KR101343367B1 - Compositions comprising lichen-forming fungi or scytalone for preventing and treating diabetes mellitus - Google Patents

Abstract

The present invention is an Anzia genus, Caloplaca genus, Cetrelia genus, Cladia genus, Evernia genus, Myelochroa genus, Nepro heavily's (Nephromopsis), a La Marina (Ramalina) in jaw gunnera Liao (Tuckneraria) in and glass topa Melia (Xanthoparmelia) extract, the extract is extracted from any one or more of lichens forming mold, selected from the group consisting of in lichens forming mold The present invention relates to a pharmaceutical composition for preventing and treating type 2 diabetes, comprising any one or more selected from the group consisting of a fraction, a scytalone compound, and a pharmaceutically acceptable salt of the citalon compound. The pharmaceutical composition of the present invention is derived from a natural lichen-forming fungus, which is harmless to the human body, has no side effects and is less concerned with resistance, and has an alpha glucosidase inhibitory activity to inhibit sugar absorption, thereby preventing and preventing type 2 diabetes. It can be usefully used for treatment.

Description

Compositions comprising lichen-forming fungi or scytalone for preventing and treating diabetes mellitus}

The present invention relates to a composition for preventing and treating type 2 diabetes, specifically an extract extracted from a lichen-forming fungus, a fraction of the extract, a scytalone compound, and a pharmaceutically acceptable compound of the citalone compound. It relates to a composition for preventing and treating type 2 diabetes comprising any one or more selected from the group consisting of salts as an active ingredient.

Diabetes is a disease that discharges sugar in the urine due to insufficient insulin secretion or normal functioning, resulting in a high blood glucose concentration. If the disease persists, diabetic neuropathy, retinopathy, etc. , Arteriosclerosis, stroke, heart disease, and other diabetes complications.

Diabetes can be classified into type 1 diabetes, which is an insulin-dependent diabetes that does not produce insulin at all, and type 2 diabetes, which is a relatively insufficient insulin-dependent diabetes that produces insulin, depending on whether or not insulin is produced. More than 90% of patients are known to have type 2 diabetes.

Type 1 diabetes occurs because beta cells of the pancreas are destroyed due to congenital or viral invasion or serious damage to the pancreas, and there is no or extremely little insulin produced in the body. It is mainly called childhood diabetes because it occurs acutely in childhood, and it is treated by controlling blood sugar by administering insulin.

Type 2 diabetes mainly occurs in the adult population, and is caused by insulin resistance, which is manifested by impaired insulin secretion to the underlying state and stimulation, increased production of endogenous glucose in the liver, and decreased sugar utilization in peripheral tissues. In addition to genetic factors, there are high-calorie and high-fat diets, lack of exercise, stress, obesity, and taking drugs due to westernization of diet. It is treated by taking blood sugar-lowering drugs along with diet or exercise.

There are three main types of hypoglycemic agents used in the treatment of type 2 diabetes. One is to stimulate the beta cells of the pancreas to promote the secretion of insulin, and there are sulfonylurea and meglitinide drugs, and the other is the insulin resistance improving agent, biguanide and There are thiazolidinedion-based drugs, and the other is an alpha glucosidase inhibitor, such as acarbose and miglitol, which suppress the absorption of sugar in the intestine by inhibiting the breakdown of sugar.

However, the above drugs are chemically manufactured, and the side effects of taking drugs such as disorders of kidney or liver function, heart disease, abdominal bloating, diarrhea, and the like, and patient tolerance are problematic, so they have fewer side effects from natural products and are effective. Has been striving to develop excellent diabetes treatments.

Accordingly, as a method of preventing or treating diabetes derived from natural products, Korean Patent Application Laid-Open No. 10-2007-0069566 discloses a composition for preventing and treating type 2 diabetes and diabetic complications containing thymosaponin A-III. , Korean Patent Registration No. 10-0963511 discloses an antidiabetic active extracellular polysaccharide derived from a culture medium of P. mushroom mycelium and a method for preparing the same, and Korean Patent Laid-Open No. 10-2010-0033064 uses pork potato powder or extract as an active ingredient. Disclosed is a composition for preventing or improving obesity or type 2 diabetes, including. And, Republic of Korea Patent Publication No. 10-2010-0088794 discloses a composition for the prevention and treatment of lipid metabolism disorders and diabetes complications caused by type 2 diabetes containing a pine oyster mushroom extract as an active ingredient. No. 0947547 discloses a composition for treating or preventing diabetes containing silk peptides as an active ingredient, and Korean Patent No. 10-0956278 discloses Yeoju, Cordyceps sinensis, Gigolpi, Sangbaekpi, Gwijeonwoo, Galgeun, Hwangjeong, Baekchul, Maekmundong, Disclosed is a composition for the treatment or prevention of diabetic complications, including cornus milk and ginseng.

However, there is not yet disclosed a method for preventing or treating diabetes using the lichen-forming mold as in the present invention.

Lichen-forming fungi (LFF) refers to a group of plants in which fungi and algae are combined to live, and Lichen-forming fungi (LFF) is a fungus belonging to lichenized fungi. It refers to fungi isolated from (thali).

Lichens have a form in which fungi surround algae, and fungi are hyphae that absorb and preserve water, and algae do photosynthesis to make nutrients. Due to this symbiotic relationship, lichens can survive in extreme environments where other plants cannot live. In addition, since it produces a variety of physiologically active substances that are difficult to find in other organisms, it has high industrial application value.

Accordingly, the present inventors have made efforts to develop a composition for preventing or treating diabetes that is harmless to the human body while having excellent effects using natural products different from the conventional ones. As a result, extracts and fractions extracted from lichen-forming fungi, and compounds isolated therefrom It was found to be very effective in the treatment of diabetes, and the present invention was completed.

Not is an object of the present invention Jia (Anzia), A knife Plaka (Caloplaca), A set trellis O (Cetrelia), A Cloud Dia (Cladia), An Eberhard California (Evernia) in, Mai Ilo Croix (Myelochroa) Extracts extracted from any one or more lichen-forming fungi selected from the group consisting of genus, Nephromopsis genus, Ramalina genus, Tuckneraria genus, and Xanthoparmelia genus lichen-forming fungi And/or to provide a pharmaceutical composition for preventing and treating type 2 diabetes comprising a fraction of the extract.

Another object of the present invention is to provide a pharmaceutical composition for preventing and treating type 2 diabetes, including a scytalone compound and/or a pharmaceutically acceptable salt of the citalone compound.

Another object of the present invention is to prevent and improve type 2 diabetes, including any one or more selected from the group consisting of an extract extracted from the lichen-forming mold, a fraction thereof, the citalone compound, and a food pharmaceutically acceptable salt thereof. It is to provide health foods for the sake of use.

In some embodiments, the invention is not regia (Anzia), A knife Plaka (Caloplaca), A set trellis O (Cetrelia), A Cloud Dia (Cladia), An Eberhard California (Evernia) in, Mai Ilo Croix (Myelochroa ), Nephromopsis , Ramalina , Tuckneraria , and Xanthoparmelia It relates to a pharmaceutical composition for preventing and treating type 2 diabetes comprising an extract and/or a fraction of the extract as an active ingredient.

The lichen-forming fungus of the present invention is a fungus belonging to the lichen-like associa bacterium, and refers to a fungus isolated from the lichen body of lichens. In particular, the lichen-forming mold of the present invention is Anzia genus, Caloplaca genus, Cetrelia genus, Cladia genus, Evernia genus, Myelocroa ( Myelochroa ) genus, Nephromopsis genus, Lamarina ( Ramalina ) genus, Tuckneraria genus and Xanthoparmelia genus refers to a lichen-forming fungus.

Examples of the lichen-forming mold are not necessarily limited thereto, but are as follows. The lichen-forming mold of the genus Anzia is, for example, Anzia opuntella (Anzia opuntiella ) lichen-forming fungi. The Lichen-forming fungus of the genus Caloplaca is, for example, Caloplaca crenuraria. crenularia ), Caloplaca Flavorubesense (Caloplaca flavorubescens ) lichen-forming fungi. The lichen-forming fungus of the genus Cetrelia is, for example, Cetrelia brown cyana (Cetrelia braunsiana ), Cetrelia japonica (Cetrelia japonica ), Cetrelia olivetorum (Cetrelia olivetorum ) lichen-forming fungi. The lichen-forming fungus of the genus Cladia is, for example, Cladia agregata (Cladia aggregata ) lichen-forming fungi. The Lichen-forming fungus of the genus Evernia is, for example, Evernia mesomorpha (Evernia mesomorpha ), Evernia prunastri ) lichen-forming fungi. And, Myelochroa (Myelochroa) Lichen-forming fungi, for example, Myelochroa aururenta (Myelochroa) aurulenta ), Myelochroa, Myelochroa entotheiochroa ), Myelochroa ribs (Myelochroa) galbina ), Myelochroa irrugans ), Myelochroa leucotyliza ) lichen-forming fungi. The lichen-forming fungus of the genus Nephromopsis is, for example, Nephromopsis unanensis. yunnanensis ), Nephromopsis pudocomplicata (Nephromopsis pseudocomplicata ) lichen-forming fungi. The lichen-forming fungus of the genus Ramalina is, for example, Lamarina conduplicans ( Ramalina conduplicans ), Lamarina Calicaris (Ramalina calicaris ), La Marina Pastigiata (Ramalina fastigiata ) lichen-forming fungi. The lichen-forming fungus of the genus Tuckneraria is, for example, Tuckneraria pudocomplicata (Tuckneraria pseudocomplicata ) lichen-forming fungi. The lichen-forming fungus of the genus Xanthoparmelia is, for example, Xanthoparmelia stenophila (Xanthoparmelia). stenophylla ), Xanthoparmelia pulla ) lichen-forming mold.

In the present invention, the extract of the lichen-forming fungus or a fraction of these extracts refers to a substance having α-glucosidase inhibitory activity obtained by extracting or fractionating the lichen-forming fungus with an organic solvent. In addition, in the present invention, the extract of the lichen-forming fungus or fractions of these extracts includes all of the extracts or fractions of these extracts as well as their purified products, the extracts, fractions, or concentrated or dried form of the purified product. The concentration and/or drying method includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, infrared drying, and the like.

The solvent for the lichen-forming mold extract is methanol, ethanol, propanol, isopropanol, butanol, pentanol, hexanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylform. Amide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, and water may be used alone or in combination of two or more, but is not limited thereto.

The solvent for the fraction fractionated from the lichen-forming mold extract is methanol, ethanol, propanol, isopropanol, butanol, pentanol, hexanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, and water may be used alone or in combination of two or more, but is not limited thereto.

The method for preparing the extract of the lichen-forming mold or the fraction of these extracts is not particularly limited, and for example, cold needle extraction, ultrasonic extraction, reflux cooling extraction, or the like may be used.

The purified product is a process of removing suspended solid particles from the extract or fraction, and the particles may be filtered using cotton, nylon, or the like, or an ultrafiltration method, a cryofiltration method, a centrifugal separation method, etc. may be used, but the present invention is not limited thereto. In addition, a separation process by various chromatography (chromatography according to size, charge, hydrophobicity or affinity) may be further included.

Since the extract of the lichen-forming fungus or fractions of these extracts has superior alpha-glucosidase inhibitory activity compared to the extracts of lichens themselves or fractions of these extracts, the extracts of lichen-forming fungi or these than the extracts of lichens themselves or fractions of these extracts It is preferred to use a fraction of the extract.

In one specific implementation, Cetrelia genus, Myelochroa genus, Nephromopsis genus, Tuckneraria genus and Xanthoparmelia genus extract of lichens and As a result of comparing the alpha glucosidase inhibitory activity using the lichen-forming fungus extract for the lichens, it was observed that the alpha glucosidase inhibitory activity of the lichen-forming fungus extract was superior to that of the lichen itself. Therefore, for preventing, improving, and treating diabetes, it is preferable in terms of effectiveness to use an extract of a lichen forming fungus rather than an extract of lichen itself.

Since the growth rate of the lichen-forming mold is slow, in order to industrially use the extract of the lichen-forming mold or the fraction of the extract, the lichen-forming mold extract or the alpha glucosida of the fraction of the lichen-forming mold is excellent while the growth rate of the lichen-forming mold is excellent. It is important to find optimal growth conditions that do not reduce the enzyme inhibitory activity.

Among the growth conditions of the lichen-forming fungi, the medium for cultivating lichen-forming fungi contains nutrients necessary for the growth of the lichen-forming fungus in the medium component, so the type is not limited as long as the lichen-forming fungus can be grown using this. Does not. In addition, the type of medium for culturing the lichen-forming fungus is preferably selected in consideration of the growth rate according to the type of the lichen-forming fungus and the alpha glucosidase inhibitory effect of the lichen-forming fungus extract or a fraction thereof.

The medium for culturing the lichen-forming fungus is, for example, PDA (potato extract agar), My (Malt-yeast extract) agar, MNT 2% (Malt-yeast extract + 　 2% mannitol) agar, MS (Murashige -Skoog) medium, S (Sabouraud) agar, MCM (Mushroom complete media) and GCM (Ganoderma complete media), etc., and both liquid or solid medium may be used, but are not necessarily limited to these.

In one specific implementation, as a result of examining the growth conditions according to the medium type of the lichen-forming fungi of Myelochroa galbina , MS (Murashige-Skoog) medium, GCM (Ganoderma complete media) and MNT 2% agar It was observed that the growth rate was excellent when cultured using MNT, and the alpha glucosidase inhibitory effect was also excellent when using MNT 2% agar. Therefore, when considering both the growth rate of myelochroa galbina lichen-forming fungus and the degree of alpha glucosidase inhibition, the lichen-forming fungus of Myelochroa galbina uses MNT 2% agar. It is desirable to culture.

Among the growing conditions of the lichen-forming fungus, the environment for culturing the lichen-forming fungus, for example, pH or culture temperature, considers the habitat environment of the lichen-forming fungus and the alpha glucosidase inhibitory effect of the lichen-forming fungus extract or a fraction thereof. It is desirable to select it. For example, in the case of lichen-forming molds collected in cold areas, it is desirable to cultivate them at a low temperature, as there is a possibility that the growth rate may be slowed when cultured at a high temperature. In the case of culturing in, the growth rate may be slow, so it is preferable to cultivate in an environment with a high pH.

In one specific embodiment, Mai Ilo Croix ribs or the result of examining the growth conditions according to the pH and culture temperature of lichens forming mold of (Myelochroa galbina), Mai Ilo Croix rib or (Myelochroa galbina) has a pH of 6 and a temperature of 15 to When cultured in an environment of 20° C., it was confirmed that although the growth rate of lichen-forming fungi was excellent, it did not inhibit the alpha glucosidase inhibitory activity of the lichen-forming fungus extract of Myelochroa galbina.

The genus Anzia , Caloplaca , Cetrelia , Cladia , Evernia , Myelochroa , cells because four pro heavily's (Nephromopsis) in La Marina (Ramalina) in jaw gunnera Ria (Tuckneraria) genus and cup topa Melia (Xanthoparmelia) in lichen forming fungi are used in life, such as a car drinking at a gathering countries Since they are not toxic, the extracts and fractions thereof extracted from them can be usefully used for diabetes, preferably type 2 diabetes, due to the inhibitory effect of alpha glucosidase.

The extract and/or fraction thereof of the lichen-forming fungus contained in the pharmaceutical composition of the present invention is contained in an amount of 0.0001 to 30% by weight, preferably 0.0001 to 10% by weight, based on the total weight of the pharmaceutical composition.

In another aspect, the present invention relates to a pharmaceutical composition for preventing and treating type 2 diabetes, comprising as an active ingredient a citalon (scytalone) compound and/or a pharmaceutically acceptable salt thereof.

Scytalone of the present invention is a compound having a structure as shown in Formula 1 below, and is an intermediate produced in the early stages of the melanin biosynthesis process, and is produced by reduction of 1,3,6,8-tetrahydroxynaphtalene. It is known to be.

The citalone compound of Formula 1 may be chemically synthesized and used, but preferably a lichen-forming mold, more preferably a lichen-forming mold in Myelochroa , and even more preferably a myelochroa galbina (Myelochroa). galbina ) can be used separately from extracts of lichen-forming fungi.

In one specific implementation, Myelochroa galbina (Myelochroa galbina ) Lichen-forming mold was extracted and fractionated with 75% ethanol and ethyl acetate, and then a substance having alpha glucosidase inhibitory activity was separated by chromatography, and mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) were used. As a result of observation, it was confirmed that it was a scytalone compound.

Pharmaceutically acceptable salts of the present invention include salts of acidic or basic groups that may be present in the compounds of the present invention unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include idobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate. , Acetate, succinate, citrate, tartrate, lactate, mantelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, It can be manufactured through.

In addition, as a pharmaceutically acceptable salt, an acid addition salt formed by a free acid is useful. The acid addition salt is prepared by a conventional method, for example, dissolving a compound in an excess acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and an acid or alcohol (eg glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be suction filtered.

At this time, organic and inorganic acids can be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, citric acid, and the like can be used as the organic acid. Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like.

In addition, a pharmaceutically acceptable metal salt can be made using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable for pharmaceutical use to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

Since the citalone compound and/or a pharmaceutically acceptable salt thereof has excellent alpha glucosidase inhibitory activity and inhibits the absorption of sugar in the body, it is useful for the prevention and treatment of diabetes, preferably type 2 diabetes, and has cytotoxicity. No, it can be used safely.

The citalone compound and/or a pharmaceutically acceptable salt thereof is included in the pharmaceutical composition in an amount of 0.0001 to 30% by weight, preferably 0.0001 to 10% by weight, based on the total weight of the pharmaceutical composition.

The pharmaceutical composition of the present invention may be formulated into a conventional formulation in the pharmaceutical field depending on the therapeutic purpose, for example, tablets, capsules, powders, granules, suspensions, emulsions, syrups, emulsions, warning agents, ointments, It can be formulated as a spray, oil, gel, alcohol, tincture, bath, liner, lotion, patch, pad, cream, and the like. In addition, it can be preferably used as an external preparation for skin for the purpose of topical administration applied directly to the affected area. In this case, an ointment, lotion, spray, gel, or the like is preferable. These external preparations for skin may also be included in a support base or matrix that can be applied directly to the treatment site, and examples of the support base include gauze or bandages.

When formulated as an ointment, the skin surface temperature, skin pH, transdermal water loss value, and total lipid value of the epidermis must be considered. Vaseline, liquid paraffin, paraffin, plastic base, silicone, pig fat (lard), vegetable oil, wax, oil-soluble substrates such as purified lanolin, water-soluble substrates, emulsion-based substrates, suspension substrates, etc. are formulated and formulated. These ointments include antioxidants (e.g., tocopherol, BHA, BHT, NDGA, etc.), preservatives (e.g., phenolic substances, chlorobutanol, benzyl alcohol, parabens, benzoic acid, etc.), moisturizing agents (e.g., glycerin, propylene clicone, sorbitol, etc.) ), solubility aids (eg, ethanol, propylene glycol), softening aids (eg, liquid paraffin, glycerin, propylene glycol, surfactants, etc.).

When formulated as a lotion, it can be prepared as a lotion, such as a solution type, a suspension type, or an emulsion type, and in the case of a lotion applied to the skin, it can be prepared to have a viscosity of, for example, 200 cps to 500 cps, and it feels soft when applied. It is preferable to prepare by blending a wetting agent such as glycerin and propylene glycol in order to give it.

When formulated as a spray, a propellant or the like may be blended with an additive so that the aqueous dispersion concentrate or wet powder is dispersed.

When formulated as a patch, an absorption accelerator may be used to increase the absorption of the compound through the skin.

The pharmaceutical composition of the present invention can be administered by various methods of administration such as oral, parenteral, topical administration, rectal administration, and intranasal administration. In the case of oral administration, for example, oral solids can be used with active substances and diluents (e.g. lactose, dextrose, sucrose, cellulose, corn starch, potato starch, etc.), lubricants (e.g. silica, talc, stearic acid). , Magnesium or calcium stearate, polyethylene glycol, etc.), binders (e.g. starch, arabic gum, gelatin methylcellulose, carboxymethylcellulose, polyvinyl pyrrolidone, etc.), disintegrants (e.g. starch, alginic acid, alginate, etc.) Sodium starch glycolate, etc.), formal mixtures, dyes, sweeteners, wetting agents (eg, lecithin, polysorbate, lauryl sulfate, etc.), and other pharmacologically inert substances commonly used in pharmaceuticals. For topical administration, excipients (e.g. lactose, starch, cellulose, lactose, polyethylene glycol, etc.), lubricants (e.g. magnesium stearate, stearic acid, glyceryl behenate, talc, etc.), preservatives (e.g. benzyl) Alkonium chloride, etc.), and the like.

Parenteral administration includes injections that produce systemic effects or injections administered directly to the affected area. Examples of parenteral administration include subcutaneous, intravenous, intramuscular, intradermal, intrathecal, intraocular and general infusion techniques.

Topical administration includes, for example, treatment of infected areas or organs that are readily accessible by topical application such as ear, vagina, open and closed wounds or closed wounds and skin, including eye, outer and middle ear infections. It also includes transdermal delivery that results in systemic effects.

Intranasal administration includes nasal aerosol or inhalation application.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount means an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective dose level is the type of disease and its severity, drug activity, patient Age, weight, health and sex of the patient, sensitivity to the drug of the patient, the time of administration of the particular extract used, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used in combination or co-use with the specific extract used, and other medicines. It can be determined according to factors well known in the field. In general, an amount of 0.1 to 1000 mg/kg per day, preferably a dose of 10 to 100 mg/kg per day, may be administered to an adult once to several times a day.

As another aspect, the present invention relates to a method of treating type 2 diabetes by administering the pharmaceutical composition for preventing and treating diabetes to an individual.

The treatment refers to a therapeutic treatment and prevention or prevention means for type 2 diabetes. Therefore, those in need of treatment include those who have already developed type 2 diabetes, as well as those who want to prevent type 2 diabetes. In addition, the individual refers to any animal that has already developed diabetes or is highly likely to develop diabetes and needs treatment. Specifically, it refers to mammals such as humans, cows, dogs, horses, pigs, rodents such as guinea pigs, hamsters, squirrels, rats, etc. , But is not limited to this.

As another aspect, the present invention is any one selected from the group consisting of the lichen-forming fungus extract, the fraction of the lichen-forming fungus extract, the citalone compound, and a food pharmaceutically acceptable salt of the citalone compound. It provides a health food for preventing and improving type 2 diabetes including the above.

Details of the extract of the lichen-forming fungus, the fraction thereof, the citalone compound, and the pharmaceutically acceptable salt thereof are as described above.

Food pharmaceutically acceptable salts of the present invention include all salts of acidic or basic groups that may be present in the compounds of the present invention, unless otherwise indicated. For example, there are hydrochloride, acetate, citrate, and lactate, but the types are not necessarily limited thereto.

In the health food of the present invention, any one or more components from the group consisting of the extract extracted from the lichen-forming mold, the fraction of the extract, the citalone compound, and the food pharmaceutically acceptable salt of the citalone compound are added as it is, or It may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

The mixing amount of the above ingredients may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of food or beverage, the ingredients of the present invention are added in an amount of 10% by weight or less, preferably 5% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and health control, it may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and all health foods in the usual sense are included.

When the health food of the present invention is a health drink, it may contain various flavoring agents or natural carbohydrates as an additional component, like a normal beverage. As the natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. . The ratio of the natural carbohydrate is generally about 0.01 to 0.4 g, preferably about 0.02 to 0.03 g per 100 ml of the health drink of the present invention.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols. , Carbonating agents used in carbonated beverages, and the like may be contained. In addition, the health food of the present invention may contain pulp for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

When the health food is consumed, it is possible to prevent and improve diabetes, particularly type 2 diabetes, by inhibiting the digestion and absorption of carbohydrates by inhibiting alpha glucosidase activity.

As described above, not Jia (Anzia), A knife Plaka (Caloplaca), A set trellis O (Cetrelia), A Cloud Dia (Cladia), An Eberhard California (Evernia) in, Mai Ilo Croix of the present invention ( Myelochroa ), Nephromopsis , Ramalina , Tuckneraria and Xanthoparmelia from any one or more lichen-forming fungi selected from the group consisting of lichen-forming fungi. A drug for preventing and treating type 2 diabetes, comprising as an active ingredient any one or more selected from the group consisting of the extracted extract, the fraction of the extract, the scytalone compound, and the pharmaceutically acceptable salt of the citalone compound The pharmaceutical composition is derived from a natural product, lichen-forming fungus, is harmless to the human body, has no side effects, has little resistance to resistance, and has alpha glucosidase inhibitory activity, so it can be usefully used in the prevention and treatment of type 2 diabetes.

1 shows the results of comparing the yield of the lichen extract and the lichen forming fungus extract of the present invention and the alpha glucosidase and alpha amylase inhibitory activities of the extracts.
Figure 2 is Myelochroa ribina of the present invention (Myelochroa galbina ) shows the growth rate according to the medium type of lichen-forming fungi and the alpha glucosidase inhibitory activity of the extract.
Figure 3 is Myelochroa ribs of the present invention (Myelochroa galbina ) It shows the growth rate of lichen-forming fungi according to pH and temperature, and the alpha glucosidase inhibitory activity of the extract.
4 is a flowchart illustrating a process for separating the citalone compound of Formula 1 from Myelochroa galbina among the lichen-forming molds of the present invention.
Figure 5 shows the alpha glucosidase inhibitory activity of the citalone compound of the present invention by concentration.
6 shows the degree of cell viability when the citalone compound of the present invention is treated by concentration in HIT-T15 cells.

The present invention will be described in more detail through the following examples. Since these examples are merely illustratively presented so that the present invention may be more understood, the scope of the present invention should not be limited to these examples.

Example 1: Collection of lichens and separation of lichen-forming molds

1-1. Collection of lichens

Lichens were collected from 2003 to 2006 in China, Korea, Japan, Bulgaria, Antarctica, Chile and Hungary. Samples were confirmed at the Chinese Academy of Sciences (Chinease Academy or Science; CAS) and the Korean Lichen Research Institute (KOLRI) of Sunchon National University. The collected lichens were stored at the Korean Lichen & Allied Bioresources Center.

1-2. Isolation of lichen-forming fungi

The lichens collected in Example 1-1 were subjected to the Yamamoto method (Yamamota et al; Tissue cultures of Usnea) within 7 days from the date of collection. rubescens and Ramalina yasudae and production of usnic acid in their cultures. Agricultural and Biological Chemistry. 49(11):3347-3348) was used to isolate the lichen-forming fungus from the lichen body or asciata of lichens. The lichens separated from lichens, each within 1 cm in width, length, and height, were washed in running water for about an hour, and then 3 to 5 ml of purified water was added and softened using a mortar and pestle. Thereafter, the suspension was filtered, and the separated pieces were checked under a microscope, and then transferred to an agar medium and cultured. Incubation was performed for 2 to 3 months in a dark environment at 18°C. As a result, it was confirmed that the lichen-forming fungus formed a mycelium having a diameter of about 2 to 5 mm, and it was passaged again to increase the mass. To compare the DNA sequence of the ITS (Internal transcribed spacer) region of the lichen-forming fungus cultured in this way and the original lichen lichen, ITS4 (5'-TCCTCCGCTT-ATTGATATGC-3'; SEQ ID NO: 1) and ITS5 (5' as primers) -GG-AAG TAAAAGTCGR AACAAGG-3'; SEQ ID NO: 2) was used as a PCR method (White et at, Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.PCR Protocols: a Guide to Methods and Applications, 315 -322.Academic Press, Orlando). As a result, it was confirmed that a total of 272 types of lichen-forming fungi were isolated from lichens.

Example 2: Measurement of alpha-glucosidase inhibitory activity of lichen-forming fungus extract

2-1. Preparation of extracts from lichen-forming fungi

Each 0.1 ml of 75% ethanol and water was added to 0.1 g of 272 types of lichen-forming molds isolated in Example 1-2, and extracted three times for 48 hours using ultrasonic waves. After that, each extract was filtered using Whatman paper and then evaporated at room temperature to prepare an extract.

2-2. Measurement of alpha glucosidase inhibitory activity of extracts of lichen-forming fungi

In order to measure the alpha glucosidase inhibitory activity of the lichen-forming fungus extract prepared in Example 2-1, the absorbance of p-nitrophenyl was measured at 410 nm (Kim et al, 2010. A novel competitive class of α-glucosidase) Inhibitors: (E)1-phenyl-3-(4styrylphenyl) urea derivatives.Chem. Bio Chem. 11: 2125-2131 and Ryu et al, 2010. Polyphonls from Broussonetia papyrifera displaying potent αglucosidase inhibition. J. Agric. Food Chem. 58:202-208). 20 µl of 0.1M phosphate buffer (pH 6.8), 20 µl of substrate solution (prepared by putting 1.5mM PNPG in 0.1mM phosphate buffer), and 20 µl of lichen-forming fungus extract were mixed and preincubated at 37°C for 10 minutes. Thereafter, 20 µl of an enzyme solution (prepared by adding 1 U/ml of alpha glucosidase in 0.1 M phosphate buffer) was added to the pre-cultured mixture, followed by incubation at 37° C. for 15 minutes. The reaction was stopped using 0.4M sodium hydroxide containing 80 µl of glycine, and the degree of inhibition of alpha glucosidase activity of the lichen-forming fungus extract was evaluated by measuring the absorbance at 410 nm using an ELISA reader and observing the amount of PNP released. . IC ₅₀ was set as the concentration at which 50% of the alpha glucosidase activity was inhibited.

And, in order to quickly qualify the alpha glucosidase inhibitory activity, the TLC bioautographic method was used (Simet al, A TLC bioautographic method for the detection of alpha and beta AG inhibitors in plant extracts.Phytochemical Analysis. 20: 511- 515). Alpha glucosidase was prepared by dissolving in 100 ml of a buffer solution (prepared by mixing 10.25 g of sodium acetate in 250 ml of water and then titrating to pH 7.5 with 0.1 M acetic acid), and the sample was dip on a TLC plate and spotted. And then dried to completely remove the solvent. After that, after spraying the enzyme solution on the TLC plate, water was used as a solvent to separate the solution, and then naphthyl glucopyranoside solution and Fast Blue B salt solution were mixed in a ratio of 1:1. It was examined whether it changed to a purple light by spraying.

The results of evaluating the alpha glucosidase inhibitory activity of the extracts extracted with 75% ethanol and water from each of the lichen-forming fungi using the above method are shown in Tables 1 to 14 below.

As a result of the experiment, it was found that the alpha glucosidase inhibitory activity was more than 80% in 75% ethanol extracts of 30 types of lichen-forming fungi and water extracts of 10 types of lichen-forming fungi, of which Cetrelia genus, My Ilo Croix (Myelochroa) and the balance in topa Melia (Xanthoparmelia) in let azepin showed excellent inhibitory activity against alpha-glucosidase in the extracts of lichens forming mold. In particular, Myelochroa galbina (Myelochroa galbina ) In the case of the 75% ethanol extract of lichen-forming fungi, the alpha glucosidase inhibitory activity was excellently observed at 97.18%.

Example 3: Comparison of alpha glucosidase and alpha amylase inhibitory activity of lichen extract and lichen forming fungus extract

In order to compare the yield of extracts extracted from lichens and lichen-forming fungi with the alpha glucosidase and alpha amylase inhibitory activity of the extracts, each lichen and lichen-forming fungi were extracted with 75% ethanol or water using the method of Example 2 Next, the inhibitory activity of alpha glucosidase of the extracts was measured. The yield was expressed as% by comparing the dry weight after extraction with respect to the dry weight before extraction of lichens and lichen-forming molds with 75% ethanol or water.

Meanwhile, the alpha amylase inhibitory activity of the extracts extracted from lichens and lichen-forming fungi was measured using porcine pancreatic alpha amylase (MC Cordeiro et al, Culture studies and secondary compounds of six Ramalina species. The British Mycological Society. 108 ( 5): 489-497, Wang et al, α-Glucosidase and α-amylase inhibitory activities of guava leaves.Food Chemistry. 123: 6-13, Nickavar et al.). After mixing 50 µl of sample and 50 µl of alpha amylase solution (prepared by dissolving 5U/ml of EC 3.2.1.1. in a phosphate buffer of pH 6.9 containing 6.7mM sodium chloride) and incubating for 30 minutes at 25°C, 100 µl of starch solution (0.5% w/v) was added as a substrate and then incubated at 25° C. for 3 minutes. Then, 100 µl of the reaction solution (prepared by mixing 20 ml of 96 mM 3,5-dinitrosalicylic acid, 8 ml of 2M sodium hydroxide and 12 ml of deionized water) was added, followed by 15 minutes in a water bath at 85°C. After reacting, it was cooled to room temperature, diluted in 900 µl of distilled water, and absorbance was measured at 540 nm to evaluate alpha amylase inhibitory activity.

The yield of the extract obtained by extracting the lichens and lichen-forming fungi with 75% ethanol or water by the method as described above and the results of measuring the alpha glucosidase and alpha amylase inhibitory activity of the extracts are shown in FIG. 1 below.

As a result of the experiment, there was no correlation between the yield of lichens or lichen-forming fungal extracts and alpha glucosidase inhibitory activity, and the formation of Parmelia sp. and Myelochroa leucotyliza or lichens thereof It was observed that the alpha glucosidase inhibitory activity of the lichen-forming fungus extract was higher than that of the lichen extract in all experimental groups except for the fungal extract.

Likewise, there was no correlation between the yield of lichens or lichen-forming fungus extracts and alpha amylase inhibitory activity, and alpha amalase inhibitory activity could not be measured in a number of experimental groups.

In summarizing the above results, Myelochroa ribina (Myelochroa) has excellent alpha-glucosidase inhibitory activity and a relatively fast growth rate. galbina ) Lichen forming fungus was selected as a strain to develop a composition for preventing and treating diabetes.

Example 4: Mai Ilo Croix or ribs (Myelochroa galbina ) Growth conditions of lichen-forming fungi and measurement of alpha glucosidase inhibitory activity of the lichen-forming fungus extract

4-1. Myelochroa ribs galbina ) Comparison of growing conditions according to the medium type of lichen-forming fungi

Myelochroa ribs galbina ) To compare the growth conditions of lichen-forming fungi, Myelochroa ribs (Myelochroa galbina ) Lichen-forming fungi were cultured for 2 months under solid and liquid shaking culture conditions, respectively, and then the dry weight was measured, and the results of measuring the alpha glucosidase inhibitory activity of the lichen-forming fungus extract after cultivation are shown in Table 15 below. .

Experimental results, Myelochroa galbina (Myelochroa galbina ) Lichen-forming fungi were hardly grown in the case of liquid shaking culture, and the alpha glucosidase inhibitory activity of the lichen-forming fungus extract after culture was 38.6%, which was only about half of that in the case of culture in a solid medium.

4-2. Myelochroa ribs galbina ) Comparison of growing conditions of lichen-forming fungi according to the type of medium

Myelochroa ribs galbina ) To compare the growth conditions of lichen-forming fungi, Myelochroa ribs (Myelochroa galbina) Lichen-forming fungi were respectively treated with PDA (potato extract agar), My (Malt-yeast extract) agar, MNT 2% (Malt-yeast extract + 2% mannitol) agar, MS (Murashige-Skoog) medium, S (Sabouraud). ) After culturing in agar, MCM (Mushroom complete media) and GCM (Ganoderma complete media) for 2 months, the results of measuring the dry weight and the alpha glucosidase inhibitory activity of the lichen-forming fungus extract after cultivation are shown below. It is shown in 2.

Experimental results, Myelochroa galbina (Myelochroa galbina ) When the lichen-forming fungus was cultured on MS agar, the dry weight was 0.179 g, which was the fastest growth rate, and when cultured on 2% MNT agar, the dry weight was 0.136 g, when cultured on MY agar. It was observed that the growth rate was about twice the dry weight of 0.061 g.

In addition, after culturing the lichen-forming fungi of Myelochroa galbina for each type of medium, the alpha glucosidase inhibitory activity was measured using the extract of the lichen-forming fungus. It was observed that the alpha glucosidase inhibitory effect of the forming fungus extract was almost 100%, which was the most excellent.

4-3. Myelochroa ribs galbina ) Comparison of growing conditions according to temperature and pH of lichen-forming fungi

Myelochroa ribs galbina ) In order to compare the growth conditions of the lichen-forming mold, the lichen-forming mold of Myelochroa galbina was used at a temperature of 15, 20 and 25°C and a pH of 4, 5, 6, 7, 8, and After culturing for 2 months under the condition of 9 persons, the values of dry weight and the results of measuring the alpha glucosidase inhibitory activity of the lichen-forming fungus extract after cultivation are shown in FIG. 3 below.

Experimental results, Myelochroa galbina (Myelochroa galbina ) Lichen-forming fungi showed the best growth rate when the pH was 6 and the culture temperature was 15 and 20°C, and the alpha glucosidase inhibitory activity of the lichen-forming fungus extract was observed to be excellent. Particularly, when the temperature was 20°C, it was observed that the alpha glucosidase inhibitory effect of the lichen-forming fungus extract was superior to that of 15°C. However, it was observed that when the temperature was 25°C, the growth rate and the alpha glucosidase inhibitory activity of the lichen-forming fungus extract were rather decreased.

Example 5: Mai Ilo Croix or ribs (Myelochroa Isolation of Active Ingredients from Lichen Forming Fungi

5-1. Myelochroa ribs galbina ) Preparation of extracts and fractions from lichen-forming fungi and measurement of alpha glucosidase inhibitory activity of these extracts and fractions

Myelochroa ribs galbina ) In order to separate the active ingredient from lichen-forming fungi, Myelochroa galbina (Myelochroa galbina ) 75% ethanol extracts of lichen-forming fungi were fractionated with petroleum ether, ethyl acetate and normal butanol, respectively, and then evaporated to measure the yield and alpha glucosidase inhibitory activity of these fractions. In addition, the fractionated residues are also freeze-dried, and the results of measuring the alpha glucosidase inhibitory activity of these residues are shown in Table 16 below.

Experimental results, Myelochroa galbina (Myelochroa galbina _{) It was observed that the IC 50} of the ethyl acetate fraction of the 75% ethanol extract of the lichen forming fungus was 0.112 mg/ml, showing the most excellent alpha glucosidase inhibitory activity, and the degree was about 4 compared to the positive control, glucobay. It turned out to be about twice as good.

5-2. Myelochroa ribs galbina ) Isolation of Active Ingredients from Ethyl Acetate Fraction from 75% Ethanol Extract of Lichen Forming Fungi

Myelochroa ribina fractionated in Example 5-1 (Myelochroa galbina ) In order to separate the active ingredient from the ethyl acetate fraction for the 75% ethanol extract of lichen-forming fungi, the ethyl acetate fraction was mixed with hexane-acetone (hexane: acetone in a ratio of 20:1, 10:1, 10:2, respectively. , 10:4 and 10:6) and ethyl acetate-methanol (ethyl acetate: methanol ratio of 10:4) were used as eluents using a silica gel chromatography column. Thereafter, the alpha amylase inhibitory activity of each fraction was measured to select an active fraction, and the selected fraction was subjected to Sephadex LH-20 column chromatography in a chloroform-methanol system (chloroform: methanol ratio of 1:1). And re-fractionated. 60% methanol was used as the eluent, and the small fraction 1 (SFr. 1) was fractionated using a step-gradient MCl column protograph in which the solvent was 20% to 100% methanol.

As a result of the experiment, an effective substance of 8.4 mg in fraction 1, 7.4 mg in fraction 2, and 3.9 mg in fraction 9 were obtained. A process for separating the active ingredient from the ethyl acetate fraction for the 75% ethanol extract of the lichen-forming mold of Myelochroa galbina is shown in FIG. 4 below.

5-3. Myelochroa ribs galbina ) Identification of active ingredients of lichen-forming fungi

In order to confirm the structure of material No. 9 among the active ingredients isolated in Example 5-2, ¹ H and ¹³ C nuclear magnetic resonance (NMR, Bruker Analytische Messentechink GMbH, Germany) spectroscopy was used to confirm the structure of the Korea Research Institute of Chemical Technology. In Table 17 below, the structure of the active ingredient confirmed using the results obtained from the mass spectrometry (Mass Spectroscopy; MS, JMS-DX303; JEOL Ltd., Japan) and the ¹ H and ^{13 C nuclear magnetic resonance is shown in Table 17 below.} It is shown in the following formula (2).

As a result of the experiment, it was confirmed that substance 9 is a scytalone compound.

Example 6 Alpha-glucosidase inhibitory activity evaluation at the time of the etalon (scytalone) compound is

6-1. Alpha Glucosidase Inhibitory Activity of Scytalone Compounds

In order to evaluate the alpha glucosidase inhibitory activity of the scytalone compound identified in Example 5-3, the results of observing the alpha glucosidase inhibitory activity using the method of Example 2-2 are shown in FIG. 5 below. Shown in.

As a result of the experiment, _{it was observed that the IC 50} for alpha glucosidase of the citalone compound was 91.42 µg/ml, which was superior to that of the positive control glucobi.

6-2. At the time of the etalon (scytalone) compound in In vitro cytotoxicity assessment

To evaluate the cytotoxicity of the citalone compound, pancreatic beta cells, HIT-T15 cells, were purchased from a Korean cell line bank, and then 10% Fetal bovine serum (FBS), 1% antibiotics, and pancrease a-amylase (PAA) were obtained. It was cultured using the included RPMI 1640 medium, and when the cells were cultured, 0.8 mM glucose was added to the medium to prevent a decrease in the insulin reserves of the cells (Shin et al, Ethanol decreases basal insulin secretion from HIT-T15 cells. Life Sciences 70: 1989-1997 and Robertson et al, Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations.The Journal of Clinical Investigation, 90(2):320-325). When the citalone compound was added to HIT-T15 cells, the degree of cell viability was measured by modifying the method of Skehan (Skehan et al, New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82: 1107-1112). After dispensing 5,000 HIT-T15 cells per well in a 96-well plate, the day after dispensing, the cells were treated with 0, 20, 40, 80, 160 and 250 µg/ml of citalone compounds, and then 5% carbon dioxide incubator Incubated for 24 hours at. Thereafter, the cells were fixed by placing the cells for an hour using cold 50% TCA in a dark environment, and then the cells were washed and dried. Next, 50 µl of a solution to which 0.4% sulforhodamine B was added to 1% acetic acid solution was added to each of the dried wells, and allowed to stand for 10 minutes to stain the cells. Thereafter, after removing the staining solution, the cells were washed and dried with 1% acetic acid solution, 100 μl of 10 mM Tris solution was added to dissolve the cells, and the absorbance was measured at 490 nm to measure the degree of cell viability. Shown in.

As a result of the experiment, it was observed that the degree of cell viability of pancreatic beta cells was not changed at all concentrations tested, so that the citalone compound did not show cytotoxicity to pancreatic beta cells.

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Claims (4)

A pharmaceutical composition for preventing and treating type 2 diabetes, including any one or more selected from the group consisting of a citalone compound represented by Formula 1 and a pharmaceutically acceptable salt of the citalon compound.

Formula 1

[Claim 2] The type 2 diabetes prevention and treatment of claim 1, wherein the citalone compound and the pharmaceutically acceptable salt of the citalon compound are isolated from liposome forming fungus of the genus Myelochroa . Pharmaceutical composition for.
According to claim 2, wherein the genus Myelo croa lichen forming fungus is Myelochroa galbina ) pharmaceutical composition for preventing and treating type 2 diabetes.
A health food for preventing and improving type 2 diabetes comprising at least one selected from the group consisting of a citalone compound represented by Formula 1 and a food acceptable salt of the citalon compound.

Formula 1

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