KR20100119922A - Anti-inflamatory composition containing betaglucan extracted from agrocybe chaxingu - Google Patents

Abstract

PURPOSE: A composition containing Agrocybe chaxingu-derived beta-glucan for anti-inflammation is provided to suppress LPS-inducing iNOS and COX-2 expression level. CONSTITUTION: A composition for anti-inflammation contains beta-glucan isolated from Agrocybe chaxingu. Beta-glucan is a pellet layer obtained by compression hot water extraction or centrifugation using ethanol on the supernatant. Beta-glucan is a fraction obtained by treating with alpha-amylase, protease and amyloglucosidase to the pellet layer. The Agrocybe chaxingu extract is contained in 0.001-10%(w/v).

Description

Anti-inflamatory composition containing betaglucan extracted from Agrocybe chaxingu}

The present invention relates to an anti-inflammatory composition containing chashingo mushroom extract beta glucan.

Progression of inflammation involves the activation of monocytes and macrophages, which secrete inflammatory mediators such as nitric oxide. Inducible nitric oxide synthase (hereafter referred to as “iNOS”) is one of three enzymes that produce nitric oxide, which mediates many physiological events. It is well known that overproduction of inducible nitric oxide synthase is associated with various human diseases such as inflammation and neurological diseases (Yu, HH et al., (2008) J. Neurosci. Res. 86 , 2963-2972, Colton , CA et al., (2006) Proc. Natl. Acad. Sci. USA 103 , 12867-12872, Dawson, VL et al., (1996) J. Neurosci. 16 , 2479-2487, Good, PF et al. , (1998) J. Neuropathol.Exp. Neurol. 57 , 338-342, Huang, Z. et al., (1994) Science 265 , 1883-1885, Iadecola, C. et al., (1997) J. Neurosci . 17, 9157-9164, Martin, LJ et al., (2007) J. Comp. Neurol. 500, 20-46, Amin, AR et al., (1999) Curr. Opin. Rheumatol. 11, 202- 209 ).

Prostaglandins (PGs) are potent proinflammatory mediators derived from arachidonic acid metabolism by cyclooxygenases (COXs) and play important functions in coordinating pathological situations, including inflammatory and allergic immune responses (Tilley, SL et al. al., (2001) J. Clin. Invest. 107 , 191-195). Two kinds of isoforms of cyclooxygenase (hereinafter referred to as "COX") enzymes are well known. COX-1 is constantly expressed and plays an important role in maintaining normal physiological function of the cell. COX-2 is significantly induced by several promoters, including cytokines, during the inflammatory response (Smith, WL and Dewit, DL (1990) Adv. Immunol. 62 , 167-215, Carey, MA et al., (2003) Prostaglandins Leukotrienes Essential Fatty Acids 69 , 157-162, Vancheri, C. et al., (2004) Trends Immunol. 25 , 40-46).

Lipopolysaccharides (hereinafter referred to as "LPS") are the major elements of endotoxins and are formed by phosphoglycolipids covalently bound to hydrophilic heteropolysaccharides (Rietschel, ET et al., (1994) FASEB J. 8 , 217-225). Lipopolysaccharides inhibit macrophage proliferation and activate macrophages to produce proinflammatory factors that play an important role in the immune response (Adams, DO and Hamilton, TA (1984) Annu. Rev. Immunol. 2 , 283- 318, Morrison, DC and Ryan, JL (1987) Annu. Rev. Med. 38 , 417-432).

Previous studies have found that 12-O-tetradecanoylpobol 13-acetic acid (mixed with tetradecanoylphobol 13-acetate, hereinafter "TPA") promotes mouse skin tumorigenesis, which is similar to the inflammatory response. These responses include edema and hypertrophy development, induction of proinflammatory cytokines, reactive oxygen species, COX-2 and iNOS expression. Thus, inhibition of COX-2 and iNOS expression induction is a new paradigm that inhibits skin diseases including inflammation and tumors (DiGiovanni, J. (1992) Pharmacol. Ther. 54 , 63-128, Slaga, TJ et al. , (1982) J. Cell Biochem. 18 , 99-119, Furstenberger, G. et al., (1981) Proc. Natl. Acad. Sci. USA 78 , 7722-7726, Furstenberger, G. et al., (1983) Science 220 , 89-91, Song, HY et al., (2008) Biochem.Pharmacol . 75 , 1348-1357, Chung, WY et al., (2007) Carcinogenesis 28 , 1224-1231, Ho, YS et al., (2007) Biochem. Pharmacol. 73 , 1786-1795, Surh, YJ et al., (2001) Mutat. Res. 480-481 , 243-268).

Mushrooms are generally known as natural products with medical potential and have been widely used in the treatment of various diseases including tumors for hundreds of years in China and other Asian countries. Mushrooms have been reported to have antitumor, antibacterial and antiviral activity depending on their type (Yoon, SY et al., (1994) Arch Pharm. Res. 17 , 438-442, Wang, SY et al., (1997). Int. J. Cancer 70 , 699-705). In vitro studies and in vivo studies have established that polysaccharide fractions of some mushrooms are involved in anticancer effects.

In addition, other studies show that the molecular mechanism of anticancer and anti-inflammatory effects is to inhibit COX-2 and iNOS expression.

Mushrooms have long been used as a dietary supplement to prevent diseases as well as to treat diseases. It has been proved through various studies that it has an immune enhancing, anticancer and anti-aging effect.

Studies on the functionality of mushrooms have been focused on the glycoprotein components of mushrooms (Pak et al. 1998, Korean J. Food Sci.Technol, 30 (5): 1236-1242; Jin et al. 1999, Korean Journal of Pharmacy, 43 (5): 635-641; Jung et al. 2002, Korean Patent 10-0340663; Cho et al. 2004, Korean Journal of Microbiology, 40 (3): 217-220). It has been reported to have various anticancer and antioxidant activities (Lin, JY and Chou, TB 1984, J. Biochem. 6: 35-40; Kawagishi, H. et. Al ., 1990, Biochim. Biophys. Acta, 1034: 247 -252; Yu, LG et. Al ., 1993, Cancer Res. 53: 4627-4632; Ye, SF et. Al ., 2003, Life Sciences. 74: 593-602), proteins having anticancer activity from various mushrooms Polysaccharides have been identified. Among them, Retinan extracted from shiitake mushrooms, Schizophyllan from squirrel mushrooms and Crestine (PSK) from cloud mushrooms have excellent activity. It has been used as adjuvants in clinical trials (Zhu D. 1987, Abstr Chin Med 1: 251-286; Hayakawa K. et al, 1993, Anticancer Res 13:...... 1815-1820; Nakajato H. et. al ., 1994, Cancer Lancet 343: 1122-1226; Hobbs C. 1995, Fenichel RL, Chiigos MA (eds) Immunomodulation agents and their mechanisms.Dekker, New York, 409-436).

Agrocybe chaxingu is a wood decay fungus that occurs in the tea tree, which belongs to the folds of the fungi, dung mushroom, and Agrocybe , which helps diuretic and spleen and stomach and brighten the eyes. It is known to have high medicinal value.

Chashingo mushroom is Agrocybe Among the mushrooms belonging to the genus, Agrocybe aegerita is known to be relatively close genetically (Gonzalez, P. and J. Labarre, 1998, Appl.Environ.Microbiol., 64 (11): 4149-4160) The functionality of mushrooms has been reported several times (Hyun, JW et. Al ., 1996, Arch. Pharm. Res., 19 (3): 207-212; Kim, BK et. Al ., 1997, Arch. Pharm Res., 20 (2): 128-137; Kim et al., 2000, Korean Journal of Mycology, 36 (1): 64-68; Kim et al., Korean Journal of Mycology, 30 (2): 124-130) There was no report on the functionality of the mushrooms.

Japanese Patent Application Laid-Open No. 2006-025725 (February 02, 2006) "Multipurpose Japanese Sauce and Manufacturing Method thereof" discloses a Japanese sauce made from chashin mushroom extract, Japanese soy sauce, kelp, bonito, and the like. In addition, Chinese Patent Publication No. 1349767 (2002.05.22.) "Flavour Chicken Recipe Using Shingo Mushroom" discloses the use of Chashingo mushroom for the purpose of adding flavor to chicken dishes.

As such, chashingo mushroom has been used only for cooking or for some folk remedies, but its anti-inflammatory effect is not known at all.

It is an object of the present invention to provide a natural product-derived anti-inflammatory composition that is safe for humans and inhibits inflammation.

In order to achieve the above object, the present inventors extracted the polysaccharide beta glucan from chashingo mushroom whose anti-inflammatory activity is not known, and then tested the anti-inflammatory activity of beta glucan of chashingo mushroom against cancer cell lines and showed excellent anti-inflammatory activity. By confirming, the present invention has been completed.

The present inventors examined the effect of chasin-derived beta glucan on inhibiting the expression of nitric oxide and COX-2 and LPA-induced mouse ear edema in LPS-induced mouse macrophage Raw 264.7 cells. The results show that the betaglucan protects LPS- and / or TPA-induced cell damage in vitro and in vivo.

Polysaccharides extracted from Chashingo mushroom of the present invention severely inhibited LPS-induced Nitrogen Oxide Synthase (iNOS) and COX-2 expression levels in cells, and when the polysaccharides were topically applied, TPA-induced ear edema in mice was significantly suppressed. It became. These results suggest that the chashin mushroom-derived polysaccharide is useful as an anti-inflammatory composition.

In order to achieve the above object, the present invention provides an anti-inflammatory composition comprising chashingo mushroom extract beta glucan.

It is preferable to extract beta glucan from chashingo mushroom from the fruiting body.

The anti-inflammatory composition may include a pressurized hot water extract obtained from high-pressure hot water extract of Chashingo mushroom or 0.001-10% (w / v) of the pellet layer obtained by centrifugation by treating ethanol to the centrifugal supernatant of Chashingo mushroom pressurized hot water extract. It is preferable to include).

In addition, the anti-inflammatory composition is more preferably characterized in that the beta glucan fraction obtained by treating the pellet layer with alpha-amylase treatment, protease treatment, amyloglucosidase treatment.

Hereinafter, the present invention will be described in detail.

The present invention relates to an anti-inflammatory composition comprising chashingo mushroom extract beta glucan.

The present inventors applied anti-inflammatory effects to the cultured skin cells by applying beta glucan to the cultured skin cells using the chashingogi fruit body extracted with hot water, an organic solvent, etc., and showed anti-inflammatory activity. Natural extract derived from shishingo mushroom is widely used in cooking and is used for folk remedies.

As used herein, the term "anti-inflammatory" refers to the ability to inhibit or relieve inflammation. It was confirmed that the inflammation of the skin cell line was relieved when the tea extract mushroom beta glucan was administered.

The composition may comprise a pharmaceutically acceptable carrier and be formulated for human or animal use.

Pharmaceutical compositions for oral administration include, for example, capsules or tablets, powders or granules, solvents, syrups or suspensions.

Suitable excipients for tablets or hard gelatin capsules are lactose, corn starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use in soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solids or liquid polyols. Excipients that can be used to prepare solvents and syrups are, for example, water, polyols and sugars. Oils, such as vegetable oils, can be used to prepare suspensions to provide oil-in-water or water-in-oil suspensions. Pharmaceutical compositions for transdermal administration may be presented as separate patches to enable intimate contact with the epidermis of the recipient for a long time.

Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes that are substantially isotonic with the blood of the recipient; And aqueous and non-aqueous sterile suspending agents which may include suspending agents and thickening agents. Excipients that can be used in the preparation of injectables include, for example, water, alcohols, polyols, glycerin and vegetable oils. Such compositions can be stored in unit-dose (single) or multi-dose (several) containers, such as sealed ampoules and vials, with the addition of a sterile liquid carrier, such as water for injection, just prior to use. Can be stored under freeze-drying conditions for use.

Instant injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

The route of administration of the compositions of the invention can be administered via a common route that can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, rectal administration, but is not limited to the above listed methods of administration. Pharmaceutical compositions can also be administered by any device capable of moving the active substance to the target cell.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the type and severity, severity, age, sex, and drug of the patient. Can be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The compositions of the present invention may be administered in a single or multiple doses. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and a pharmaceutically effective amount can be easily determined by those skilled in the art.

Pharmaceutical formulations comprising the composition of the present invention can be applied orally, parenterally, ie, subcutaneously, intramuscularly, or topically, as desired. The dosage is a daily dosage of 0.001 μg˜10 mg / kg. It may be administered in several divided doses.

The present invention relates to a composition having anti-inflammatory activity extracted from chashingo mushroom, chasingo mushroom extract has excellent anti-inflammatory activity and can be usefully used as a prophylactic and therapeutic agent for inflammatory reactions, particularly skin inflammatory reactions.

The anti-inflammatory composition of the present invention comprises chashingo mushroom extract, which is obtained by extracting and extracting chashingo mushroom with hot water and an organic solvent.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are intended to describe the present invention in more detail, and the scope of the present invention is not limited by the description of the embodiments.

<Material>

Fetal bovine serum (FBS), DMEM and penicillin-streptomycin antibiotics were purchased from Gibco BRL (Grand Island, USA). Primary antibodies against COX-2, iNOS and actin were purchased from Santa Cruz Biotechnology company (Santa Cruz, CA, USA). The other reagents used a first grade product.

Experimental tea shingo mushroom samples were used for the experiment while preserving the fruiting bodies of Agrocybe chaxingu fruit grown in the Agricultural Products Research Center of the Institute of Agricultural Technology, Gangwon-do, and hot-air dried at 50 ° C.

<Example 1-1: Preparation of Pressurized Hot Water Extract>

After distilled water 2000ml was added to 100g of crushed tea Shingo mushrooms and extracted at 121 ° C for 2 hours, the pressurized hot water extract was centrifuged at 6,000 rpm for 40 minutes, and 4 times (v / v) 95% ethanol was added to the supernatant. After standing at 4 ° C. for 24 hours, the pellet layer was recovered by centrifugation at 6,000 rpm for 40 minutes. The recovered pellet layer was sufficiently dissolved in d- H ₂ O, dried using a lyophilizer, and the freeze-dried pressurized hot water extract sample was used under cold dark conditions.

Example 1-2: α-amylase Treatment

In order to decompose the glucan of α-1,4 binding contained in the extract, the pressurized hot water extract was dissolved in 5 ml of 20 mM sodium acetate buffer (pH 6.0), and the α-amylase was treated with 5U and reacted at 55 ° C. for 10 minutes. After the reaction, 4 times (v / v) of ethanol was added to precipitate, followed by centrifugation at 4, 6,500 rpm for 30 minutes to obtain a precipitate.

Example 1-3 Protease Treatment

In order to decompose and remove the protein contained in the extract, it was dissolved in 5 ml of 20 mM Tris-HCl buffer (pH 7.5), and the protease was treated with 2.8 × 10 ⁻² U and reacted at 37 ° C. for 10 minutes. Then, 5 ml of 10% trichloroacetic acid was added to precipitate the protein, followed by centrifugation at 6,500 rpm and 4 ° C. for 10 minutes to obtain a precipitate.

Example 1-4 Amyloglucosidase Treatment

To remove the α-1,4 or α-1,6 binding glucose contained in the extract, it was dissolved in 5 ml of 20 mM sodium acetate buffer (pH 4.3) and treated with amyloglucosidase at 1 × 10 ^-4 U. After reacting at 55 ° C. for 10 minutes, 4 times (v / v) ethanol was added thereto, followed by centrifugation at 6,500 rpm and 4 ° C. for 10 minutes to obtain a precipitate.

Example 1-5: Separation by Ultrafiltration

After re-dissolving in lyophilized pressurized hot water extract, the ultrafiltration system (Ultra Filtration System) was used as shown in Table 1 to determine the distribution characteristics of useful substances according to molecular weight.

Ultrafiltration Membrane Flow concentrators 10,000 MWCO ^a) PES (polyethersulfone)
3,000 MWCO PES (polyethersulfone)
1,000 MWCO PES (polyethersulfone) Spin concentrators 0.2 mm polyethersulfone (PES) ^a) molecular weight cut off,

Example 1-6: Component Analysis of Separation Materials

One component of the isolated material was analyzed according to AOAC. Moisture content was measured by 105 ℃ atmospheric pressure drying method, protein content was analyzed by geldal method. Beta-glucan content of the material separated by the above method was measured using a megazyme beta glucan assay kit (Megazyme Pty. Ltd., Australia).

Example 1-7 Determination of Molecular Weight of Separation Material

Gel filtration chromatography was performed to determine the molecular weight of the material separated in the same manner as above. A Ø15 × 230 mm glass column (Bio-rad, USA) was filled with Sepharose CL-6B (molecular weight range: 10,000 to 100,000), loaded with 1 ml of sample, and developed at a flow rate of 0.8 ml / min. 5 mL fractions were collected using an auto fraction collector. In addition, the molecular weight was calculated by comparing the retention time of the isolated polysaccharide with the retention time of the standard dextran under the same conditions as described above (FIG. 5).

Example 2: Cell Culture and MTT Analysis

Rat macrophage Raw 264.7 cells were treated at 37 ° C. in DMEM medium containing 20 mM HEPES / NaOH (pH 7.4), 5 mM NaHCO ₃ , 10% fetal calf serum (FBS) and antibiotics (100 μg / ml streptomycin, 100 U / ml penicillin). Incubated in a humid atmosphere with 95% air and 5% CO ₂ .

The biological activity of Chashingo mushroom polysaccharides was analyzed by measuring the viability of Raw 264.7 cells treated with LPS (100 ng / ml) for 12 hours. Cells were incubated in 6-well plates until 70%. Cells were first pretreated with polysaccharides (10-100 μg) for one hour and then treated with LPS. Cell viability was analyzed by staining with MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-dipheyltetrazolium bromide}.

Example 3: Nitric Oxide Production Measurement

Raw 264.7 rat macrophages were incubated for 12 h to 70% in 24-well plates. Cultured cells were pretreated with polysaccharides (10-100 μg) for one hour and then treated with LPS (100 ng / ml) for 12 hours and harvested culture medium. Nitric oxide production was analyzed by measuring nitric oxide nitrite (34, 35). Briefly, 100 μl of cell culture medium was incubated for 10 min at room temperature in the same volume of Griess reagent (5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride with 1% sulfanylamide) and then 540 nm. Absorbance was measured with a microplate reader. NaNO ₂ was used as a standard solution.

Example 4: Western Blot Analysis

Raw 264.7 cells were treated with lysate buffer at 4 ° C. for 30 minutes to obtain lysate. Protein concentration was measured by Bio-Rad protein measurement kit. Protein (40 μg) was developed on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred to nitrocellulose membrane. The membranes were blocked for 2 hours with 5% skim milk dissolved in Tris buffer saline (TBS; 20 mM Tris, 0.2 M NaCl, pH 7.5) (TBST) containing 0.05% Tween-20 and then dissolved in TBST for 1 hour at room temperature. Incubated with -COX-2, anti-iNOS, and anti β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1: 500 dilution). After washing, the membranes were incubated with horseradish peroxidase-bound secondary antibody (1: 0000 dilution in TBST) for one hour. The membrane was incubated with luminescent chemicals and then exposed to Hyperfilm ECL (Amersham Biosciences, Piscataway, NJ, USA).

Example 5 COX-2 and iNOS Expression Measurement

Raw 264.7 rat macrophages were incubated for 12 h to 70% in 24-well plates. Cells were pretreated with polysaccharides (10-50 μg) for one hour and then incubated for 12 hours with LPS (100 ng / ml) and the medium harvested. COX-2 and iNOS protein levels were western bloated using anti-COX-2 and anti-iNOS antibodies, respectively.

Example 6: RT-PCR Analysis

Total RNA isolated from Raw 264.7 rat macrophages was isolated using Trizol reagent kit (Invitrogen, Gaithersburg, MD, USA) according to manufacturer's instructions (36). RNA (2 μg) was reversibly transcribed using 10,000 U reverse transcriptase and 0.5 μg / μl oligo- (dT) initiator. PCR amplification of the cDNA fractions was performed using the following sense and antisense initiators:

COX-2 antisense initiator, 5'-TGGACGAGGTTTTT CCACCAG-3 ';

COX-2 sense initiator, 5'-AAAGGCCTCCATTGACCAGA-3 ';

Betaactin antisense initiator, 5'-GGACAGTGAGGCCAGGATGG-3 ';

Betaactin sense initiator, 5'-AGTGTGACGTTGACATCCGTAA AGA-3 ';

iNOS antisense initiator, 5'-CTGTCAGAGCCTCGTGGCTTT-3 ';

iNOS sense initiator, 5'-ATGGCT CGGGATGTGGCTAC-3 '.

PCR was performed by heating 50 μl of 10 mM Tris-HCl (pH 8.3), 25 mM MgCl ₂ , 10 mM dNTP, 100 U Taq DNA polymerase and 0.1 μM of each initiator to 70 ° C. for 15 minutes. PCR products were run on 1% agarose gel and observed with UV light for EtBr treatment.

Example 7: TPA-Induced Skin Inflammation

6-8 week old male ICR mice were obtained from Hallym University Experimental Animal Center. Animals were bred at a constant temperature (23 ° C), moderate relative humidity (60%) for 12 hours light: 12 hours dark cycle, and free food and water intake. Animals used in the present invention were bred according to the "Laboratory Animal Protection Rules" (NIH Publication No. 86-23).

Skin inflammation was induced in the right ear of the mouse by topical application of TPA. Five were in one group. TPA (1.0 μg) was dissolved in 20 μl acetone and applied to the inner and outer surfaces of the mouse ears every 24 hours for 2 days. Polysaccharide (300 μg) was applied topically to mouse ears after one hour of TPA treatment. The second day, TPA was applied and one hour after the polysaccharide was applied to the same site. Twenty four hours after the last application of polysaccharides, ear skin thicknesses were measured with a digital thickness meter (Mitutoyo Corporation, Japan). The ears of the mice were punched (diameter 5 mm, Kai Industries Co. Ltd., Gifu, Japan) and weighed.

Example 8: Histological Analysis

Ear biopsies were ear fixed with 10% (v / v) buffered formalin, inserted into paraffin, prepared into sections of 4 μm, and stained with hematoxyline and eosin. Stained tissue sections were assayed for infiltration of inflammatory cells using standard bright-field optics (Zeiss, AXIOIMAGER M1, Gottingen, Germany).

Statistical analysis

Results are expressed as mean ± standard deviation of at least 3 independent experiments. Values were evaluated through Duncan's multiple range tests using one-way ANOVA and later GraphPad Prism 4.0 software (GraphPad Software Inc, San Diego, Calif., USA). The difference is significant when P <0.05.

<Result 1: Extraction rate and composition of Chashingo mushroom extract>

The results analyzed by Example 1-6 are shown in Table 2 below. It was confirmed from Table 2 that the purity and content of the beta glucan was increased through the purification process of Example 1.

Crude extract Purified Compound Extraction rate (% by weight) 7.34 4.23 Composition (% by weight)

Beta Glucan 40.4 87.5 protein 23.8 9.24 moisture 6.8 3.21

The analysis results of Examples 1-7 are shown in Table 6.

<Result 2: Effects of Chashingo Mushroom Extract Beta-glucan on Cell Viability and LPS-Induced Nitric Oxide Production

The anti-inflammatory effect of edible mushroom Agrocybe chaxingu beta glucan is not known (Fig. 1a). Thus, we tested the anti-inflammatory effects of beta glucan extracted from the mushrooms in vitro and in vivo.

Macrophages play an important role in the initiation and maintenance of inflammation. Nitric oxide concentrations are important for measuring inflammation and cell viability. Since the level of nitric oxide is important in dealing with the degree of inflammation and cell viability, we investigated the effect of polysaccharides on inhibiting nitric oxide expression and cell viability. To determine if the polysaccharides were cytotoxic to Raw 264.7 cells, cells were treated with polysaccharides of various concentrations (10-100 μg / ml) and incubated for 12 hours. As shown in Figure 1b, chashingo mushroom extract beta glucan did not affect the cell viability at various concentrations. It is well known that nitric oxide plays an important role in the pathophysiology of many diseases. Raw 264.7 cells were incubated with lipopolysaccharide (LPS, 100ng / ml) for 12 hours in order to investigate the effect of beta glucan extracted from S. mushroom on nitric oxide production. At this time, one group was cultured in the presence of chasingo mushroom extract beta glucan of various concentrations (10-100㎍ / ml), the other group was cultured in the state without adding the chashingo mushroom extract beta glucan. Cell culture medium was harvested to determine nitrite levels. Chashingo mushroom extract beta glucan reduced the concentration of nitric oxide production (Fig. 1c).

<Result 3: Effect of Chashingo Extract Extracted Beta Glucan on Lipopolysaccharide-Induced iNOS and COX-2 Expression Levels in Raw 264.7 Cells>

Lipopolysaccharides are major endotoxins and play an important role in the immune response by inhibiting macrophage proliferation and activating proinflammatory factors. Thus, we tested the effect of polysaccharides on COX-2 and iNOS expression levels under lipopolysaccharide exposure. Raw 264.7 cells were incubated for 12 hours with Lipopolysaccharide (100ng / ml) with and without Chashingo mushroom extract betaglucan (10-50㎍ / ml). Polysaccharides inhibited lipopolysaccharide-induced COX-2 and iNOS protein expression levels concentration dependently (FIG. 2). We also tested the effect of Chashingo mushroom extract betaglucan on COX-2 and iNOS mRNA expression levels in lipopolysaccharide-induced cells by RT-PCR. As shown in Fig. 3, the extract from beta glucan was concentration-dependently inhibited the lipopolysaccharide-induced COX-2 and iNOS mRNA expression levels. These results suggest that inhibition of COX-2 and iNOS mRNA expression levels by Chashingo mushroom extract betaglucan is associated with inhibition of COX-2 and nitric oxide production. It is well known that COX-2 and nitric oxide produced in macrophages play a critical role in inflammatory diseases. COX-2 and iNOS proteins have been reported to be closely associated with skin inflammation, cell proliferation and skin tumor promotion, which can be rapidly induced by proinflammatory mediators. In addition, other reports suggest that inhibition of COX-2 and iNOS expression is important for alleviating inflammation and suppressing tumors. Therefore, inhibition of COX-2 and iNOS expression by chashingo mushroom extract beta glucan may be an effective new strategy of inflammation treatment and disease inhibition.

<Result 4: Effect of Chashingo Mushroom Extract Beta-glucan on Mouse TPA-Induced Ear Edema>

TPA induced mouse ear edema was used as an inflammation model. Topical application of TPA to mouse ears induced chronic dermatitis, such as edema, epidermal hyperplasia, and infiltration of inflammatory cells. TPA was applied to mouse ears twice over two days to induce inflammatory cell infiltration resulting in an increase in ear thickness and ear weight. As shown in FIG. 4, the TPA-induced increased ear thickness and weight were significantly reduced as a result of applying TPA to the mouse ears once a day for 2 hours and treating the Shingo mushroom extract betaglucan for one hour. In addition, cha shingo mushroom extract beta glucan inhibited the skin penetration of monocytes, one of the early stages of inflammation in the skin. Although the exact mechanism should be further revealed, the chashingo mushroom extract betaglucan of the present invention effectively inhibited TPA-induced ear edema.

In summary, Agrocybe chaxingu extracted beta glucan effectively inhibited LPS-induced nitric oxide and COX-2 expression levels and cell death in Raw 264.7 cells. In addition, it was suggested that chashingo mushroom extract beta glucan of the present invention exhibits local anti-inflammatory activity by showing a protective effect against TPA-induced skin inflammation. The exact mechanism should be further revealed, but the chashingo mushroom extract betaglucan of the present invention is useful as a topical application for inflammatory skin diseases such as skin tumors.

Figure 1a is the structure of the tea Shingo mushroom derived polysaccharides.

Figure 1b shows the effect of chashingo mushroom extract beta glucan on cell viability and LPS-induced nitric oxide production, Raw 264.7 cells incubated with Chashingo mushroom extract betaglucan (10-100 ㎍ / ml) for 12 hours Afterwards, cell viability was obtained by measuring color development using MTT. Raw 264.7 cells were first treated with Chashingo mushroom extract beta glucan (10-100 µg / ml) for one hour and then treated with lipopolysaccharide (LPS, 100ng / ml) for 12 hours.

Figure 1c is the result of measuring the nitrite level. Measurement was performed using the Griess reaction in lipopolysaccharide-stimulated cell culture medium. Each bar represents the mean ± standard deviation of five experiments. P represents the beta glucan extracted chacha of the present invention.

2 shows the effect of Chashingo mushroom extract betaglucan on LPS-induced iNOS (A) and COX-2 (B) protein expression. Raw 264.7 cells were first treated with Chashingo mushroom extract beta glucan for 1 hour and then treated with lipopolysaccharide (LPS, 100ng / ml) for 12 hours. Cell lysates were prepared and iNOS and COX-2 protein expression levels were measured by western blotting. P represents the beta glucan extracted chacha of the present invention.

Figure 3 shows the inhibitory effect of Chashingo mushroom extract betaglucan on LPS-induced iNOS (A) and COX-2 (B) mRNA levels in Raw 264.7 cells. Cells were previously treated with Chashingo mushroom extract betaglucan for 1 hour and then treated with lipopolysaccharide (LPS, 100ng / ml) for 12 hours. Total RNA was extracted and iNOS and COX-2 mRNAs were RT-PCR analyzed using specific initiators. P represents the beta glucan extracted chacha of the present invention.

Figures 4a, 4b, 4c shows the inhibitory effect of Chashingo mushroom extract beta glucan on TPA-induced ear edema. Mouse ears were treated with TPA (1 μg) once a day for two days. After one hour after TPA treatment, beta glucan extracted from tea mushrooms was applied topically. Mice were sacrificed 24 hours after the last tea extract extract betaglucan treatment.

Figure 4a was analyzed by hematoxylin and eosin staining the inhibitory effect of chashingo mushroom extract beta glucan on TPA-induced ear edema.

4B analyzes the change in ear thickness. P represents the beta glucan extracted chacha of the present invention.

4C analyzes the change in ear weight. P represents the beta glucan extracted chacha of the present invention.

Figure 5 illustrates one method of extracting beta glucan from chashingo mushroom.

6 is a graph showing the molecular weight analysis results of each fraction according to Example 1-7.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Anti-inflamatory composition containing betaglucan extracted from          Agrocybe chaxingu <130> HallymU-chaxingu2 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of cyclooxygenase-2 <400> 1 tggacgaggt ttttccacca g 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer of cyclooxygenase-2 <400> 2 aaaggcctcc attgaccaga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of beta-actin <400> 3 ggacagtgag gccaggatgg 20 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sense primer of beta-actin <400> 4 agtgtgacgt tgacatccgt aaaga 25 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of iNOS <400> 5 ctgtcagagc ctcgtggctt t 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer of iNOS <400> 6 atggctcggg atgtggctac 20  

Claims (5)

Anti-inflammatory composition comprising beta glucan extracted from chashingo mushroom. The method according to claim 1, The beta glucan is an anti-inflammatory composition, characterized in that the pellet layer obtained by centrifugation by treating ethanol to the centrifugal supernatant of the pressurized hot water extract or chashingo mushroom pressurized hot water extract extracted high temperature hot water. The method according to claim 2, The beta glucan is an anti-inflammatory composition, characterized in that the fraction obtained by treating the pellet layer with alpha-amylase, protease and amyloglucosidase. The method according to any one of claims 1 to 3, The chashingo mushroom is an anti-inflammatory composition, characterized in that Chashingo mushroom fruiting body. The method according to any one of claims 1 to 3, The tea Shingo mushroom extract is contained 0.001 ~ 10% (w / v), characterized in that the anti-inflammatory composition comprising a pharmaceutically possible carrier.

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