative (FIG.
  • the pharmaceutical composition of the present invention is useful in the prevention and treatment of diseases attributable to the accumulation of excess active oxygen, including liver diseases such as alcoholic hepatitis, vascular diseases such as cerebral apoplexy, myocardial infarction, diabetic vascular diseases, hyperlipidemia, acute inflammation, rheumatoid diseases and cancer [Arthritis. Res. Ther. 6(6): 265-78 (2004), J. Neuropathol. Exp. Neurol. 65(7): 631-41 (2006), Trends. MoI. Med. 12(11): 521-8 (2006), Int. J. Biochem. Cell. Biol. 39(1); 44-84 (2007)] as well as in the prevention of aging .
  • liver diseases such as alcoholic hepatitis
  • vascular diseases such as cerebral apoplexy, myocardial infarction, diabetic vascular diseases, hyperlipidemia, acute inflammation, rheumatoid diseases and cancer
  • composition of the present invention may further comprise one or more ingredients identical or similar in function to that of the extract from Clltocybe aurantlaca or the novel clitocybin derivative.
  • the composition in accordance with the present invention may be used in oral or non-oral forms. It is usually formulated in combination with a diluent or an excipient, such as a filler, a thickening agent, a binder, a wetting agent, a disintegrant, a surfactant, etc.
  • a diluent or an excipient such as a filler, a thickening agent, a binder, a wetting agent, a disintegrant, a surfactant, etc.
  • Solid agents intended for oral administration of the composition of the present invention may be in the form of tablets, pills, powders, granules, capsules, and the like. In these solid agents, the extract from Clltocybe aurantlaca or the novel clitocybin derivative is formulated in combination with at least one excipient, such as starch, calcium carbonate, gelatine, etc.
  • Liquid agents intended for oral administration include suspensions, internal use solutions, emulsion, syrups, and the like.
  • various excipients such as wetting agents, sweetening agents, aromatics, preservatives, and the like, may be contained in the liquid agents for the oral administration of the extract or the novel derivative of the present invention.
  • non-oral dosage forms of the extract or the novel derivative of the present invention include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, and freeze-dried agents.
  • Non-aqueous solutions and suspensions made from propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.
  • the effective dosage of the extract or the derivative in accordance with the present invention depends on various factors, including the patient's weight, age, gender, state of health, diet, the time of administration, route of administration, excretion rate, severity of diseases, etc.
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivative in accordance with the present invention may be administered in a single dose, and preferably in multiple doses per day at a daily dose ranging from 0.1 to 100 mg/day, and preferably from 5 to 50 mg/kg.
  • the composition according to the present invention may be used alone or in combination with other therapies, including surgery, hormonal therapy, chemical therapy and/or biological reaction regulators .
  • the present invention provides an anti- aging cosmetic composition comprising the extract from Clitocybe aurantiaca or the novel clitocybin derivative as an active ingredient.
  • the form of the cosmetic produced as the anti-aging cosmetic composition according to the present invention includes lotions, ointments, gel, creams, patches and sprays, but is not limited thereto.
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivative in accordance with the present invention may be contained in an amount from 1 to 10 parts by weight, and preferably in an amount from 2 to 10 parts by weight, based on 100 parts by weight of the preparation.
  • the cosmetic preparation for external application to the skin may comprise lipids, organic solvents, dissolving agents, thickening agents, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, aromatics, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, UV blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators, liposomes, and/or other general supplements used in the skin science field. These ingredients may be used in amounts that are generally- accepted in the skin science field.
  • Clitocybe aurantiaca (KCTC 11143BP) was inoculated into 100 ml of yeast-malt extract (YPS) broth in a 500 ml Erlenmeyer flask and cultured at 27 0 C for 5 days in an incubator with rotational agitation at 140 rpm.
  • YPS yeast-malt extract
  • Into a 5 L jar fermentor (Korea Fermentor Co. Ltd) containing 3 liters of sterilized yeast-malt extract (YPS) broth were transferred 100 ml of the culture. After incubation for 7 days under the same condition, the culture was mixed with 3 liters of 70% acetone and then subjected to reflux condensation at 100 0 C for 2 hrs .
  • the resulting extract was filtered through a filter paper (Advantec No. 2) and the filtrate was concentrated in a vacuum using an evaporator (EYELA, N-1000, Japan) to afford an acetone extract from Clitocybe aurantiaca (48.3 g) .
  • Example 1-1 The same procedure as in Example 1-1 was repeated, with the exception that water was used instead of acetone, to afford a water extract from Clitocybe aurantiaca (42.5 g ) -
  • the acetone extract (48.3 g) from Clitocybe aurantiaca prepared in Example 1-1 was dispersed in water and added with ethyl acetate.
  • the ethyl acetate layer was concentrated in a vacuum to give an ethyl acetate fraction of Clitocybe aurantiaca (15.4 g) .
  • Example 2 The fraction (15 g) obtained in Example 2 was subjected to silica gel column chromatography eluting with a gradient of a mixture of chloroform and methanol (50:1 ⁇ 1:1) to give an active fraction (8.3 g) .
  • Example 3 The active fraction obtained in Example 3 was purified using Sephadex LH-20 column chromatography eluting with 100 % methanol to produce a pure compound as a light brown powder (6.5 mg) . This was identified to be pure as analyzed by reverse-phase column (ODS) chromatography eluting with 35% methanol . The compound thus obtained was named clitocybin A.
  • Clitocybin A As seen in Table 1, Clitocybin A according to the present invention was produced as a pale brownish powder and was found to have (M+H + ) at m/z 258.07571, which is almost the same as the calculated value m/z 258.07608, as analyzed by high resolution electrospray ionization mass spectrometry (HR-ESI-MS) . Therefore, the molecular formula was determined to be Ci 4 HnO 4 N. As for UV spectra, maximum absorption was read at 211 nm and 286 ran while a band corresponding to a hydroxy group was observed at 3399 cm "1 on the IR spectra. Taking these results into consideration, Clitocybin A was identified as a novel iso-indolone-based compound.
  • Clitocybin B according to the present invention was produced as a pale yellowish powder and was found to have (M+H + ) at m/z 272.0923, which is almost the same as the calculated value m/z 272.0923, as analyzed by high resolution electrospray ionization mass spectrometry (HR- ESI-MS) . Therefore, the compound was determined to have a molecular formula of Ci 5 Hi 3 O 4 N. As for UV spectra, maximum absorption was read at 211 nm and 286 nm while a band corresponding to a hydroxy group was observed at 3399 cm x on the IR spectra. Taking these results into consideration, Clitocybin B was identified as a novel iso-indolone-based compound.
  • Clitocybin C was produced as a pale yellowish powder and was found to have (M+H + ) at m/z 286.1079, which is almost the same as the calculated value m/z 286.1079, as analyzed by high resolution electrospray ionization mass spectrometry (HR- ESI-MS) . Therefore, the compound was determined to have a molecular formula of C 16 H 15 O 4 N. As for UV spectra, maximum absorption was read at 211 run and 286 nm while a band corresponding to a hydroxy group was observed at 3399 cm "1 on the IR spectra. Taking these results into consideration, Clitocybin B was identified as a novel iso-indolone-based compound.
  • Clitocybin A - C were NMR analyzed for 1 H and 13 C NMR, HMQC ( (lH-detected Heteronuclear Multiple-Quantum Coherence) and HMBC (Heteronuclear Multiple-Bond Coherence) spectra using an NMR apparatus (Burker AMX 300, Burker, USA) . These are shown in FIGS . 1 - 7 and Table 2.
  • FIG. 1 for 13 C NMR spectrum
  • FIG. 2 for 1 H NMR spectrum
  • FIG. 3 for an HMBC spectrum.
  • Xanthine oxidase catalyzes the oxidation of xanthine into uric acid and superoxide. Superoxide reduces NBT, with the concomitant formation of blue formazan, which allows ELISA to measure absorbance at 560 nm.
  • DMSO only was used as a negative control, while ferulic acid or catechin was used as a positive control .
  • Radical scavenging activity was calculated on the basis of the difference in absorbance between the test group and the control group in accordance with the following Mathematical Formula 2.
  • ⁇ A SaiI pie a change of the absorbance measured in well containing sample compound.
  • the extracts from Clitocybe aurantiaca and the novel clitocybin derivatives in accordance with the present invention were measured to have an EC50 ranging from 6.4510.32 to 10.26+0.23 ⁇ M against an ABTS + radical, and from 3.6110.38 to 15.42+3.22 ⁇ M against a superoxide radical, indicating far superiority in radical scavenging activity over the positive control group (trolox, BHA, ferulic acid, catechin) , which are known as excellent radical scavengers.
  • the compounds of the present invention showed radical scavenging activity comparable with that of BHA.
  • the excellent radical scavenging activity enables the extracts from Clitocybe aurantiaca or the novel clitocybin derivatives according to the present invention to be effectively applied not only to the prevention and treatment of diseases, attributable to the accumulation of excess active oxygen, but also to the prevention of aging.
  • lymphocytes isolated from the blood sample from healthy adult women were incubated in media with PBS alone as a negative control, or with 100 ⁇ l of the extract from Clitocybe aurantiaca, the ethyl acetate fraction thereof, the novel clitocybin derivative, or H 2 O 2 , or with a combination of the extract or the novel compound according to the present invention and H 2 O 2 .
  • the amount of the tail DNA produced was measured to quantitatively analyze cellular DNA damage while the cells were subjected to image analysis using a fluorescence microscope (Leica DMLB, Germany) . The bigger the tail DNA was, the more serious the DNA damage was .
  • FIG. 8 and Table 4 In the fluorescence microphotographs of FIG. 8, the lymphocytes which were treated with PBS only, as a negative control, and with hydrogen peroxide only, as a positive control, are shown in panels (a) and (e) , respectively.
  • the lymphocytes which were incubated with the novel clitocybin compounds A, B and C alone are shown in panels (b) , (c) and (d) , respectively.
  • the lymphocytes were incubated with the novel clitocybin compounds A, B and C in accordance with the present invention after exposure to hydrogen peroxide, or with the novel compounds and hydrogen peroxide simultaneously in panels (f) , (g) and (h) , respectively.
  • Fluorescence microscopy displays lymphocytes with damaged DNA, evidenced by a comet upon exposure to H 2 O 2 (e) , and lymphocytes with a small number of tail DNA, visible as significantly shortened comets, upon treatment with the clitocybin derivative according to the present invention (f ⁇ h) .
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivatives in accordance with the present invention can reduce the oxidative DNA damage induced by potent reactive oxygen species, such as hydrogen peroxide, which leads to aging, by about 40%, and thus can be used as an active ingredient of an anti-aging cosmetic composition.
  • potent reactive oxygen species such as hydrogen peroxide
  • Erythema Reduction (AU) A °°"*°' ⁇ A ⁇ control wherein AU is arbitrary unit, and A is a chromameter value
  • FIG. 9 erythema reduction obtained after treatment with the extract from Clitocybe aurantiaca or the novel clitocybin derivatives in accordance with the present invention (a ⁇ c) and with the control, known as an antioxidant, are plotted.
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivatives in accordance with the present invention have erythema reduction effects comparable with those of the control, which is known to have excellent erythema reduction effects. Also, histochemical staining analysis with H&E showed no significant differences in erythema reduction between the control and the extract from Clitocybe aurantiaca or the novel clitocybin derivatives in accordance with the present invention (not shown) .
  • the extract from Clitocybe aurantiaca or the novel clitocybin derivatives in accordance with the present invention are therefore useful as an ingredient for functional cosmetic compositions .
  • novel clitocybin derivatives according to the present invention were assayed for acute toxicity as follows .
  • an autopsy was carried out to check for abnormalities in the abdominal and thoracic organs with the naked eye.
  • novel clitocybin derivative in accordance with the present invention was formulated in combination with auxiliary agents, such as excipients, binders, lubricants, disintegrants, diluents, etc., into pharmaceutical preparations as follows.
  • a tablet comprising the novel clitocybin derivative or a pharmaceutically acceptable salt thereof in accordance with the present invention as an active ingredient was prepared as follows .
  • the compound according to the present invention was sieved, mixed with lactose, starch and pre-gelatinized corn starch and granulized with a suitable volume of distilled water. After being dried, the granules thus obtained were mixed with magnesium stearate and pressurized into a tablet.
  • the tablet comprised the following ingredients.
  • Novel Clitocybin derivative 5.0 mg Lactose BP 150.0 mg
  • a capsule comprising the novel clitocybin derivative or a pharmaceutically acceptable salt thereof in accordance with the present invention as an active ingredient was prepared as follows.
  • the compound of the present invention was mixed with a predetermined amount of excipients and magnesium stearate, and the resulting mixture was loaded into a gelatin capsule to afford a capsule medicine.
  • the capsule comprises the following ingredients.
  • Novel Clitocybin derivative 5.0 mg Starch 1500 100.0 mg
  • An injection comprising the novel clitocybin derivative or a pharmaceutically acceptable salt thereof in accordance with the present invention as an active ingredient was prepared as follows .
  • novel clitocybin derivative was dissolved in a suitable volume of injectable sodium chloride BP, and was adjusted to a pH of 3.5 using dilute hydrochloric acid BP.
  • Injectable sodium chloride BP was further added to achieve a desired volume, and the solution was sufficiently mixed.
  • the mixture solution was then put into a 5-ml type I ampule made of transparent glass.
  • the glass was melted to seal the ampule, which was subsequently autoclaved at 120 0 C for 15 min, thereby giving an injectable solution.
  • Novel Clitocybin derivative 100 ⁇ g/ml Dilute Hydrochloric acid BP to pH 3.5 Injectable NaCl BP Up to 1 ml FORMULATION EXAMPLE 2: Preparation of External Application to the Skin

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Abstract

L'invention concerne de nouveaux dérivés de clitocybine et leurs procédés de préparation. De même, l'invention concerne une composition pharmaceutique ou cosmétique anti-vieillissement comprenant les dérivés de clitocybine comme ingrédient actif. L'extrait de Clitocybe aurantiaca (KCTC 11143BP) ou les nouveaux dérivés de clitocybine ont une excellente capacité à désactiver les radicaux d'oxygène, à inhiber les dommages liés à l'oxydation de l'ADN et à réduire l'érythème provoqué par les UV, de sorte qu'ils puissent être utiles dans la prophylaxie et le traitement de maladies imputables à l'accumulation d'oxygène actif en excès et puissent être appliqués à la préparation de compositions pharmaceutiques ou cosmétiques pour la prévention du vieillissement.
PCT/KR2008/000804 2008-02-12 2008-02-12 Nouveaux dérivés de clitocybine, leur procédé de préparation et composition contenant l'extrait de clitocybe aurantiaca kctc 11143bp ou les nouveaux dérivés de clitocybine comme ingrédient actif pour la prévention du vieillissement WO2009102083A1 (fr)

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PCT/KR2008/000804 WO2009102083A1 (fr) 2008-02-12 2008-02-12 Nouveaux dérivés de clitocybine, leur procédé de préparation et composition contenant l'extrait de clitocybe aurantiaca kctc 11143bp ou les nouveaux dérivés de clitocybine comme ingrédient actif pour la prévention du vieillissement

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PCT/KR2008/000804 WO2009102083A1 (fr) 2008-02-12 2008-02-12 Nouveaux dérivés de clitocybine, leur procédé de préparation et composition contenant l'extrait de clitocybe aurantiaca kctc 11143bp ou les nouveaux dérivés de clitocybine comme ingrédient actif pour la prévention du vieillissement

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KR101376246B1 (ko) 2012-12-18 2014-03-26 대한민국 노루궁뎅이 버섯 물질을 포함하는 항암용 약학적 조성물
KR101398026B1 (ko) * 2012-05-14 2014-05-27 한국생명공학연구원 클라이토싸이빈 유도체를 함유하는 혈관평활근세포 이상 증식성 질환의 예방 또는 치료용 조성물
EP2821404A4 (fr) * 2012-03-02 2015-11-04 Tms Co Ltd Dérivé de chromane

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2821404A4 (fr) * 2012-03-02 2015-11-04 Tms Co Ltd Dérivé de chromane
KR101398026B1 (ko) * 2012-05-14 2014-05-27 한국생명공학연구원 클라이토싸이빈 유도체를 함유하는 혈관평활근세포 이상 증식성 질환의 예방 또는 치료용 조성물
KR101376246B1 (ko) 2012-12-18 2014-03-26 대한민국 노루궁뎅이 버섯 물질을 포함하는 항암용 약학적 조성물
WO2014098307A1 (fr) * 2012-12-18 2014-06-26 대한민국(농촌진흥청장) Composition pharmaceutique anticancéreuse

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