KR20060060376A - A novel fungal species of micromucor ramannianus var. angulisporus crm000232 and protein tyrosin phosphatase 1b inhibitor comprising depside compounds isolated and purified from them - Google Patents

Abstract

The present invention provides a protein tyrosine dephosphorylation enzyme using micromuco ramanianus var. Angulisporus CRM000232 (KCTC 10696BP), and a depth-based compound isolated and purified therefrom as an active ingredient. 1Ｂ inhibitors.

Depth-based compounds isolated and purified from the fungal strain CRM000232 (KCTC 10696BP) according to the present invention is excellent in protein tyrosine dephosphorylation enzyme 1B inhibitory activity, and excellent in hypoglycemic effect can be useful for the prevention and treatment of diabetes mellitus, In particular, the therapeutic effect is excellent for insulin-independent diabetes mellitus type 2 diabetes.

Description

A novel fungal species of Micromucor ramannianus var. A novel fungal species of Micromucor ramannianus var. A novel fungal strain Micromuco ramananias bar Angolith sporcus CrM000232, and a depseed compound isolated and purified therefrom as an active ingredient. angulisporus CRM000232 and protein tyrosin phosphatase 1B inhibitor comprising depside compounds isolated and purified from them}             

1 is a diagram showing the blood glucose level of a depseed compound isolated and purified from the fungus strain Micromuco Ramananias bar Angoulosporus CRM000232 (KCTC 10696BP) according to the present invention.

The present invention relates to a protein tyrosine dephosphorase 1 Ｂ inhibitor comprising the novel fungal strain Micromuco Ramananias bar Angulosporus CRM000232 and a depseed compound isolated and purified therefrom as an active ingredient.

Diabetes mellitus is a non-infectious chronic disease in which the insulin secreted by the pancreatic β cells is insufficient or does not function properly, and glucose is not used as an energy source in the body and remains high in the blood and excreted in the urine. .

Diabetes mellitus can be classified into insulin dependent diabetes mellitus (IDDM), a type 1 diabetes mellitus, and non-insulin dependent diabetes mellitus (NIDDM), a type 2 diabetes mellitus, depending on the cause of the disease and a method of treating the symptoms. Are classified.

Type 1 diabetes is a state in which the pancreatic β cells are degraded due to genetic causes, viral infections, etc., and little insulin is secreted. Blood sugar is controlled only by supplying insulin, and suddenly develops in the 10s and 20s. Also called pediatric diabetes.

Type 2 diabetes is caused by family history, obesity, stress, eating errors, or drugs that antagonize insulin, and the like. Blood sugar is not controlled by insulin supply, and blood sugar can be controlled by blood sugar lowering agents or diets. Appears later, and is also known as adult diabetes. Insulin secretion is sufficient in the pancreas, but blood glucose is not normalized despite hyperinsulinemia due to differences in insulin resistance and glucose utilization from normal individuals. In Korea, more than 90-95% of diabetics are type 2 diabetics.

Increasing living conditions improves living conditions, and the overall diet improves calorie intake significantly, leads to lack of exercise, and continues to be exposed to excessive stress, which leads to a decrease in insulin performance. As obesity develops, the resulting type 2 diabetes (insulin non-dependent diabetes) is increasing. If type 2 diabetes persists for a long time, in addition to the increase in blood sugar, blood lipid levels increase, resulting in cardiovascular diseases such as fatty liver, arteriosclerosis, and coronary heart disease, diabetic nephropathy and retinal diseases, diabetic peripheral neurosis, Deterioration of sensory function will cause complications, the development of its therapeutics is urgent situation.

Currently used diabetic drugs are roughly divided into oral hypoglycemic agents and insulin injections. Generally, insulin injections are administered to insulin-dependent diabetic patients who have no insulin secretion in the body, gestational diabetics, or insulin-independent diabetes patients whose blood glucose control is poorly controlled by oral hypoglycemic agents, despite the combination of diet and exercise therapy. It is a common practice to take oral hypoglycemic drugs for insulin-independent diabetics who do not have adequate glycemic control.

Commonly used oral hypoglycemic agents are sulfonylureas, biguanides, acarbose, and thiazolidinedione depending on the mechanism of action and the point of action of the drug. It can be divided into drugs.

Sulfonylurea-based drugs include glypizide, glyclazide, glyquidone, glybenclamide, chlorpropamide, and the like, which promote insulin secretion in the pancreas. Therefore, there is a problem that they can not be used in insulin-dependent diabetes patients with no insulin secretion in the pancreas, insulin-independent diabetes patients with relatively reduced insulin secretion in the pancreas, women of childbearing age. In addition, these drugs can cause hypoglycemia when administered in an overdose or on an empty stomach, and also have side effects such as skin rash, jaundice, anorexia, nausea (nausea), and diarrhea. In particular, long-lived drugs (12-36 hours), such as chlorpropamide, have a high risk of developing hypoglycemia due to accumulation in the body. In addition, since most sulfonylurea drugs are metabolized in the liver and excreted in the kidney, they should be used with caution in patients with hepatic and renal dysfunction.

Biguanide-based drugs include metformin and the like, but the mechanism of action is not accurate, but it has been found that there is no effect of increasing insulin secretion in the pancreas, and decreases glucose neosynthesis in the liver. Biguanide-based drugs have a weaker hypoglycemic effect than sulfonylurea-based drugs, but do not cause hypoglycemia. However, due to the high incidence of side effects of the digestive system, nausea, vomiting, diarrhea and rash appear early in the treatment, and cause lactic acidosis, which can cause life-threatening side effects, so be careful for the elderly and patients with cardiovascular disease. do. Currently used only as an experimental drug in the United States.

Acarbose-based drugs have been shown to interfere with the digestion of carbohydrates after ingesting carbohydrates and thus to raise blood sugar levels. Acarbose-based drugs are alpha-glucoamylase inhibitors that reversibly inhibit alpha-D-glycosidase activity, and mainly inhibit maltase activity in the mucous membranes of small intestine cells to prevent blood glucose spikes after meals. . The side effects are mild and are used to treat mild diabetes.

Thiazolidinedione-based drugs have been recently developed, and have an effect of improving insulin resistance by activating PPI-gamma (PPAR-Υ, Peroxisome proliferator activated receptor Υ), which is a nuclear membrane receptor and gene transcription promoter.

However, since oral hypoglycemic agents alone do not drop blood sugar effectively and most of the known side effects occur, there is an urgent need to develop safer diabetes or diabetes prevention agents.

Recently, the association with protein tyrosin phosphatase 1B (PTP1B), which is responsible for negative regulation in type 2 diabetes and insulin signaling, has been revealed. This is emerging. Kennedy and Ramachandran have reported susceptibility to insulin in mice lacking the protein tyrosine dephosphorase 1B gene (Elchevly et al., Science, 283, 1544-1548, 1999). . In other words, when insulin was administered to rats, it was shown that the phosphorylation of insulin receptors in the liver and muscle cells increased and lasted for a long time. Has been shown to be very effective in treating type 2 diabetes. In addition, it has been reported that the protein tyrosine dephosphorylase 1B inhibitor is effective in type 2 diabetes, such as hyperglycemia and overcoming insulin resistance in type 2 diabetes model mice (J. Med. Chem. 42, 3199-3202, 1999; J. Biol. Chem. 275, 10300-10307, 2000).

Therefore, efforts have been actively made to develop inhibitors of protein tyrosine dephosphoryase 1B as therapeutic agents for diabetes [Nature Review Drug Discovery, 1,696 to 709].

In general, among the various methodologies for the development of drugs with new ingredients, there is a high possibility of finding new drug components in various metabolites produced by natural soil microorganisms rather than efforts by experimental modifications of existing drugs. Many drugs such as anticancer drugs have been developed in microorganisms. However, reports of compounds isolated from microorganisms as protein tyrosine dephosphatase 1B inhibitors to date have been extremely poor.

Accordingly, the present inventors searched for a substance that inhibits the activity of protein tyrosine dephosphorase 1B, which has little side effects and is effective for treating type 2 diabetes, from various microorganisms. Forus [ Micromucor ramannianus var. angulisporus ) CRM000232 (KCTC 10696BP)] was selected, and the depth-based compound isolated and purified therefrom inhibited the activity of the protein tyrosine dephosphorylase 1B to confirm that it is effective in lowering blood sugar and completed the present invention.

The present invention seeks to provide a novel fungal strain Micromucor ramannianus var. Angulisporus CRM000232 (KCTC 10696BP).

In addition, the present invention is to provide a protein tyrosine dephosphorylation enzyme 1 Ｂ inhibitor comprising as an active ingredient a depseed compound separated and purified from the micromuco Ramanania Barus Angoulosporus CRM000232 culture solution.

The present invention provides a novel fungal strain Micromucor ramannianus var. Angulisporus CRM000232 (KCTC 10696BP).

The fungal strain micromuco ramananias bar Angulosporus CRM000232 of the present invention is isolated from the soil, and the separation method is as follows.

Add the soil sample to sterile saline solution, stir and dilute it with 10 ^-2 ~ 10 ^-4 times, spread the diluted solution on the potato dextrose agar medium, and incubate at 25 ℃ for 7 ~ 10 days. Purely separate colonies. The isolated mycobacterial fungi are cultured in various solid media to produce spores.

The mature spore sac is shaped like a crater, and its edge is characterized by a spiny wall with a pointed face. A large number of sporangiospores (C) are found in the spore sac and are particularly filled with spore cysts with various facets of isodiametric, which can be determined as a micromucor fungus belonging to the Mucoales neck. It is identified as Micromucor ramannianus var. Angulisporus . Therefore, the new strain isolated from the soil of the present invention is named Micromucor ramannianus var. Angulisporus CRM00232 (Accession Number: KCTC 10696BP).

In another aspect, the present invention provides a protein tyrosine dephosphorylation enzyme 1 Ｂ inhibitor comprising the compound represented by the following formula (1) separated and purified from the micromuco Ramananias bar Angoulosporus CRM000232 culture medium as an active ingredient.

, 또는 -(CH₂)₂COCH₃ 이다.)(In Formula 1, R is

Or-(CH ₂ ) ₂ COCH ₃ )

Depth-based compound represented by the formula (1) according to the present invention can be used in the form of a pharmaceutically acceptable salt, and includes all salts, hydrates and solvates prepared by conventional methods.

Depth-based compound represented by the formula (1) according to the present invention can be obtained by any conventional method, can be used commercially available reagents, in the present invention the new fungal strain micromuco Ramannians Bar Angulosporus CRM000232 Separate and purify from

The method for separating and purifying the depth-based compound from the novel fungal strain Micromuco ramanianus var. Angulisporus CRM000232 according to the present invention is as follows.

Yeast extract (0.3%), Malt as a seed medium and production medium of the fungus strain Micromuco Lamanniaus bar Angulolisporus CRM000232, to cultivate micromuco Lamanniaus bar Angilliosporus CRM000232 (KCTC 10696BP) isolated from soil. A medium containing an extract (0.3%), peptone (0.5%), glucose (2%), magnesium sulfate, 7H ₂ O (0.05%) and potassium dihydrogen phosphate (0.1%) is used.

After dispensing the seed medium into the Erlenmeyer flask and autoclaving for 20 minutes at 121 ° C, inoculate CRM000232 strains cultured in potato dextrose or YM medium with platinum, and incubate at 26 ° C for 4 days for seed culture of the incubator. do.

The incubator containing the production medium was autoclaved at 121 ° C. for 1 hour and then inoculated with the above culture medium, and then incubated for 7 days under aerobic conditions of 1.0 vvm with agitation at a speed of 300 rpm at 26 ° C.

The growth of CRM000232 reaches the highest value at 7 days of culture in liquid medium, and the production of the seed compound is maximized at 7 days of culture. The optimum incubation temperature for production is 26 ℃ and when the incubation at pH 6.8 began to drop to about pH 5.5 which is slightly acidic after 3 days and gradually increased over time to pH 7.5 ~ 8.0 at the end of incubation.

The culture solution of the micromuco Ramananias bar Angillius spore CRM000232 (KCTC 10696BP) is extracted with ethyl acetate and concentrated. The concentrated solution was subjected to ODS RP-18 column chromatography, and eluted with methanol / water (1/9 to 10/0, v / v) as a mixed solvent step by step while increasing the methanol concentration. In this case, the active fraction containing Compound 1 elutes in 90% methanol, and the active fraction containing Compound 2 elutes in 80% methanol.

After concentration of each active fraction and concentration under reduced pressure, the fraction containing Compound 1 was purified by high performance liquid chromatography using acetonitrile and water as a solvent at a concentration gradient of 60:40 to 100: 0 at an elution flow rate of 2 ml / min. Elution separates Compound 1 showing 254 nm UV absorption peak at 26 minutes.

In the case of the fraction containing Compound 2, acetonitrile and water were eluted with a solvent gradient of 50:50 to 100: 0 at a concentration gradient of 2 ml / min using a high performance liquid chromatography to obtain a UV absorption peak of 254 nm. Separate compound 2 visible in minutes.

The concentration (IC ₅₀ ) that inhibits the activity of protein tyrosine dephosphatase 1B by 50% of the depth-based compound according to the present invention is 4.9 μM for compound 1 and 69.9 μM for compound 2 to inhibit protein tyrosine dephosphatase 1B. It can be seen that the activity is excellent.

In addition, the blood glucose level of the depth-based compound according to the present invention shows a level (430 mg / dL) lowered by 16% after 1 week in the case of Compound 1 compared to the blood sugar level (500-542 mg / dL) of the control group, 4 After week, 8% is lowered to 500 mg / dL. In case of Compound 2, it is weak compared to Compound 1, but shows a 10% lower level (450 mg / dL) after 1 week, and 4% lower level (480 mg / dL) after 4 weeks. Therefore, it can be seen that the blood sugar lowering effect of the depth compound according to the present invention is excellent.

Therefore, the depth-based compound isolated and purified from the fungal strain CRM000232 (KCTC 10696BP) according to the present invention has excellent protein tyrosine dephosphorylation enzyme 1B inhibitory activity, and can be used effectively for preventing and treating diabetes due to its excellent hypoglycemic effect. have.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the depth compound.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The composition of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dose of the depth compound of Formula 1 is about 10 to 500 mg / kg, preferably 20 to 300 mg / kg, and more preferably administered once to several times a day.

As a result of conducting a toxicity experiment by orally administering the seed compound according to the present invention, 50% lethal dose (LD ₅₀ ) by the oral administration toxicity test is determined to be a safe substance of at least 10g / kg or more.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of diabetes.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Example 1  : Isolation of Mold Strain CRM000232 (KCTC 10696BP)

1 g of dry soil sample was added to 10 ml of sterile physiological saline, stirred for 30 minutes, diluted 10 ^-2 to 10 ^-4 times, and 0.1 ml of the diluted solution was plated on potato dextrose agar medium and then 25 ° C. Incubated at 7 to 10 days in the colony was isolated pure.

The isolated mycobacterial fungi were cultured in various solid media (V-8 juice, YM, Chaipec, malt medium, potato dextrose) to produce spores, followed by observation of spores, growth degree, colony shape, The shape and formation of the pigment were examined.

The morphological and culture characteristics of the isolated fungus 000232 are shown in Table 1.

badge Cell growth Colony shape Surface medium color Back shape and badge color V-8 juice Great Spore Formation of Powder Severe Wrinkles Center Brown Peripheral White Severe wrinkle off-white YM Great Spore Formation of Wrinkles White Wrinkle off-white Chai Peck Good Powder cream color cream color Malt badge Good Powder Core Cream Periphery Off White Off White Potato Dextrose Good Powder Off White Off White

As shown in Table 1, the shape of the wall of the mature sporangia is crater-shaped, and its edges are characterized by an echinulate wall with a pointed face. A large number of sporangiospores (C) are found in the spore sac and are particularly filled with spore cysts with various facets of isodiametric, which can be determined as a micromucor fungus belonging to the Mucoales neck. It was identified as Micromucor ramannianus var. Angulisporus .

Therefore, the new strain isolated from the present invention was named as micromucor ramanianus var. Angulisporus CRM00232. As of September 17, 2004, the Korea Biotechnology Research Institute, a depository institution for Korean patent strains, It was deposited with the Gene Bank and was given accession number KCTC 10696BP.

Example 2  : Depth Compounds from Mold Strain CRM000232 (KCTC 10696BP) Separation and Purification

1. Incubation of Mold Strain CRM000232 (KCTC 10696BP)

In order to culture the fungal strain CRM000232, a medium containing a nutrient source commonly used by microorganisms was prepared.

Yeast extract (0.3%), malt extract (0.3%), peptone (0.5%), glucose (2%), magnesium sulfate, 7H ₂ O (0.05%), potassium di as species and production medium of the fungal strain CRM000232 A medium containing hydrogen phosphate (0.1%) was used.

After dispensing 200 ml of seed medium into a 1 L Erlenmeyer flask and autoclaving for 20 minutes at 121 ° C, inoculate CRM000232 strains cultured in potato dextrose or YM medium with platinum and incubate at 26 ° C for 4 days for incubation. Species culture of.

15 l fermentor containing 10 l of production medium was autoclaved at 121 ° C. for 1 hour, and then inoculated with 200 ml of the above culture medium, followed by stirring at 300 rpm at 26 ° C. with aeration of 1.0 vvm and aerobic under aeration. Incubated for 7 days under conditions.

2. Isolation and Purification of Depsid Compounds from Mold Strain CRM000232 (KCTC 10696BP)

The culture solution of mold CRM000232 (KCTC 10696BP) cultured in 1 was extracted with the same amount of ethyl acetate solvent, and the extract was concentrated using a reduced pressure evaporator under reduced pressure. The concentrated solution was adsorbed to ODS Al 18 and subjected to ODS RP-18 column chromatography. At this time, methanol / water (1/9 to 10/0, v / v) was mixed with a solvent. Eluting with increasing methanol concentration step by step. At this time, the active fraction containing Compound 1 eluted in 90% methanol, the active fraction containing Compound 2 eluted in 80% methanol.

After concentration of each active fraction and concentrated under reduced pressure, the fraction containing Compound 1 was subjected to high performance liquid chromatography (column: Capcell C18, length 250 mm, diameter 10 mm) using acetonitrile and water as a solvent. Elution was carried out under the elution flow rate of 2 ml / min under a 100: 0 concentration gradient to separate Compound 1 showing a UV absorption peak of 254 nm at 26 minutes.

For fractions containing Compound 2, elution flow rate 2 ml of acetonitrile and water at a concentration gradient of 50:50 to 100: 0 using high performance liquid chromatography (column: Capcell C18, length 250 mm, diameter 10 mm). Compound 2 was eluted under the conditions of / min to show a UV absorption peak of 254 nm at 22 minutes.

3. Structural Analysis

Compound 1 and Compound 2 isolated from the culture medium of the fungus CRM000232 (KCTC 10696BP) were determined by using an ESI mass spectrometer (Electrospray Ionization mass spectrometer) or FAB-MS mass spectrometer to determine the molecular weight and molecular formula. Nuclear Magnetic Resonance (NMR) analysis (Bruker AMX 300) also revealed ¹ H NMR, ¹³ C NMR, Homo-Cozy (HOMO-COSY), 1M-Detected heteronuclear Multiple-Quantum Coherence (HMQC), and Heteronuclear Multiple-Bond (HMBC). Coherence) and DEPT (Distortionless Enhancement by Polarization) spectra were obtained and molecular structure was determined.

The measurement results are as follows. As a result of comparative analysis with the published literature, the material isolated from the culture solution of the fungus CRM000232 (KCTC 10696BP) was identified as Compound 1 and Compound 2 (European Patent 0 335 386 A1, (1989). 3. 30)].

Compound 1: [bis (2,4-dihydroxy-3,5,6-trimethyl-benzoic acid 3-hydroxy-2,4,5-trimethyl-6-carbonyl-phenyl ester) disulfide]

1) Physical property: white powder

2) Molecular Weight: 778

3) Molecular formula: C ₄₀ H ₄₂ O ₁₂ S ₂

4) ¹ H-NMR (500 MHz, CDCl ₃ ) δ 11.35 (1H, s, 2,2'-OH), 2.47 (3H, s, H-10, 10 '), 2.31 (3H, s, H- 18, 18 '), 2.18 (3H, s, H-19, 19'), 2.13 (3H, s, H-9, 9 '), 2.12 (3H, s, H-8, 8'), 2.00 ( 3H, s, H-20, 20 ')

5) ¹³ C-NMR (125 MHz, CDCl ₃ ) δ 188.5 (C-17,17 '), 170.5 (C-7,7'), 161.1 (C-2,2 '), 157.8 (C-4, 4 '), 154.8 (C-15,15'), 143.7 (C-11,11 '), 138.8 (C-6,6'), 133.6 (C-13,13 '), 124.1 (C-12, 12 '), 121.1 (C-14,14'), 115.4 (C-5,5 '), 114.8 (C-16,16'), 107.5 (C-3,3 '), 104.6 (C-1, 1 '), 19.1 (C-10,10'), 16.8 (C-18,18 '), 12.0 (C-9,9'), 11.9 (C-19,19 '), 9.9 (C-20, 20 '), 8.1 (C-8,8')

6) ESI-MS (rel. Int.) M / z [M + Na] ⁺ = 801

Compound 2: [2,4-dihydroxy-3,5,6-trimethyl-benzoic acid 3-hydroxy-2,4,5-trimethyl-6- (3-oxo-butylsulfanylcarbonyl) -phenyl ester ]

1) Physical property: white powder

2) Molecular Weight: 460

3) Molecular formula: C ₂₄ H ₂₈ O ₇ S

4) ¹ H-NMR (500 MHz, CDCl ₃ ) δ 11.43 (1H, s, 2′-OH), 5.34 (1H, s, 4-OH), 5.05 (1H, s, 15-OH), 3.06 ( 2H, m, H-21), 2.54 (3H, s, H-10), 2.51 (2H, m, H-22), 2.21 (3H, s, H-18), 2.18 (3H, s, H- 9), 2.16 (3H, s, H-9), 2.14 (3H, s, H-8), 2.04 (3H, s, H-20), 1.93 (3H, s, H-24)

5) ¹³ C-NMR (125 MHz, CDCl ₃ ) δ 206.2 (C-23), 194.7 (C-17), 170.5 (C-7), 161.1 (C-2), 157.8 (C-4), 153.8 (C-15), 143.4 (C-11), 138.5 (C-6), 132.4 (C-13), 126.4 (C-12), 120.9 (C-14), 115.5 (C-5), 114.8 ( C-16), 107.6 (C-3), 107.6 (C-3), 104.8 (C-1), 43.0 (C-22), 29.5 (C-24), 23.5 (C-21), 19.1 (C -10), 16.8 (C-18), 12.0 (C-9), 11.9 (C-19), 9.9 (C-20), 8.1 (C-8)

6) FAB-MS (rel. Int.) M / z [M + Na] ⁺ = 483

Experimental Example 1  : Determination of Protein Tyrosine Dephosphatase 1B Inhibitory Activity

In order to determine the protein tyrosine dephosphatase 1B inhibitory activity of the depseed compound isolated and purified from the fungal strain CRM000232 (KCTC 10696BP) according to the present invention, the following experiment was performed.

Protein tyrosine dephosphorylase 1B was purchased from Biomol (BIOMOL) was used in the experiment.

To spectroscopically measure enzyme activity, 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) at 0.5 μg / ml concentration ), An inhibitor, 20 mM pNPP was added and gently shaken, and then reacted at 30 ° C. for 1 hour, and then absorbance was measured at 410 nm.

Enzyme inhibition rate was calculated by the following equation (1), IC ₅₀ value was determined by the concentration of the inhibitor to the inhibition rate of the enzyme activity 50%.

The results are shown in Table 2.

% Inhibition = [(A-B) / (A-C)] × 100

※ A: absorbance after the reaction of the enzyme

   B: absorbance after the reaction with the inhibitor,

   C: The absorbance after the reaction between the inhibitor and the enzyme.

IC ₅₀ (μM) Compound 1 4.9 Compound 2 69.9

As shown in Table 2, the concentration (IC ₅₀ ) that inhibits the activity of the protein tyrosine dephosphatase 1B of compound 1 by 50% (IC ₅₀ ) was 4.9 μM and compound 2 was 69.9 μM. Therefore, it can be seen that the protein tyrosine dephosphorylation enzyme 1B inhibitory activity of the depsid compound isolated and purified from the fungal strain CRM000232 (KCTC 10696BP) according to the present invention is excellent.

Experimental Example 2  : Diabetic model animal Leprdb / Leprdb  Measurement of hypoglycemic effect in mice

In order to determine the hypoglycemic effect of the depseed compound isolated and purified from the fungal strain CRM000232 (KCTC 10696BP) according to the present invention, the following experiment was performed.

Leprdb / Leprdb mice, a model animal that causes obesity and diabetes beyond the leptin receptor, an appetite-controlling hormone, are accompanied by very high hyperglycemia and consequent diabetes symptoms. Therefore, in order to investigate the hypoglycemic effect of the depsid-based compound, an experiment was conducted on 21 adult Leprdb / Leprdb mice of 2 months of age.

Seven rats were put in each of the two experimental groups, and the depth compound obtained in Example 2 was diluted in dimethylsulfoxide (dimethylsulfoxide, DMSO) and administered intraperitoneally once a day at a concentration of 30 mg / kg. In the case of only the same amount of DMSO was administered.

Blood was collected from retro-orbital sinus using a heparin-treated capillary tube at weekly intervals after administration and blood glucose (ACCU-CHEK Active blood glucose monitor, Roche, Germany) blood glucose) was analyzed to determine the difference between the control and experimental groups.

The results are shown in FIG.

As shown in Figure 1, compared to the blood sugar level of the control group (500 ~ 542 mg / ㎗), Compound 1 showed a 16% lower level (430 mg / ㎗) after 1 week, 8% after 4 weeks Lowered 500 mg / dL.

In addition, Compound 2 was weaker than Compound 1 but showed a 10% lower level (450 mg / dL) after one week, and 4% lower (480 mg / dL) after 4 weeks.

Therefore, it can be seen that the hypoglycemic effect of the depseed compound isolated and purified from the fungal strain CRM000232 (KCTC 10696BP) according to the present invention is excellent.

Experimental Example 3  : Acute Toxicity in Oral Administration in Mice

In order to determine the acute toxicity of the depth-based compound according to the invention, the acute toxicity test was carried out as follows.

Acute toxicity test was performed using two month old Leprdb / Leprdb mice. 10 animals per group were dissolved in dimethylsulfoxide (DMSO), each of which was dissolved in dimethyl sulfoxide (DMSO), and diluted once with water at a dose of 10 g / kg / ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings.

Therefore, all of the compositions of the present invention did not show a change in toxicity even in mice up to 10 g / kg, oral administration minimum dose (LD ₅₀ ) was determined to be a safe substance of more than 10 g / kg depth compound.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation example  : Preparation of Pharmaceutical Formulations

1. Preparation of powder

2 g of the depth compound of Formula 1

1g lactose

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

2. Preparation of Tablets

100 mg of the seed compound of Formula 1

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

100 mg of the depth compound of Formula 1

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Depth-based compounds isolated and purified from the fungal strain CRM000232 (KCTC 10696BP) according to the present invention is excellent in protein tyrosine dephosphorylation enzyme 1B inhibitory activity, and excellent in hypoglycemic effect can be useful for the prevention and treatment of diabetes mellitus, In particular, the therapeutic effect is excellent for insulin-independent diabetes mellitus type 2 diabetes.

Claims (5)

Fungal strain Micromuco ramannianus var. Angulisporus isolated from soil CRM000232 (KCTC 10696BP). A protein tyrosine dephosphorase 1B inhibitor comprising the depth compound represented by the following formula (1) as an active ingredient. <Formula 1>

(In Formula 1, R is

Or-(CH ₂ ) ₂ COCH ₃ ) 3. The protein tyrosine dephosphatase 1B inhibitor according to claim 2, wherein the depth-based compound is isolated and purified from the fungal strain Micromuco Lamanniaus bar Angulosporus CRM000232 (KCTC 10696BP) of claim 1. 3. The protein tyrosine dephosphatase 1B inhibitor according to claim 2, wherein the inhibitor is useful for the prevention and treatment of diabetes. 1) extracting and concentrating the culture solution of micromuco Ramananias bar angilisporus CRM000232 (KCTC 10696BP) of claim 1 with ethyl acetate, 2) eluting the concentrate by column chromatography using methanol / water as a mixed solvent, and 3) Separating the eluate under reduced pressure, separating the acetonitrile and water as a mixed solvent by high-performance liquid chromatography separation method of the depth compound represented by the formula (1). <Formula 1>

(In Formula 1, R is

Or-(CH ₂ ) ₂ COCH ₃ )

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