KR100957203B1 - Extract of Stereocaulon alpinum Having Inhibitory Effect of Protein Tyrosine Phosphatase 1B Activity and Usnic Acid Derivatives Isolated Therefrom - Google Patents

Abstract

Disclosed are antarctic lichen stereocaulon alpinum extract and a usnic acid derivative isolated therefrom that have an activity inhibiting activity of the protein tyrosine dephosphorylase 1B. Stereocaulon alpinum extract according to the present invention and the usnic acid derivative isolated therefrom have a hypoglycemic effect on hyperglycemia, a major symptom of diabetes.

Stereocaulon alpinum, protein tyrosine dekinase 1B (PTP1B)

Description

Extract of Stereocaulon alpinum Having Inhibitory Effect of Protein Tyrosine Phosphatase 1B Activity and Usnic Acid Derivatives Isolated Therefrom}

The present invention relates to a stereo kaolin alpinum extract having an activity inhibiting activity of the protein tyrosine dephosphatase 1B and a usnic acid derivative isolated therefrom, and more particularly, by inhibiting the activity of the protein tyrosine dephosphatase 1B, diabetes Antarctic lichen Stereocaulon with hypoglycemic effect on hyperglycemia, a major symptom of alpinum ) extract and the Usnic acid derivative isolated therefrom.

Lichens are a symbiotic complex of fungi and algae or cyanobacteria, and are classified into about 17,000 species. It was once proposed to use lichen secondary metabolites as antimicrobial agents or antiherbivore agents. In addition, some lichen extracts have been used in a variety of therapeutics in the private sector, and lichen metabolites include antibiotic agents, antimycobacterial, antiviral, analgesic, antipyretic properties, etc. It has been found that has a variety of biological activities. Therefore, lichen metabolites are of considerable interest as a source of potential pharmacological agents.

On the other hand, diabetes is a chronic wasting disease characterized by glucose metabolic dysfunction due to relative or absolute deficiency of insulin, causing systemic lethargy and lowered resistance, vascular disorders, cerebral infarction, myocardial infarction and other complications. Diabetes mellitus is divided into two types according to its pathogenesis and symptoms. Insulin-dependent type 1 diabetes caused by destruction of beta cells, insulin secreting cells by lymphocyte immunological mechanisms, and the function of beta cells secreting insulin There is type 2 diabetes, which is insulin-independent, caused by depression and increased resistance to insulin in peripheral target organs. Due to the aging of society and changing lifestyles, adult type 2 diabetes accounts for more than 90% of all patients, and the number is increasing steadily.

Insulin resistance refers to a condition in which the response to a certain amount of insulin is less than normal. Generally, it is caused by genetic and environmental factors. It is a major cause of the development of type 2 diabetes. The main target is to improve the sensitivity.

Current drug treatments can be divided into several types according to the treatment mechanism. The sulfonylurea system indirectly increases insulin secretion and enhances insulin action, but may have side effects such as hypoglycemia and loss of insulin secretion. There. The biguanide system acts to inhibit the angiogenesis in the liver and to reduce the absorption of sugar in the gastrointestinal tract, but has side effects such as gastrointestinal disorders and renal toxicity. The alpha-glucosidase inhibitor system is used in the small intestine. Inhibit the action of glucose-producing enzymes to suppress the rise of blood sugar, while diarrhea, abdominal pain and other side effects.

In recent years, the development of insulin-increasing agents has been in full swing, and there are drugs of thiazolidinedione (TZD), which activates PPAR-γ (peroxisome proliferator activated receptor γ). PPAR binds to the retinoid X receptor (RXR), one of the nuclear hormone receptors, forms a heterodimer and acts as a transcription factor to produce fat. It is important for cell differentiation, carbohydrates, and fat metabolism. In particular, PPAR-gamma (PPAR-γ) is mainly present in adipocytes, and is responsible for adipogenesis, glucose uptake, and PPAR-γ agonist. Is used as a treatment for type 2 diabetes and many drugs of thiazolidinedione (TZD) class are used [ Diabetes , 47: 507 ~ 514, 1998].

In the current situation of drug treatments, the development of drugs that can fundamentally solve the insulin resistance is urgent, because of the lack of treatment for the underlying cause of diabetes such as the development of insulin resistance and various side effects.

Protein tyrosine phosphatase 1B (PTP1B) is known to act as a negative regulator of insulin signaling through dephosphorylation of activated insulin receptors (IR) [ Mol. Cell. Biol. , 182: 101-108, 1998]. In rats from which the protein tyrosine phosphatase 1B (PTP1B) gene was removed, insulin resistance was eliminated and type 2 diabetes was not induced and obesity was suppressed [ Science , 283: 1544 ~ 1548, 1999]. It has been observed that substances that inhibit protein tyrosine phosphatase 1B (PTP1B) show antidiabetic effects [ J. Med. Chem. , 43: 995-1010, 2000].

However, there are still no clinically successful examples of inhibitors of protein tyrosine dekinase 1B, and studies on this have been inadequate. Thus, there is a problem in that no effective inhibitor of protein tyrosine dephosphatase 1B has been found.

Accordingly, the present inventors have made diligent efforts to solve the problems of the prior art, antarctic lichen stereocaulon Alpinum ( Stereocaulon) alpinum ) extract was confirmed that the activity of the protein tyrosine dephosphatase 1B activity inhibited, to complete the present invention.

After all, an object of the present invention, the stereocaulon ( Stereocaulon) having the activity of inhibiting the activity of the protein tyrosine dephosphatase 1B alpinum ) extracts.

Another object of the present invention is to provide a usnic acid derivative having an activity inhibiting activity of the protein tyrosine dephosphatase 1B isolated from stereocaulon alpinum .

Still another object of the present invention is to provide a method for separating usnic acid and its derivatives from Stereocaulon alpinum having the activity of inhibiting protein tyrosine dephosphoryase 1B.

Still another object of the present invention is a stereocaulon ( Stereocaulon) having the activity of inhibiting the activity of the protein tyrosine dephosphatase 1B alpinum ) extract, the usnic acid isolated therefrom and its derivatives to provide a pharmaceutical composition for hypoglycemic containing as an active ingredient.

In order to achieve the above object, the present invention has a stereocaulon ( Stereocaulon) having the activity of inhibiting the activity of the protein tyrosine dephosphatase 1B alpinum ) extract.

The present invention also comprises the steps of (a) extracting a stereocaulon alpinum with a solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms and mixtures thereof; (b) eluting the stereocowlon alpinum extract extracted in step (a) with 100% methanol on a silica gel column; And (c) eluting the fraction eluted in step (b) with a solvent in which the ratio of hexane and dichloromethane is 0 to 90: 100 to 10 on a silica gel column. Provided is a method for separating usnic acid represented by the following Chemical Formula 1 having the activity of inhibiting the activity of the protein tyrosine dephosphatase 1B from Stereocaulon alpinum .

[Formula 1]

The present invention also provides a steronic acid derivative derived from stereo cowlon alpinum represented by any one of the following Chemical Formulas 2 to 4 having the activity inhibitory activity of the protein tyrosine dephosphoryase 1B.

[Formula 2]

(3)

[Formula 4]

The present invention also comprises the steps of (a) extracting a stereocaulon alpinum with a solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms and mixtures thereof; (b) eluting the stereo cowlon alpinum extract extracted in step (a) with an aqueous 60-70% methanol solution (methanol: water = 60-70: 40-30) on a silica gel column; And (c) eluting the fraction eluted in step (b) with acetonitrile (CH ₃ CN) aqueous solution (acetonitrile: water = 40-91: 60-9) on reverse phase HPLC. Provided is a method for separating a usnic acid derivative represented by any one of the above Chemical Formulas 2 to 4 having the activity of inhibiting the activity of the protein tyrosine dephosphatase 1B from Stereocaulon alpinum .

The present invention also provides a stereocaulon ( Stereocaulon) alpinum ) provides a pharmaceutical composition for lowering blood sugar, which contains the extract as an active ingredient.

The present invention also provides a pharmaceutical composition for lowering blood sugar containing at least one compound of Formulas 1 to 4 as an active ingredient.

Antarctic lichen stereocaulon alpinum according to the present invention ( Stereocaulon) alpinum ) extract, and its acidic acid and its derivatives are excellent in inhibiting the activity of protein tyrosine dekinase 1B, and thus can be used as a therapeutic agent for diabetes.

In one aspect, the present invention relates to a stereocaulon alpinum extract having an activity inhibiting activity of the protein tyrosine dephosphatase 1B.

Stereocaulon alpinum extract according to the present invention can be prepared according to a conventional extraction method of stereo cowlon alpinum which is a kind of antarctic lichen.

In the present invention, as a solvent for extracting stereocaulon alpinum , a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixtures thereof may be used. Examples of the lower alcohol having 1 to 4 carbon atoms include methanol, ethanol, propanol, butanol, and the like, and methanol or methanol aqueous solution is preferably used. In this case, the stereo cowlon alpinum may be used as it is, or may be used after pulverization, and the amount of the solvent used may be 0.1 to 5 L per 100 g of the dried stereo cowlon alpinum. The temperature for the extraction may be 4 to 120 ° C, but is not limited thereto. In addition, the extraction time is not particularly limited, but may be 10 minutes to 30 days, and a conventional extraction device, an ultrasonic mill extractor, or a fractionator may be used. The prepared extract can then be filtered under reduced pressure or / and freeze-dried to remove the solvent.

In another aspect, the present invention relates to a usnic acid derived from stereo cowlon alpinum represented by the following general formula (1) having an activity inhibitory activity of the protein tyrosine dephosphoryase 1B.

[Formula 1]

Usnic acid represented by the formula (1) is a major compound isolated from the stereo cowlon alpinum. Typical spectroscopic properties of usnic acid include 1,3-keto-enolic moiety, 4 separate methyl groups, 2 phenolic hydroxyl groups, and enolic hydroxyl groups. Corresponding ¹ H and / or ¹³ C NMR signals.

The method for separating the usnic acid represented by Formula 1 having the activity inhibiting activity of the protein tyrosine dephosphatase 1B from the stereocaulon alpinum according to an embodiment of the present invention Method for separating the usnic acid represented by the following formula (1) having the activity of inhibiting the activity of protein tyrosine dephosphatase 1B from the stereocaulon alpinum : (a) Stereocaulon alpinum 1 to Extracting with a solvent selected from the group consisting of lower alcohols of 4 and mixtures thereof; (b) eluting the stereocowlon alpinum extract extracted in step (a) with 100% methanol on a silica gel column; And (c) eluting the fraction eluted in step (b) with a solvent in which the ratio of hexane and dichloromethane is 0 to 90: 100 to 10 on a silica gel column.

In another aspect, the present invention is a usonic acid derivative derived from stereocaulone alpinum represented by any one of the following Chemical Formulas 2 to 4 having the activity inhibitory activity of the protein tyrosine dephosphoryase 1B (Formula 2: usimine A, Formula 3: usimine B, formula 4: usimine C).

[Formula 2]

(3)

[Formula 4]

It was confirmed that the compound represented by Formula 2 to 4 derived from stereo cowlon alpinum is a usnic acid derivative through an analysis result such as NMR.

Usimine A represented by the formula (2), unlike the usnic acid represented by the formula (1) does not have a ketone functional group (C-10), carbonyl carbon (carbonyl carbons), methoxy group (methoxy group), -CH -CH ₂ -CH ₂ -further includes a unit. Usimine B represented by Formula 3 is a compound having a mass (CH ₂ ) of less than usimine A, and a methoxy group of usmine A is substituted with a hydroxyl group. In addition, usimine C represented by Formula 4 is a stereoisomer of the usimine B.

Method for separating a usnic acid derivative represented by any one of the formulas (2) to (4) having the activity of inhibiting the activity of the protein tyrosine dephosphatase 1B from the stereocaulon alpinum according to an embodiment of the present invention (A) extracting stereocaulon alpinum with a solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms and mixtures thereof; (b) eluting the stereo cowlon alpinum extract extracted in step (a) with an aqueous 60 to 70% methanol solution (methanol: water = 60 to 70:40 to 30) on a silica gel column; And (c) eluting the fraction eluted in step (b) with acetonitrile (CH ₃ CN) aqueous solution (acetonitrile: water = 40-91: 60-9) on reverse phase HPLC.

In another aspect, the present invention provides a stereocaulon. alpinum ) relates to a blood sugar-lowering pharmaceutical composition containing the extract as an active ingredient.

The pharmaceutical composition for lowering blood sugar according to the present invention is the stereocaulon alpinum ( Stereocaulon alpinum ) extract, preferably a compound selected from the group consisting of Usnic acid (Formula 1), Usnic acid derivative (Formula 2: usimine A, Formula 3: usimine B, Formula 4: usimine C), and mixtures thereof It may be included as a component, and may further include a pharmaceutically acceptable carrier, excipient, and diluent according to the formulation, method of use, and purpose of use. When provided in a mixture, the active ingredient may be included in the pharmaceutical composition for lowering blood sugar in an amount of 0.001 to 99.9% by weight, but is usually included in an amount of 0.1 to 50% by weight.

The hypoglycemic pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be. 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 may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspending agents, solvents, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. 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.

In addition, the dosage of the pharmaceutical composition for lowering blood glucose to the human body varies depending on the condition and weight of the patient, the extent of the disease, the drug form, the route of administration and the duration, and 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 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, the following examples can be modified in many different forms, it is common knowledge in the art that the scope of the present invention is not to be construed as limited by these examples. It will be obvious to those who have.

Example 1 Preparation of Stereo Cowlon Alpinum Extract

Stereocaulon alpinum was built around the Barton Peninsular, King George Island, King Sejong Station (S 62 ° 13.3 ', W 58 ° 47.0') in 2003. Collected in January, Voucher specimens (reference L-5) have been deposited with the Korea Polar Research Institute.

The dried stereo cowlon alpinum (50 g) was first extracted with 1 L of methanol (MeOH) for 24 hours, and then extracted with 1 L of methanol. After mixing the primary and secondary extracts, filtered, concentrated under reduced pressure to evaporate the solvent, to prepare a stereo cowlon alpinum extract (yield: 11.8%).

Example 2 Usnic Aicd Separation from Stereo Cowlon Alpinum

5.9 g of the stereocaulone alpinum methanol extract of Example 1 was loaded on a silica gel-filled column (Aldrich octadecyl-functionalized silica gel (C ₁₈ ), 3 × 15 cm), followed by 20%, 40%, and 60% of each 400 mL. %, 70%, 80%, 90% and 100% methanol (MeOH) were injected sequentially and each fraction was obtained. The fraction obtained by using 100% methanol as a mobile phase of the obtained fraction (414.2 mg) was loaded on a silica gel-filled column (Aldrich octadecyl-functionalized silica gel (C ₁₈ ), 2.5 × 50 cm), followed by hexane and declination. By diluting 400 mL each of a mixed solvent having a ratio of chloromethane (dichlorometnane, CH ₂ Cl ₂ ) of 90:10, 70:30, 50:50, 30:70, 10:90, and 0: 100, A fraction (225.9 mg) having excellent activity inhibitory activity of tyrosine dephosphoryase 1B was obtained.

The obtained fraction was in the form of yellow needles, Perkin Elmer 341 digital polarimeter, NMR, JEOL JNM ECP-400 spectrometer, ¹ H = 400MHz, ¹³ C = 100MHz, solvent : pyridine- d ₅ and CDCl ₃ ) and electrospray ionization mass spectrometry (ESI-MS, Perseptive Biosystem, USA) were used as a result of the determination as usnic acid ([α] ²⁵ _D + 300 °). (c 0.54, CH ₂ Cl ₂ ); ¹ H, ¹³ C NMR data: ¹ H NMR (CDCl ₃ , 400 MHz) 13.30 (OH-7), 11.01 (OH-9), 5.92 (1H, s, H-4 ), 2.67 (3H, s, H3-13), 2.65 (3H, s, H3-11), 2.30 (2H, m, H-2 '), 2.10 (3H, s, H3-14), 1.75 (3H , s, H3-15); ¹³ C NMR (CDCl ₃ , 100 MHz) 201.9 (C-10), 200.4 (C-12), 191.8 (C-3), 179.5 (C-4a), 164.0 (C-7 ), 157.6 (C-9), 155.3 (C-5a), 109.4 (C-8), 104.0 (C-9a), 98.4 (C-4), 101.62 (C-6), 59.2 (C-9b) , 32.2 (C-15), 31.4 (C-13), 28.0 (C-11), 7.6 (C-14); ESIMS m / z 345 (M + H) ⁺ ).

Example 3 Isolation of Usnic Acid Derivatives (usimine A) from Stereocaulone Alpinum

Of the fractions obtained in Example 2, the fraction obtained by using 70% methanol as a mobile phase (76 mg) was subjected to semi-preparative reversed-phase HPLC (Shiseido Capcell Pak C ₁₈ column, 10 × 250 (inner diameter × height) mm , 5 μm particle size, UV detector wavelength: 254 nm), and 2 mL / of acetonitrile (CH ₃ CN) aqueous solution (acetonitrile: water = 40-91: 60-9) containing 0.1% of formic acid. A fraction (7.8 mg) having excellent activity inhibitory activity of protein tyrosine dephosphatase 1B was isolated at 29.2 minutes (retention time) while gradient elution at min rate.

Fractions were in the form of yellow gums, Perkin Elmer 341 digital polarimeter, Biochrom 1300 UV / Visible spectrophotometer, NMR, JEOL JNM ECP-400 spectrometer, ¹ H = 400 MHz, ¹³ C = 100 MHz, Solvent: pyridine- d ₅ ), HMBC (Heteronuclear Multiple Bond Coherence, ⁿ J _CH = 8 Hz), High Resolution Electrospray Ionization Mass Spectrometer (HRESI-MS, Perseptive Biosystem, USA) Was determined to be a novel usnic acid derivative, which was named usimine A {[α] ²⁵ _D + 39 ° (c 0.77, CH ₂ Cl ₂ ); UV (CH ₃ OH) λ max (log ε) 297 (4.1), 216 (4.0); ¹ H, ¹³ C NMR data: see Table 1, FIGS. 1 and 2; HMBC data (CDCl ₃ , 400 MHz) H-4 → C-1, C-4a, C-9b, C-15; _{H 3 -13 → C-6,} C-12 H 3 -14 → C-7, C-8, C-9 H 3 -15 → C-1, C-4a, C-9a, C-9b; H-1 ′ → C-2 ′, C-5 ′, H-2 ′ → C-1 ′, C-3 ′, C-4 ′, C-5 ′, H-3 ′ → C-1 ′, C-2 ′, C-4 ′, 7-OH → C-6, C-7, C-8, 9-OH → C-8, C-9, OCH ₃ → C-4 ′; HRESIMS m / z 488.1552 (M + H) ⁺ (calc. For C ₂₄ H ₂₆ NO ₁₀ , 488.1557)}.

TABLE 1

^a: measured at 400 MHz, ^b: measured at 100 MHz, ^c: correlation of weak four bonds, carbon multiplicities measured with DEPT.

Table 1 shows that usimine A does not have a ketone functional group (C-10) unlike usonic acid, and chemical shifts have been changed from a methyl group α to a ketone moiety. Chemical shifts are based on Tetramethylsilane (TMS) peaks (0 ppm) for ¹ H and ¹³ C, and chemical shift values are in parts per million (ppm). ) Is expressed in units. In addition, the ¹ H and ¹³ C NMR spectra of usimine A compared with carbonic carbons, methoxy groups, -CH-CH ₂ -CH ₂ -units It can be seen that further indications. These structural properties have 13 unsaturation equivalents and show linear coupling with other structural units at the C-10 position of the basonic acid backbone, the binding of these units confirmed by COSY and HMBC analytical data. . ¹ H-detected heteronuclear multiple quantum coherence (HMQC) and heteronuclear multplebond correlation (HMBC) analyzes according to FIGS. 3 and 4 were optimized at ¹ J _CH = 140 Hz and ⁿ J _CH = 8 Hz, respectively.

As shown in FIG. 5, the spin system of C-1 ′ to C-3 ′ of usimine A was confirmed by COSY analysis. Carbonyl carbon (δ _C 173.0) is bound to H-1 ′ / C-5 ′ and C-1 ′ with HMBC correlation with H ₂ −2 ′ / C-5 ′. From the HMQC data, it was confirmed that the methyl group protons at δ _H 3.59 and δ _C 51.7 are correlated with each other, and from the HMBC data, it was confirmed that methyl ester groups were present in the carbonyl carbon at δ _C 172.9. From the HMBC correlation of H ₂ -3 ′ and C-4 ′, it was confirmed that the methyl ester group was linked to C-3 ′, and based on the HMBC correlation with C-2 and C-10 of H ₃ -11, δ It was confirmed that methyl carbon (C-11) and C-2 are linked to _C 175.1 (C-10). In addition, from the HMBC correlation of H-1 ′ and C-10, the presence of nitrogen in the molecule, and the chemical shifts of C-10, C-1 ′ and C-5 ′, C-10 represents an imine group of carbon ( C = N).

In FIG. 2 showing the ¹³ C NMR spectrum of usimine A, 19 distinct carbon signals were observed. It is judged that the signals of C-2, C-3, and C-10 do not appear, and they are caused by imine-enamine tautomerism containing the hydroxyl group of C-3. Was supposed to. Location of imine of usimine A was confirmed by nuclear overhauser enhancement spectroscopy (NOESY) as shown in FIG. ₃ H -11 and H-1 'of the correlation C-11 and C-1', and are joined in cis for groups imine (imine), therefore, an imine is E -geometry - was identified as (E three-dimensional arrangement) .

Example 4 Separation of Usnic Acid Derivatives (usimine B) from Stereo-Caulon Alpinum

Among fractions obtained in Example 2, a fraction obtained by using 60% methanol as a mobile phase (56 mg) was subjected to semi-preparative reversed-phase HPLC (Shiseido Capcell Pak C ₁₈ column, 10 × 250 (inner diameter × height)). 2 ml of acetonitrile (CH ₃ CN) aqueous solution (acetonitrile: water = 40 to 69:60 to 31) containing 0.1% of formic acid after loading at 5 μm particle size and ultraviolet detector wavelength: 254 nm). Fraction 1 (3.6 mg) and Fraction 2 (2.0 mg) were isolated at 22.2 minutes (retention time) and 27.2 minutes (retention time) with excellent activity inhibitory activity of protein tyrosine dephosphatase 1B, while gradient elution was performed at / min. It was.

Fraction 1 was in the form of yellow gums, Perkin Elmer 341 digital polarimeter, Biochrom 1300 UV / Visible spectrophotometer, Nuclear Magnetic Resonance Device (NMR, JEOL JNM ECP-400 spectrometer, ¹ H = 400 MHz, ¹³ C = 100 MHz, solvent: pyridine- d ₅ ), Heteronuclear Multiple Bond Coherence ( ⁿ J _CH = 8 Hz), high resolution electrospray ionization mass spectrometer (HRESI-MS, Perseptive Biosystem, USA) It was determined using a new usonic acid derivative, which was named usimine B {([α] ²⁵ _D + 159 ° (c 0.46, MeOH); UV (CH ₃ OH) λ max (log ε) 297 ( 4.2), 216 (4.3); ¹ H, ¹³ C NMR data: see Table 2, FIGS. 7 and 8; HMBC data (pyridine-d ₅ , 400 MHz) H-4 → C-2, C-4a, C- _{9b, C-15; H 3} -11 → C- 2, C-10; H 3 -13 → C-6, C-12; H 3 -14 → C-7, C-8, C-9; H _{3 -15 → C-1, C} -4a, C-9a, C-9b; HRESIMS m / z 473.1392 (M + H) + (calc for C 23 H 24 NO 10, 474.1400.)}.

Fraction 2 was in the form of yellow gums, Perkin Elmer 341 digital polarimeter, Biochrom 1300 UV / Visible spectrophotometer, Nuclear Magnetic Resonance Device (NMR, JEOL JNM ECP-400 spectrometer, ¹ H = 400 MHz, ¹³ C = 100 MHz, solvent: pyridine- d ₅ ), Heteronuclear Multiple Bond Coherence ( ⁿ J _CH = 8 Hz), high resolution electrospray ionization mass spectrometer (HRESI-MS, Perseptive Biosystem, USA) It was determined using a new usonic acid derivative, which was named usimine C {([α] ²⁵ _D + 162 ° (c 0.43, MeOH); UV (CH ₃ OH) λ max (log ε) 297 ( 4.2), 216 (4.3); ¹ H, ¹³ C NMR data: see Table 2, FIGS. 9 and 10; HRESIMS m / z 473.1392 (M + H) ⁺ (calc. For C ₂₃ H ₂₄ NO ₁₀ , 474.1400) }.

TABLE 2

^a: measured at 400 MHz, ^b: measured at 100 MHz, carbon multiplicities measured in DEPT.

From Table 2, ¹ H and ¹³ C NMR spectra of usimine B was confirmed to and is consistent with the ¹ H and ¹³ C NMR spectra of usmine A except for the absence of the signal of the methoxy (methoxy) group. Thus, it was assumed that the methoxy group of usmine A was replaced with the hydroxyl group of usmine B. COSY, HMQC, and HMBC data for usmine usmine A and B was analyzed to be consistent with each other, migration positions of usmine B is from the correlation of the H ₃ -11 and H-1 'through the NOESY (nuclear overhauser enhancement spectroscopy) analysis it was confirmed that the (three-dimensional arrangement E) E -geometry.

In addition, the ¹ H and ¹³ C NMR spectra of usimine C were found to be in close agreement with usimine B, and it was confirmed that the notable difference is a fine chemical shift signal of C-2 ′ and C-3 ′. Therefore, usimine C is a geometrical isomer of usimine B, and it is assumed that the imine group is Z -geometry ( Z -stereometry), which is H ₃ -11 and H- by analysis of nuclear overhauser enhancement spectroscopy (NOESY). It was confirmed from the correlation of 1 '.

Example 5 Determination of PTP1B Inhibitory Activity

PTP1B (human, recombinant) was purchased from BIOMOL Research Laboratories, Inc and used in the experiment. Enzyme activity was measured in a mixture containing 2 mM p-nitrophenyl phosphate (pNPP), 0.1M NaCl, 1 mM EDTA and 1 mM dithiothreitol (DTT) contained in 50 mM citrate (pH 6.0). That is, inhibitors (stereocaulone alpinum extract concentration: 30 μg / mL, Usnic acid, concentrations of usimine A, B, C: 3.75 μg / mL, 7.5 μg / mL, 15.0 μg / mL, respectively) were added to the mixture, After shaking gently, the reaction was carried out at 30 ° C., and after 30 minutes, 1N NaOH was added to stop the reaction. The amount of p-nitrophenol produced in the reaction was measured by absorbance at 405 nm. As a control group, RK-682, a well-known phosphatase inhibitor, was used. Non-enzymatic hydrolysis of 2 mM pNPP was corrected with absorbance measured at 405 nm for the mixture without PTP1B enzyme. The enzyme activity inhibition rate is calculated as shown in Equation 1 shown in Table 3 below. In addition, the IC ₅₀ value was determined by the concentration of the inhibitor of 50% inhibition rate of enzyme activity, the results are shown in Table 4 below. The experiment was performed twice.

[Equation 1]

A: absorbance after the reaction of the enzyme

B: Absorbance after the reaction with the inhibitor

C: absorbance after reaction between inhibitor and enzyme

[Table 3]

[Table 4]

From Table 4, it can be seen that Usnic acid and its derivatives (Usimine A, B, C), in particular, the PTP1B inhibitory activity of Usimine A is excellent.

Experimental Example 1 Decomposition of Usimine A

Usimine A (1.5 mg) was dissolved in a mixture of tetrahydrofuran (THF, 1 mL) and 3N hydrochloric acid (HCl, 1.5 mL), and then reacted at 40 ° C. for 12 hours. After confirming the presence of usnic acid through HPLC analysis, the reaction was concentrated, and the concentrated reaction was partitioned between dichloromethane (CH ₂ Cl ₂ ) and an aqueous acid solution (pH = 2).

The dried dichloromethane fraction (900 mg) was separated and purified in a C18 cartridge (cartridge, mobile phase: aqueous methanol solution (methanol: water = 10-100: 90-0)) to obtain usnic acid (0.4 mg). It was confirmed that the optical rotation value of the obtained compound was [α] 25 _D + 125 ° (c0.04, CH ₂ Cl ₂ ).

[Example 2] mapi (Marfey) Preparation and analysis of the derivative

Usimine A (1.5 mg) was added to 6N hydrochloric acid (HCl, 1 mL), and then hydrolyzed at 110 ° C. for 24 hours. Next, the hydrolyzate was lowered, dried under reduced pressure, and dissolved again in distilled water (H ₂ O, 50 µl). A solution of FDAA solution (100 μl, acetone containing 1% Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide)) and sodium bicarbonate (NaHCO ₃ ) in the dissolved acid hydrolysate (1M, 20 μl) was added and mixed, followed by reaction at 40 ° C. for 1 hour. Next, hydrochloric acid (HCl, 2N, 10 µl) was added to stop the reaction to prepare a derivative, and then the solvent of the derivative (reactant) was dried under reduced pressure, and the residue was again washed with an aqueous methanol solution (MeOH / H ₂ O, 1: 1; 1 mL).

Next, an aqueous solution of methanol derivative (20 µl) was loaded on HPLC (Capcell Pak C ₁₈ column, UV detector wavelength: 340 nm), and then acetonitrile (CH ₃ CN) aqueous solution containing 0.1% of formic acid (acetonitrile: Water = 30-60: 70-40) was linear gradient elution for at least 60 minutes at 1 mL / min. Standards (10 μl, FDA-induced L-Glu and D-Glu) were also analyzed by HPLC in the same manner as the aqueous solution of the derivative derivative methanol. As a result, the retention time (min) of the FDAA amino acid derivative used as a reference material was L-Glu (32.7) and D-Glu (33.9). -Residence time of Glu) was found to be 32.4 minutes.

1 is a ¹ H NMR spectrum (400 MHz, pyridine- d ₅ ) of usimine A.

Figure 2 is a ¹³ C NMR spectrum (100MHz, pyridine- d ₅ ) of usimine A.

Figure 3 is the HMQC spectrum of usimine A (400MHz, pyridine- d ₅ ).

4 is an HMBC spectrum of usimine A (400 MHz, pyridine- d ₅ ).

5 is a COSY spectrum of usimine A (400 MHz, pyridine- d ₅ ).

Figure 6 is a NOESY spectrum (400MHz, pyridine- d ₅ ) of usimine A.

Figure 7 is a ¹ H NMR spectrum (400MHz, pyridine- d ₅ ) of usimine B.

FIG. 8 is ¹³ C NMR spectrum (100MHz, pyridine- d ₅ ) of usimine B. FIG.

9 is a ¹ H NMR spectrum (400 MHz, pyridine- d ₅ ) of usimine C. FIG.

FIG. 10 is ¹³ C NMR spectrum of usimine C (100 MHz, pyridine- d ₅ ).

Claims (7)

delete delete A method for separating usnic acid represented by the following Chemical Formula 1 having the activity of inhibiting the activity of the protein tyrosine dephosphatase 1B from the Stereocaulon alpinum comprising the following steps: (a) extracting Stereocaulon alpinum with a solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms and mixtures thereof; (b) eluting the stereocowlon alpinum extract extracted in step (a) with 100% methanol on a silica gel column; And (C) eluting the fraction eluted in step (b) with a solvent in which the ratio of hexane and dichloromethane is 0 to 90: 100 to 10 on a silica gel column. [Formula 1]

Usnic acid derivatives derived from stereocaulone alpinum represented by any one of the following Chemical Formulas 2 to 4 having the activity inhibitory activity of the protein tyrosine dephosphatase 1B. [Formula 2]

(3)

[Formula 4]

Separation method of the usnic acid derivative represented by any one of the following Chemical Formulas 2 to 4 having the activity of inhibiting the activity of the protein tyrosine dephosphatase 1B from the stereocaulon alpinum comprising the following steps: (a) extracting Stereocaulon alpinum with a solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms and mixtures thereof; (b) eluting the stereo cowlon alpinum extract extracted in step (a) with an aqueous 60-70% methanol solution (methanol: water = 60-70: 40-30) on a silica gel column; And (c) eluting the fraction eluted in step (b) with acetonitrile (CH ₃ CN) aqueous solution (acetonitrile: water = 40-91: 60-9) on reverse phase HPLC. [Formula 2]

(3) [Formula 4]

Stereocaulon alpinum ) A pharmaceutical composition for lowering blood sugar, which contains the extract as an active ingredient. A pharmaceutical composition for lowering blood sugar, which contains at least one compound of Formulas 1 to 4 as an active ingredient. [Formula 1]

[Formula 2]

(3)

[Formula 4]

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