KR100523461B1 - Pharmaceutical composition comprising cerebroside, 5-dihydroergosterol, 2-methoxyfatty acid or cerevisterol isolated from Ganoderma applanatum for treating or preventing diabetes - Google Patents

Abstract

The present invention is a cerebroside, 5-dihydroergosterol isolated from Ganoderma applanatum having a prophylactic and therapeutic activity of diabetes diseases, 5-dihydroergosterol, 2-methoxyfatty acid (2-methoxyfatty It relates to a composition containing at least one compound selected from acid) or cerevisterol compounds.

Since the compound of the present invention has excellent inhibitory activity against aldose reductase, it can prohibit the accumulation of glycation end products and the like to provide a medicine useful for the prevention and treatment of diabetes-related diseases.

Description

Pharmaceutical composition comprising cerebroside, 5-dihydroergosterol, 2- containing cerebroside, 5-dihydroergosterol, 2-methoxyfatty acid or cerevisterol methoxyfatty acid or cerevisterol 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 cerebroside, 5-dihydroergosterol, 2-methoxyfatty acid or cerevisterol compound 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 (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 ergostane-based steroid compounds isolated from Jangnabi stool (Strigina LI et al ., Phytochemistry , 10 , pp2361-2365, 1991: Ripperger and Budzikiewicz, Phytochemistry , 14 , pp2297-2298, 1975: Protiva et al ., Collection Czechoslov.Chem.Commun. , 45 , pp2710-2713, 1980: Kac, D. et al., Phytochemistry , 23 , pp2686-2687, 1984: Gan KH et al ., J. Nat. Prod ., 61, pp1421-1422, 1998) and the free Delrin (friedelin) and a pre Doolin-5-ene (friedoolean-5-en) tree Interface Variable Name cannabinoid (triterpenoid) type compound, palmitic acid (palmitic acid and the like) ( Protiva et al ., Collection Czechoslov.Chem.Commun. , 45 , pp2710-2713, 1980), ganoderenic acid A, AP, F, G, H, I, furanoganoderenic acid, compound B8 (Nishitoba et al ., Phytochemistry , 28 , pp193-197, 1989), applanoxidic acid A, B, C, D (Chairul SM et al ., Phytochemistry , 30 , pp4105-4109, 1991), Flanoxide Acid EH from Hairul SM et al ., Phyt ochemistry , 35 , pp 1305-1308, 1994), 24-methyl-5α-lanosta-25-one (Gan, KH et al. , J. Nat. Proc. , 61 , pp1421-1422, 1998) Lanostane-based triterpenes and the like and heterogalactan (Usui, T. et al ., Carbohydr. Res. , 92 , pp102-114, 1981) and β-D-glucan ( Polysaccharides such as Usui, T. et al ., Carbohydr.Res. , 115 , pp273-280, 1983). Recently, ergosterol, beta-amyrenone, 2-hydroxy-hexacosanoic acid and beta-amyrin acetate acetate), 2,5-dihydroxybenzoic acid, ganoderenic acid A, D, G and ganoderma aldehyde have been reported (Ming, DS et al ., Fitoterapia , 73 , pp147-). 152, 2002), their activity has not been reported.

In this regard, the present inventors confirmed that the inhibitory activity of aldose reductase was excellent in the ethyl acetate fraction and methylene chloride fraction of Jannabi stool mushroom in order to develop a therapeutic agent for diabetes disease using a safe natural agent. The bioassay-guided fraction was performed as a result, and as a result, cerebroside, 5-dihydroergosterol, 2-methoxyfatty acid or isolated from ethylacetate fraction or methylene chloride fraction The present invention was completed by confirming that the cerevisterol compound has excellent therapeutic and prophylactic efficacy in diabetic diseases.

An object of the present invention is diabetes mellitus containing 5-dihydroergosterol, 2-methoxyfatty acid or cerevisterol isolated from a sperrebroside compound isolated from a soluble fraction of Jannabidae mushroom ethylacetate or a soluble fraction of Jannabidae mushroom methylene chloride. It is to provide a pharmaceutical composition effective for the prevention and treatment of diseases.

In accordance with the above object, the present invention provides a method for the prevention and treatment of diabetes diseases containing at least one compound selected from cerebroside, 5-dihydroergosterol, 2-methoxyfatty acid and cerevisterol isolated from Jangnabi It provides a pharmaceutical composition.

The present invention relates to a diabetic disease comprising a cerebroside compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof isolated from the soluble fraction of Jangana stool mushroom ethyl acetate and containing a pharmaceutically acceptable carrier or excipient. It provides a pharmaceutical composition for the prevention and treatment of.

(Ⅰ)

N is an integer of 7 to 9.

The present invention also provides a pharmaceutically acceptable carrier or excipient comprising a 5-dihydroergosterol compound represented by the following structural formula (II) or a pharmaceutically acceptable salt thereof, isolated from the soluble fraction of Jangnabinchi mushroom methylene chloride. Provided are pharmaceutical compositions for the prevention and treatment of diabetes mellitus containing.

(Ⅱ)

The present invention also provides a pharmaceutically acceptable carrier or excipient comprising a 2-methoxy fatty acid compound represented by the following general formula (III) or a pharmaceutically acceptable salt thereof, isolated from the soluble fraction of Jangnabinchi mushroom methylene chloride. Provided are pharmaceutical compositions for the prevention and treatment of diabetes mellitus containing.

(Ⅲ)

N is an integer from 12 to 15.

In addition, the present invention is a diabetic comprising a cerevisterol compound represented by the following structural formula (IV) or a pharmaceutically acceptable salt thereof isolated from the soluble fraction of mite chloride marrow chloride and containing a pharmaceutically acceptable carrier or excipient Provided are pharmaceutical compositions for the prevention and treatment of diseases.

(Ⅳ)

The composition for preventing and treating diabetic diseases of the present invention comprises 0.5 to 50% by weight of the compound relative to the total weight of the composition.

Pharmaceutical compositions comprising the compounds of the present invention as active ingredients can be used for the treatment and prevention of diabetes related diseases.

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. .

In addition, the present invention provides a method for separating a cerebromide compound, 5-dihydroergosterol, 2-methoxyfatty acid or cerevisterol compound, as follows.

Hereinafter, the present invention will be described in detail.

The cerebroside compound, 5-dihydroergosterol, 2-methoxyfatty acid or cerevisterol compound of the present invention is pulverized by drying the dried Janna beetle mushroom, and then about 1 to 15 times the weight, preferably About 5 to 10 times the volume of water, lower alcohol or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 3, 20 to 100 ° C, preferably 40 to Extraction method such as ultrasonic extraction, reflux extraction, etc. for about 0.5 hours to 2 days, preferably about 0.5 hours to 1 day at an extraction temperature of 60 ℃ is repeated once to 5 times, preferably 2 to 5 times Obtaining crude extract which is a soluble extract according to a lower alcohol or a mixed solvent thereof; After the crude extract is suspended in a polar solvent such as water or methanol, it is added with a nonpolar solvent such as hexane, chloroform, methylene chloride, ethyl acetate and the like to about 1 to 100 times, preferably about 1 to 5 times the volume of 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 cerebromide compound from the zanabi sperm 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, water and the like, and obtaining a polar solvent soluble extract;

Subsequently, column chromatography, liquid chromatography, and TLC are performed on three steps of the polar solvent soluble extract, wherein the silica gel column has hexane: ethyl acetate: methanol 1: 1: 1 to 50: 10: 0.5 (w / w). A developing solvent having a ratio, preferably 10: 3: 1, was developed on the column to obtain 15 fractions, and the third fraction was subjected to silica gel column chromatography (1.5 × 10 cm, Merck, ASTM 7729, Germany). ), Eluting with a solvent having a ratio of chloroform: methanol: water 50: 10: 0.5 to 0.5: 10: 50 (w / w), preferably 7: 1: 0.5, wherein the R _f value is 0.51 ( Developing solvent: chloroform: methanol: water = 7: 2: 0.5).

In addition, a process for separating 5-dihydroergosterol, 2-methoxyfatty acid or cerevisterol compound from Janna snail 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, water and the like, and obtaining a polar solvent soluble extract;

Subsequently, two steps of non-polar solvent soluble extracts are subjected to recrystallization, column chromatography, liquid chromatography, and TLC, wherein the silica gel column is eluted with gradient with chloroform: methanol (1, 2, 3, 5, 10, 50, 100). %) was obtained after the 16 fractions, the 6th and 9, respectively carried out by recrystallization of the second fraction with R _f value is 0.91 (hexane: ethyl acetate 1: 10) of 2-methoxy-fatty acid or R _f value is 0.76 5-dihydroergosterol, (hexane: ethyl acetate 1:10), can be obtained, or the 12th fraction is purified by silica gel column chromatography (1.5 × 10 cm, Merck, ASTM 7729, Germany). After eluting a solvent having a ratio of 1: 1, 8: 5, and 5: 8 to 1:10 (w / w), the R _f value was 0.16 (developing solvent: hexane: ethyl acetate = 1: 10). It consists of a fourth step of obtaining levesterol.

Compositions comprising a compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

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

The compositions comprising the compounds according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions according to conventional methods. Carriers, excipients and diluents which may be used in combination with the compound, and which may be included in the composition comprising the compound 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. 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, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. 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 the compound of the present invention may vary depending on the age, sex, and weight of the patient, but generally, an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, may be administered once or several times a day. Can be. The dosage of the compound can also be increased or decreased depending on the route of administration, the severity of the 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 various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention will be 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 Otario, Canada) was chopped and powdered, and 5 liters of methanol solution was added thereto, followed by extracting with warm water seven times at 50 ° C. for 1 hour, and then combining them 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

Suspended 85 g of the crude butterfly extract of Example 1 of the above-mentioned mushrooms in 1.5 L of water, 1.5 liters of hexane was added to the separatory funnel, and fractionated into a hexane insoluble layer (lower layer) and a hexane soluble layer (upper layer). Collected. The same volume of the 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 (15 g), methylene chloride soluble extract (25 g), ethyl acetate soluble extract (30 g) and butanol soluble extract (15 g). .

Example 3 Isolation of Cerebroside Compounds

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 third fraction was separated by silica gel column chromatography (1.5 × 10 cm, Merck, ASTM 7729, Germany) using an eluent (chloroform: methanol: water = 7: 1: 0.5), and TLC. 5 mg of cerebroside having R _f of 0.51 on (developing solvent; chloroform: methanol: water = 7: 2: 0.5) was obtained by ¹ H-NMR, ¹³ C-NMR and (+)-FABMS analysis. It was identified as the side and used as a sample.

(Ⅰ)

Cerebroside

when n = 7, [(2S, 3R, 4E, 8E) -1-O-β-D-glucopyranosyl-3-hydroxy-2-[(R) -2'-hydroxypalmitoyl] amino -9-methyl-4,8-octadecadiene,

when n = 8, (2S, 3R, 4E, 8E) -1-O-β-D-glucopyranosyl-3-hydroxy-2-[(R) -2'-hydroxyheptadecanoyl] amino -9-methyl-4,8-octadecadiene,

when n = 9, (2S, 3R, 4E, 8E) -1-O-β-D-glucopyranosyl-3-hydroxy-2-[(R) -2'-hydroxystearoyl] amino -9-methyl-4,8-octadecadiene.

Melting Point: 155-164 ℃,

¹ H-NMR (300 MHz, pyridine-d ₅ ) d: 0.85 (3H, t, J = 6.9 Hz, CH ₃ ), 1.25, 1.27 [(CH ₂ ) n], 1.60 (3H, br s, 9- CH ₃ ), 2.00 (2H, m, H-10), 2.14 (4H, m, H-6, 7), 3.88 (1H, m, H-5 "), 4.01 (1H, dd, J = 7.5, 9.0 Hz, H-2 "), 4.19 (1H, t, J = 8.7 Hz, H-3"), 4.22 (1H, dd, J = 3.6, 11.4 Hz, H-1a), 4.33 (1H.dd, J = 5.4, 11.7 Hz, H-6 "a), 4.49 (1H, dd, J = 2.4, 11.7 Hz, H-6" b), 4.56 (1H, dd, J = 3.6, 7.2 Hz, H-2 '), 4.69 (1H, dd, J = 5.7, 10.2 Hz, H-1b), 4.77 (1H, m, H-3), 4.81 (1H, m, H-2), 4.90 (1H, d, J = 7.8 Hz, H-1 "), 5.24 (1H, t-like, J = 5.7 Hz, H-8), 5.92 (1H, dt, J = 5.7, 15.3 Hz, H-5), 6.00 (1H, dd, J = 5.4, 15.3 Hz, H-4), 8.33 (1H, d, J = 8.4 Hz, NH);

¹³ C-NMR (75.5 MHz, pyridine-d ₅ ) d: 14.3 (18-CH ₃ ), 16.1 (19-CH ₃ ), 22.9 (C-17), 25.9 (C-4 '), 28.2 (C- 11), 28.3 (C-7), 29.5, 29.6, 29.9, 30.0, 32.1, 33.0 (C-6), 35.6 (C-3 '), 40.0 (C-10), 54.6 (C-2), 62.6 (C-6 "), 70.1 (C-1), 71.5 (C-4"), 72.3 (C-2 '), 72.5 (C-3), 75.1 (C-2 "), 78.4 (C-3 "), 78.5 (C-5"), 105.6 (C-1 "), 124.1 (C-8), 131.9 (C-5), 132.3 (C-4), 135.8 (C-9), 175.6 (C -One');

(+)-FABMS m / z 750 [(M + Na) ⁺ , C ₄₁ H ₇₇ O ₉ N], 764 [(M + Na) ⁺ , C ₄₂ H ₇₉ O ₉ N], 778 [(M + Na ) ⁺ , C ₄₃ H ₈₁ 0 ₉ N], 562 (M-179).

Example 4 Isolation of 5-dihydroergosterol Compounds

25 g of the soluble extract of Jangnabinchi mushroom methylene chloride of Example 2 was eluted with gradient chloroform-methanol (1, 2, 3, 5, 10, 50, 100%) to silica gel column chromatography (10 × 45 cm, Merck) , ASTM 7729, Germany), to obtain 16 small fractions. Of the 16 fractions, the 9th fraction was recrystallized to give 25 mg of 5-dihydroergosterol having R _f of 0.76 on TLC (developing solvent; hexane: ethyl ether = 1: 10), and EI-Mass, ¹ It was identified as 5-dihydroergosterol through H-NMR and ¹³ C-NMR analysis and used as a sample.

5-dihydroergosterol [(22E, 24R) -Ergosta-7,22-diene-3β-ol]

Melting point: 170-3 ℃,

¹ H-NMR (300 MHz, CDCl ₃ ) δ: 0.54 (3H, s, 18-CH ₃ ), 0.80 (3H, s, 19-CH ₃ ), 0.82 (3H, d, J = 6.9 Hz, 26- CH ₃ ), 0.84 (3H, d, J = 6.6 Hz, 27-CH ₃ ), 0.91 (3H, d, J = 6.9 Hz, 28-CH ₃ ), 1.02 (3H, d, J = 6.9 Hz, 21 -CH ₃ ), 3.60 (1H, m, H-3), 5.19 (2H, m, H-22,23);

¹³ C-NMR (75.5 MHz, CDCl ₃ ) δ: 37.1 (C-1), 31.5 (C-2), 71.1 (C-3), 38.0 (C-4), 40.3 (C-5), 29.6 ( C-6), 117.5 (C-7), 139.5 (C-8), 49.5 (C-9), 34.2 (C-10), 21.5 (C-11), 39.5 (C-12), 43.3 (C -13), 43.3 (C-14), 55.1 (C-15), 22.9 (C-16), 56.0 (C-17), 12.1 (C-18), 13.0 (C-19), 40.5 (C- 20), 21.1 (C-21), 135.7 (C-22), 131.9 (C-23), 42.8 (C-24), 33.1 (C-25), 19.9 (C-26), 19.6 (C-27 ), 17.6 (C-28);

EI-MS m / z (rel. Int.,%) 398 [M] ⁺ (16.5), 383 [M-CH ₃ ] ⁺ (7.4), 355 [M-C ₃ H ₇ ] ⁺ (2.5) 300 ( 8.3), 285 [300-CH ₃ ] ⁺ (3.3), 273 [M-SC (C ₉ H ₁₇ )] ⁺ (17.4), 271 [M-(SC + 2H)] ⁺ (54.5), 255 [M -(SC + H ₂ O)] ⁺ (18.2), 229 (9.9), 213 [M-(ring D + H ₂ O)] ⁺ (7.4), 55 (100).

Example 5. Isolation of 2-methoxyfatty acid compound

25 g of the soluble extract of Jangnabinchi mushroom methylene chloride of Example 2 was eluted with gradient chloroform-methanol (1, 2, 3, 5, 10, 50, 100%) to silica gel column chromatography (10 × 45 cm, Merck) , ASTM 7729, Germany), to obtain 16 small fractions. Of the 16 fractions, TLC subjected to recrystallization to sixth fractions; to afford the R _f is 0.91 in 2-methoxy on fatty acid 18mg (developing solvent: hexane: ethyl ether = 1 10), EI-Mass , 1 H 2-methoxy fatty acids were identified and used as samples through -NMR and ¹³ C-NMR analysis.

(Ⅱ)

2-methoxy fatty acids (n = 12-15)

Melting point: 64-65 ℃,

¹ H-NMR (300 MHz, pyridine-d ₅ ) δ: 0.86 (3H, d, J = 6.6 Hz, CH ₃ ), 1.25, 1.30 [each s, (CH ₂ ) _n ], 3.71 (3H, s, 2-OCH ₃ ), 4.55 (1H, doublet of doublets, J = 5.1, 7.4 Hz, H-2);

¹³ C-NMR (75.5 MHz, pyridine-d ₅ ) δ: 175.9 (C-1), 71.2 (C-2), 51.5 (OCH ₃ ), 35.2, 32.1, 30.0, 29.9, 29.7, 29.6, 25.7, 22.9 , 14.3 (CH ₃ ); EI-MS m / z (rel. Int.,%) 412 [M] ⁺ (2-methoxypentacosanoic acid, 0.8), 398 [M] ⁺ (2-methoxylignoceric acid, 5.8), 384 [M] ⁺ (2- methoxytricosanoic acid, 1.5), 370 [M] ⁺ (2-methoxybehenic acid, 2.1), 90 [C ₃ H ₆ O ₃ ] ⁺ (23.1).

Example 6 Separation of cerevisterol Compounds

25 g of the soluble extract of Jangnabinchi mushroom methylene chloride of Example 2 was eluted with gradient chloroform-methanol (1, 2, 3, 5, 10, 50, 100%) to silica gel column chromatography (10 × 45 cm, Merck) , ASTM 7729, Germany), to obtain 16 small fractions. Of the 16 fractions, the 12th fraction was subjected to silica gel column chromatography (1.5 × 10 cm, Merck, ASTM) using an eluent (hexane: ethyl acetate = 10: 1, 8: 5, 5: 8 to 1:10). 7729, Germany), and on a TLC (developing solvent; hexane: ethyl ether = 1: 10), 7 mg of cerevisterol having an R _f of 0.16 was obtained, followed by EI-Mass, ¹ H-NMR and ¹³ C-NMR analysis. It was identified as cerevisterol represented by the following structural formula through was used as a sample.

Cerevisterol [(22 E , 24 R ) -ergosta-7,22-diene-3β, 5α, 7β-triol]

Melting Point: 224-226 ℃,

¹ H-NMR (300 MHz, pyridine-d ₅ ) δ: 0.65 (3H, s, 18-CH ₃ ), 0.84 (3H, d, J = 6.6 Hz, 26-CH ₃ ), 0.85 (3H, d, J = 6.9 Hz, 27-CH ₃ ), 0.94 (3H, d, J = 6.9 Hz, 28-CH ₃ ), 1.05 (3H, d, J = 6.3 Hz, 21-CH ₃ ), 1.54 (3H, s , 19-CH ₃ ), 4.33 (1H, br s, H-6), 4.84 (1H, m, H-3), 5.16 (1H, dd, J = 7.8, 15.3 Hz, H-22), 5.24 ( 1H, d, J = 6.9, 15.3 Hz, H-23), 5.74 (1H, t, J = 2.4 Hz, H-7);

EI-MS m / z (rel. Int.,%) 412 [M-H ₂ O] ⁺ (20.5), 397 [M-H ₂ O-CH ₃ ] ⁺ (6.6), 394 [M-2H ₂ O ] ⁺ (10.6), 382 [M-H ₂ O-2CH ₃ ] ⁺ (11.5), 379 [M-2H ₂ O-CH ₃ ] ⁺ (18.0), 376 [M-3H ₂ O] ⁺ (3.3) , 369 [M-H ₂ O-C ₃ H ₇ ] ⁺ (2.5), 287 [M-H ₂ O-SC (C ₉ H ₁₇ )] ⁺ (2.5), 269 [M-(SC + 2H ₂ O )] ⁺ (14.8), 251 [M-(SC + 3H ₂ 0)] ⁺ (27.9), 107 (29.5), 105 (59.5), 81 (43.4), 69 (100), 55 (94.3).

Experimental Example 1. Measurement of intracellular aldose reductase activity of compounds

In order to measure the intracellular aldose reductase activity of the cerebroside, 5-dihydroergosterol, 2-methoxyfatty acid and cerevisterol compound of the present invention, the following experiment was performed.

In order to prepare the raw material of aldose reductase (AR), the rat's lens was removed from Sprauge-Dawley (SD) for standard weights of 250 to 280 g. Frozen and surface areas of flat rat lens 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 enzymatic activity was calculated from the logarithmic regression line of log concentration plotted against residual activity.

As a result, D-mannitol isolated from soluble fraction of ethyl acetate soluble fraction of Xavier locust mushroom showed little activity, and cerebromide showed activity with IC ₅₀ value of 30 µg / ml (see Table 1). .

Inhibitory Activity of the Compounds Isolated from the Extract of Janna Beetle Mushroom sample Concentration (µg / ml) Inhibition Rate (%) IC ₅₀ (μg / ml) Cerebroside 100 6.85 30.0 50 94.27 10 66.31 5-dihydroergosterol 100 18.90 2-methoxy fatty acid 100 20.73 Cerevisterol 100 40.3 TMG (tetramethylene glutanoic acid) 10 91.47 0.62 One 62.94 0.1 18.36

Experimental Example 2. Toxicity Test

Toxicity for 24 hours after intraperitoneal administration of 20 mg / kg, 10 mg / kg, 1 mg / kg of cerebrose compound of the present invention by dividing ICR mice and Sprague Dawley into three groups of 10 mice each 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.

Cerebroside, 5-dihydroergosterol, 2-methoxy fatty acid and cerevisterol compound isolated from the soluble fraction of Jannabi stool mushroom ethylacetate according to the present invention significantly inhibited the activity of aldose reductase to prevent diabetic diseases. It can be effectively used for prevention and treatment.

Claims (2)

One selected from 5-dihydroergosterol represented by the following formula (II), 2-methoxy fatty acid represented by the following general formula (III), and cerevisterol represented by the following formula (IV) Pharmaceutical composition for the prevention and treatment of diabetic retinopathy containing the above compounds. (Ⅱ) (Ⅲ) N is an integer of 12 to 15 (Ⅳ) delete