KR20100111632A - Pharmaceutical composition for preventing or treating diabetes or obesity containing compounds derived from stereocaulon alpinum - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing Stereocaulon alpinum extract for preventing or treating diabetes or obesity is provided to suppress tyrosine phosphatase 1B and to ensure anti-diabetes effect. CONSTITUTION: A pharmaceutical composition for preventing or treating diabetes or obesity contains Stereocaulon alpinum extract as an active ingredient. The extract is isolated using water, low alcohol of 1-4 or mixture thereof. The extract contains compounds of chemical formulas 1-3 as active ingredients. A pharmaceutical composition for preventing or treating diabetes or obesity contains the compound selected from compounds of chemical formulas 1-3 as an active ingredient.

Description

Pharmaceutical Composition for Preventing or Treating Diabetes or Obesity Containing Compounds Derived from Stereocaulon alpinum}

The present invention relates to a pharmaceutical composition for preventing or treating diabetes or obesity, and more specifically, to an antarctic lichen, a stereocaulon alpinum extract and a compound isolated from the extract such as lobaric acid as an active ingredient. A pharmaceutical composition for preventing or treating diabetes or obesity, and stereocaulon alpinum ) extract and a functional food containing the compound isolated from the extract.

Lichens are known to produce higher plants and other unique secondary metabolites (Ingolfsdottir, K., Phytochemistry , 61: 729, 2002), and the secondary metabolites produced by these lichens are mostly depside and depsidone ( depsidone) and dibenzfurane, and these compounds are believed to be associated with low growth rates of lichens (Kumar, KCS et. al ., J. Nat . Prod ., 62: 817, 1999; Huneck, S., Naturwissenschaften , 86: 559, 1999). In addition, various biological activities of lichen metabolites, including antibiotics, antimycobacterial, antiviral, analgesic and antipyretic activities, have been identified by the screening process (Ingolfsdottir, K., Phytochemistry , 61: 729, 2002; Kumar, KCS et al ., J. Nat . Prod ., 62: 817, 1999). Therefore, there is a growing interest in the development of drugs using metabolites of lichens.

On the other hand, diabetes is a metabolic disorder including hyperglycemia caused by a deficiency of insulin action, insulin secretion, or both, and is a disease with a high possibility of future vascular complications. Type 1 (insulin dependent) diabetes is caused by beta-cell destruction of the pancreatic islets due to immunity and the absolute deficiency of insulin. Type 2 (insulin-independent) diabetes is produced by insulin, but not in sufficient amounts. It is caused by the body's inability to utilize insulin secreted effectively. In the state of 'insulin resistance' in which the cells of the body do not work effectively, the energy source in the body, especially sugars, is not used well, so the necessary energy is insufficient. Accumulated and eventually discharged into the urine is one of the chronic degenerative diseases that do not heal fundamentally.

The World Health Organization (WHO) and the United Nations (UN) will have about 246 million people with diabetes worldwide by the end of 2007, and deaths from diabetes are increasing year by year. Emphasis is placed on prevention, strict blood sugar control and the prevention of complications. In addition, according to a survey by the Korean Diabetes Association and the Korea Health Insurance Review & Assessment Service, in 2003, there were 401 million diabetic patients in Korea, and in 2030, 7.2 million diabetic patients will be 1 per 7 people. . In particular, the rapid increase in medical expenses is closely related to the explosive increase in the number of diabetics, the continuous increase in complications due to diabetes, and the increase in the average life expectancy of diabetics. As life expectancy changes due to rapid economic development, life expectancy is increasing, while chronic degenerative diseases such as diabetes are increasing.

Types of diabetes mellitus in Korea account for more than 99%, and the incidence of type 1 diabetes is less than about 1%. Type 2 diabetes reported in foreign countries is about 90%, and about 10% It is different from type 1 diabetes. The causes of diabetes are intertwined with various factors, and important factors include heredity (approximately 20% of family history), environment, age (approximately 60% between 40-49 years old), obesity, reduced body resistance, drug abuse And stress stimulation. The pathogenesis of diabetes has not yet been elucidated, but with the exception of a few specific diabetes mellitus (eg, MODY), it is a disease that is caused by a multigenic cause. In other words, the onset of diabetes is complicated by a number of genes and a large number of new genes continue to be identified.

Diabetes mellitus is caused by a variety of mechanisms, so the treatment also has to vary, moreover, the existing conventional treatments alone do not see a satisfactory effect, so a new treatment is required. Diabetes treatment research has been actively developed with the focus on Type 2 diabetes treatment, which accounts for more than 90% of patients with diabetes (Tables 1 and 2).

Current Status of Diabetes-Related Drug Development Company Name Development theme name Development stage Remarks Samjin Pharmaceutical Co., Ltd. Developed curator diabetes treatment with natural products Preclinical New Drug Development Differentiation from Embryonic Stem Cells to Pancreatic Beta Cells quest New Drug Development SK Corporation Diabetes Applications New Drug Development Youyu Co., Ltd. YYGG quest New Drug Development Yuhan Corporation Biochip Applications Improvement, diagnosis Chong Kun Dang Genetically modified human insulin Phases III Improved drug Neomarl Tablet Product Release Improved drug Hanmi Pharmaceutical Co., Ltd. HM 80200 Development Drug substance

Source: 2004-White Paper Pharmaceutical Industry, Korea Health Industry Development Institute, 2004, 12

Insulin secretagogues (pirogliride, linogliride, 2,4-diaminino-5-cyan-bromorididine, incretin, repaglinide, nateglinide), insulin enhancers (troglitazone), insulin resistance enhancers, and insulin-like effects in target tissues Indicating drugs (pirogliride, linogliride, dichloroacetate, insulin lispro, insulin aspart), glucosynthesis inhibitors (lipase inhibitors, carnitine transferase inhibitors, beta antioxidants), drugs that delay carbohydrate absorption (dietary fiber, alphaglucocyte) A lot of researches have been carried out on the (izase inhibitor), amylin analogue (pramlintide).

Some of these are currently on the market, but many are still in experimental or toxicological testing that are insufficient for human use. In particular, fast-acting insulin secretagogues and insulin resistance-improving agents considering biorhythms will be one of the effective methods for the treatment of diabetes mellitus.

In addition, studies on the pathogenesis of diabetes mellitus have been conducted for the past 10 years, presuming that the cause of insulin resistance may be a problem with the insulin receptor, and the direction of the research is shifting toward the insulin signaling system.

Development of New Drugs for Diabetes Treatment Mechanism of action Clinical stage Leading Company L Ⅲ Ⅱ I P α-Glucosidase inhibitor 3 One Bayer, Takeda, Chong Kun Dang Insulin agonist 13 5 11 4 27 Chiron, Eli Lilly, IDEAZymo-Genet ics, Aventis, Novo Nordisk, Akzo Nobel, Biobras, Alkermes, Merk KGaA Glucagon like peptide-1 agonist One 4 One 4 Amylin, Eli Lilly, Novo Nordisk, Restoragen, Zealand Pharmaceuticals β3-Adrenoreceptor agonist One 2 One Dainippon, Asahi Kasei, GlaxoSmithKline Dipeptidyl peptide Ⅳinhibitor 2 One Bristol-Myers Squibb, Novatis Peroxisome proliferator activated receptorα agonist 2 2 6 Novatis, Kyorin, BMS, GlaxoSmithKline Protein tyrosine phosphatase-1B inhibitor One 7 Wyeth, ISIS Pharmaceutical Leptin stimulator One One Amgen, Tularik Melanocortin-4 agonist One Neurocrine Biosciences AMPKstimulant 2 2 One One Andrx, Merck KGaA, Flamel Technologies Peroxisome proliferator activated receptor γagonist 3 4 9 GlaxoSmithKline, Samchundang, BMS, Japan Tabacco, Dr Reddy's, Kyorin

Source: Health Industry Technology Trends, "Recent Trends in Diabetes Treatment," 2003.

The activity of PTP-1B in adipocytes of obese type and non-obese type 2 diabetes mellitus was examined. It increased fold and activity was reported to appear 71%, 88%. In addition, recent experimental results showed that mice knocked out protein tyrosine phosphatase-1B (PTP1B) showed increased insulin sensitivity and resistance to high fat diet. In addition, according to a number of recently published studies, substances that inhibit the activity of PTP-1B may overcome insulin resistance by increasing insulin sensitivity in target cells. In Korea, randomized bulk screening is performed to develop inhibitors of PTP1B activity from tens of thousands of compounds not yet developed as drugs by the Korea Compound Bank.

On the other hand, leptin is released from the adipocytes into the blood and passes through the cerebral blood membrane and acts on receptors in the central nervous system, inhibits food intake, reduces weight, and promotes energy consumption. As new findings suggest that PTP1B modulates leptin activity itself, it is expected to have synergistic effects when PTP1B is used with leptin agonists (Koren, S., Best Pract . Res . Clin . Endocrinol . Metab ., 21: 621, 2007).

Therefore, the importance of inhibitors to PTP-1B is increasing in the development of treatment of obesity and obesity type diabetes, and there is a report on the leading substance recently discovered through the HTS (hight throughput screening). To date, there have been no clinically successful studies of PTP-1B and its inhibitors. However, as shown in Table 3, it is known that many research groups and companies are proceeding with interest.

PTP-1B inhibitor under development Mechanism Drug name Development company step Etc protein tyrosine phosphatase 1B inhibitor Ertiprotafib Wyeth phase Ⅱ Benzenepropanoic acid (discontinued) SIS-113718 ISIS Pharmaace uticals preclinical 2nd-generation antisense PTP-1B inhibitor OC-86839 Ontogen preclinical selective non-peptide inhibitor of PTP-1B PTP1B inhibitor Abbott preclinical phosphatase-1B inhibitor PTP1B inhibitor Array BioPharma preclinical PTP-1B inhibitor PTP1B inhibitor Structural Bioinformatics preclinical orally-active selective PTP-1B inhibitor PTP1B inhibitor Kaken Pharmaceutical preclinical orally-active PTPase inhibitor

Source: Pharmaproject, 2002

However, most of the inhibitors of PTP1B have been difficult to selectivity and bioavailability because they have been developed as non-hydrolyzable phosphotyrosine mimetics that target active sites of positively charged PTP1B (Liu, S. et al ., J. Am . Chem . Soc ., 130: 17075, 2008).

Accordingly, the present inventors have made diligent efforts to develop an effective therapeutic agent for the treatment of diabetes and obesity. As a result, the Antarctic lichen stereocaulon alpinum ( Stereocaulon) alpinum ) extracts and compounds isolated from the extracts confirmed that effectively inhibit PTP1B, stereocaulon ( Stereocaulon alpinum ) was isolated from the compound having a PTP1B inhibitory activity and the structure was identified, and confirmed that the anti-diabetic effect when the disease model animal administration, and completed the present invention.

An object of the present invention is stereocaulon ( Stereocaulon) alpinum ) extract or a compound isolated from the extract as an active ingredient to provide a pharmaceutical composition and functional food for the prevention or treatment of diabetes or obesity.

In order to achieve the above object, the present invention is a stereocaulon ( Stereocaulon) alpinum ) provides a pharmaceutical composition and functional food for the prevention or treatment of diabetes or obesity containing the extract as an active ingredient.

The present invention also provides a pharmaceutical composition and functional food for preventing or treating diabetes or obesity containing a compound selected from the group consisting of compounds represented by Formulas 1 to 3 as an active ingredient.

[Formula 1]

Here, R ₁ , R ₂ , R ₃ are each independently H, C ₁ ~ C ₄ Alkyl, OH And H ₃ CO)

[Formula 2]

Where R ₁ , R ₂ , R ₃ , R ₄ is each independently selected from the group consisting of H, C ₁ to C ₄ alkyl, OH, COOH and H ₃ CO)

(3)

(Wherein R ₁ , R ₂ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO)

The present invention also provides a pharmaceutical composition and a functional food for preventing or treating diabetes or obesity containing the compound 8 or 9 as an active ingredient.

[Compound 8]

[Compound 9]

The invention also provides a process for the preparation of a compound selected from the group consisting of the following compounds 1-4 comprising the following steps:

(a) extracting Stereocaulon alpinum with methanol;

(b) Stereocaulon obtained in step (a) alpinum ) eluting the extract in an aqueous methanol solution using column chromatography; And

(c) eluting the fraction eluted in step (b) with acetonitrile (CH ₃ CN) aqueous solution using reverse phase high performance liquid chromatography to obtain a fraction containing a compound selected from the group consisting of compounds 1-4. .

[Compound 1]

[Compound 2]

[Compound 3]

[Compound 4]

The present invention also provides a process for preparing the following compound 8 or 9 comprising the following steps:

(a) adding a hexane solution containing trimethylsilyldiazomethane (TMSCHN ₂ ) to a methanol solution containing Compound 1 and mixing; And

(b) injecting into semi-preparative reverse-phase HPLC, and then eluting with methanol (MeOH) solution with a concentration gradient to obtain a fraction containing compound 8 or 9.

[Compound 8]

[Compound 9]

The present invention also provides compounds represented by the following compounds 2-3 or 8-9.

[Compound 2]

[Compound 3]

[Compound 8]

[Compound 9]

Stereocaulon according to the present invention ( Stereocaulon alpinum ) extract and the compound isolated from the extract is excellent in the inhibitory activity of the protein tyrosine phosphatase 1B, it is effective in the prevention or treatment of diabetes and obesity.

1 shows a nonlinear regression graph and a Lineweaver-Burke graph of a compound represented by Chemical Formula 1. FIG.
Figure 2 shows a nonlinear regression graph and a Lineweaver-Burke graph of the compound represented by the formula (2).
3 is a graph showing the kinetics of PTP-1B according to the amount of compound 1 (Lobaric acid), showing Vmax (A) and Km (B).
4 is treated with compound 1 (Lobaric acid) to PTP1B, and the degree of phosphorylation of the insulin receptor was verified by Western blot.
5 is a graph measuring the change of Glut 4 and Glut 2 according to compound 1 (Lobaric acid) treatment.
Figure 6 is a graph measuring the blood glucose change after the compound 1 (Lobaric acid) intraperitoneal administration.
Figure 7 is a graph of the results of blood glucose measurement after 6 hours fasting according to Compound 1 (Lobaric acid) intraperitoneal administration.
8 is a graph measuring the change in weight according to compound 1 (Lobaric acid) treatment.
FIG. 9 is a graph showing changes in blood glucose levels of Group (A) not administered Compound 1 (B) and Group (B) administered Compound 1 daily for 27 days.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, the extract of Stereocaulon alpinum has excellent activity of inhibiting the activity of PTP1B, it was confirmed that it is effective in the prevention or treatment of diabetes or obesity. Thus, the present invention in one aspect, Stereocaulon ( Stereocaulon alpinum ) relates to a pharmaceutical composition for preventing or treating diabetes or obesity containing the extract as an active ingredient. In addition, the stereocaulon Alpinum according to the present invention ( Stereocaulon alpinum ) extract may be provided as a functional food containing it as an active ingredient, and thus, in another aspect, the present invention provides a stereocaulon alpinum ) relates to a functional food for preventing or improving diabetes or obesity containing the extract as an active ingredient.

In one aspect of the invention, lichen stereocaulon ( Stereocaulon alpinum (Hedw.) GL Sm.) was collected from Barton Peninsular around King Sejong Station (S 62 ° 13.3 ', W58 ° 47.0') on King George Island in Antarctica in January 2003.

In the present invention, the extract may be characterized in that the extract of the solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms and mixtures thereof.

In the present invention, the extract may be characterized in that it contains at least one compound selected from the group consisting of compounds represented by Formulas 1 to 3 as an active ingredient.

In one embodiment of the present invention Stereocaulon ( Stereocaulon alpinum ) The extract was extracted with dried Stereocaulon alpinum with methanol for 24 hours, the solvent was distilled to obtain an extract, and the extract was flash column chromatography filled with silica gel (C ₁₈ ). , 5 × 25 cm), followed by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% (v / v) methanol (MeOH) Injection was carried out and each fraction was obtained to confirm PTP1B inhibitory activity.

Meanwhile, in one embodiment of the present invention, a compound having excellent PTP1B inhibitory activity was isolated by selecting a fraction having excellent PTP1B inhibitory activity among the fractions. Therefore, in another aspect, the present invention relates to a pharmaceutical composition for preventing or treating diabetes or obesity, which contains one or more compounds selected from the group consisting of compounds represented by the following Chemical Formulas 1 to 3 as an active ingredient. In addition, at least one compound selected from the group consisting of compounds represented by the following Chemical Formulas 1 to 3 according to the present invention may be provided as a functional food containing the same as an active ingredient, and thus the present invention provides the following Chemical Formula 1 It relates to a functional food for the prevention or improvement of diabetes or obesity containing at least one compound selected from the group consisting of the compound represented by ~ 3 as an active ingredient.

[Formula 1]

(Wherein R ₁ , R ₂ , R ₃ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO)

[Formula 2]

Where R ₁ , R ₂ , R ₃ , R ₄ is each independently selected from the group consisting of H, C ₁ to C ₄ alkyl, OH, COOH and H ₃ CO)

(3)

(Wherein R ₁ , R ₂ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO)

In the present invention, a preferred example of Formula 1 is R ₁ And Compound ₂ wherein R ₂ is OH and R ₃ is H ₃ CO, but is not limited thereto.

[Compound 1]

.

.

The compound 1 is a stereo cowl theory to the over (Stereocaulon alpinum) Alfie extracted with methanol, to obtain the stereo cowl Rhone Alfie over (Stereocaulon alpinum ) extract can be obtained by eluting with methanol solution using column chromatography, and eluted fractions with acetonitrile (CH ₃ CN) solution using reversed phase high performance liquid chromatography, a compound known as lobaric acid. .

The lobaric acid is the inhibitory activity of antimycobacterial and 5- and 12-lipoxygenases (Ingolfsdottir, K. et al ., Eur , J. Pharm . Sci . 6: 141, 1998; Bucar, F. et al ., Phytomedicine, 11: 602, 2004), but has not been reported on the inhibitory activity of PTP1B, and has not been reported for the treatment of diabetes and obesity, the lobaric acid of the present invention It is the first to reveal the inhibitory activity of PTP1B.

In the present invention, a preferred example of Formula 2 is R ₁ is H, R ₂ is OH, R ₃ and R ₄ is H ₃ CO and 2 and R ₁ is COOH, R ₂ is OH, R ₃ and R ₄ may be a compound 3 which is H ₃ CO, but is not limited thereto.

[Compound 2]

.

.

[Compound 3]

.

.

Compounds 2 and 3 may be obtained by the same method as Compound 1 above.

In the present invention, a preferred example of Formula 3 may be characterized in that R ₁ is H ₃ CO, R ₂ is OH compound 4, but is not limited thereto.

[Compound 4]

.

.

 Compound 4 may be obtained by the same method as Compound 1 above.

Compound 4, a compound known as atranorin, has been reported for antifungal activity (Athukoralage, PS et. al ., Fitoterapia , 72: 565, 2001), the inhibitory activity of PTP1B has not been reported, and there has been no report on the therapeutic effect of diabetes and obesity, and thus, the present invention discloses the inhibitory activity of atranorin's PTP1B for the first time.

Meanwhile, Compounds 8 and 9 were added to a methanol solution containing Compound 1, mixed with hexane solution containing trimethylsilyldiazomethane (TMSCHN ₂ ), mixed, and injected into semi-preparative reverse-phase HPLC. , Aqueous methanol (MeOH) solution can be obtained by eluting with a concentration gradient.

[Compound 8]

[Compound 9]

In the present invention, compound 8 is a substance prepared by replacing the COOH group of compound 1 with a methyl ester group, compound 9 is a substance prepared by replacing the OH group of the phenol ring of compound 8 with a methoxy group. In one embodiment of the present invention it was confirmed that compounds 8 and 9 have a PTP1B inhibitory activity of a superior or similar degree compared to Ulsolic acid (Ursolic acid) known as a PTP1B inhibitor. Therefore, in another aspect, the present invention relates to a pharmaceutical composition for preventing or treating diabetes or obesity containing the following compound 8 or 9 as an active ingredient. In addition, the following compound 8 or 9 according to the present invention may be provided as a functional food containing it as an active ingredient, and thus the present invention, in another aspect, prevention of diabetes or obesity containing the following compound 8 or 9 as an active ingredient Or to a functional food for improvement.

[Compound 8]

[Compound 9]

The following compounds 2, 3, 8, and 9 isolated from the present invention are novel compounds, and thus the present invention relates to compounds represented by any one of the following compounds 2, 3, 8, and 9 from another aspect.

[Compound 2]

[Compound 3]

[Compound 8]

[Compound 9]

In the present invention, liposome metabolites that inhibit PTP1B derived from methanol extract ( Sterile degree of inhibition of 90% or more, 30 µg / ml) of Stereocaulon alpinum , Compound 1 is depsidone, Compounds 2 and 3 are pseudodepsidone , Compound 4 belongs to the depside system.

In one embodiment of the present invention, as a result of comparing the inhibitory activity of PTP1B of compounds 1-9 with Ulsolic acid, a PTP1B inhibitor, the IC ₅₀ of Ulsolic acid is 3.08 μM, compound 1 showed the most excellent effect to inhibit PTP1B IC ₅₀ = 0.87μM, compound 2 and compound 3 showed a better or higher PTP1B inhibitory effect than PTP1B so similar to each IC ₅₀ = 6.86μM and IC ₅₀ = 2.48μM . In contrast, compound 4 showed low inhibitory ability with IC ₅₀ = 63.5 μM. In addition, more In order to examine the importance of the acid contained in the respective compounds in the PTP1B inhibitory effect of confirming the PTP1B inhibitory ability of the compound 8 converts the carboxyl group of the compound 1-methyl ester result, as IC ₅₀ = 3.02μM of Compound 81 The carboxyl group was found to be an important factor for the inhibitory activity, and the inhibitory activity of PTP1B of compound 9, which converted the phenol hydroxyl group of compound 8 to the methoxy group, was found to be IC ₅₀ = 7.42 μM, indicating a carboxyl group and a phenol hydroxyl group. Confirmed that it is involved. As a result of confirming the PTP1B inhibitory activity of compounds 1 to 9, it was confirmed that compounds 1 to 4, compound 8 and compound 9 is a substance capable of pharmaceutical treatment and prevention of diabetes and obesity.

In another embodiment of the present invention, compound 1, ie, lobaric acid, is injected into a db / db mouse, which is a disease animal model, to measure changes in dietary intake, weight change, blood glucose concentration, and insulin concentration by measuring body weight, blood sugar, and insulin. The antidiabetic effect was verified by checking the correlation with resistance.

The pharmaceutical compositions comprising the compounds according to the invention, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, external preparations, suppositories, and sterile injectable solutions according to conventional methods. Can be formulated and used. Carriers, excipients, and diluents that 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 include at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 60, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.1 to 1000 µg / kg, preferably at 1 to 100 µg / kg. Administration may be administered once a day or may be divided orally. The dosage does not limit the scope of the invention in any aspect.

The functional food of the present invention includes various foods, candy, chocolate, beverages, gums, teas, vitamin complexes, health supplements, and the like, and can be used in the form of powders, granules, tablets, capsules or beverages.

The extract of the present invention may be added to food or beverages for the purpose of preventing diabetes and obesity. At this time, the amount of the extract in the food or beverage is generally added to the health functional food composition of the present invention 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total food weight, the health beverage composition is 100 ml It can be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g as a reference.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of natural carbohydrates are monosaccharides such as glucose, fructose and the like, disaccharides such as maltose, sucrose and the like, conventional sugars such as polysaccharides such as dextrin, cyclodextrin and xylitol Sugar alcohols such as sorbitol and erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention, in addition to the above, the functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors. Flavoring agents, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks Carbonic acid used, etc. The functional food of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. It may be used either individually or in combination. It is common ratio of the additive is selected from the range of the composition per 100 parts by weight 0 to about 20 parts by weight of the present invention, but not so important.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Lichen Stereo cowlon Alpinum ( Stereocaulon alpinum ) Preparation of Extract

Lichen Stereocaulon alpinum (Hedw.) GL Sm.) Was collected at Barton Peninsular around King Sejong Station (S 62 ° 13.3 ', W58 ° 47.0') in King George Island, Antarctica, in January 2003. It is lichen that can be done.

Dried Stereocaulon alpinum ) 50 g was extracted twice with 1 L of methanol for 24 hours to obtain 3.6 g of methanol extract. The obtained extract was loaded into flash column chromatography (5 × 25 cm) filled with silica gel (C ₁₈ ), 10%, 20%, 30%, 40%, 50%, 60%, 70 Concentration gradients of%, 80%, 90% and 100% (v / v) methanol (MeOH) yielded each fraction. Methanol extract inhibited the activity of PTP1B by more than 90% at a concentration of 30 μg / ml.

At this time, the protein tyrosine dephosphorylase 1B (PTP1B) was purchased from BIOMOL (USA) and used in the experiments, the protein tyrosine dephosphorase of about 0.2 ㎍ / ㎖ concentration spectroscopically to measure the enzyme activity 1B, protein tyrosine dephosphatase 1B buffer (50 mM citrate, pH 6.0, 0.1 M NaCl, 1 mM EDTA, 1 mM DTT), inhibitor (methanol extract), 4 mM pNPP was added and gently shaken for 30 minutes After the reaction at 37 ℃ absorbance was measured at 405 nm.

Lichen Stereo cowlon Alpinum ( Stereocaulon alpinum ) From the extract lobaric  Preparation of Acid (Compound 1)

204.6 mg of the fraction obtained by elution with 80% methanol obtained in Example 1 was again loaded on flash column chromatography (2.5 × 30 cm) filled with silica gel (C ₁₈ ), and obtained by TLC analysis. 200 ml of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% methanol (in CH ₂ Cl ₂ ), respectively, to obtain eight major fractions. Solution and 100% (v / v) methanol were injected to give each fraction.

After injecting 59 mg of the fraction eluted with 9% methanol into semi-preparative reverse-phase HPLC, acetonitrile (CH ₃ CN) aqueous solution containing 0.1% formic acid was 75 to 83 Compound 1 was isolated by elution for at least 30 minutes giving a concentration gradient of% (22.9 mg; t _R = 39 minutes).

As a result of NMS and MS analysis, compound 1 was found to be lobaric acid, and lobaric acid was found to exhibit inhibitory activity of antimycobacteria and 5- and 12-lipoxygenases, but there was no report on the inhibitory activity of PTP1B. (Ingolfsdottir, K. et al ., Eur , J. Pharm . Sci . 6: 141, 1998; Bucar, F. et al ., Phytomedicine, 11: 602, 2004).

[Compound 1]

Results of the NMS and MS are as _{^{follows: 1 H NMR (DMSO- d 6}} , 400 MHz) d 6.98 (d, J = 2.2 Hz, H-3), 7.11 (d, J = 2.2 Hz, H-5) , 2.86 (t, J = 7.3, H ₂ -9), 1.15 1.60 (m, H ₂ -10 and 11), 0.89 (t, J = 6.9 Hz, H ₃ -12), 3.91 (s, OCH _3- 3), 6.65 (br s, H-3 '), 2.90 (m, H ₂ -8'), 1.15-1.60 (m, H ₂ -9 ', 10' and 11 '), 0.88 (t, J = _{6.9 Hz, H 3 -12 ')} ; _{^{13 C NMR (DMSO- d 6,}} 100 MHz) d 111.6 (C-1), 163.3 (C-2), 106.7 (C-3), 164.6 (C-4), 112.0 (C-5), 140.8 ( C-6), 162.7 (C-7), 203.7 (C-8), 41.6 (C-9), 25.9 (C-10), 22.5 (C-11), 14.4 (C-12), 57.0 (OCH ₃ -3), 120.1 (C-1 '), 155.1 (C-2'), 106.2 (C-3 '), 149.0 (C-4'), 144.9 (C-5 '), 135.3 (C-6 '), 169.0 (C-7'), 31.9 (C-8 '), 31.0 (C-9'), 27.6 (C-10 '), 22.0 (C-11'), 14.39 (C-12 ') ; ESIMS m / z 455 [M−H] ⁻ .

Lichen Stereo cowlon Alpinum ( Stereocaulon alpinum ) Pseudodepsidone-type from extracts metabolite (Compound 2), methyl orsellinate (Compound 5), methyl haematommate (Compound 6) and 2 ', 6'- dihydroxy -4' methoxy Preparation of -3'-methylacetophenone (Compound 7)

150.2 mg of the fraction eluted with 70% methanol obtained in Example 1 and 260.7 mg of the fraction eluted with 90% methanol were mixed, followed by flash column chromatography (2.5 × 30 cm) filled with silica gel (C ₁₈ ). ), Each of CH ₂ Cl ₂ to obtain seven major eluents. Each fraction of (in MeOH) solution was obtained.

In addition, 22.8 mg of the fraction eluted with 1% methanol was injected into a semi-preparative reverse-phase HPLC, followed by acetonitrile (CH ₃ CN) aqueous solution containing 0.1% formic acid. Compound 2 (1.0 mg; t _R = 42.1 min), Compound 5 (1.5 mg; t _R = 19.4 min), Compound 6 (3.2 mg; t _R = 33.3) eluting for at least 46 minutes with a concentration gradient of 40-86% Min) and compound 7 (1.0 mg; t _R = 25.1 min) were separated.

As a result of NMS and MS analysis, it was confirmed that Compound 2 is pseudodepsidone, Compound 5 is methyl orsellinate, Compound 6 is methyl haematommate, and Compound 7 is 2 ', 6'-dihydroxy-4'methoxy-3'-methylacetophenone.

[Compound 2]

The results of NMS and MS are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) d 6.16 (d, J = 2.0 Hz, H-3), 6.54 (d, J = 2.0 Hz, H-5), 2.16 (m, H-9), 1.96 (m, H-9), 1.35 1.53 (m, H ₂ -10), 1.27-1.39 (m, H ₂ -11), 0.86 (t, J = 6.6, H ₃ -12), 3.77 (s, OCH ₃ -4), 3.13 (s, OCH ₃ -8), 6.40 (d, J = 2.7, H-3 '), 6.30 (d, J = 2.7, H-5' ), 2.35 (t, J = 8.1, H ₂ -7 '), 1.44-1.56 (m, H ₂ -8'), 1.13-1.23 (m, H ₂ -9 '), 1.11 1.21 (m, H ₂ -10 '), 0.78 (t, J = 6.2 Hz, H 3 -11'); ¹³ C NMR (CDCl ₃ , 100 MHz) d 108.4 (C-1), 157.9 (C-2), 102.4 (C-3), 167.3 (C-4), 100.8 (C-5), 151.6 (C- 6), 166.4 (C-7), 109.5 (C-8), 38.3 (C-9), 25.2 (C-10), 22.7 (C-11), 14.0 (C-12), 56.2 (OCH _3- 4), 51.1 (OCH ₃ -8), 133.2 (C-1 '), 149.3 (C-2'), 102.2 (C-3 '), 154.0 (C-4'), 108.5 (C-5 ') , 137.1 (C-6 '), 30.1 (C-7'), 29.3 (C-8 '), 31.7 (C-9'), 22.4 (C-10 '), 13.9 (C-11'); ESIMS m / z 443 [M−H] ⁻ .

[Compound 5]

The results of NMS and MS are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) d 6.27 (d, J = 2.6, H-3), 6.22 (d, J = 2.6, H-5), 2.48 (s _{, H 3 -8), 11.73 (} s, OH-2), 3.95 (s, OCH 3 -7); ¹³ C NMR (CDCl ₃ , 100 MHz) d 105.8 (C-1), 160.3 (C-2), 101.4 (C-3), 165.5 (C-4), 111.3 (C-5), 144.1 (C- 6), 172.1 (C-7), 24.4 (C-8), 52.0 (OCH _3-7 ); ESIMS m / z 183 [M−H] ⁺ .

[Compound 6]

The results of NMS and MS are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) d 6.29 (s, H-5), 2.52 (s, H ₃ -8), 10.34 (s, CHO-3), 12.88 (s, OH-2), 12.41 (s, OH-4), 3.95 (s, OCH _3-7 ); ESIMS m / z 209 [M−H] ⁻ .

[Compound 7]

The results of NMS and MS are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) d 6.20 (s, H-5), 2.10 (s, H ₃ -8), 2.45 (s, H ₃ -9), 12.03 (s, OH-6), 3.91 (OCH _3-4 ); ESIMS m / z 195 [M−H] ⁻ .

Lichen Stereo cowlon Alpinum ( Stereocaulon alpinum ) Pseudodepsidone-type from extracts metabolite Preparation of (Compound 3)

57.5 mg of the fraction eluted with 20% methanol obtained in Example 1 and 57.5 mg of the fraction eluted with 40% methanol were again injected into semi-preparative reverse-phase HPLC, followed by 0.2% formic acid. acetonitrile (CH ₃ CN) aqueous solution containing acid) was eluted for at least 33 minutes with a concentration gradient of 55-73% (6.3 mg; t _R = 27 minutes).

As a result of analysis of NMS and MS, Compound 3 was confirmed to be pseudodepsidone-type metabolite.

[Compound 3]

The results of NMS and MS are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) d 6.11 (br s, H-3), 6.53 (br s, H-5), 2.17 (m, H-9), 1.96 (m, H-9) , 1.35-1.53 (m, H 2 -10), 1.22-1.38 (m, H 2 -11), 0.88 (t, J = 6.6, H 3 -12), 3.77 (s , OCH ₃ -4), 3.18 (s, OCH ₃ -8), 6.55 (br s, H-3 '), 2.70 (m, H ₂ -8'), 1.21-1.42 (m, H ₂ -9 ' ), 1.01 1.23 (m, H 2 -10 '), 1.04-1.17 (m, H 2 -11'), 0.69 (t, J = 6.2 Hz, H 3 -12 '); ¹³ C NMR (CDCl ₃ , 100 MHz) d 108.3 (C-1), 157.8 (C-2), 102.1 (C-3), 167.3 (C-4), 100.8 (C-5), 151.7 (C- 6), 167.4 (C-7), 110.0 (C-8), 38.4 (C-9), 25.1 (C-10), 22.6 (C-11), 13.9 (C-12), 56.2 (OCH _3- 4), 51.3 (OCH ₃ -8), 102.9 (C-1 '), 156.1 (C-2'), 104.1 (C-3 '), 154.8 (C-4'), 133.1 (C-5 ') , 141.1 (C-6 '), 163.9 (C-7'), 28.2 (C-8 '), 30.8 (C-9'), 32.2 (C-10 '), 22.1 (C-11'), 13.9 (C-12 '); ESIMS m / z 487 [M−H] ⁻ .

Lichen Stereo cowlon Alpinum ( Stereocaulon alpinum ) Preparation of atranorin (Compound 4) from Extracts

833.2 mg of the fraction eluted with 100% methanol obtained in Example 1 was again loaded into flash column chromatography (2.5 × 30 cm) filled with silica gel (C ₁₈ ), followed by hexane (in CH ₂ Cl _2). Eluting with 50% hexane solution gave 50% hexane fraction.

After injecting 10.7 mg of the 50% hexane fraction into a semi-preparative reverse-phase HPLC, the aqueous solution of acetonitrile (CH ₃ CN) containing 0.1% formic acid (CH ₃ CN) was 75 to 91%. Compound 4 was isolated by elution for at least 26 minutes with a gradient of (13.2 mg; t _R = 24 min).

The analysis of NMS and MS confirmed that compound 4 was atranorin, and there was a report on the antifungal activity of atranorin, but there was no report on the inhibitory activity of PTP1B (Athukoralage, PS et. al ., Fitoterapia , 72: 565, 2001).

[Compound 4]

Results of the NMS and MS are as _{^{follows: 1 H NMR (CDCl 3,}} 400 MHz) d 6.40 (s, H-5), 10.35 (s, H-8), 2.68 (s, H 3 -9), 12.50 (s, OH-2), 12.55 (OH-4), 6.51 (s, H-6 '), 2.07 (s, H 3 -7'), 2.53 (s, H 3 -9 '), 11.95 (OH -3 '), 3.98 (s, OCH 3 -8'); ¹³ C NMR (CDCl ₃ , 100 MHz) d 102.9 (C-1), 169.1 (C-2), 108.6 (C-3), 167.6 (C-4), 112.9 (C-5), 152.5 (C- 6), 169.8 (C-7), 193.9 (C-8), 25.7 (C-9), 151.9 (C-1 '), 116.9 (C-2'), 162.9 (C-3 '), 110.4 ( C-4 '), 140.0 (C-5'), 116.1 (C-6 '), 9.5 (C-7'), 172.3 (C-8 '), 24.1 (C-9'), 52.4 (OCH ₃ -8'); ESIMS m / z 373 [M−H] ⁻ .

Preparation of Compound 8 and Compound 9 from Compound 1

6-1: Preparation of Compound 8

To a 2 ml methanol solution containing 2.6 mg of compound 1, 20 μl hexane containing ₂ M trimethylsilyldiazomethane (TMSCHN ₂ ) is added, rotated for 24 hours, mixed and semi-preparative reverse-phase After injecting into HPLC, Compound 8 was isolated by elution of methanol (MeOH) solution containing 0.1% formic acid over 80 minutes with a concentration gradient of 80-95% (0.3 mg; t _R = 28.7 minutes). ).

[Compound 8]

The results of NMS and MS are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) d 6.72 (d, J = 2.2 Hz, H-3), 6.73 (d, J = 2.2 Hz, H-5), 2.77 (t, J = 7.3, H ₂ -9), 1.35-1.80 (m, H ₂ -10 and 11), 3.89 (s, OCH ₃ -4), 0.93 (t, J = 6.9 Hz, H ₃ -12 ), 6.79 (br s, H-3 '), 3.14 (m, H ₂ -8'), 1.35-1.80 (m, H ₂ -9 ') 10', and 11 '), 0.94 (t, J = _{6.9 Hz, H 3 -12 ')} , 11.18 (s, OH-2'), 3.97 (s, OCH 3 -7 '); ESIMS m / z 469 [M-H] ⁻ .

6-2: Preparation of Compound 9

100 μl hexane containing 2M trimethylsilyldiazomethane (TMSCHN ₂ ) was added to a 2 ml methanol solution containing 1.5 mg of compound 1, mixed for 3 hours, mixed, and semi-preparative reverse-phase. After injection into HPLC, Compound 9 was isolated by elution of aqueous methanol (MeOH) solution containing 0.1% formic acid over 30 minutes with a concentration gradient of 80-95% (0.5 mg; t _R = 30.7 minutes). ).

[Compound 9]

The results of NMS and MS are as follows: ¹ H NMR (CDCl ₃ , 400 MHz) d 6.700 (d, J = 2.2 Hz, H-3), 6.704 (d, J = 2.2 Hz, H-5), 2.76 (t, J = 7.3, H ₂ -9), 1.34-1.80 (m, H ₂ -10 and 11), 0.92 (t, J = 6.9 Hz, H ₃ -12), 3.89 (s, OCH ₃ -4 ), 6.72 (br s, H-3 '), 2.72 (m, H ₂ -8'), 1.34-1.80 (m, H ₂ -9 '), 10' and 11 '), 0.91 (t, J = _{6.9 Hz, H 3 -12 ')} , 3.78 (OCH 3 -2'), 3.87 (OCH 3 -7 '); ESIMS m / z 507 [M−H] ⁻ .

Stereo cowlon Alpinum ( Stereocaulon alpinum Of compounds derived from PTP1B  Inhibitory Activity Assay

In order to analyze the protein tyrosine dephosphatase 1B (PTP1B) inhibitory activity, as in Example 1, protein tyrosine dephosphorase 1B (PTP1B) was purchased from BIOMOL (USA) was used in the experiment, spectroscopic Protein tyrosine dephosphatase 1B, protein tyrosine dephosphatase 1B buffer solution (50 mM citrate, pH 6.0, 0.1 M NaCl, 1 mM EDTA, 1 mM DTT), inhibitor (Compound 1-9 or Ulsoric acid as a control), 4 mM pNPP was added and gently shaken, and then reacted at 37 ° C. for 30 minutes, and the absorbance was measured at 405 nm.

The inhibitory activity of compounds 1-9 against PTP1B was compared with Ulsolic acid, a PTP1B inhibitor, whereas Compound ₅₀ had an IC ₅₀ of 3.08 μM, whereas Compound 1 had an IC ₅₀ = 0.87. showed the best PTP1B inhibitory effect in μM, compound 2 and compound 3, the inhibitory effects against a similar or even better PTP1B hayeoseo wool sorbic acid and compared to a known PTP1B inhibitor in each IC ₅₀ = 6.86μM and IC ₅₀ = 2.48μM Indicated. In contrast, compound 4 showed low inhibitory ability with IC ₅₀ = 63.5 μM. In addition, Compounds 5 to 7 exhibited no inhibitory effect on PTP1B at IC ₅₀ > 200 μM.

In addition, more In order to examine the importance of the acid contained in the respective compounds in the PTP1B inhibitory effect of confirming the PTP1B inhibitory ability of the compound 8 converts the carboxyl group of the compound 1-methyl ester result, as IC ₅₀ = 3.02μM of Compound 81 It appeared low, indicating that the carboxyl group is an important factor for the inhibitory activity. It was confirmed that the degree of PTP1B inhibitory activity was decreased through IC ₅₀ = 7.42 μM of compound 9 in which phenol hydroxyl group of compound 8 was converted to methoxy group. From this, it was confirmed that acidic protons on molecules that provide oxygen bonds in correlation with PTP1B are important in the inhibition mechanism (Table 4).

Inhibitory activity against PTP1B of compounds represented by compounds 1 to 9 compound Inhibitory Activity of PTP1B
(IC ₅₀ = μM) One 0.87 2 6.86 3 2.48 4 63.5 5 ＞ 200 6 ＞ 200 7 ＞ 200 8 3.02 9 7.42 Ulsolic acid 3.08 ※ Ulsoric acid: control

As the concentration of Compound 1 increased, the reaction rate of PTP1B confirmed that Compound 1 inhibited PTP1B, and that Non-linear regression analysis confirmed that Compound 1 was an uncompetitive inhibitor, and Lineweaver-Burke Replotting of the data by transformations once again confirmed that this is a typical model of noncompetitive inhibitors (FIG. 1). In addition, p -nitrophenyl phosphate (p NPP) for use as a substrate decreased the V _max of the Compound (2), the level of PTP1B in the K _m was confirmed that not changed on the experiment (Fig. 2).

Compounds 1 and 2 in the present invention are non-competitive inhibitors of PTP1B, and the two compounds have confirmed the possibility of binding the enzyme-substrate complex and PTP1B to the homozygous site, and the appropriate sensitivity and bioavailability of PTP1B inhibitor are Characterization of non-competitive inhibitors targeting homozygous and active sites for inactive loci.

Compound 1 ( lobaric acid Molecular molecular characteristics of

8-1: Compound 1 PTP1B kinetics

Compound 1 (Lobaric acid), which was found to have the best PTP1B inhibitory activity, was further examined for PTP1B kinetics. The inhibition activity of PTP1B was determined by the method of Example 7.

As a result, as shown in Figure 3, by examining the kinetics of PTP-1B according to the amount of compound 1 (Lobaric acid), Vmax was reduced, Km was kept constant. This, together with the results of Example 7, indicates that Compound 1 is a noncompetitive inhibitor.

8-2: of compound 1 PTB1B  Insulin receptor phosphorylation at treatment

PTP1B was treated with Compound 1 and then phosphorylation of the insulin receptor was measured. Insulin receptor-overexpressed hepatocytes (HepG2) were treated with compound 1 at 0.1, 1, 10, and 100 μM for 6, 12, and 24 hours, and then the change of p-insulin Rβ was measured by Western blot.

As a result, as shown in Figure 4, when the compound 1 (Lobaric acid) to the PTP1B it was observed that the phosphorylation of the insulin receptor increases. This means that the sensitivity of the insulin receptor to insulin (sensitization), through which it can be seen that Compound 1 can inhibit insulin resistance (insulin resistance).

8-3: according to the treatment of compound 1 Glut  4, 2 change measurement

Signaling that occurs when insulin binds to receptors increases the expression of Glucose transporters (Glut2 and Glut4) and transports them to cell membranes, whereby glucose is absorbed into cells, causing blood sugar to fall. Insulin receptor overexpressed hepatocytes (HepG2) was treated with compound 1 at 0.1, 1, 10, 100μM for 6, 12, 24 hours, and the changes of Glut 4, 2 were measured by Western blot.

As a result, as shown in Figure 5, after the treatment of Compound 1 (Lobaric acid) it was confirmed that the expression of Glut 2 and Glut 4 increased. This means that the compound 1 increases the sensitivity of the insulin receptor to insulin (sensitization), it can be seen that through the compound 1 can inhibit the insulin resistance (insulin resistance).

Disease Model Animals  For Compound 1 ( lobaric acid Validation of)

9-1: PTP1B  Changes in blood glucose after intraperitoneal administration of an inhibitor

Doses (expressed in mg / kg body weight of test animals) were determined based on preliminary, potent, toxicological, and data tests.

Seven-week-old male db / db mice (type 2 diabetes model animals, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) were treated with PBS as a control and Compound 1 (Lobaric acid) 10, a inhibitor of PTP1B activity, Blood glucose was measured twice a week before 20 mg / kg intraperitoneal administration.

Changes in blood glucose following intraperitoneal injection of PTP1B inhibitor compound 1 (Lobaric acid) in 7-week-old male db / db mice (type 2 diabetes model animals, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) Same as FIG. 6.

Referring to FIG. 6, in the control group (n = 6), the mean 0 days 439 mg / dL, 3 days 500 mg / dL, 7 days 535 mg / dL, 10 days 561 mg / dL, 14 days 559 mg / dL, 17 days 583 mg / dL and 21 days 568 mg / dL showed a rapid increase in blood glucose.

In contrast, in the experimental group intraperitoneally injected with 10 mg / kg of compound 1 (Lobaric acid) (n = 7), the mean was 0 days 414 mg / dL, 3 days 452 mg / dL, 7 days 482 mg / dL, 10 days 477 mg / dL. , 514 mg / dL at 14 days, 508 mg / dL at 17 days, and 537 mg / dL at 21 days showed lower blood glucose increase than the control group.

In addition, in the experimental group intraperitoneally injected with 20 mg / kg of Compound 1 (Lobaric acid) (n = 7), on average 0 days 442 mg / dL, 3 days 296 mg / dL, 7 days 320 mg / dL, 10 days 297 mg / dL , 263 mg / dL on 14 days, 308 mg / dL on 17 days, 320 mg / dL on 21 days showed significantly lower blood glucose levels than the control group, and 10 mg / kg of Compound 1 (Lobaric acid) was intraperitoneally injected. Compared to the experimental group, the blood glucose increase was significantly lower than that of the experimental group.

9-2: PTP1B  Changes in blood glucose after fasting for 6 hours after intraperitoneal administration of an inhibitor

For more accurate antidiabetic effect on PTP1B inhibitor compound 1 (lobaric acid), blood glucose was measured after fasting for 6 hours after intraperitoneal injection of PTP1B inhibitor compound 1 at 10, 20 mg / kg.

As a result of measuring the change in blood glucose following the intraperitoneal injection of PTP1B activity inhibitor compound 1 (Lobaric acid) in type 2 diabetic animals, as shown in FIG. 7, in the control group (n = 6), the mean was 167 mg / dL on day 0, 3 days of 203 mg / dL, 7 days of 237 mg / dL, 10 days of 345 mg / dL, 14 days of 344 mg / dL, 17 days of 395 mg / dL, 21 days of 401 mg / dL. Could.

Meanwhile, in the experimental group (n = 7) injected with Compound 1 (Lobaric acid) at 10 mg / kg intraperitoneally, average 0 days were 160 mg / dL, 3 days were 163 mg / dL, 7 days were 180 mg / dL, and 10 days were 194 mg / dL. , 226 mg / dL at 14 days, 237 mg / dL at 17 days, and 289 mg / dL at 21 days showed lower blood glucose increase than the control group.

In the experimental group (n = 7) with 20 mg / kg intraperitoneal injection of compound 1, on average 0 days 152 mg / dL, 3 days 151 mg / dL, 7 days 151 mg / dL, 10 days 136 mg / dL, 14 days 136 mg / dL, 17 days 147 mg / dL, 21 days 131 mg / dL showed a significantly lower blood glucose than the control group, and also significantly lower than the experimental group intraperitoneally injected with 10 mg / kg Compound 1 (Lobaric acid) Blood glucose increase was seen.

9-3: PTP1B  Observation of weight change after intraperitoneal administration of an inhibitor

To observe the weight change after intraperitoneal injection of PTP1B activity inhibitor Compound 1 (Lobaric acid), the weight was measured using an animal scale.

As a result of measuring the change in body weight following the intraperitoneal injection of PTP1B activity inhibitor compound 1 (Lobaric acid) in type 2 diabetic animals, as shown in FIG. 8, in control group (n = 6), the mean 0 days were 36.5 g, 3 days 37.1 g, 7 days of 36.7 g, 10 days of 37.5 g, 14 days of 37.2 g, 17 days of 39.8 g, 21 days of 40.5 g, and 10, 20 mg / kg intraperitoneally injected compound 1 (Lobaric acid) n = 7) on average 0 days 36.0, 37.2 g, 3 days 37.0, 37.4 g, 7 days 37.7, 37.4 g, 10 days 38.2, 38.6 g, 14 days 39.9, 38.9 g, 17 days 41.7, 39.9 g, 21 days 42.6, 42.0 g of weight change was possible.

This indicates that there was no significant change in body weight in type 2 diabetic animals treated with PTP1B inhibitor.

9-4: PTP1B  Validation of glucose tolerance test after 28 days of active inhibitor substance treatment

In order to measure the intraperitoneal glucose tolerance test (IPGTT, Intraperitoneal glucose tolerance test) in an animal model of compound 1 (Lobaric acid), the following experiment was performed.

After fasting for 16 hours without drug compound 1 in a 7-week-old type 2 diabetic model animal, glucose (500 mg / ml, administration volume 200 μl) was injected by intraperitoneal injection, followed by 0, 15, 30, Blood glucose changes were observed in blood samples taken from the tail vein at 60, 90 and 120 minutes.

As a result of measuring glucose tolerance according to glucose intraperitoneal administration in type 2 diabetic animals, as shown in FIG. 9A, the change in blood glucose according to glucose administration in all experimental groups was 0 min 169, 199, 197 mg / dL, 15 Min 470, 463, 393 mg / dL, 30 min 462, 440, 440 mg / dL, 60 min 373, 371, 347 mg / dL, 90 min 302, 267, 265 mg / dL, 120 min 227, 217, 207 In mg / dL, there was no change in blood glucose increase trend and blood glucose drop behavior among individuals.

On the other hand, PTP1B activity inhibitor compound 1 (Lobaric acid) (10, 20 mg / kg, administration volume 200 μl) was injected by the method of daily intraperitoneal administration for 27 days, glucose (500 mg / ㎖, A blood glucose change was observed in blood samples taken from the tail vein at 0, 15, 30, 60, 90, and 120 minutes after the injection of 200 μl of the administration volume.

After that, glucose tolerance change according to subcutaneous glucose administration was measured in type 2 diabetic animals. As shown in FIG. 9B, in the case of the control group (physiological saline injection), 0 minutes after administration of glucose 252 mg / dL, 15 PTP1B, showing a rapid hypoglycemic trend and a very slow hypoglycemic behavior, with 730 mg / dL min, 756 mg / dL 30 min, 680 mg / dL 60 min, 584 mg / dL 90 min, 515 mg / dL 120 min Inhibitors Compounds 1 (Lobaric acid) 10, 20 mg / kg, 0 min 242, 194 mg / dL, 15 min 562, 482 mg / dL, 30 min 580, 520 mg / dL, 60 min Drug concentration dependence at 410, 311 mg / dL, 90 minutes 273, 174 mg / dL, 120 minutes 269, 163 mg / dL confirmed the normalization of blood glucose through low and high blood glucose levels.

The results of Examples 1 to 4 of Example 9 show that Lobaric acid of Compound 1 according to the present invention has a very good antidiabetic effect.

The specific parts of the present invention have been described in detail above, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Unsigned

Claims (25)

Stereocaulon alpinum ) Pharmaceutical composition for the prevention or treatment of diabetes or obesity containing the extract as an active ingredient.
The pharmaceutical composition for preventing or treating diabetes or obesity of claim 1, wherein the extract is an extract of a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixtures thereof.
According to claim 1, wherein the extract is a pharmaceutical composition for the prevention or treatment of diabetes or obesity, characterized in that it contains as an active ingredient at least one compound selected from the group consisting of compounds represented by Formula 1 to 3
[Formula 1]

(Wherein R ₁ , R ₂ , R ₃ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO)
(2)

Where R ₁ , R ₂ , R ₃ , R ₄ is each independently selected from the group consisting of H, C ₁ to C ₄ alkyl, OH, COOH and H ₃ CO)
(3)

(Wherein R ₁ , R ₂ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO).
A pharmaceutical composition for preventing or treating diabetes mellitus or obesity containing a compound selected from the group consisting of compounds represented by formulas 1 to 3 as an active ingredient
[Formula 1]

(Wherein R ₁ , R ₂ , R ₃ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO)
(2)

Where R ₁ , R ₂ , R ₃ , R ₄ is each independently selected from the group consisting of H, C ₁ to C ₄ alkyl, OH, COOH and H ₃ CO)
(3)

(Wherein R ₁ , R ₂ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO).
The method according to claim 4, wherein in Formula 1 R ₁ And R ₂ is OH, and R ₃ is H ₃ CO, wherein the pharmaceutical composition for preventing or treating diabetes or obesity.
The pharmaceutical composition for preventing or treating diabetes or obesity of claim 4, wherein in Formula 2, R ₁ is H, R ₂ is OH, and R ₃ and R ₄ are H ₃ CO.
5. The pharmaceutical composition for preventing or treating diabetes or obesity of claim 4, wherein in Formula 2, R ₁ is COOH, R ₂ is OH, and R ₃ and R ₄ are H ₃ CO.
The pharmaceutical composition for preventing or treating diabetes or obesity of claim 4, wherein in Formula 3, R ₁ is H ₃ CO, and R ₂ is OH.
A pharmaceutical composition for preventing or treating diabetes or obesity containing the following Compound 8 as an active ingredient
[Compound 8]

.
A pharmaceutical composition for preventing or treating diabetes or obesity containing the following compound 9 as an active ingredient
[Compound 9]

.
Method for preparing a compound selected from the group consisting of the following compounds 1 to 4 comprising the following steps:
(a) extracting Stereocaulon alpinum with methanol;
(b) Stereocaulon obtained in step (a) alpinum ) eluting the extract in an aqueous methanol solution using column chromatography; And
(c) eluting the fraction eluted in step (b) with an aqueous solution of acetonitrile (CH ₃ CN) using reverse phase high performance liquid chromatography to obtain a fraction containing a compound selected from the group consisting of the following compounds 1-4: step
[Compound 1]

[Compound 2]

[Compound 3]

[Compound 4]

.
Process for the preparation of the following compound 8 or 9 comprising the following steps:
(a) adding a hexane solution containing trimethylsilyldiazomethane (TMSCHN ₂ ) to a methanol solution containing Compound 1 and mixing; And
(b) injecting into semi-preparative reverse-phase HPLC, and then eluting with methanol (MeOH) solution with a concentration gradient to obtain a fraction containing the following compound 8 or 9
[Compound 1]

[Compound 8]

[Compound 9]

.
Compound represented by the following compound 2
[Compound 2]

.
Compound represented by the following compound 3
[Compound 3]

.
Compound represented by the following compound 8
[Compound 8]
.
Compound represented by the following compound 9
[Compound 9]

.
Stereocaulon alpinum ) Functional food for the prevention or improvement of diabetes or obesity containing the extract as an active ingredient.
The functional food according to claim 17, wherein the extract contains at least one compound selected from the group consisting of compounds represented by Formulas 1 to 3 as an active ingredient.
[Formula 1]

(Wherein R ₁ , R ₂ , R ₃ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO)
(2)

Where R ₁ , R ₂ , R ₃ , R ₄ is each independently selected from the group consisting of H, C ₁ to C ₄ alkyl, OH, COOH and H ₃ CO)
(3)

(Wherein R ₁ , R ₂ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO).
Functional food for the prevention or improvement of diabetes or obesity containing a compound selected from the group consisting of compounds represented by the formula 1 to 3 as an active ingredient
[Formula 1]

(Wherein R ₁ , R ₂ , R ₃ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO)
(2)

Where R ₁ , R ₂ , R ₃ , R ₄ is each independently selected from the group consisting of H, C ₁ to C ₄ alkyl, OH, COOH and H ₃ CO)
(3)

(Wherein R ₁ , R ₂ are each independently H, C ₁ -C ₄ alkyl, OH And H ₃ CO).
The method of claim 19, wherein in Formula 1 R ₁ And R ₂ is OH, and R ₃ is H ₃ CO.
The functional food according to claim 19, wherein in Formula 2, R ₁ is H, R ₂ is OH, and R ₃ and R ₄ are H ₃ CO.
The functional food according to claim 19, wherein in Formula 2, R ₁ is COOH, R ₂ is OH, and R ₃ and R ₄ are H ₃ CO.
The functional food according to claim 19, wherein in Formula 3, R ₁ is H ₃ CO, and R ₂ is OH.
Functional food for preventing or improving diabetes or obesity containing the following compound 8 as an active ingredient
[Compound 8]

.
Functional food for preventing or improving diabetes or obesity containing the following compound 9 as an active ingredient
[Compound 9]

.

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