KR101602799B1 - Anti-inflammatory active composition and pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to an antiinflammatory active composition and a pharmaceutical composition for preventing or treating inflammatory diseases, and more particularly, to a composition for preventing or treating inflammatory diseases, Reduction of NF-κB activity in the nucleus, or inhibition of the activity of AP-1, and a pharmaceutical composition for preventing or treating inflammatory diseases containing the same.

Description

TECHNICAL FIELD The present invention relates to an anti-inflammatory active composition and a pharmaceutical composition for preventing or treating an inflammatory disease,

The invention anti-inflammatory activity compositions, and relates to a pharmaceutical composition for inflammatory disease prevention or treatment comprising the same, more specifically to the intracellular nitric oxide (NO) expression level decreased, intracellular prostaglandin (PGE _2; prostaglandin E ₂₎ Reduction of NF-κB activity in the nucleus, or inhibition of the activity of AP-1, and a pharmaceutical composition for preventing or treating inflammatory diseases containing the same.

Inflammatory reactions are caused by enzymatic activation of various inflammatory mediators and immune cells in the local blood vessels and body fluids when the tissues (cells) are damaged or infected with external infectious agents (bacteria, fungi, viruses, various allergens) Inflammatory mediator secretion, fluid infiltration, cell migration, tissue destruction, and a combination of physiological responses such as erythema, edema, fever and pain. Specifically, when foreign bacteria invade a specific tissue and proliferate, the leukocyte of the living body recognizes it and actively attacks the propagated external bacteria. The death of leukocyte during this process is accumulated in the invaded tissue At the same time, the cell debris of invading microorganisms killed by leukocytes melts into the invading tissues and abscess forms.

In normal cases, the inflammatory reaction removes the external infectious agent and regenerates the damaged tissue to regenerate the organism's function. However, if the antigen is not removed or the internal substance causes the inflammatory reaction to occur excessively or continuously, Inflammation, inflammation, inflammation in the joints such as rheumatoid arthritis, skin diseases in the form of psoriasis, and allergic inflammatory diseases such as bronchial asthma. In addition, they become obstacles in transfusion, drug administration and transplantation.

With the recent development of molecular biology, inflammatory diseases have been attempted to be understood at the molecular level of cytokine, and the factors affecting these diseases are also being clarified one by one. Among these factors, nitric oxide (NO) plays a role in maintaining homeostasis by defending against pathogenic DNA as a mediator of inflammatory processes (Kou and Schroder, Annuals of Surgery 221, 220-235, 1995 ).

NO is produced from L-arginine by three major nitric oxide synthase (NOS) isomers, nNOS (neuronal NOS), eNOS (endothelial NOS) and iNOS (inducible NOS). nNOS and eNOS are controlled by Ca ^{2 +} / calmodulin (calmodulin), but, iNOS is regulated at the transcriptional level by inflammatory stimuli such as IL (interleukin), IFN (interferon), LPS. NO produced by nNOS or eNOS plays a normal physiological function such as vasodilation, neurotransmission, cell destruction to pathogen, etc. However, overexpressed NO produced by macrophages in iNOS is involved in various pathophysiological , Which reacts with superoxide to form peroxynitrite which acts as a powerful oxidant to damage cells and activate NF-κB in macrophages activated by inflammatory stimuli (Lawrence et al., Nat Med., 7: 1291-1297, 2001), which is known to be involved in chronic diseases such as inflammation, cancer, and arteriosclerosis.

Poster prostaglandin E ₂ (PGE ₂₎ and leukotriene also as inflammatory mediators produced from Araki donik acid, in particular PGE ₂ is cycloalkyl oxide synthase (COX-2; cyclooxygenase-2 enzyme) is produced by, mainly macrophages and monocytes It is produced in many cells.

Currently, anti-inflammatory drugs include dexamethasone and cortisone using corticosteroids. However, they act as anti-inflammatory drugs, but they are toxic and cause side effects such as edema. In addition, they sometimes fail to act selectively for the cause of inflammation and cause severe immunosuppression [Check W.A. and Kaliner M. A., Am. Rev. Respir. Dis., 141, pp. 44-51. 1990].

Therefore, although anti-inflammatory compositions containing an extract of Pseudomonas fluorescens as an active ingredient are disclosed in an anti-inflammatory drug alternative to corticosteroids, There is a problem that the extraction yield is low.

In addition, steroid drugs using the existing antiinflammatory drug, corticosteroid, exhibit severe side effects such as edema, and it is urgent to develop a nonsteroidal drug having no side effects.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a non-steroidal ingredient having no adverse effects such as a conventional chemical anti-inflammatory drug, reducing the expression level of NO (NO), production of PGE ₂ (prostaglandin E ₂ ) , Anti-inflammatory activity through the inhibition of NF-κB translocation in the nucleus, or inhibition of the activity of AP-1, and is not cytotoxic as a natural product-derived substance, and a pharmaceutical composition for preventing or treating inflammatory diseases containing the same .

The present invention provides an anti-inflammatory active composition comprising at least one compound selected from the group consisting of a compound represented by the following formula (1), a compound represented by the following formula (2), and a salt thereof:

(Wherein, R ₁ is alkenyl Diallo giim of C ₁₆ ~ C _18.)

According to a preferred embodiment of the present invention, the compound represented by the formula (1), the compound represented by the formula (2), or a salt thereof may be derived from the mushroom.

According to another preferred embodiment of the present invention, the antiinflammatory activity composition is an amount of nitric oxide (NO) expressed in a cell; Within the cell prostaglandin (PGE _2; prostaglandin E ₂₎ production; Nuclear translocation of NF-κB; And < RTI ID = 0.0 > AP-1. ≪ / RTI >

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising the above-mentioned anti-inflammatory active composition.

According to a preferred embodiment of the present invention, the pharmaceutical composition may contain an anti-inflammatory active agent at a concentration of 50 to 200 μM.

Furthermore, the present invention provides an anti-inflammatory functional cosmetic composition comprising the above-mentioned anti-inflammatory active composition.

In addition, the present invention provides a health functional food composition for preventing or ameliorating an inflammatory disease comprising the above-mentioned anti-inflammatory active composition.

In addition, the present invention provides a quasi-drug composition for the prevention or amelioration of an inflammatory disease comprising the above-mentioned anti-inflammatory active composition.

According to a preferred embodiment of the present invention, the quasi-drug composition is at least one of the group consisting of disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment agent, coating agent, .

Hereinafter, terms used in this specification will be briefly described.

"Inflammation" of the present invention means a pathological condition of an abscess formed by the infiltration of external infectious agents (bacteria, fungi, viruses, various kinds of allergens).

The term " treatment "or" improvement "as used herein refers to all actions that inhibit or delay the onset of symptoms caused by the administration of the composition by an inflammatory disease. Means any act that improves or alleviates the symptoms caused by.

Furthermore, the "quasi-drug product" of the present invention refers to a drug, a drug or the like which is used for treating, alleviating, treating or preventing a disease of a human or an animal, An article which is not an appliance or a machine and which is similar to a product used for sterilization, insecticide and similar uses for the prevention of infectious diseases and which is intended to diagnose, treat, alleviate, Means an article, except machinery, machinery or equipment, which is not an apparatus, machine or apparatus, used for the purpose of causing pharmacological effects on the structure and function of humans or animals;

In addition, the term "anti-inflammatory activity" of the present invention refers to inhibition of inflammation. The inflammation is one of biological tissue defense responses to certain stimuli, and is a complex reaction involving three kinds of tissue degeneration, circulatory disorder and exudation, Lesion. More specifically, inflammation is part of congenital immunity and, like in other animals, human congenital immunity recognizes a pattern of cell surfaces that are specifically present in a pathogen. Phagocytes recognize cells with such surfaces as non-magnetic and attack pathogens. If pathogens break through the physical barriers of the body, an inflammatory reaction occurs. Inflammation is a nonspecific defense that creates hostile environments for microorganisms entering the wound. In the inflammatory response, white blood cells that are responsible for the early stage of immune response, when wounded or infected, enter the body to express cytokines. Therefore, the expression level of intracellular cytokine is an index of inflammatory response activation.

The present invention relates to a non-steroidal composition which is free of side effects such as a conventional chemical anti-inflammatory agent, and is a non-steroidal component that reduces the expression of NO in the cell, decreases the amount of PGE ₂ (prostaglandin E ₂ ) Or anti-inflammatory activity through inhibition of the activity of AP-1, and has no cytotoxicity as a natural-substance-derived substance, and a pharmaceutical composition for preventing or treating inflammatory diseases containing the same.

1 is a graph showing the results of ¹ H NMR spectral analysis among spectroscopic analysis results of Compound 1 of Experimental Example 1. FIG.
FIG. 2 is a graph showing the results of ^{13 C} NMR spectral analysis among spectroscopic analysis results of Compound 1 of Experimental Example 1. FIG.
3 is a graph showing the results of ESI-MS spectral analysis among spectroscopic analysis results of Compound 1 of Experimental Example 1. FIG.
4 is a graph showing the results of IR spectral analysis among spectroscopic analysis results of Compound 1 of Experimental Example 1. FIG.
5 is a graph showing the results of ¹ H NMR spectral analysis among spectroscopic analysis results of Compound 2 of Experimental Example 1. FIG.
FIG. 6 is a graph showing the results of ^{13 C} NMR spectral analysis among spectroscopic analysis results of Compound 2 of Experimental Example 1. FIG.
7 is a graph showing the results of ESI-MS spectral analysis among spectroscopic analysis results of Compound 2 of Experimental Example 1. FIG.
FIG. 8 is a graph showing the results of IR spectral analysis among spectroscopic analysis results of Compound 2 of Experimental Example 1. FIG.
FIG. 9 shows the cytotoxicity measurement results of cells treated with Compound 1 or Compound 2 in Experimental Example 2. FIG.
FIG. 10 shows the results of measurement of the amount of nitric oxide expressed in the cells treated with the extract of Mushroom mushroom extract of Example 1-1 of Example 1-2 in Experimental Example 3-1 by concentration.
FIG. 11 shows the results of measurement of the amount of nitric oxide expressed in cells treated with the fraction of Norwegian mushroom nucleic acid or the fraction of methylene chloride of Norwegian mushroom by concentration in Experimental Example 3-1.
FIG. 12 shows the results of measurement of the amount of nitric oxide expressed in cells treated with the concentrations of Comparative Examples 1 to 4, Compound 1 or Compound 2 in Experimental Example 3-1.
FIG. 13 shows the results of measurement of the amount of nitric oxide expression in cells treated with Compound 1 or Compound 2 at different concentrations in Experimental Example 3-1.
14 shows the results of measurement of PGE2 expression level of cells treated with Compound 1 or Compound 2 in Experimental Example 3-2.
15 shows the results of measurement of NF-κB or AP-1 protein expression of cells treated with Compound 1 in Experimental Example 4. FIG.
16 shows the results of measurement of NF-κB or AP-1 protein expression of cells treated with Compound 2 in Experimental Example 4. FIG.

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

As described above, there are no severe side effects such as edema of steroid medicines using the existing anti-inflammatory drug, corticosteroid, and there is no cytotoxicity and high anti-inflammatory activity. Thus, development of a naturally occurring substance suitable for use in the prevention or treatment of inflammatory diseases There was this urgent problem.

The present invention has been made to solve the above-mentioned problems by providing an anti-inflammatory active composition containing at least one compound selected from the group consisting of a compound represented by the following formula (1), a compound represented by the following formula (2) This is a non-steroidal component that has no adverse effects such as conventional chemical anti-inflammatory drugs. It is a non-steroidal component that decreases the expression of NO in the cells, decreases the amount of PGE ₂ (prostaglandin E ₂ ), decreases the NF- Or anti-inflammatory activity through inhibition of the activity of AP-1, and has no cytotoxicity as a natural-substance-derived substance, and a pharmaceutical composition for preventing or treating inflammatory diseases containing the same.

[Chemical Formula 1]

(Wherein, R ₁ is alkenyl Diallo giim of C ₁₆ ~ C _18.)

(2)

The present invention provides an anti-inflammatory active composition comprising at least one compound selected from the group consisting of the compounds represented by the general formulas (1), (2) and their salts.

)일 수 있다.R 1 of the compound represented by the formula (1) may be a C ₁₆ -C ₁₈ dialkenyl group, more preferably a C ₁₇ dialkenyl group, and most preferably a

).

The compounds represented by the general formula (1) or the compounds represented by the general formula (2) can be obtained by general chemical synthesis, separated from natural products or sold, and preferably those separated from natural products, More preferably, it is possible to use those derived from Mushroom mushroom.

In one embodiment of the present invention, the compound represented by the formula (1) and / or the compound represented by the formula (2) can be obtained by methanol extraction, organic solvent fractionation and chromatography.

The salt of the compound represented by the formula (1) or the compound represented by the formula (2) may be an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, metal sulfonic acid, acetic acid, glycolic acid, Acid, 4-toluenesulfonic acid, galacturonic acid, embic acid, glutamic acid, citric acid, and sputanic acid.

The addition salt according to the present invention can be obtained by a conventional method, that is, dissolving the compound represented by Formula 1 or the compound represented by Formula 2 in a water-miscible organic solvent such as acetone, methanol, ethanol or acetonitrile, By adding an organic acid or by adding an aqueous acid solution of an inorganic acid, followed by precipitation or crystallization, or by evaporating a solvent or an excess of an acid, followed by drying or precipitating the salt by suction filtration.

The present invention encompasses the compounds represented by the above general formula (1), the compounds represented by the general formula (2) or their respective salts as well as solvates, hydrates and stereoisomers which exhibit the same effects as those which can be prepared therefrom.

As shown in Experimental Example 4 and FIGS. 15 to 16, the antiinflammatory activity composition exhibits a decrease in nuclear translocation of NF-κB (nuclear factor kappa B), which is a major transcription factor involved in the inflammatory reaction, 1 (activating protein 1), thereby confirming the remarkable anti-inflammatory activity.

Furthermore, as will be confirmed in Experimental Example 2 and figures 12 to 14, wherein the anti-inflammatory activity composition intracellular nitric oxide (NO) reduction of the expression level and intracellular prostaglandin; through (PGE _2, prostaglandin E ₂₎ reduction in the amount And excellent anti-inflammatory activity was confirmed.

In addition, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising the above-mentioned anti-inflammatory active composition.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But may include calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, But is not limited thereto.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, And the like. When administered orally, the protein or peptide is extinguished and the oral composition should be formulated to coat the active agent or protect it from degradation from above. The pharmaceutical composition may also be administered by any device capable of transferring the active agent to the target cell.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001 to 100 mg / kg. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat vascular disease and / or an amount sufficient to inhibit angiogenesis.

The pharmaceutical composition of the present invention may be administered as an individual prophylactic agent or a therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

The pharmaceutical composition of the present invention is not particularly limited in its concentration and / or content as long as it contains the anti-inflammatory active ingredient as described above, but it may preferably be contained at a concentration of 50 to 200 μM.

If the concentration is less than 50 μM, as shown in Experimental Example 3 and FIGS. 13 to 14, the inhibitory activity of intracellular nitric oxide and intracellular prostaglandin formation is low, so that it can be used for prevention or treatment of inflammatory diseases Incompatible problems can occur, and toxicity problems that can kill the cells themselves can occur if they are included at concentrations above 200 μM.

In addition, the present invention provides an anti-inflammatory functional cosmetic composition comprising an anti-inflammatory active ingredient as described above.

The cosmetic composition is not particularly limited in its concentration and / or content as long as it contains the above-mentioned anti-inflammatory active composition.

The cosmetic composition is not particularly limited so long as it is a formulation suitable for use in the prevention or improvement of inflammatory diseases. For example, it may be formulated into solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, But is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

The ingredients contained in the cosmetic composition of the present invention include, in addition to the active ingredient and the carrier ingredient, the ingredients conventionally used in cosmetic compositions and include conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, .

The present invention also provides a health functional food composition for preventing or ameliorating an inflammatory disease comprising the anti-inflammatory active ingredient as described above.

The kind of health functional food is not particularly limited as long as it is usually produced and / or sold. For example, dairy products such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums and ice cream, soups, drinks, tea, drinks, alcoholic beverages, And can be used in the form of pills, powders, granules, infusions, tablets, capsules or beverages, all of which include health functional foods in the conventional sense.

The health beverage composition of the present invention is not particularly limited to a liquid ingredient except that it contains the above-mentioned anti-inflammatory active composition and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages.

Usually, the amount of the anti-inflammatory active composition contained in the health functional food composition may include 0.1 to 50% by weight, preferably 1 to 40% by weight of the total food. In addition, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

Furthermore, the present invention provides a quasi-drug composition for preventing or improving an inflammatory disease comprising the above-mentioned anti-inflammatory active composition.

That is, the anti-inflammatory active composition of the present invention may be added to a quasi-drug composition for the purpose of preventing or improving an inflammatory disease. When the anti-inflammatory active ingredient of the present invention is used as a quasi-drug additive, the anti-inflammatory active composition may be added as it is or may be used together with other quasi-drugs or quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

The quasi-drug composition is not particularly limited as long as it is for preventing or ameliorating an inflammatory disease. For example, the quasi-drug composition can be used in the manufacture of disinfectant cleaners, shower foams, gags, wet tissues, detergent soaps, handwashes, humidifier fillers, masks, ointments, coatings or filter fillers.

Hereinafter, the present invention will be described in more detail with reference to the following Examples. However, these drawings and the following embodiments are only illustrative of the contents and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the technical idea of the present invention based on these examples.

[ Example ]

Example  One: Mushroom mushroom  Separation of single compounds derived

1-1. Mushroom  Extract preparation

Three kilograms of dried mushroom (Pocheon mushroom), which was dried with the entire fruiting body, was pulverized, and 20 liters of 80% methanol was added and extracted twice at 25 ° C for 24 hours. The mixture was filtered through a filter paper, and the filtrate was concentrated under reduced pressure to obtain 300 g of Mushroom extract.

1-2. Spindle  Organic solvent from mushroom extract Fraction  Produce

300 g of the mushroom extract of Example 1 prepared in Example 1-1 was suspended in 2 L of distilled water, 2 L of hexane was further suspended, and the mixture was partitioned into hexane, methylene chloride, ethyl acetate and butanol in sequence. The hexane, methylene chloride, Ethyl acetate and butanol were evaporated and concentrated to give hexane, methylene chloride, ethyl acetate and butanol fractions.

1-3. Organic solvent From the fraction  activation Fraction  And preparation of compounds

The hexane, methylene chloride, ethyl acetate and butanol fractions obtained in Example 1-2 above were subjected to chromatography using a silica gel column to obtain another fraction. That is, a stepwise gradient solvent system consisting of a hexane-ethyl acetate mixed solvent (99%: 1%, 50%: 50%) and a methylene chloride- methanol mixed solvent (98%: 2%, 50%: 50% (HEH1 to HEH6), 9 fractions of methylene chloride fractions (HEM1 to HEM9) and fractions of ethyl acetate fraction (HEE1 to HEE5) were obtained, respectively.

Active fractions were obtained by confirming that HEH2, HEM2, HEM6, HEM7 and HEE3 in the fraction showed inhibitory activity in a concentration dependent manner on acetylcholinesterase.

1-4. activation From the fraction  Separation of single compounds

Compounds 1 and 2 were separated and purified by HPLC analysis of HEM2 and HEM6, respectively, as fractions of the active fractions HEH2, HEM2, HEM6, HEM7 and HEE3 of Example 1-3.

Comparative Example  One.

As the extract of Aspergillus oryzae, the compound represented by the following formula (3) is regarded as the present comparative example.

Comparative Example  2.

As the extract of Aspergillus oryzae, the compound represented by the following general formula (4) is regarded as the present comparative example.

Comparative Example  3.

As the extract of Aspergillus oryzae, a compound represented by the following formula (5) was used as the present comparative example.

Comparative Example  4.

As the extract of Aspergillus oryzae, a compound represented by the following formula (6) was used as the present comparative example.

Experimental Example  1: Analysis of the structure of Compound 1 and Compound 2.

The molecular weights and molecular weights of Compound 1 and Compound 2 isolated in Example 1-4 were determined using an LC / MS spectrometer (manufacturer: Thermo finnigan LCQ deca plus) and analyzed with a nuclear magnetic resonance (NMR) analyzer (Verian 500 MHz) ≪ ¹ > H NMR and < ¹³ > C NMR spectra were obtained, and the molecular structure was determined. ¹ H NMR results are shown in Figs. 1 and 5, ¹³ C NMR results are shown in Figs. 2 and 6, ESI-MS spectral analysis results are shown in Figs. 3 and 7, and IR spectral analysis results are shown in Figs. Figs. 1 to 4 are the results of the analysis of Compound 1, and Figs. 5 to 8 are the results of the analysis of Compound 2. Fig.

As a result, the chemical structures of Compound 1 and Compound 2 were determined as follows.

Compound 1:

1) Properties: Gum

2) Molecular weight: 594

3) Molecular formula: C ₃₇ H ₅₄ 0 ₆

^{4) 1 H NMR (500MHz,} CD 3 OD): δ12.45 (1H, s, OH), 10.14 (1H, s, CHO), 6.71 (1H, s, H-6), 6.12 (1H, s, H-6 '), 5.33 ( 8H, m, H-9 ", H-10", H-12 ", H-13"), 5.32 (2H, s, H-1-CH 2), 5.32 (1H , t, J = 7.0 Hz, H-2 '), 3.91 (3H, s, OCH 3), 3.48 (2H, d, J = 7.5 Hz, H-1'), 2.99 (2H, s, H-4 '), 2.76 (2H, dd , J = 6.5, 6.5 Hz, H-11 "), 2.32 (2H, dd, J = 7.5, 7.5 Hz, H-2"), 2.07 (3H, s, H-9 M, H-8 ", H-14"), 1.83 (3H, s, H-8 '), 1.74 H-3 "), 1.60 ( 4H, m, H-7", H-15 "), 1.25 (10H, m, others), 0.87 (3H, t, J = 7.0 Hz, H-18")

5) ¹³ C NMR (125 MHz, CD ₃ OD): δ200.4 (C-5 ', 194.0 (CHO), 174.1 (C-1 "), 163.4 (C-7 '), 139.8 (C-1), 129.7 (C-3'), 130.6,129.9,127.9,127.8 '), 122.5 (C-6 '), 116.6 (C-4), 112.8 (C-2), 105.6 (C-6), 62.7 (C-1-CH 2), 55.4 (OCH 3), 54.9 ( C-4 '), 33.7 (C-2), 28.9 (C-8'), 21.1 18 "), 31.8, 31.4, 29.5, 29.4, 29.2, 28.8, 26.9, 26.5, 25.3, 24.8, 22.4 (C-3" -15 ", 11", 14 "-17").

6) ESI-MS data at m / z 617 [M + Na] ⁺ (calcd. For C ₃₇ H ₅₈ 0 ₆ Na: 617)

7) Structure analysis technology for compounds

¹ methyl group (δH = 2.07, 1.83, 1.74 and 0.87), one methoxy group signal (δH = 3.91) and one aromatic proton (δH = δH = 6.71), two methylene proton signals (δH = 3.48, 2.99), and an oxymethylene proton (δH = 5.32).

¹³ through the C NMR spectrum was found to 37 carbon signals one ketone (ketone) (δC = 200.4) , 6 of aromatic carbon (aromatic carbons) (δC = 163.4 , 162.4, 139.8, 116.6, 112.8 and 105.6), 4 Methylene carbons (δC = 28.9, 19.6, 15.3 and 13.2), two methylene carbons (δC = 54.9 and 21.1), oxygenated methylene carbons (δC = 62.7), 1 Four methoxy groups (? C = 55.4) and four olefinic carbons (? C = 156.3, 129.7, 126.4 and 122.5) were identified.

Compound 2:

1) Properties: Gum

2) Molecular weight: 332

3) Molecular formula: C ₁₉ H ₂₄ 0 ₅

4) ¹ H NMR (500 MHz, CD ₃ OD):? 6.92 (1H, s, H-7), 5.54 (1H, br d, J = 8.5 Hz, H-6 '), 4.39 (1H, dt, J = 7.5, 8.5 Hz, H-5'), 3.87 (3H, s, OCH 3), 3.45 (1H, dd, J = 13.5, 7.0 Hz , H-1'b), 3.39 (1H, dd, J = 13.5, 7.0 Hz, H-1'a), 2.21 (1H, dd, J = 12.5, 6.0 Hz, H H-4 '), 1.76 (3H, s, H-8'), 2.03 (1H, dd, J = 12.5, 6.0 Hz, H- 1.58 (3H, s, H-10 ')

5) ¹³ C NMR (125 MHz, CD ₃ OD),? 171.7 (C-1), 160.1 (C-6), 149.7 ), 127.9 (C-7a), 127.0 (C-6 '), 124.8 (C-3a), 124.0 3), 66.8 (C-5 '), 55.2 (OCH 3), 47.8 (C-4'), 28.7 (C-8 '), 24.5 (C-1'), 16.9 (C-10 '), 15.7 (C-9 ').

6) ESI-MS data at m / z 331 [MH] - (calcd for C 19 H 24 0 5:. 332)

7) Structure analysis technology for compounds

¹ methyl group (δH = 1.80, 1.76 and 1.58), one methoxy group signal (δH = 3.87) and one aromatic proton (δH = J = 13.5, 7.0 Hz), 3.39 (dd, J = 13.5, 7.0 Hz),? H = 2.21 (dd, J = 12.5, 6.0Hz) and 2.03 (dd, J = 12.5, 6.0Hz)).

^{The 19} C-NMR spectra revealed 19 carbon signals, including one carbonyl unit (δC = 171.7), three methyl carbons (δC = 28.7, 16.9 and 15.7), two methylene carbon , one methylene carbons (delta C = 47.8 and 24.5), one methoxy group (delta C = 55.2) and four olefinic carbons (delta C = 139.3, 134.2, 127.0 and 124.0).

Preparation Example  1. Cell culture

(GIBCO) supplemented with 10% fetal bovine calf serum (Weljin, Daegu, Korea) and glutamine and antibiotics (penicillin plus streptomycin) in a 100 mm diameter culture dish were incubated with macrophages (RAW264 .7, purchased from ATCC, USA). Then, the cells were cultured in a 5% carbon dioxide incubator at 37 ° C for 18 hours, and the cells were then separated using a scraper to conduct experiments at a concentration of 2 × 10 ⁶ cells / ml. Trypan blue dye was used as a reference for cell activity and the concentration of dead cells was maintained at less than 1%.

Experimental Example  2: Cytotoxicity measurement

The macrophage (RAW 264.7) cultured for 18 hours in Preparation Example 1 was divided into 2 × 10 ⁶ cells in a 96-well, and the extracts or compounds 1 and 2 obtained in Example 1-1 were mixed with 0 Cytotoxicity of the compounds 1 to 2 was measured by measuring the viability of the cells after treatment at a concentration of 50 μM, 25 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM or 600 μM.

The cell viability was evaluated by a commercially available MTT assay, and the cultured cells of Preparation Example 1 were treated with the extract of Mushroom Mushroom, Compound 1 or Compound 2 of Example 1-1 and incubated for 3 hours at 37 ° C in a 5% carbon dioxide incubator After 10 μl of MTT solution (10 mg / ml in phosphate buffered saline, pH 7.4; PBS) was added and further cultured for 3 hours.

15% sodium dodecyl sulphate (SDS) was added to each well to dissolve the formazan produced by the culture, and the culture was stopped. The absorbance was measured at 570 nm using a microplate reader (Spectramax 250), and the measurement results are shown in FIG.

As can be seen from Fig. 9, even when Compound 1 and Compound 2 were treated to 100 [mu] M, the cell viability was 70% or more, indicating that the compound was not cytotoxic.

Experimental Example  3. Anti-inflammatory activity measurement

3-1: Nitric oxide inhibitory activity

The extracts of Mushroom mushroom extract obtained in Example 1-1, Mushroom nucleic acid fractions obtained in Example 1-2, Mushroom methylene chloride fractions of Example 1-2, Compounds 1 to 4, Examples Compound 1 or Compound 2 obtained in 1-4 was treated with macrophages (RAW 264.7) cultured in Preparation Example 1, and then the amount of NO ₂ ^- present in the cell culture was measured using a Griess reagent, The nitrogen inhibitory activity was measured.

Specifically, the same macrophages as in Preparation Example 1 were divided into 96-wells at 2 × 10 ⁶ cells, treated with 1 μg / ml of lipopolysaccharide (LPS), and cultured in a 5% CO 2 incubator at 37 ° C. And cultured for 24 hours. Then, the macrophage cells were cultured in the same manner as in Example 1-1 except that 200 μg / ml, 300 μg / ml, 400 μg / ml or 600 μg / ml of the extracts of Mulberry Mushroom, 37.5 μg / ml, 75 μg / Ml or 300 쨉 g / ml of the Herba sp. Fractions of Example 1-2 or the fractions of Example 1-2, methylene chloride fraction, 25 袖 M, 50 袖 M or 100 袖 M of Compound 1 or Compound 2, 100 袖 M Comparative Examples 1 to 4, Compound 1 or Compound 2 were added. 100 μl of the cell culture supernatant and 100 μl of the Griess reagent were mixed and reacted on a 96-well plate for 10 minutes, and the absorbance was measured at 540 nm.

The grease was prepared by adding 5% (v / v) phosphoric acid and 0.1% (w / v) naphthylethylenediamine-HCl to 1% (w / v) sulfanilamide It is an added reagent.

10 to 13 show the result of treatment with the extract of Aspergillus oryzae of Example 1-1, and Fig. 11 shows the results of treatment with the fractions of the Aspergillus oryzae mushroom nucleic acid fractions of Example 1-2 FIG. 12 shows results of treatment of Comparative Examples 1 to 4, Compound 1 or Compound 2, and FIG. 13 shows the results of treatment of Compound 1 or Compound 2 by concentration.

As shown in FIG. 10, it was found that the extract of Mushroom mushroom extract of Example 1-1 was treated with 600 μg / ml or more to exhibit a 50% inhibitory effect on nitrogen oxides.

As can be seen from FIG. 11, the nucleic acid fractions of Example 1-2 had a low ability to inhibit nitric oxide, so that treatment with 300 μg / ml or more required 50% inhibition of nitric oxide. On the other hand, the methylene chloride fraction of Example 1-2 showed remarkable nitric oxide inhibition ability at 150 占 퐂 / ml or more.

As can be seen in Fig. 12, the remarkably high nitric oxide inhibition of Compound 1 and Compound 2 was found as compared with other compounds.

As shown in FIG. 13, when Compound 1 and Compound 2 were treated with 100 [mu] M, they had an inhibitory effect on nitrogen oxides of 60 to 70%, and excellent anti-inflammatory activity was confirmed.

3-2: PGE ₂  Measurement of inhibitory activity

Compound 1 or Compound 2 obtained in Example 1-4 was treated with macrophages (RAW 264.7) cultured in Preparation Example 1, and then the amount of liberated PGE ₂ present in the cell culture was measured using a Griess reagent It was measured according to the protocol of the _{PGE 2 EIA system (Amersham, RPN222} ).

Cell culture and cell culture were obtained in the same manner as in Experimental Example 3-1.

The measurement result, PGE ₂ inhibitory activity, is shown in FIG.

As can be seen in FIG. 14, Compound 2 showed a remarkably high PGE ₂ inhibitory activity of 50% or more when treated with 50 μM or more, and Compound 1 showed PGE ₂ inhibitory activity when treated with 100 μM or more 60% or more.

Further, 50% inhibitory concentration of nitric oxide or PGE ₂ of Compound 1 and Compound 2 was calculated and shown in Table 1 below.

division IC ₅₀ ([mu] M) Nitrogen oxides (NO) PGE ₂ Compound 1 78.95 + - 4.73 85.20 ± 0.30 Compound 2 67.36 + - 2.53 60.10 + - 0.42

Experimental Example  4. Cytoplasm and Within the nucleus NF -KB protein expression and AP -1 protein expression

Western blotting was performed by separating cytoplasmic and nuclear NF-κB proteins (p50, p65) to examine the effect of Compound 1 or Compound 2 on the nuclear migration of NF-κB protein by LPS .

Specifically, the test group treated with 100 μM of Compound 1 or Compound 2 and the control group were collected and washed twice with phosphate buffered saline (PBS). Then, 0.4 ml of cell lysis buffer [10 mM HEPES 7.9), 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 2.0 [mu] g / l aprotinin]. After incubation for 15 minutes at 4 ° C, 25 μl of 10% NP40 was added and vigorously vortexed for 10 seconds with a voltex. The reaction mixture was centrifuged at 4 ° C and 1,300 rpm for 2 minutes to obtain a supernatant as a cytoplasmic protein. Then, the supernatant was washed with 50 μl of ice-cold nuclear extraction buffer (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 μM PMSF, 2.0 μg / μl leupeptin and 2.0 μg / μl aprotinin] was added to each well and allowed to stand at 4 ° C. for 15 minutes while intermittently shaking. The reaction solution was then centrifuged at 1,300 rpm For 2 minutes to obtain a supernatant which is a nuclear protein. The resulting cytoplasmic and nuclear protein solutions were quantitated using Pro-measure ™ reagent and 30 μg of proteins were mixed with sample buffer, heated at 95 ° C. for 5 minutes, and stored at -20 ° C. The completed sample was electrophoresed on a 12% SDS-acrylamide gel and the protein was transferred to a PVDF membrane. After the transfer, the membrane was blocked for 1 hour at room temperature in a skim milk solution of 5%, and the NF-κB p65 primary antibody was diluted to a predetermined ratio in a 5% skim milk solution. Respectively. The next day, the cells were washed three times for 5 minutes each with TBST, and the anti-mouse secondary antibody was reacted at room temperature for 1 hour. After washing three times for 10 minutes with TBST, the cells were developed with BCIP-NBT solution (Nakanai Tesque, Japan). The measurement results are shown in Figs. 15 to 16, Fig. 15 shows the result of treating Compound 1, and Fig. 16 shows the results of treating Compound 2.

As can be seen in FIG. 15, Compound 1 showed significant c-Jun inhibitory activity after 30 minutes of treatment and after 60 minutes of treatment, and the inhibitory activity of p65 was confirmed 60 minutes after Compound 1 treatment.

As can be seen in FIG. 16, Compound 2 showed excellent inhibition of c-Jun 30 minutes after treatment, and inhibited p65 after 60 minutes of treatment with Compound 2, indicating that anti-inflammatory activity was high.

Thus, Compound 1 and Compound 2 of the present invention are useful for the reduction of the expression level of NO in the cells, the decrease of PGE2 (prostaglandin E2) production, the decrease of NF-KB in the nucleus, It is obvious that it is highly applicable in various fields such as anti-inflammatory active composition, pharmaceutical composition for prevention or treatment of inflammatory disease, anti-inflammatory health functional food, and the like.

Claims (9)

An anti-inflammatory active composition comprising at least one compound selected from the group consisting of a compound represented by the following formula (1), a compound represented by the following formula (2)
[Chemical Formula 1]

(Wherein, R ₁ is alkenyl Diallo giim of C ₁₆ ~ C _18.)
(2)

The anti-inflammatory active composition according to claim 1, wherein the compound represented by the formula (1), the compound represented by the formula (2) The composition of claim 1, wherein the anti-inflammatory active composition
The amount of nitric oxide (NO) expressed in the cells;
Within the cell prostaglandin (PGE _2; prostaglandin E ₂₎ production;
Nuclear translocation of NF-κB; And
Activity of AP-1;
≪ / RTI > or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for preventing or treating an inflammatory disease comprising the anti-inflammatory active composition according to any one of claims 1 to 3. [Claim 5] The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition comprises an antiinflammatory activity composition at a concentration of 50 to 200 [mu] M. An anti-inflammatory functional cosmetic composition comprising the anti-inflammatory active composition of any one of claims 1 to 3. A health functional food composition for preventing or ameliorating an inflammatory disease comprising the anti-inflammatory active composition according to any one of claims 1 to 3. A quasi-drug composition for preventing or ameliorating an inflammatory disease comprising the anti-inflammatory active ingredient according to any one of claims 1 to 3. The composition according to claim 8, wherein the quasi-drug composition is at least one of the group consisting of disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment agent, coating agent, A quasi-composition for preventing or improving inflammatory diseases.

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