KR20040069415A - Pharmaceutical composition comprising the extract or the polyphenol fraction isolated from Ganoderma applanatum for treating or preventing diabetes - Google Patents

Abstract

The present invention relates to a composition containing ethyl acetate soluble extract or polyphenol fraction of Ganoderma applanatum having a prophylactic and therapeutic activity for diabetic disease.

Ethyl acetate soluble extract and polyphenol fraction of the present invention has excellent inhibitory activity against aldose reductase, and thus inhibits the accumulation of glycation end products, which is useful for the prevention and treatment of diabetes-related diseases. Can be provided.

Description

Pharmaceutical composition comprising the extract or the polyphenol fraction isolated from Ganoderma applanatum for treating or preventing diabetes}

The present invention relates to a pharmaceutical composition effective for the prevention and treatment of diabetic diseases containing a non-polar solvent soluble extract or polyphenol fraction isolated from Ganoderma applanatum .

Diabetes is a disease caused by abnormally high levels of glucose in humans and mammals, which increases the level of hemoglobiin in plasma and chronic hyperglycemia. ), Atherosclerosis, microangiopathy, kidney disease, heart disease, diabetic retinopathy and other eye diseases.

Diabetes mellitus is characterized by two types: insulin dependent diabetes (IDDM) is caused by a lack of secretion of insulin, a glucose-regulating hormone in the blood, mainly in the 10s to 20s. It is called juvenile diabetes because it occurs in younger ages. Type II (non-insulin dependent diabetes, NIDDM) occurs mainly after the age of 40, and occupies most of the diabetic patients in Korea. Unlike type I, it is called adult-type diabetes and the cause of the disease is not known yet, but it is known to be caused by genetic factors and environmental factors. The etiology of type II diabetes has been observed in both pancreatic beta cells with impaired insulin secretion and in target cells with insulin action (insulin resistance), but it is not yet clear which changes are of primary importance.

Agents currently used for complications such as type II diabetes and insulin resistance include five groups of compounds: biguanides, thiazolidinediones, sulfonylureas, and benzoic acid. Derivative compounds and α-glucosidase inhibitors are used. Among these, biguanide compounds such as metformin are known to prevent excessive blood gluconeogenesis. Thiazolidinedione compounds are thought to act to increase glucose consumption in peripheral nerves, benzoic acids such as sulfonylureas, repaglinide compounds such as tolbutamide and glyburide A-glucosidase inhibitors, such as derivatives and acarbose, act to stimulate insulin secretion and lower plasma glucose.

The sulfonylurea compound cannot be administered to insulin-dependent type I diabetic patients, and non-insulin-dependent type II diabetic patients reduce insulin secretion, abnormal fetal birth, abortion and May cause side effects such as stillbirth. In addition, most sulfonylurea compounds should be administered carefully to patients with impaired liver and kidney function due to the metabolism of sulfonylureas.

Although the mechanism of biguanide-containing preparations such as metformin has not been elucidated, although biguanide compounds are unable to increase pancreatic insulin secretion, they lower blood sugar more effectively than sulfonylureas and the frequency of hypoglycemia induction. low. However, it can cause nausea, vomiting, diarrhea and rash at the beginning of administration, and can cause side effects such as fatal lactic acidosis, so it can only be used as a clinical reagent in the United States.

Since sulfonylurea and biguanide-containing preparations have the above drawbacks and side effects, there is a need for an excellent hypoglycemic agent with low side effects and high safety.

The jannabi stool mushroom used in the present invention (Ganoderma applanatum (Fr.) Pat. = Elfvingia applanata (Pers.) Karst.) Is belonging to the genus jannabi stool mushroom perennial wood white rot caused to saengnamu or the old tree of hardwoods in the summer and autumn It is a fungus (Wood white rot fungus), which is distributed worldwide. The tissue is cork 1-5 cm thick, and the fruiting layer is yellowish white to white but turns brown when in contact.

Several types of ergostane-based steroid compounds isolated from Jangnabi (Strigina L. I.et al.,Phytochemistry,10, pp2361-2365, 1991: Ripperger and Budzikiewicz,Phytochemistry,14, pp2297-2298, 1975: Protivaet al.,Collection Czechoslov. Chem. Commun.,45, pp2710-2713, 1980: Kac, D. et al.,Phytochemistry,23, pp2686-2687, 1984: Gan K. H.et al.,J. Nat. Prod.,61, pp1421-1422, 1998) and compounds of triterpenoids, such as Friedelin and Friedoolean-5-en, palmitic acid (Protivaet al.,Collection Czechoslov. Chem. Commun.,45, pp2710-2713, 1980), ganoderenic acid A, AP, F, G, H, I, furanoganoderenic acid, compound B8 (Nishitobaet al.,Phytochemistry,28, pp 193-197, 1989), applanoxidic acid A, B, C, D (Chairul S. M.et al.,Phytochemistry,30, pp 4105-4109, 1991), aplanoxide acid E-H (Chairul S. M.et al.,Phytochemistry,35, pp 1305-1308, 1994), 24-methyl-5α-lanosta-25-one (Gan, K. H.et al.,J. Nat. Proc.,61, pp1421-1422, 1998) Lanostane-based triterpenes such as heterogalactan (Usui, T.et al.,Carbohydr. Res.,92, pp 102-114, 1981) and β-D-glucan (Usui, T.et al.,Carbohydr. Res.,115, pp273-280, 1983). Recently, ergosterol, β-amyrenone, 2-hydroxy-hexacosanoic acid, and β-amyrin acetate from Canadian Janavier stool acetate), 2,5-dihydroxybenzoic acid, ganoderenic acid A, D, G and ganoderma aldehyde have been reported (Ming, DS)et al.,Fitoterapia,73, pp 147-152, 2002), their activity has not been reported.

In this regard, the present inventors confirmed that the ethanol extract of Jannabe stool mushroom ethyl acetate was excellent in inhibiting activity of aldose reductase in order to develop a therapeutic agent for diabetes disease using a safe and natural product. The bioassay-guided fraction was carried out, and as a result, the nonpolar solvent soluble extract and the polyphenol fraction isolated therefrom were excellent in the treatment and prevention of diabetes disease. It was.

Disclosure of Invention It is an object of the present invention to provide a pharmaceutical composition effective for the prevention and treatment of diabetic diseases, which comprises a soluble extract of Jangnabi stool mushroom nonpolar solvent or a polyphenol fraction isolated therefrom.

In accordance with the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of diabetic diseases, including a soluble extract of pinnacle stool mushroom non-polar solvent and containing a pharmaceutically acceptable carrier or excipient.

The nonpolar solvent means an extract which is soluble in hexane, chloroform, methylene chloride, non-polar solvent such as ethyl acetate, and more preferably ethyl acetate.

The present invention provides a pharmaceutical composition for the prevention and treatment of diabetic disease, comprising a polyphenol fraction isolated from Jangnabindung mushroom and containing a pharmaceutically acceptable carrier or excipient.

Such diabetes-related diseases include a combination of one or more of chronic hyperglycemia, atherosclerosis, microangiopathy, kidney disease, heart disease, diabetic retinopathy and other ophthalmic diseases. .

The present invention also provides a production method for separating the non-polar solvent soluble extract from the pinnacle mushroom.

The present invention also provides a production method for separating the polyphenol fraction from Janna butterfly.

Hereinafter, the present invention will be described in detail.

The non-polar solvent soluble extract or polyphenol fraction of the present invention is pulverized by drying the dried Janna beetle mushroom, and then about 1 to 15 times by weight, preferably about 5 to 10 times the volume of water, lower alcohol or their A mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 3, preferably about 0.5 hour to 2 days at an extraction temperature of 20 to 100 ° C, preferably 40 to 60 ° C. Preferably, the extraction method such as ultrasonic extraction, reflux extraction, etc. for about 0.5 hours to 1 day is repeated once to five times, preferably two to five times, to obtain a crude extract of water, lower alcohol, or a mixed solvent thereof. Obtaining an extract; After the crude extract is suspended in a polar solvent such as water or methanol, it is added to a nonpolar solvent such as hexane, chloroform, dichloromethane, ethyl acetate, and the like in an amount of about 1 to 100 times, preferably about 1 to 5 times the suspension. Fractionating the nonpolar solvent soluble layer into the polar solvent soluble layer once to 10 times, preferably 2 to 5 times to obtain a nonpolar solvent soluble portion and a polar solvent soluble portion; The non-polar solvent soluble layer can be separated using silica gel column chromatography, liquid chromatography (LC) and thin layer column chromatography (TLC).

Hereinafter, a process of separating the non-polar solvent soluble extract or the polyphenol fraction from the Janna butterfly stool mushroom,

For example, the first step of slicing the dried Jangnabi stool mushroom, hot-extracted with a polar solvent such as water, methanol or butanol, followed by filtration and concentration under reduced pressure to obtain a crude extract soluble in the polar solvent;

A second step of dissolving the crude extract sequentially with a nonpolar solvent such as hexane, chloroform, methylene chloride, ethyl acetate, and then suspending and fractionating with a separatory funnel to obtain a nonpolar solvent soluble extract;

A third step of sequentially fractionating and filtering the aqueous layer using a polar solvent such as butanol and water to obtain a polar solvent soluble extract;

Subsequently, column chromatography is performed on the two-phase nonpolar solvent soluble extract, wherein the silica gel column has a hexane: ethyl acetate: methanol ratio of 1: 1: 1 to 50: 10: 0.5 (w / w), preferably 10 Polyphenol fractions of fractions 8 and 9 and nonpolar solvent soluble extracts of 15 fractions obtained by developing a developing solvent having a ratio of 3: 3 on a column were subjected to reverse phase chromatography using Sephadex LH-20. And a fourth step of obtaining the polyphenol fractions of subfractions 2 and 4 out of the four subfractions obtained by gradient eluting with methanol-water.

The present invention provides a pharmaceutical composition for the treatment and prevention of diabetes-related diseases comprising the non-polar solvent soluble extract or polyphenol fraction of the present invention obtained by the above method as an active ingredient.

The composition for preventing and treating diabetic diseases of the present invention, the extract or fraction based on the total weight of the composition by 0.5 to 50% by weight.

Compositions comprising extracts or fractions of the invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts or fractions of the present invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active fractions, as well as in any suitable collection.

Compositions comprising extracts or fractions according to the invention are in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents which may be used in formulated compositions comprising extracts or fractions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber , Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil have. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate and sucrose in the extract or fraction. It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of extract or fraction of the present invention may vary depending on the age, sex, and weight of the patient, but in general, the amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, is divided once to several times daily. May be administered. In addition, the dosage of the extract or fraction may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention is described in more detail based on the following examples and experimental examples, but the present invention is not limited thereto.

Example 1 Preparation of Crude Buttercup Mushroom Crude Extract

2.1 kg of dried Jannabi stool mushroom (gathered near Ontario, Canada) was chopped and powdered, and 5 liters of methanol solution was added thereto, followed by extraction with warming for 7 hours at 50 ° C. for 1 hour, and then combined with a filter paper (Whatman, USA). After filtrating, the resultant was concentrated under reduced pressure using a vacuum concentrator (Eyela Co., NN Series, Japan) at 50 ° C. or lower to obtain 85 g of Jangnabi Snail Mushroom.

Example 2. Preparation of Jangnabi Stool Mushroom Polar Solvent and Nonpolar Solvent Soluble Extract

After extracting the crude butterfly extract of Example 1, the crude extract of the hibiscus in 1.5 L of water, 1.5 L of hexane was added to the separatory funnel, and the hexane insoluble layer (lower layer) and the hexane soluble layer (upper layer) were collected. It was. The same volume of hexane solvent as the lower layer (water layer) was repeated twice in the same manner as described above to obtain a substance that can be dissolved in the hexane layer as much as possible until the color of the solution became light, fractionated and dried, and sequentially methylene chloride. , Ethyl acetate and butanol were carried out in the same manner as above to obtain hexane soluble extract (15g), methylene chloride soluble extract (25g), ethyl acetate soluble extract (30g) and butanol soluble extract (15g). .

Example 3. Preparation of Polyphenol Fractions

30 g of the residue of the ethanol ethanol acetate extract of Example 2 was subjected to silica gel column chromatography (12 × 60 cm, Merck, ASTM 7734, Germany) using an eluent (hexane: ethyl acetate: methanol = 10: 3: 1). 15 fractions were obtained by TLC pattern. Of the 15 fractions, the eighth fraction (1.5 g) and the ninth fraction (2.5 g) were designated as polyphenol fractions A and B, respectively. Meanwhile, 2.5 g of ethyl acetate soluble extract was subjected to Sephadex LH-20 column chromatography (2.5x60 cm, Pharmacia, Sweden) using an eluent (methanol: water = 3: 7 → 10: 0) and concentrated. Fractionation into four small fractions. The second fraction (0.3 g) and the fourth fraction (0.2 g) were named polyphenol fractions C and D, respectively.

Experimental Example 1. Measurement of aldose reductase activity of extracts

In order to observe the anti-diabetic activity of each of the hexane soluble extract, methylene chloride soluble extract, ethyl acetate soluble extract and butanol soluble extract of Example 2, the activity against aldose reductase was measured.

For the raw material of aldose reductase (AR), the lens of the rat was removed from Sprauge-Dawley (SD) for a standard weight of 250-280 g and stored frozen before use. Surface areas of flat rat lenses were prepared using the methods described in the literature (Hayman, S. & Kinoshita, JH; J. Biol. Chem. , 240 , pp877-882, 1965). Enzyme inhibition activity was tested using a partially purified enzyme with a specific activity of 6.5 U / mg, and aldose reductase activity was absorbed by NADPH for 5 minutes using DL-glyceraldehyde as a substrate. The reduction in the band 340 nm was measured (Sato, S and Kador, PF, Biochem. Pharmacol. , 40 , pp 1033-1042, 1990). Equal amounts of enzyme, 0.10 M sodium phosphate buffer (pH 6.2) and 0.3 mM NADPH were placed in each 1.0 ml cuvette, the treated group treated with 10 mM substrate and inhibitor, and the untreated group did not contain substrate and inhibitor. The concentration of inhibitor (IC ₅₀ ) showing 50% inhibition of enzyme activity was calculated from the logarithmic regression line of log concentration plotted against residual activity.

From the results of Table 1, it was confirmed that IC ₅₀ values of methylene chloride soluble extract and ethyl acetate soluble extract of Janna butterfly cultivars were 1.66 µg / ml and 0.66 µg / ml, respectively. It showed similar activity to the tetramethylene glutaric acid (tetramethylene glutaric acid, TMG) control group, it was confirmed that it has an excellent aldose reductase inhibitory activity.

sample Concentration (µg / ml) Inhibition Rate (%) IC ₅₀ (μg / ml) TMG 10 82.1 0.63 One 53.7 0.1 29.8 n-hexane extract 100 91.1 4.98 10 52.7 One 32.7 Methylene chloride extract 10 87.2 1.66 5 68.2 One 41.2 Ethyl acetate extract 100 100 0.66 10 90.5 One 66.6 0.5 37.7 Butanol extract 10 94.4 1.29 One 37.7 0.5 35.4

Experimental Example 2 Measurement of Intracellular Aldose Reductase Activity of Polyphenol Fractions

In order to measure the intracellular aldose reductase activity of the polyphenol fractions of Example 3, which was isolated from the ethyl acetate soluble extract of the Jangnabindae mushroom of the present invention, the experiment was performed by the activity measuring method of Experimental Example 1.

As a result, the polyphenol fractions A and C of the Rana mushrooms showed IC ₅₀ values of 0.89 µg / ml and 0.73 µg / ml, respectively, and had an aldose reductase inhibitory activity. TMG) showed a similar activity, it was confirmed that it has an excellent aldose reductase inhibitory activity (see Table 2).

Inhibitory Activity of Polyphenol Fractions Isolated from Extracts sample Concentration (µg / ml) Inhibition Rate (%) IC ₅₀ (μg / ml) Polyphenol Fraction A 100 97.4 0.89 10 81.2 One 48.5 Polyphenol Fraction B 100 89.0 11.7 10 51.4 One 30.8 Polyphenol Fraction C 100 99.2 0.73 10 86.4 One 49.4 Polyphenol Fraction D 100 97.0 2.0 10 62.6 One 45.0 TMG (tetramethylene glutanoic acid) 10 91.47 0.62 One 62.94 0.1 18.36

Experimental Example 3. Toxicity Test

Toxicity for 24 hours after intraperitoneal administration of the polyphenol fraction A of the present invention at a dose of 20 mg / kg, 10 mg / kg or 1 mg / kg, divided into three groups of 10 mice each with ICR-based mice and Sprague Dawley Whether or not was observed.

As a result of the experiment, no deaths were observed in all three groups, and no symptoms were found in the weight gain and feed intake.

The polyphenol fraction of the present invention can be administered in the following formulations, and the formulation examples below are merely to illustrate the invention, whereby the content of the invention is not limited.

1. Preparation of powder

Polyphenol Fraction A 100mg

Corn Starch 100mg

Lactose 100mg

Talc 10mg

The above ingredients are mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

Polyphenol Fraction A 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components and tableting according to the manufacturing method of the conventional tablet to prepare a tablet.

3. Preparation of Capsule

Polyphenol Fraction A 100mg

Lactose 50mg

1 mg magnesium stearate

The above ingredients are mixed and compressed into tablets according to a conventional method for preparing capsules to fill gelatin capsules.

4. Manufacture of liquid

Polyphenol Fraction A 100mg

10 g of isomerized sugar

10g per book

Lemon flavor

Purified water

According to the conventional method for preparing a liquid solution, each component is added to the purified water, dissolved, and lemon flavor is added, and then purified water is added to adjust the total amount to 100 ml, and then filled into a brown bottle to prepare a liquid solution.

Pine mushroom ethanol acetate soluble extract and polyphenol fractions according to the present invention significantly inhibit the activity of aldose reductase can be effectively used for the prevention and treatment of diabetes diseases.

Claims (5)

The first step of obtaining a crude extract soluble in polar solvent by slicing dried dried Ganoderma applanatum , immersing in a polar solvent such as water, methanol or butanol, and filtration and concentrating under reduced pressure. The second step of sequentially dissolving and suspending with a non-polar solvent, such as hexane, chloroform, methylene chloride, ethyl acetate, and fractionation and filtration with a separatory funnel, and then a polar solvent such as butanol, water, etc. Fraction and filtration using, and the method of producing a non-solvent soluble extract of Jangnabi stool mushroom, characterized in that it comprises a third step of obtaining a polar solvent soluble extract. The non-polar solvent soluble extract obtained by the method of claim 1 is subjected to column chromatography. The silica gel column has a hexane: ethyl acetate: methanol ratio of 1: 1: 1 to 50: 10: 0.5 (w / w). Four small fractions obtained by eluting the developing solvent with a column to obtain 15 fractions, of which polyphenol fractions of fractions 8 and 9 and ethyl acetate soluble extract were eluted with methanol-water by reverse phase chromatography. A method for producing a polyphenol fraction isolated from Janna butterfly stool, characterized in that it comprises a second step of obtaining a polyphenol fraction of the small fractions 2 and 4. The process according to claim 1 or 2, wherein the nonpolar solvent is ethyl acetate. A pharmaceutical composition for the prevention and treatment of diabetic disease, comprising a non-polar solvent soluble extract prepared by the method of claim 1 and containing a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition for the prevention and treatment of diabetic disease, comprising the polyphenol fraction prepared by the method of claim 2 and containing a pharmaceutically acceptable carrier or excipient.