KR20120021959A - A composition for inflammatory disorder comprising the polyphenol extracts from phellinus baumii - Google Patents

Abstract

PURPOSE: A composition containing Phellinus baumii-derived polyphenol extract for preventing and treating inflammatory diseases is provided to ensure excellent inflammation. CONSTITUTION: A composition for preventing or treating inflammatory diseases contains Phellinus baumii-derived polyphenol extract as an active ingredient. A composition for preventing or treating inflammatory diseases contains active compounds of Phellinus baumii-derived styrylpyrones as an active ingredient. The active compound is isolated from the polyphenol extract. The active compounds include interfugin A, baumin-Pb211, davallialactone, hispidin, hypholomine B, phelligridin D, or inoscavin A. A food composition contains the polyphenol extract.

Description

A composition for Inflammatory Disorder comprising the polyphenol extracts from Phellinus baumii}

The present invention relates to a composition for the prevention and treatment of inflammatory diseases containing a polyphenol extract derived from Baumi situation mushroom as an active ingredient, more particularly, a food composition for the treatment and prevention of inflammatory diseases with excellent anti-inflammatory activity The present invention relates to a Phellinus baumii extract useful as a pharmaceutical composition and a composition for the prevention and treatment of inflammatory diseases containing a styrylpyrone-based compound isolated from the extract as an active ingredient.

The pharmacological effects of edible mushrooms have been announced in various fields so far, and the role that can be achieved by the mushroom diet that promotes health by eating in the modern age of aging society is expected.

For millions of years, mushrooms have been adapted to the environment by producing defenses in the body to fight off invaders such as viruses and bacteria.

From medicinal mushrooms known to date, the so-called defense substances, the secondary metabolites they produce, have been known to humans as safe and effective medicines for thousands of years.

In particular, thousands of mushroom fungi are known to produce unique and diverse compounds that could be used as a good material for new drug development.

However, research on natural products from mushrooms is still lower than that of other microorganisms. In particular, there are no clear studies on the exact components and contents of traditional medicinal mushrooms such as Ganoderma lucidum, shiitake, and chaga, and their efficacy. The development of natural medicines based on mushrooms is incomplete.

The present invention is to prevent the inflammatory disease containing a polyphenol extract derived from Baumi situation mushroom and a styrylpyrone-based compound contained in the extract as an active ingredient in order to solve the above problems and It is an object to provide a therapeutic composition.

In order to achieve the object of the present invention as described above, the present invention

It provides a composition for the prevention or treatment of inflammatory diseases comprising a polyphenol extract derived from Baumi situation mushroom as an active ingredient.

In addition, the present invention provides a composition for the prevention or treatment of inflammatory diseases, which comprises a styrylpyrone-based active compound derived from the Baumi situation mushroom.

The active compound is preferably isolated from the polyphenol extract derived from Baumi situation mushroom.

The active compound of the styrylpyrone series is Interfungin A, Baumin (Baumin = Phoebe 211 (Pb211)), Davallialactone, Hispidin (Hispidin), Hipolomin B ( It is preferable that it is at least 1 type selected from the group which consists of Hypholomine B), Pelligliin D, and Inoscavin A.

The inflammatory disease is preferably arthritis or pulpitis.

Also provided is a food composition or a pharmaceutical composition comprising a polyphenol extract derived from Baumi situation mushroom.

According to the present invention, a composition comprising a polyphenol extract derived from Baumi situation mushroom as an active ingredient is used as a food composition or a pharmaceutical composition.

In addition, the composition comprising a styrylpyrone-based compound contained in the extract as an active ingredient may also be used as a food composition or pharmaceutical composition for the treatment and prevention of inflammatory diseases.

Figure 1 shows a crude extract manufacturing process of the situation mushroom polyphenol group.
Figure 2 is contained in the polyphenol extract Pauminus baumii mushroom (Phellinus baumii)
Is a diagram showing the chemical formula of seven compounds of the styrylpyrone series.
3 is a diagram showing an Interfungin A compound.
a: hydrogen nuclear magnetic resonance spectrum b: carbon nuclear magnetic resonance spectrum
4 is a diagram showing a Baumin (= Pb211) compound.
a: hydrogen nuclear magnetic resonance spectrum b: carbon nuclear magnetic resonance spectrum
5 is a diagram showing a Davallialactone compound.
a: hydrogen nuclear magnetic resonance spectrum b: carbon nuclear magnetic resonance spectrum
6 is a diagram showing a Hispidin compound.
a: hydrogen nuclear magnetic resonance spectrum b: carbon nuclear magnetic resonance spectrum
7 shows a Hypholomine B compound.
a: hydrogen nuclear magnetic resonance spectrum b: carbon nuclear magnetic resonance spectrum
8 is a diagram showing a Phelligridin D compound.
a: hydrogen nuclear magnetic resonance spectrum
9 is a diagram showing an Inoscavin A compound.
a: hydrogen nuclear magnetic resonance spectrum b: carbon nuclear magnetic resonance spectrum
Figure 10 shows an HPLC chromatogram of the dissolved fraction of ethyl acetate of the methanol extract of Baumi Situation Mushroom (1. Interfungin A; 2. Davallialacton 3. Hypholomine B)
FIG. 11 shows inflammation inhibition by polyphenols derived from Baumyi mushrooms in a collagen-induced arthritis model. The severity of arthritis symptoms was assessed by AI during four consecutive weeks of treatment.
FIG. 12 shows the effect on anti-collagen II antibodies by polyphenols from Baumi S. mushrooms in the collagen-induced arthritis model.
Figure 13 shows the results of histopathology obtained using the joints.
FIG. 14 is a graph in which Bauminus baumii polyphenol extract reduces the AI of collagen-induced arthritis.
15 shows Hematological Parameters.
16 shows the total cell numbers of lymph and joint.
17 shows fluorescence flow cytometry analysis of lymph and joint.
18 shows Type II collagen IgG in serum.
19 shows RA factor IgG in serum.
20 shows RA factor IgM in serum.
21 shows the levels of cytokines in serum.
22 shows histological analysis through the joints of mice.
23 shows the weight of the thrombus.
24 shows cytotoxicity against pulp cells of davalalactone (DAVA).
25 shows inhibition of cell adhesion molecules by davalalactone (DAVA).
Figure 26 shows inhibition of inflammation related signaling and free radical transmission by davalalactone (DAVA).
Figure 27 shows the effect of inhibiting free radicals by davalalactone (DAVA).
Figure 28 shows the inhibitory effect of davalalactone (DAVA).

The present inventors have conducted various studies to develop functional foods, food additives, or pharmaceutical compositions for the treatment and prevention of inflammatory diseases using natural substances, and as a result, Phellinus baumii polyphenol extracts are anti-inflammatory, It was confirmed that the anti-inflammatory, anti-arthritis effect.

In one embodiment of the present invention provides a composition for the prevention or treatment of inflammatory diseases, characterized in that it comprises a polyphenol extract derived from Baumi situation mushroom as an active ingredient.

Baumi Situation Mushroom ( Phellinus) baumii ), also known as long- lived situation mushroom, is one of wild wild situation mushrooms, which contains the most protein polysaccharide and is one of the rare perennial mushrooms of rare basidiomycete.

These bauhinia mushrooms ( Phellinus baumii ) has been recently grown in many farms because of the possibility of artificial cultivation and harvesting in a year or two. It appears that Baumi situation mushrooms contain 0.10 to 0.16% of betiglucan. Natural Killer) has been reported to have the effect of activating cells and human body.

In addition, an embodiment of the present invention provides a composition for the prevention and treatment of inflammatory diseases, characterized in that it comprises a styrylpyrone-based active compound isolated from the polyphenol extract derived from Baumi situation mushroom as an active ingredient. do.

Baumi situation mushroom ( Phellinus) exhibiting the same effects as described above baumii ) was identified as a major component of the anti-inflammatory, anti-inflammatory and anti-arthritis effects, and the compounds of the styrylpyrone family were isolated.

The separated compounds were identified by chemical analysis through structural analysis. As a result, styrylpyrone-based compounds such as Interfungin A, Baumin (Baumin = Phoebe211 (Pb211)), and davallialactone A total of seven compounds, including Hispidine, Hypholomine B, Phelligridin D, and Inoscavin A, were identified as main components. In addition to these, styrylpyrone-based polyphenol compounds are contained, but are contained as a minor component and cannot be separated by conventional methods. Therefore, at least one selected from the group consisting of the styrylpyrone-based active compounds is preferable.

In particular, the seven compounds are Phellinus baumii ) was the first compound isolated.

Therefore, in the present invention, the anti-inflammatory effect of the Baumi situation mushroom polyphenol extract and the styrylpyrone-based compound contained in the extract was completed as an indicator.

Hereinafter, the composition for the prevention and treatment of inflammatory diseases of the present invention will be described in detail.

In the present invention, the inflammatory disease is not limited thereto, but is preferably chronic neurological inflammation. The chronic neuroinflammatory symptoms may be associated with a large, irrelevant group of diseases based on various human diseases. Non-limiting examples of diseases that exhibit chronic neurological inflammation as symptoms include, without limitation, acne, acid reflux / heartburn, Alzheimer's disease, appendicitis, arteritis ), Arthritis, asthma, atherosclerosis, autoimmune diseases, balanitis, blepharitis, bronchiolitis, bronchitis, bursitis, cancer, heartitis, celiac disease, vesicular inflammation (cellulitis), cervicitis ( cervicitis, cholangitis, cholecystitis, chorioamnionitis, chronic obstructive pulmonary disease (COPD), cirrhosis, colitis, conjunctivitis, cystitis, common cold, lacrimitis, dementia, dermatitis, dermatitis, emphysema, encephalitis, endocarditis, endometritis , Enteritis, colitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, glomerulonephritis, phlebitis, heart disease, hepatitis, hidradenitis suppurativa, hypertension, ileum , Inflammatory neuropathy, insulin resistance, interstitial cystitis, iris, ischemic heart disease, keratitis, keratoconjunctivitis, laryngitis, mastitis, mastoiditis, meningitis, metabolic syndrome (syndrome X), migraine, myelitis, myocarditis, myositis , Nephritis, obesity, ovitis, ovarian inflammation, testicles, osteochondritis, osteopenia, osteoporosis, osteoarthritis, otitis, pancreatitis, Parkinson's disease, parotitis, pelvic inflammatory disease, pericarditis, peritonitis, pharyngitis, Phlebitis, pleurisy, interstitial pneumonia (pneumonitis), proctitis, prostatitis, pulpitis, pyelonephritis, pylephlebitis, rheumatism fever, rhinitis, salpingitis, salivaitis, sinusitis, spastic colon ), Stomatitis, synovitis, tendinitis, tendinosis, hay salt, thrombophlebitis, tonsillitis, trigonitis, tumors, urethritis, uveitis, vaginitis, vasculitis, and vulvitis It includes.

In one embodiment of the present invention, arthritis or pulpitis is more preferable among the inflammatory diseases.

Medically, arthritis is an inflammatory change in the joints that is confined to the joints, the joints are swollen, and the range of motion of the joints decreases. In particular, degenerative joint disease is osteoarthritis, the most commonly known arthritis, and is often inflammatory with age. It is known that cartilage is lost without alteration and joint deforms and local degenerative change occurs. Cartilage is changed and destroyed by various biological factors. Osteoarthritis includes osteoarthritis, rheumatoid arthritis and polyarthritis. Osteoarthritis is the most common cause of chronic inability after middle age due to pain and stiffness, and the underlying pathological process is degenerative, unlike inflammatory rheumatoid arthritis (Govan, ADT et al., Pathology Illustrated 3rd ed., Pp 811-821 , Churchill Livingstone, 1991).

In general, rheumatoid refers to chronic rheumatoid arthritis, which is known to be a refractory disease that is difficult to identify as the disease is caused by connective tissues distributed throughout the body centered on the joint.

On the other hand, pulpitis is a disease in which dental caries (mainly streptococcus mutans) corrode teeth. Pulpitis is a disease caused by tooth decay or periodontitis, which is caused by infection of bacteria in the pulp (the nerves in the center of it or the passage of blood vessels). If the treatment is delayed, the inflammation spreads to the jaw and blood, causing osteoarthritis and sepsis.

In order to separate the Baumi situation mushroom polyphenol extract and the active substance from the extract, the Baumi situation mushroom is extracted with an organic solvent selected from alcohols, acetates, ethers, acetones, hydrocarbons, and the like. A well-known method used for separation extraction of mushroom components, such as water distribution and column chromatography, can be easily obtained by combining individually or suitably.

At this time, the Baumi situation mushroom may be used as it is, or may be extracted using mushroom fruiting bodies and mycelium nutrient solution, and crude extracts may be further purified according to the conventional method as needed.

Baumi situation mushroom polyphenol extract according to the present invention and a preferred method for efficiently separating the seven compounds of the styrylpyrone (styrylpyrone) contained in the extract is as follows.

1) A process for extracting Baumi situation mushroom extract with a solvent selected from aliphatic alcohols having 1 to 6 carbon atoms and aliphatic hydrocarbons having 1 to 10 carbon atoms 2) Polyphenol fraction by partition extraction of the solvent extract using hexane and ethyl acetate solvent 3) a process of silica gel column chromatography of the polyphenol extract using chloroform-methanol eluent 4) a Sepadex LH-20 column chromatography of the eluent using methanol or the eluate 40 ~ Preparative high performance liquid chromatography (preparative HPLC) using 60% methanol consists of separating and obtaining each compound.

In addition, in one embodiment of the present invention, a composition containing seven compounds of the styrylpyrone series contained in the polyphenol extract derived from the Baumi situation mushroom is also useful as a food composition or a pharmaceutical composition. Can be used.

In particular, the polyphenol extract and the styrylpyrone compound isolated from the Baumi situation mushroom of the present invention when contained as an active ingredient of the composition for the prevention and treatment of inflammatory diseases, 0.0001 ~ It is contained in 10.0% by weight, preferably 0.1 to 10.0% by weight characterized in that it is contained.

When the content of the active ingredient is less than 0.0001% by weight, the anti-inflammatory effect is hardly seen. When the content of the active ingredient is more than 10% by weight, the effect of increasing the content is insignificant.

In addition, when the polyphenol extract derived from the Baumi situation mushroom of the present invention and the styrylpyrone compound isolated to the extract as a pharmaceutical composition, it can be prepared by a known method in the pharmaceutical field, It can be prepared and used in the form of powder, granules, tablets, capsules or injections, etc., by themselves or in admixture with pharmaceutically acceptable carriers, excipients, diluents and the like, which can be administered orally or parenterally.

In addition, in addition to the active ingredient described above for administering the pharmaceutical composition may be prepared by including one or more pharmaceutically acceptable carriers.

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.

In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The compositions of the present invention may be parenterally administered (eg, applied intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status of the patient, 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 effective dosage of the pharmaceutical composition according to the present invention may be appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, and once to several times a day. It is more preferable to administer separately.

In addition, the polyphenol extract derived from the Baumi situation mushroom of the present invention and the styrylpyrone compound isolated on the extract can be used as a food, beverage, or beverage additive for health, the composition is a food It is preferable to use in the form of a soft or hard capsule filled with a functional beverage for the antioxidant effect or by dry powdering the composition component for the purpose of antioxidant by mixing with water at 0.01% to 10% by weight relative to the total weight.

The composition containing the active ingredient of the present invention can be used in a variety of food and beverages for the anti-inflammatory effect. Examples of the food to which the present composition can be added include various foods, beverages, gums, candy, confectionery, tea, vitamin complexes, and health functional foods.

Hereinafter, the present invention will be described in detail through examples. The following examples are only illustrative of the present invention, and the scope of the present invention is not limited thereby.

< Example  1> Situation Mushroom ( Baumijong , Phellinus baumii ) Polyphenolic  For animal efficacy assays Crude extract  secure

A crude polyphenol group extract was prepared to verify the prevention and treatment of arthritis of the antioxidant polyphenol group isolated from Baumi species, which are cultivated in farmhouses. 1 kg of domestic situation mushrooms purchased from Geumsan Herbal Market were extracted by stirring with a homogenizer for 24 hours with methanol. After drying under reduced pressure, the extract was partitioned and extracted with hexane to remove the oil and fat, and the remaining water layer was partitioned and extracted again with ethyl acetate. Ethyl acetate layer was dried under reduced pressure, and the dried product was ground and ground to prepare 50 g of crude extract (see FIG. 1).

< Example  2> Situation Mushroom ( Phellinus baumii A) of extract quality control  ( QC Indicator components and establish simple analysis method

1. Selection of Indicator Components

Index components for QC were set from crude polyphenol extracts of the situation mushrooms. The index component was selected as interfungin A, davallialactone and hypholomine B, which are the main antioxidants of situational mushrooms. These compounds can not only characterize situational mushroom Baumi species, but are also active in biological activity. Therefore, these components were selected as indicator components for QC (see FIG. 10).

2. Establish simple analysis method

Through the detection of the selected indicator components, the HPLC simple analysis method for QC of the crude extract of polyphenol group of the situation mushroom was established. In other words, the crude extract was made into a concentration of 10 mg / ml, HPLC was performed using an analytical column, and HPLC setting conditions for easily analyzing the index components were as follows. Column (Column: reverse phase column (150 x 4.6 mm; Cosmosil, Nacalai tesque, Japan), Mobile phase: A linear gradient solvent system (solvent A, 5% (v / v) methanol, 0.04% (v / v) TFA; Solvent B, Methanol): 02 minutes, 20% B; 210 minutes, 2045% B; 10-20 minutes, 45% B; 2025 minutes, 70% B; 2532 minutes, 70% B; 3236 minutes, 100% B; 3640 min, 100% B; 4043 min, 20% B), flow rate (Flow rate: 1 ml / min).

<Application 1> Situation Mushroom ( Phellinus baumii ) Arthritis Improvement of Polyphenol Extracts in vivo  Active evaluation

Situation mushroom polyphenol extract has anti-inflammation activity based on strong antioxidant activity, according to a previous study. In this experiment, we investigated the effects of these polyphenols on inflammatory responses in arthritis model animals and examined their mechanism of action.

1. Research method

One) Rheumatism  Arthritis Animals ( CIA A) model manufacture

CIA induction was performed by mixing 100 μg of bovine type II collagen and 0.1 ml Complete Freund's Adjuvants in 8-week-old DBA / 1 OlaHsd rats subcutaneously, and boosting the same amount after 21 days to prepare a CIA model. .

2) Arthritis index  ( AI ) and incidence  analysis

After boosting with Type II collagen, arthritis index was determined based on the following signs of arthritis in each of the four paws of DBA / 1 OlaHsd rats, and the degree of CIA was induced once a week as shown in Table 1 below. Recorded.

CIA index Yu Horned Dildo 0 no arthritis One small degree of arthritis 2 light swelling 3 medium swelling 4 severe swelling 5 severe swelling and non-weight-bearing

3) Arthritis Evaluation Items

Determination of total cell count in the draining lymph node (DLN) and joints.

Fluorescent flow cytometry analysis of PBMCs, lymph, and joints

Cytokine Measurement in Serum and Splenocyte Culture Supernatants of Mice

Inflammatory Cytokines and Rheumatoid Factor (RF) Analysis

Measurement of collagen II antibody in serum

Serum liver function test (LFT) analysis

Pathological examination

2. Findings

Arthritis of Situation Mushroom Polyphenol Extract by Arthritis Index Suppression degree  evaluation

Two weeks after the second collagen injection, the arthritis index was measured at the start of the experiment (0 weeks) and for 4 weeks. The arthritis-induced control group showed that the arthritis index increased from 11.8 ± 1.9 to 17.8 ± 0.5 at 4 weeks after the start of the experiment. However, 50 mg / kg of the contextual mushroom polyphenol extract showed a decrease in arthritis index from 11.6 ± 1.9 to 10.4 ± 0.9 after 4 weeks on the start of the experiment (0 weeks). The arthritis index at the start of day (0 weeks) was 11.8 ± 1.2 and 8.4 ± 0.8 after 4 weeks. As a positive control, methotrexate (MTX) was observed at 3 mg / kg for the same period, and as shown in FIG. 11, arthritis index was strongly inhibited after 4 weeks of experiment.

2) formed in serum type II collagen  Effects of Situary Mushroom Polyphenol Extracts on Antibodies

As a result of confirming the degree of collagen type II antibody formation in serum during the action of the inhibitory action on arthritis-inducing animals, it was confirmed that the antibody was inhibited in a concentration-dependent manner in collagen-induced arthritis-inducing animals as shown in FIG. 12 (1,174,270 unit / ml Reduced to 867,815 units / ml and 667,088 units / ml).

3) pathological biopsy

13 shows the results of pathological examination obtained using the joint after the end of the experiment. In collagen-induced arthritis model animals (CIA control I and CIA control II), damage to joint tissues and infiltration of inflammatory cells was observed in comparison with normal control group. 50 mg / kg and 150 mg / The kg treatment group showed comparable inhibition to the positive control group treated with methotrexate (MTX) 3 mg / kg.

4) Rheumatism  Arthritis mice CIA Situation mushroom () in the model Phellinus baumii Effects of Polyphenol Extracts ( CIA  Arthritis index of induced mice ( AI ) Impact

MTX and situation mushroom polyphenol extracts were administered to the CIA mice for 5 weeks, and then the arthritis index (AI) was observed. As a result, MTX and P. baumii (50 mpk, or 150 mpk) were treated compared to the control group. After 2 weeks, the index value decreased significantly, and after 5 weeks, MTX and Situary mushroom polyphenol extract 150mpk treatment group showed more than 50% inhibition effect (p <0.001). In addition, the treated group such as situation mushroom polyphenol extract (50mpk) also showed a significant inhibitory effect of more than 30% compared to the control group (see Fig. 14).

5) hematological analysis ( Hematological Parameters )

Leukocytes are divided into granulocytes, lymphocytes, monocytes, and in peripheral fluids usually have an immune function against foreign antigens. Recent studies have shown that the number of leukocytes increases as the number of granulocytes increases in peripheral blood of patients with rheumatoid arthritis. The characteristics of these granulocytes are granulocytes are names used collectively as neutrophils, eosinophils, and basophils having granules characteristic of the cytoplasm. These cells are made in the bone marrow and distributed through the blood to systemic tissue. From the promyelocytic stage, specific granules of each cell appear and classify the cells into neutrophils, eosinophils, basophils. As the granulocytes mature, peroxidase-positive azurophilic granules decrease, more peroxidase-specific granules appear, and cell surface Fc receptors and receptors for complement such as CRI and CR3 appear. Neutrophils are 2 to 6%, and the number of bacteria increases dramatically as soon as they invade the human body. They interact with macrophages to recognize and bind to common components on the surface of various bacteria, acting as phagocytosis, called cytokines. It secretes biologically active molecules, and these active molecules have inflammatory effects, such as arthritis, which dilate nearby blood vessels to increase local blood flow and increase fluid leakage, while mobilizing other white blood cells nearby. Monocytes are 2-9% of the blood cells that go around and differentiate into macrophages.The largest and most motility of white blood cells, when invading pathogens or toxins, the neutrophils move out of the blood vessels. do. Eosiniphil is 1? 5% is concentrated in the area that is in contact with the outside, such as the inner wall of the skin or digestive system or the inner wall of the respiratory tract, and plays an important role in detoxifying toxic substances that enter the body from the outside, and increases in allergic diseases such as atopy and asthma, to the lungs. Calm down and play an important role in asthma induction. Basophil is 0? 2% is known to play an important role in the production of inflammation-related substances, asthma, allergies and hypersensitivity.

In addition, in recent studies, the number of platelets has been increased in chronic diseases due to inflammatory reactions in the body and an increase in eosinophils.

In this example, the WBC count in peripheral blood was increased in the control group compared to the normal DBA / 1 mouse group that did not induce arthritis. The WBC count of the positive control group administered with 3mpk MTX showed a decrease compared to the control group, and the WBC count of the experimental group administered with P.baumii also decreased compared to the control group.

In addition, the frequency of neutrophils, eosinophils, and basophils in WBC was increased in the control group compared to the normal DBA / 1 mouse group that did not induce arthritis, and the frequency (%) of neutrophils in the positive control group administered with 3mpk MTX The decrease was shown. In addition, the frequency (%) of neutrophils, eosinophils, and basophils of the experimental group treated with P.baumii showed a slight decrease compared to the control group (see FIG. 15).

The frequency (%) of lymphocytes and monocytes in WBC was relatively inversely proportional to the frequency of neutrophils.

6) lymph ( draining lymph node , DLN ) And joints ( joints Total of) Cell number

Drugs were terminated by treatment with DBA / 1 OlaHsd mice for 5 weeks, and the animals were anesthetized with ethyl ether, and lymph and joints were isolated to determine the total immune cell count.

Lymphoid T cells are activated by antigen-sensitized APC cells, which amplify the cell number by cell proliferation and differentiation. These amplified active cells are a tissue lesion, the joint-homing T cell acts as a migration.

Thus, arthritis-induced CIA control in DLN increased the total number of cells more than 7 times compared to normal DBA / 1 mice, and drug administration inhibited T cell activity, enhanced Treg cell activity, inhibited differentiation of Th17 cells, or Th2-Th1. Counter-regulation reduces total cell counts closer to normal than in control.

In addition, in the joint area, the immuno-inflammatory cells in DLN and PBMC were accumulated into arthritis tissues by various recruiting facters, and the total number of cells in the control group increased more than 4 times compared to the normal group. Alleviation showed a decrease in total cell numbers (see FIG. 16).

7) lymphatic and jointed Fluorescent flow cells  analysis

Figure 17 A), B) after administration of MTX, P.baumii for 4 weeks in the CIA mouse model, the immune cells were isolated from each DLN and joint tissues labeled with immunofluorescence staining and analyzed by FACS as a result of analysis of lymph and joint. The total absolute number in the DLN and the total absolute number in the joint are shown.

17A) shows CD4 + CD25 + (Th activated cells), CD3 + CD69 + (initial T activated cells), CD4 + (Th cells), CD8 + (cytoxtic / supression T cells), B220 + CD23 + (B activated cells), MHC-II4 + CD11c + (DC activated cells), all of which cause arthritis in DLN, showed a significant increase in total absolute No. of the control group compared to the normal group, and showed a tendency to decrease close to the normal group due to drug administration. .

17B) also showed that the total absolute no. Of CD3 + (T cells) and CD19 + (B cells) in DLN was significantly increased compared to the normal group, and that the total absolute no. Of the control group was significantly decreased compared with the normal group. Indicated.

Fig. 17C) shows that after four weeks of MTX and P.baumii administration to CIA mouse models, the immune cells were isolated from each joint tissue, labeled with immunofluorescence staining, and analyzed by FACS. The total absolute number in the.

Both CD3 + (T cells) and CD11b + Gr-1 + (gralurocytes, neutrophils, eosinophil, basophil, and mast cells) in the joints are the cells that cause arthritis, which increases the number of cells due to inflammation in the joints. Compared to the group, the total absolute No. of the control group was significantly increased, and the drug administration showed a tendency to decrease to the normal group.

8) in serum Cytokine  ( cytokine ) And anti - type  Ⅱ collagen antibody level  Measure

Measuring cytokines (TNF-α, IL-1β, IL-6) in the serum of arthritis-induced animals and measuring the level of antigen-specific antibodies such as total antibody or anti-type II collagen antibody using the ELISA kit It was measured by.

Figure 18 is one of the most accurate efficacy measures to measure the antibody level of the anti-type II collagen antibody to the therapeutic effect of the drug in animal models induced by Th1 arthritis autoimmune disease by sensitizing Collagen type II, serum after the end of the experiment As a result of separation and measurement by ELISA, the control group increased more than 100 times compared to the normal group, and the antibody level of anti-type II Collagen antibody decreased according to the therapeutic efficacy of the drug.

19 shows that B-cells responsible for humoral immunity with total antibody as a therapeutic effect in the animal model induced by Th1 arthritis autoimmune disease by sensitizing Collagen type II secrete anti-type II collagen antibody against CII antigen. As a result of measuring the total RA factor IgG antibody level during the process, the serum was separated and measured by ELISA after the end of the experiment, and the control group increased more than two times compared to the normal group, and according to the therapeutic effect of the drug, Antibody levels tended to decrease.

FIG. 20 shows that B cells responsible for humoral immunity with total antibody as the therapeutic effect in the animal model induced by Th1 arthritis autoimmune disease by sensitizing Collagen type II secrete anti-type II collagen antibody against CII antigen. As a result of measuring the total RA factor IgM antibody level during the process, the serum was separated and measured by ELISA after the end of the experiment, the control group increased compared to the normal group, and the antibody level of the total RA factor IgM according to the therapeutic efficacy of the drug It showed a tendency to decrease.

9) cultured In splenocytes cytokine level  Measure

Prepare the cell suspension by removing the spleen of arthritis-induced animal spleen under aseptic condition and remove red blood cells using lysis buffer. The required number of cells in RPMI 1640 medium is incubated in a well plate treated with a certain amount of type II collagen for a certain time. After the supernatant was taken, cytokines (IFN-g, TNF-α, IL-1β, IL-6) levels were measured using an ELISA kit.

However, the cell cultured for a predetermined time in a well plate was used directly when measuring by flowcytometry.

Inflammatory cytokine measurement in arthritis-induced mice is an important criterion for judging the efficacy test for arthritis treatment.

Serum levels of IL-1b, IL-6, TNF-a and IFN-g, which are serum inflammatory cytokines of arthritis mice, were measured by ELISA, and the control group increased compared to the normal group. The cytokines IL-1b, IL-6, TNF-a and IFN-g serum levels tended to decrease.

However, IL-10 is a cytokine inhibitory factor, which acts to regulate inflammation by increasing IL-10 levels as a therapeutic effect by drugs.

Our results also showed a significant increase in P.baumii 150mpk compared to the control (see Figure 21).

10) joints of mice ( joint Organizational analysis

MTX, P.baumii (150mpk) and P.baumii (50mpk) were administered to the CIA mouse model for 5 weeks, and after the experiment, the joints of the mice were extracted and tissues were analyzed by H & E staining and MT staining (Fig. 22). As a control of the CIA, the infiltration of macrophages, granulocytes, monocytes, synovial inflammatory cells, and synovial hyperplasia in CII-induced arthritis-induced mice showed loss of joint architecture due to cartilage and bone subsidence. . However, in MTX treated group and P.baumii (150mpk) group, inflammatory cells were significantly reduced in SM (synovial membrane) and ST (synovial tissue). (erosion) and synovial cell damage were restored to near normal group. P.baumii (50mpk) was slightly reduced.

<Application Example 2> Situation Mushroom ( Phellinus baumii Evaluation of pulp salt improvement of polyphenol extract

The inhibitory effect of pulp inflammation was investigated using a davalaclactone compound, a polyphenolic compound isolated from the Baumi situation mushroom.

Cytotoxicity Test

To investigate the inhibitory effect of pulp inflammation that causes dental caries, we first examined the cytotoxicity on the pulp cells of davalalactone. It was isolated from the third molars of the tooth and treated with each concentration of davalalactone in normal cultured pulp cells (1 X 10 ⁵ cells). Then, cytotoxicity was observed by performing MTT assay after incubation for 24 hours and 48 hours in 5% CO ₂ incubator, respectively. As a result, daalilactone did not show cytotoxicity in primary pulp cells isolated and cultured. However, cell growth was slightly inhibited at 100 μM. In this experiment, 1 μM and 10 μM without cytotoxicity were used (FIG. 24).

In order to induce inflammation of the pulp cells, after 18 hours of starvation of the cells in a medium containing 0.5% FBS, the cells were treated with davalaclactone at each concentration, and after 1 hour, 10 μg / ml of LPS was recovered. It was. Then, the expression of cell adhesion factor was examined by Western blot.

Zymography in MMP Expression of -9,2

Cell adhesion molecules induced by LPS were inhibited by davalalactone and NAC. MMP-9,2 expression was also inhibited. This means that davalalactone inhibits pulp inflammation molecules (FIG. 25).

Inflammation-related signaling pathways and cytokine Expression

APST and ERK signals were activated by LPS, but davalaclactone inhibited both signaling pathways. Davalalactone also inhibited the activity of cytokine induced during inflammatory reactions, which resulted in inhibition of inflammatory signaling pathways and inhibition of free radical pathways, resulting in inhibition of both signaling pathways. It is thought to be (Figure 26).

In davalalactone  Free radical suppression effect

Normally cultured 1X10 ⁶ Dog pulp cells were pretreated with 10 and 20 μM of davalalactone and after 1 hour, 200 μM of H ₂ O ₂ and 10 μg of LPS, respectively. After the reaction in 5% CO ₂ incubator for 24 hours, DCFH-DA 5 μM was treated and the cells were harvested after the reaction for 30 minutes. Then, the amount of free radicals formed per 10,000 cells was measured using flow cytometry. As a result, when treated with 200 μM of H ₂ O ₂ and 10 μg of LPS, more free radicals were induced compared to normal cultured pulp cells. However, in the group treated with davalalactone, the formation of reactive oxygen was inhibited in a concentration dependent manner (FIG. 27).

Macrophage  In a cell LPS  By induction Of davalalactone  Anti-inflammatory effect

The inhibitory effect of davalalialactone by LPS induction was investigated first in macrophage cells that showed activation during inflammatory reactions such as bacterial infections. In order to induce inflammation in Raw 264.7 cells, cells were treated with 10 μM of davalalactone after starvation for 18 hours in a medium containing 0.5% FBS, and the protein of cells was recovered 12 hours after treatment with 10 μg / ml of LPS. . Afterwards, we observed inflammation-related cytokine expression with Western blot. As a result, inflammation-associated cytokines induced by LPS were inhibited by davalalactone. It is believed that davalalactone has effects on various inflammation-related responses as well as pulpitis (FIG. 28).

Although embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (6)

A composition for the prevention or treatment of inflammatory diseases comprising a polyphenol extract derived from Baumi situation mushroom as an active ingredient. A composition for the prevention or treatment of inflammatory diseases comprising a styrylpyrone-based active compound derived from Baumi situation mushroom. The method of claim 2,
The active compound is a composition for the prevention or treatment of inflammatory diseases, characterized in that separated from the polyphenol extract derived from Baumi situation mushroom. The method of claim 2,
The active compound of the styrylpyrone series is Interfungin A, Baumin (Baumin = Phoebe 211 (Pb211)), Davallialactone, Hispidin (Hispidin), Hipolomin B ( Hypholomine B), Fegligridin D (Phelligridin D) and Inoscavin A (Inoscavin A) A composition for preventing or treating inflammatory diseases, characterized in that at least one selected from the group consisting of. 5. The method according to any one of claims 1 to 4,
The inflammatory disease is a composition for the prevention or treatment of inflammatory diseases, characterized in that the arthritis or pulp. A food composition comprising a polyphenol extract derived from Baumi situation mushroom.

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