KR20130056467A - New acetylenic acid analogue from the edible mushroom chanterelle (cantharellus cibarius) and its use - Google Patents

Abstract

PURPOSE: A novel acetylenic acid derivative isolated from Cantharellus cibarius is provided to minimize side effects and to prevent and treat diabetes or inflammatory diseases as a PPAR-gamma agonist. CONSTITUTION: A pharmaceutical composition for preventing and treating diabetes contains a Cantharellus cibarius extract as an active ingredient. A method for preparing the derivative comprises: a step of extracting Cantharellus cibarius with methanol; a step of fractioning the extract with ethyl acetate and hexane; a step of preparing a fraction using a mixture solvent of chloroform and methanol(CHCl_3-MeOH) and by concentration gradient silica gel chromatography; a step of preparing a fraction from the fraction using acetonitrile and water(MeCN-H_20) and a reverse phase column; and a step of performing chromatography of the fraction using a mixture of chloroform and acetone(CHCl_3-acetone).

Description

New acetylenic acid analogue from the Edible Mushroom Chanterelle (Cantharellus cibarius) and its use}

The present invention relates to novel acetylene acid derivatives isolated from edible oriole mushrooms and their use. More specifically, the present invention serves as an effector of the peroxysome proliferator-activated receptor gamma, which is useful for the prevention or treatment of diabetes or inflammatory diseases. A novel acetylene acid derivative isolated from oriole mushroom and its use.

Peroxysome proliferator-activated receptors (hereinafter referred to as "PPARs") play a key role in regulating central nervous system and metabolic processes, cell differentiation, inflammatory response and immune response among ligand-activated transcription factors. It belongs to the nuclear receptor superfamily. When PPARs bind to a ligand, they form heterodimers with 9-cis retinoic acid receptors (RXRs) in the nucleus. These heterodimers bind to Perxisome proliferator-response elements (PPREs) in target genes and promoters to regulate transcription and expression of these genes. A total of three PPAR isoforms, alpha (α), delta (δ), and gamma (γ), were identified.

PPARs have been reported to function as regulators of lipid and lipid protein metabolism, glucose homeostasis, and cell differentiation, as well as involved in the regulation of inflammatory responses (Issemann and Green, Nature, 347). : 645-650, 1990; Daynes and Jones, Nat rev Immunol., 2: 748-859, 2002).

Ligands that bind to receptors to produce physiological results are called agonists. Evidence has been reported that PPAR gamma effectors have potential clinical uses for a variety of diseases. PPAR gamma modulates inflammatory responses and PAPR gamma agonists have been shown to exert anti-inflammatory effects by inhibiting the expression of proinflammatory genes such as cytokines, metalloproteases and acute phase response genes (Delerive et al., H. Endocrinol., 169 (3): 453-459, 2001; Gerlman et al., Cell.Mol.Life.Sci., 55: 932-943, 1999). As such, it has been reported that PPAR gamma agonists may be used as therapeutic agents for type 2 diabetes, inflammatory chronic diseases and cancer (Murphy and Holder, Trends Pharmacol. Sci., 21: 469-474, 2000).

PPAR gamma is activated by binding to compounds such as thiazolidinedions, prostaglandin J2 and analogs of PPAR. PPAR gamma activation by ligand binding contributes to gene expression changes important for glucose and lipid metabolism (Olefsky and Saltiel, Trends Endocrinol Metab., 11 (9): 362-368, 2000; Kooners and Vrana, Physiol. Res. , 47: 215-225, 1998). Based on these changes, thiazolidinediones, or glitazones, act as insulin senitizers, which have been used successfully in the treatment of type 2 diabetes. However, glitazone drugs cause fluid retention with peripheral edema, severe cases of heart failure, and weight gain. In particular, troglitazone has a problem such as death from deadly idiopathic hepatotoxicity.

Diabetes, which are metabolic disorders of carbohydrates, have high blood sugar levels and glucose is excreted in the urine, resulting in abnormal production, secretion, or use of insulin. It can't produce or your cells don't respond to insulin, so glucose doesn't get into your cells and remains in your blood and drains into your urine. The prevalence of diabetes was estimated to be less than 1% in Korea in 1970, but increased to 25.65 per 1,000 people in 2001. Diabetes is classified into type 1 diabetes and type 2 diabetes. Type 1 diabetes is caused by genetic factors, viral infections, environmental toxicity, or other causes such as environmental factors and autoimmunity due to the consumption of milk instead of breast milk during infancy. Type 1 diabetes is called pediatric diabetes and our body's immune system, which has to fight infections from the outside, attacks and destroys beta cells that secrete insulin in the pancreas, resulting in no or no insulin secretion. Onset. Type 2 diabetes is caused by environmental factors such as genetic factors, lack of exercise, obesity, stress, drug abuse, high blood pressure, and hypercholesterolemia. Type 2 diabetes, also known as insulin-independent diabetes, accounts for 90% of all diabetes, usually after age 40, and most patients are obese. In type 2 diabetes, the pancreas secretes insulin, but blood sugar levels increase because the body does not effectively utilize the insulin secreted by the body.

Oriole mushroom is an edible mushroom that is distributed all over the world and bears fruit from autumn to early spring. Fruiting body of Oriole mushroom has the effect of protecting against various diseases through its antioxidant effect. Several fatty acids, phenolic compounds, organic acids, carotenoids and steroids isolated from oriole mushrooms showed antioxidant and anti-inflammatory effects. However, nothing related to the transcriptional activity of PPAR-γ has yet been reported.

As a background technology of the present invention, Korean Patent Publication No. 10-0702185 (Nov. 27, 2006) describes a polyvinide consumed from penicillium fungi acting as a PPAR gamma effector, and a Korean patent Hygrophoropsis is published in Korean Patent Publication No. 10-0998570 (December 7, 2010) aurantiaca It is described with respect to the anti-aging composition comprising a strain extract and fractions thereof as an active ingredient. However, the above documents show that Edible Oriole Mushroom ( Cantharellus) The transcriptional activity of PPAR-γ by cibarius ) extract is not disclosed at all.

Republic of Korea Patent Publication No. 10-0702185 (2006.11.27) Republic of Korea Patent Publication No. 10-0998570 (2010.12.07)

The present inventors have studied the biologically active agents isolated from natural products and found that the extract of Oriole mushroom has important activity in the reporter gene assay of PPAR-γ. Furthermore, the inventors of the PPAR-γ reporter gene acetylene black process, new acid in the melodious mushroom extract showed activity (10E, 14 Z) -9- oxooctadeca-10,14-dien-12-ynoic acid with a known of ( 10E, 14 Z) was separated to -9-hydroxyoctadeca-10,14-dien- 12-ynoic acid, thereby completing the present invention reveals the isolation, structure identification, and biological activity of the compounds.

Accordingly, it is an object of the present invention to provide a new acetylenic acid derivative isolated from an oriole mushroom extract and an oriole mushroom extract with few side effects acting as a PPAR-γ agonist.

Another object of the present invention is to provide new uses of such derivatives.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing and treating diabetes containing the extract of Oriole mushroom as an active ingredient.

According to another aspect of the present invention, the present invention provides an acetylene acid derivative represented by the following formula (1).

According to another aspect of the present invention, the present invention comprises a first step of extracting the oriole mushroom extract with methanol; A second step of fractionating the extract extracted in the first step into ethylacetate (ethylacetate: EtOAc) and hexane; A third step of performing fractional gradient silica gel chromatography on the fractions dissolved in the ethyl acetate using a mixed solvent of chloroform and methanol (CHCl ₃ -MeOH) in a second step to obtain a fraction; A fourth step of obtaining a fraction using a reverse phase (RP) column using acetonitrile and water (MeCN-H ₂ 0) from the fraction obtained in the third step; And performing a chromatography on the fraction obtained in the fourth step using a mixed solvent of chloroform and acetone (CHCl ₃ -acetone) to obtain the acetylene acid derivative of Formula 1, It provides a method of manufacturing.

According to another aspect of the invention, the present invention provides a pharmaceutical composition for preventing and treating diabetes containing the acetylene acid derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing and treating inflammatory diseases containing the acetylene acid derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present inventors have already described the new acetylene acid ( 10E , 14 Z ) -9-oxooctadeca-10,14-dien-12-ynoic acid (Formula 1) in extracts of oriole mushrooms that were active during the reporter gene assay of PPAR-γ. Known ( 10E , 14 Z ) -9-hydroxyoctadeca-10,14-dien-12-ynoic acid (Formula 2) was isolated, and the compounds of formula 1 were identified as troglitazone It could be confirmed that it had activity similar to the effect.

Therefore, the present invention provides a pharmaceutical composition for preventing and treating diabetes containing the extract of Oriole mushroom as an active ingredient.

As an extraction solvent for preparing an oriole mushroom extract, which is an active ingredient of the composition of the present invention, a solvent which can be generally used in natural product extraction can be used, and anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetone, and ethyl acetate It is preferred to extract using a solvent selected from the group consisting of butyl acetate and 1,3-butylene glycol, more preferably methanol.

In another aspect, the present invention provides an acetylene acid derivative represented by the formula (1) isolated from the oriole mushroom extract.

The acetylene acid derivative of Formula 1 may be used in the form of a pharmaceutically acceptable salt, and includes salts, hydrates, and solvates prepared by conventional methods.

The compound of Formula 1 according to the present invention is preferably obtained by extraction and purification from the oriole mushroom strain, it can be obtained through organic synthesis.

The method for separating and purifying novel acetylene acid derivatives according to the present invention is as follows.

A first step of extracting the oriole mushroom extract with methanol; A second step of fractionating the extract extracted in the first step into ethylacetate (ethylacetate: EtOAc) and hexane; A third step of performing fractional gradient silica gel chromatography on the fractions dissolved in the ethyl acetate using a mixed solvent of chloroform and methanol (CHCl ₃ -MeOH) in a second step to obtain a fraction; A fourth step of obtaining a fraction using a reverse phase (RP) column using acetonitrile and water (MeCN-H ₂ 0) from the fraction obtained in the third step; And a fifth step of subjecting the fraction obtained in the fourth step to chromatography using a mixed solvent of chloroform and acetone (CHCl ₃ -acetone) to obtain an acetylene acid derivative of Chemical Formula 1.

One embodiment of the preparation method of the novel acetylene acid derivative according to the present invention is as follows. The dry matter of Oriole mushroom is extracted three times with methanol at room temperature. The solvent was removed in vacuo resulting in 2 kg of methanol extract. Methanol extract is suspended in distilled water and fractionated successfully with ethylacetate (EtOAc) and hexane (hexane). The dissolved layer (30 g) in EtOAc gave CHCl ₃ -MeOH (1: 0, 80: 1, 50: 1, 30: 1, 17: 1, 10: 1, 7: 1, 3: 1, gradient) mobile phase Silica gel chromatography was performed using, and 10 fractions were obtained. Fraction 5 (2.3 g) of the obtained fraction was passed through an RP column using MeCN-H ₂ 0 (50: 50-100: 0, gradient) as a mobile phase to obtain 13 fractions. Fraction 7 of the obtained fraction was converted to CHCl ₃ -acetone (1: 0, 80: 1, 50: 1,30: 1, 20: 1, 10: 1, 5: 1, 3: 1, 2: 1, gradient). Use as mobile phase to separate Compound 1 (10.4 mg) and Compound 2 (33.5 mg).

The compound 1 of the present invention is a colorless oil form, and the molecular formula is C ₁₈ H ₂₆ O ₃ according to the molecular weight of the negative HRESIMS (m / z 289.1952 [M−H] ⁻ ; C ₁₈ H ₂₅ O ₃ , 289.1960) It can be seen that. ¹³ C-NMR and HSQC NMR spectra revealed the presence of nine saturated methylene carbons, one primary methyl group, four olephic carbons, one acetylene group, one carboxyl group, and one ketone group. ^The presence of a carbonyl carbon signal of δ _C 178.17 and a broad methylene signal at δ _H 1.34 in the ¹³ C-NMR spectrum indicates that Compound 1 is a fatty acid. The presence of acetylene groups was inferred from two quaternary carbon signals, δ _C 96.4 and δ _C 91.0. To establish the connectivity of the C-18 to C-14 used the ¹ H- ¹ H COSY and HMBC NMR spectrum. In the HMBC NMR spectrum, H-10 (δ _H 6.49) and C-12 (δ _C 91.0) are correlated, and C-11 (δ _C 123.3) and C-14 (δ _C 108.7) signals Shifting indicates that one acetylene group is located between two double bonds. The location of the ketone group was inferred from the correlation of H-8 (δ _H 2.55) and H-10 (δ _H 6.49) with C-11 (δ _C 199.3) in the HMBC NMR spectrum. These correlations clearly show that the ketone group is located at C-9. In addition, ¹ H- ¹ H NMR COSY found a correlation of the H-2 / H-3, H-3 / H-4 through the spectrum. We found that the compounds have E and Z geometries through the coupling constants of two double bonds, C-10 / C-11 and C-14 / C-15, at 16.0 Hz and 11.0 Hz. From these results, the structure of the new acetylene acid (10 E , 14 Z ) -9-oxooctadeca-10,14-dien-12-ynonic acid was identified.

The present invention provides a PPAR gamma effector containing the oriole mushroom extract or the novel acetylene acid derivative of Formula 1 isolated therefrom as an active ingredient.

The PPAR transcriptional activity of compounds 1 and 2 was investigated in CHO cells transfected with PPAR-γ expression vector and PPRE-driven luminescent enzyme gene. Compounds 1 and 2 did not activate PPAR-α or PPAR-δ at concentrations of 20 μg / mL. However, while the EC ₅₀ value of Compound 2 exceeded 20 μg / mL and showed no activity, the new acetylene acid (Compound 1) selectively activated PPAR-γ with an EC ₅₀ value of 1.88 μM. The activity of troglitazone, an effector of PPAR-γ, which is known to positively regulate PPAR-γ, was examined under the same experimental conditions, and the EC ₅₀ value was 0.44 μM.

Next, quantitative real-time PCR was performed to analyze the effect of compound 1 on target gene transcription of PPAR-γ in HepG2 hepatocytes. PPAR-γ expression was shown to be upregulated by 2.43 fold. Although Compound 1 did not affect the expression of SCD-1, it reduced the expression of two glucose producing genes, G6Pase and PEPCK. This shows an activity comparable to that of troglitazone. These results show that oriole mushroom containing acetylene acid with strong PPAR-γ transcriptional activity can provide a pharmaceutical composition for preventing and treating diabetes or inflammatory disease (see FIG. 3).

Furthermore, the present invention provides a pharmaceutical composition for preventing and treating diabetes containing the oriole mushroom extract, a novel acetylene acid derivative of Formula 1 isolated therefrom or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating inflammatory diseases containing the oriole mushroom extract, a novel acetylene acid derivative of Formula 1 isolated therefrom or a pharmaceutically acceptable salt thereof as an active ingredient.

As described above, oriole mushroom extract or a novel acetylene acid derivative of Formula 1 isolated therefrom acts as a PPAR gamma effector, which is useful as a pharmaceutical composition for preventing and treating diabetes, or a pharmaceutical composition for preventing and treating inflammatory diseases. Can be used.

Pharmaceutically acceptable salts of Formula 1 include salts of hydroxy groups which may be present in compounds of formula, unless otherwise indicated, and salts of hydroxy groups include alkali metal salts such as lithium, sodium, potassium, magnesium And alkali earth metal salts such as calcium. Preferably, physiologically acceptable salts with sodium, potassium, calcium and the like can be cited, and these salts can be produced through the production method or the production process of salts known in the art.

In addition, Chemical 1 of the present invention may be prepared with pharmaceutically acceptable non-toxic salts and solvates according to methods conventional in the art. Pharmaceutically acceptable salts include acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

Pharmaceutically acceptable salts of the acetylene acid derivatives of the present invention include salts of acidic or basic groups which may be present in the acetylene acid derivatives of the present invention, unless otherwise indicated. For example, pharmaceutically acceptable salts include the sodium, calcium, and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, But are not limited to, salts known in the art such as, for example, salts of phosphoric acid, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p- toluenesulfonate (tosylate) And can be manufactured through a manufacturing method or a manufacturing process.

 In the composition of the present invention, it may preferably comprise 0.0001 to 90% by weight of the acetylene acid derivative relative to the total weight of the composition. In the composition of the present invention, preferably, the composition may have a form of powder, granule, tablet, capsule, injection, cream, gel, patch, spray or ointment formulation. The pharmaceutical composition containing the acetylene acid derivative of the present invention as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

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, as necessary. And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added and formulated into injectable solutions, 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 by using the method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Eastern PA. have.

The compositions of the present invention may be parenterally administered (eg, applied intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method.

Preferred dosages of the extracts or compounds of the present invention vary depending 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, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound of the present invention is preferably administered at 0.0001 to l00 mg / kg, preferably at 0.001 to 10 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

According to the present invention configured as described above, the oriole mushroom extract according to the present invention and the novel acetylene acid derivative isolated therefrom act as a PPAR gamma effector, and as a natural extract component, have relatively low side effects and toxic expression rate and are biocompatible. Diabetes or inflammatory diseases can be effectively prevented and treated.

1 is a view showing the structure of compounds 1 and 2 according to the present invention.
FIG. 2 is a diagram showing selected HMBC (→) and COSY ( − ) correlations of compound 1. FIG.
3 shows the expression of PPAR-γ target genes in HepG2 hepatocytes. Cells were treated with troglitazone (10 μM) or various concentrations (μM) of Compound 1, respectively, and gene expression was analyzed by real-time PCR (NC: nontreated control; PEPCK: phosphoenolpyruvate carboxylkinase; SCD-1: stearoryl CoA desaturase; G6Pase: glucose -6 phosphatase. *, P <0.05 compared with NC).

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples illustrate the present invention, and the scope of the present invention is not limited by the following examples.

Example  1. From Oriole Mushroom Extract Acetylene acid  Derivative Separation

Oriole mushrooms were purchased from a forest wild mushroom company in Chengdu, China, and confirmed by one of the inventors, H. Z. Jin. The control sample is kept in the Department of Biotechnology, Korea University, Seoul, Korea.

The dry matter of Oriole mushroom was extracted three times with methanol at room temperature. The solvent was removed in vacuo to give 2 kg of methanol extract. Methanol extract was suspended in distilled water and was successfully fractionated using ethylacetate (EtOAc) and hexane. The dissolved layer (30 g) in EtOAc gave CHCl ₃ -MeOH (1: 0, 80: 1, 50: 1, 30: 1, 17: 1, 10: 1, 7: 1, 3: 1, gradient) mobile phase Silica gel chromatography was performed using, and 10 fractions were obtained. Fraction 5 (2.3 g) of the obtained fraction was obtained by passing through a RP column using MeCN-H ₂ 0 (50:50-100: 0, gradient) as a mobile phase. Fraction 7 of the obtained fraction was converted to CHCl ₃ -acetone (1: 0, 80: 1, 50: 1,30: 1, 20: 1, 10: 1, 5: 1, 3: 1, 2: 1, gradient). Compound 1 (10.4 mg) and Compound 2 (33.5 mg) were separated using the mobile phase.

Example  2. Structure Analysis of Compounds

In order to analyze the structures of the compounds 1 and 2 obtained in Example 1, the following analysis was carried out. NMR spectra were obtained with TMS as an internal standard using a Varian 500 MHz NMR spectrometer and chemical shifts were expressed as δ values. ESI-MS was measured using a Waters Q-TOF micro mass spectrometer. UV and IR spectra were obtained using Mecasys Optizen 2120 UV and Varian 640-IR, respectively.

As a result of comparing the results of the instrumental analysis with the literature, the compound of the present invention is represented by the formula (1), it was confirmed that the acetylene acid derivative (C ₁₈ H ₂₆ O ₃ ), a new compound that has not yet been reported.

Looking more specifically, Compound 1 and Compound 2 were analyzed as follows.

[Compound 1]

(10 E , 14 Z ) -9-oxooctadeca-10,14-dien-12-ynoic acid ( 1 ): colorless oil, measured by MeOH with UV spectrum, showed maximum absorption wavelength at 218, 309 nm As a result of measuring the spectrum, bands were observed at 3420, 2927, 2856, 2360, 1735, 1245, and 761 cm ⁻¹ . ¹ H and ¹³ C NMR data are shown in Table 1. In the voice ESI-MS 289.2 [M - H ] -, 579.3 [2M - H] - was the value of the observed 289.1952 in voice high-resolution ESI-MS [M - H] - (C 18 H 25 O 3, estimated at 289.1960) Was obtained and the molecular formula is C ₁₈ H ₂₅ O ₃ . Accordingly, (10 E , 14 Z ) -9-oxooctadeca-10,14-dien-12-ynoic acid ( 1 ) was identified as a novel compound.

[Compound 2]

(10 E , 14 Z ) -9-hydroxyoctadeca-10,14-dien-12-ynoic acid ( 2 ): colorless oil and photoluminescence measurement [a] _D ²⁷ -3.5 ° ( c 0.3, CHCl ₃ ) Is obtained. UV spectra were measured using MeOH and showed maximum absorption wavelengths at 266 and 280 nm. The ¹ H and ¹³ C NMR data are shown in Table 1. In the voice ESI-MS 291 [M - H ] -, 583 [2M - H] - , the value of was observed.

^a The assignments were based on DEPT, HSQC, and HMBC experiments.

Example  3. PPAR  Efficacy Analysis for Gamma Activity

The PPAR transcriptional activity of compounds 1 and 2 was investigated in CHO cells transfected with PPAR-γ expression vector and PPRE-driven luminescent enzyme gene.

More specifically, CHO cells were cultured at a concentration of 1 × 10 ⁵ per 1-well in 24-well plates. The next day, using Hilymax, cells were transtransformed with the report vector pGL3-PPRE3-TK-luc (including firefly luciferase under the control of PPRE), an expression vector encoding human PPAR-γ, and an expression vector encoding β-galactosidase. Specified. The medium was removed after 4 hours of incubation and replaced with DMEM containing high glucose. After 18 hours the cells were treated with compound (0, 5, 20, 40, 60, 80, or 100 μM) or troglitazone (10 μM) for 24 hours and lysed in firefly luciferase lysis buffer. Luciferase activity in cell lysates was measured by a firefly luciferase measurement set according to the manufacturer's instructions. The activity of β-galactosidase was measured by the β-galactosidase enzyme measurement system. Luciferase activity was expressed in relation to the activity of β-galactosidase in the same lysate to normalize the results for the transfection effect.

Total RNA was isolated from cells regulated or treated using the RNAiso Plus set. During cDNA synthesis, 2 μg of RNA was reverse transcribed with PrimeScript reverse transcriptase at 20 μL of oligo (dT). Human gene-specific primers for PCR were designed by the NCBI's nucleotide BLAST tool. PCR was performed using these primers and the iQ SYBR Green Supermix reagent from the iQ5 iCycler system. PCR conditions consisted of 60 rotations of 10 seconds at 95 ° C., 15 seconds at 60 ° C., and 20 seconds at 72 ° C. after initial denaturation (95 ° C., 3 minutes). To determine the specificity of the primer, the 71 rotation denaturation curve started at 55 ° C. and rose 0.5 ° C. every 10 seconds. Gene expression levels were normalized against the levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and analyzed using iQ5 system software.

As a result, Compounds 1 and 2 did not activate PPAR-α or PPAR-δ at a concentration of 20 μg / mL. However, while the EC ₅₀ value of Compound 2 exceeded 20 μg / mL and showed no activity, the new acetylene acid (Compound 1) selectively activated PPAR-γ with an EC ₅₀ value of 1.88 μM. The activity of troglitazone, an effector of PPAR-γ, which is known to positively regulate PPAR-γ, was examined under the same experimental conditions, and the EC ₅₀ value was 0.44 μM.

Next, quantitative real-time PCR was performed to analyze the effect of compound 1 on target gene transcription of PPAR-γ in HepG2 hepatocytes. PPAR-γ expression was shown to be upregulated by 2.43 fold. Although Compound 1 did not affect the expression of SCD-1, it reduced the expression of two glucose producing genes, G6Pase and PEPCK. This shows an activity comparable to that of troglitazone. These results show that oriole mushroom containing acetylene acid with strong PPAR-γ transcriptional activity can provide a pharmaceutical composition for preventing and treating diabetes or inflammatory disease (see FIG. 3).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is obvious that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

Claims (6)

Pharmaceutical composition for the prevention and treatment of diabetes mellitus containing the extract as the active ingredient. An acetylene acid derivative represented by the following formula (1).
[Formula 1]

The acetylene acid derivative according to claim 2, wherein the acetylene acid derivative is isolated from an oriole mushroom. A first step of extracting the oriole mushroom extract with methanol;
A second step of fractionating the extract extracted in the first step into ethylacetate (ethylacetate: EtOAc) and hexane;
A third step of performing fractional gradient silica gel chromatography on the fractions dissolved in the ethyl acetate using a mixed solvent of chloroform and methanol (CHCl ₃ -MeOH) in a second step to obtain a fraction;
A fourth step of obtaining a fraction using a reverse phase (RP) column using acetonitrile and water (MeCN-H ₂ 0) from the fraction obtained in the third step; And
The acetylene acid derivative according to claim 2, wherein the fraction obtained in the fourth step is chromatographed using a mixed solvent of chloroform and acetone (CHCl ₃ -acetone) to obtain an acetylene acid derivative of Formula 1. How to prepare. A pharmaceutical composition for preventing and treating diabetes, comprising the acetylene acid derivative of claim 2 or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for preventing and treating inflammatory diseases comprising the acetylene acid derivative of claim 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

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