KR101325094B1 - A composition comprising extract of Pleurotus eryngii for treating or preventing atopic dermatitis - Google Patents

Abstract

The present invention relates to a composition for the prophylaxis or treatment of atopic dermatitis, and more particularly, to a Pleurotus eryngii extract with excellent activity for treating or alleviating the symptoms of various skin diseases such as atopic dermatitis.
In the present invention, the Pleurotus eryngii extract is shown to reduce the expression of cytokines and proteins known to be increased in atopic dermatitis, TARC / CCL17, MDC / CCL22, CTACK / CCL27, ICAM-1, the extract of atopic dermatitis It can be confirmed that it can be used as a therapeutic agent.

Description

A composition comprising extract of Pleurotus eryngii for treating or preventing atopic dermatitis}

The present invention relates to a composition for the prophylaxis or treatment of atopic dermatitis, and more particularly, to a Pleurotus eryngii extract with excellent activity for treating or alleviating the symptoms of various skin diseases such as atopic dermatitis.

Atopic dermatitis is a chronic inflammatory disease that occurs before the development of asthma and allergic diseases. The symptoms vary depending on the level of chemokine, quantitative changes, and activity (Pivarcsi and Homey, 2005). Lymphocytes invade the lesional skin (Grew et). al ., 1998).

One of Th2 chemokines, TARC / CCL17 (thymus and activation-regulated chemokine), is constantly expressed in the thymus and is also produced in keratinocytes (Vestergaard et. al ., 1999). The TARC chemokine plays a role in attracting lymphocytes and is preferentially expressed in Th2 lymphocytes, basophils, and natural killer cells (Sallusto et. al ., 1998; Nickel et al ., 1999; Godisk et al ., 1997). A chemokine that is closely related to TARC is a macrophage-derived chemokine (MDC / CCL22), which is known to be produced in keratinocytes (Hino et. al ., 2007), which are characterized by excessive expression of TARC and MDC in the serum of patients with atopic dermatitis (Shimada et al., 2004; Hashimoto et. al ., 2006). In addition, CTACK / CCL27 (cutaneous T cell-attracting chemokine) is a chemokine expressed in the skin (Reiss et al ., 2001) are known to be involved in skin inflammatory diseases (Kakinuma et. al ., 2003).

Another symptom of atopic dermatitis is the overexpression of ICAM-1 (intercellular adhesion molecule-1) (Campbell et. al ., 1999), ICAM-1 acts as a receptor for the lymphocyte surface antigen, thereby mediating the adhesion of cells to cells.

Atopic dermatitis is a phenomenon in which keratinocytes proliferate abnormally due to immunological factors. When keratinocytes are exposed to IFN-γ (tumor necrosis factor-α), TARC It can be confirmed that abnormal expression of chemokines, such as MDC, CTACK. This is due to an increase in the infiltration of monocytes / T cells into the skin as an inflammatory reaction occurs (Sebastiani et al. al ., 2002).

Since the treatment of atopic dermatitis is difficult to find and treat its source, it is common to avoid causing factors and to control the symptoms of atopic dermatitis through proper treatment rather than aiming at cure. Prescriptions for atopic dermatitis are mainly pharmacotherapy such as steroids, antihistamines, and antibiotics. The steroids (adrenal cortex hormones) have anti-inflammatory and immunosuppressive effects and are excellent, but they can weaken the skin after long-term application. Side effects such as systemic hormonal symptoms and addictive symptoms may occur. Antihistamines help prevent the release of histamine in mast cells, thus alleviating the itchy symptoms, but they are used as a temporary measure. There are side effects such as burning, anxiety and loss of appetite when taken over a long period of time. Therefore, there is a need for a new concept of atopic dermatitis treatment composition that is effective against atopic dermatitis and has no side effects.

Pleurotus eryngii is a type of mushroom widely used as a food ingredient and traditional medicine in Korea. Pleurotus eryngii contains little fat or carbohydrates, but is higher in protein than most vegetables. It is also rich in vitamins B, D, K, A and C and suitable for low calorie meals (Kurtzman, 1997; Manzi and Aguzzi, 2001; Mattila et. al ., 2001; Yildiz et al ., 1998). The antimicrobial and antioxidative effects of Pleurotus eryngii (Kalyoncu et al. al ., 2010), anti-inflammatory and anti-allergic effects are known, but the therapeutic effect and other functions for atopic dermatitis are not widely known.

In contrast, the present inventors have been able to complete the present invention by discovering that the ethanol extract (PEE) of Pleurotus eryngii has a remarkable effect on the alleviation and inhibition of atopic dermatitis.

An object of the present invention to provide a composition for the prevention or treatment of atopic dermatitis containing Pleurotus eryngii extract.

The present invention relates to a composition for the prophylaxis or treatment of atopic dermatitis, which contains Pleurotus eryngii extract with excellent activity for treating or alleviating the symptoms of various skin diseases such as atopic dermatitis.

The Pleurotus eryngii extract is extracted with a polar solvent such as ethanol aqueous solution, propanol, butanol, isopropanol, ethyl acetate, ether, acetone, chloroform, and a mixed solvent thereof, and then the hexane and water distribution, diion HP-20 A well-known method used for separation extraction of plant components, such as a method of using an adsorption resin such as resin, or a method by column chromatography, can be easily obtained by combining alone or suitably. The crude extract can be further purified according to the conventional method, if necessary.

Chromatography used in the present invention includes silica gel column chromatography, LH-20 column chromatography, thin layer chromatography (TLC) and high performance liquid chromatography high performance liquid chromatography, etc.) can be used.

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis, containing Pleurotus eryngii mushroom extract or a pharmaceutically acceptable salt thereof as an active ingredient.

Pharmaceutical compositions for the prevention or treatment of atopic dermatitis comprising the Pleurotus eryngii extract according to the present invention, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It can be formulated in the form of suppositories and sterile injectable solutions. Carriers, excipients and diluents that may be included in the composition comprising the Pleurotus eryngii extract are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin in the Pleurotus eryngii extract. The mixture is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The dosage of the Pleurotus eryngii extract will vary depending on the age, sex and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 0.1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or several times. The dose is not intended to limit the scope of the invention in any way. Pleurotus eryngii mushroom extract of the present invention can be administered to various mammals such as rats, livestock, humans. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

In particular, the Pleurotus eryngii mushroom has been used as a food or medicinal herb for a long time. When the Pleurotus eryngii extract of the present invention is administered to the human body, it is considered that there is no concern of side effects compared to other synthetic medicines. No effect was found.

In addition, the present invention provides a health functional food for the prevention or improvement of atopic dermatitis, including Pleurotus eryngii mushroom extract and a food supplement acceptable food supplement. The health functional food of the present invention includes tablets, capsules, pills or liquids, and the like, and the health functional foods include dairy products including soups, meat, sausages, breads, candy, snacks, noodles, ice cream, soups, Beverages containing ionic beverages, nutritional products including alcoholic beverages and vitamin complexes may be included.

The present invention relates to a composition for the prophylaxis or treatment of atopic dermatitis, and more particularly, to a Pleurotus eryngii extract with excellent activity for treating or alleviating the symptoms of various skin diseases such as atopic dermatitis.

In the present invention, the Pleurotus eryngii extract is shown to reduce the expression of cytokines and proteins known to be increased in atopic dermatitis, TARC / CCL17, MDC / CCL22, CTACK / CCL27, ICAM-1, the extract of atopic dermatitis It could be confirmed that it can be used as a therapeutic agent.

1 is a WST-1 assay result showing that the Pleurotus eryngii mushroom extract (PEE) of the present invention is not cytotoxic.
2 is an immunoassay result showing that the Pleurotus eryngii mushroom extract (PEE) of the present invention has an effect of inhibiting the expression of TARC / CCL17 increased to TNF-α / IFN-γ.
Figure 3 is a result of immunoassay showing that the hot water extract of Pleurotus eryngii (PEW), unlike the Pleurotus eryngii mushroom extract (PEE) of the present invention, does not inhibit the expression of TARC / CCL17 that is increased to TNF-α / IFN-γ.
4 is a result of RT-PCR showing that the Pleurotus eryngii mushroom extract (PEE) of the present invention has an effect of inhibiting the expression of TARC / CCL17, MDC / CCL22 and CTACK / CCL27, which are increased to TNF-α / IFN-γ.
5 is a Western blotting result showing that the Pleurotus eryngii mushroom extract (PEE) of the present invention has an effect of inhibiting the expression of ICAM-1 increased by TNF-α / IFN-γ.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

<Example 1. Preparation of Ethanol Extract (PEE) of Pleurotus eryngii>

The extraction method of Pleurotus eryngii is [Seo et. al ., 2005].

Pleurotus eryngii drops are washed and washed by tap or drinking water, and then cut into 2 ± 1cm in width and 0.5 ± 1cm in thickness, respectively, and then using an electric hot air dryer. Dried for 36 hours. Thereafter, the dried Pleurotus eryngii mushroom (130 g) was extracted twice with 60 ml of ethanol for 4 hours at 60 ° C. Thereafter, the extract was concentrated in a rotary condenser (EYELA, SB-1000) at 55 ° C. at 30 rpm and frozen, and then lyophilized to obtain a powder (PEE, Pleurotus). eryngii ethanol extracts, powder recovery rate 0.23%), and the powder was dissolved in DMSO (dimethyl sulfoxide).

In addition, to compare the effect of the ethanol extract of Pleurotus eryngii of the present invention was prepared hot water extract of Pleurotus eryngii. For this purpose, Pleurotus eryngii mushroom (130 g) was extracted for 8 hours at 90 ℃ using water 1000ml. Thereafter, the extract was concentrated in a rotary condenser (EYELA, SB-1000) at 55 ° C. at 30 rpm and frozen, and then freeze-dried to obtain a powder (PEW, Pleurotus). eryngii water extracts, powder recovery rate 0.22%), and the powder was used by dissolving in dimethyl sulfoxide (DMSO).

<Example 2. Checking cytotoxicity of Pleurotus eryngii extract>

Cytotoxicity was used for the WST-1 assay kit, and no toxicity was confirmed due to the treatment of the Pleurotus eryngii extract of the present invention. To this end, HaCaT cells, a human keratinocyte cell line, were first prepared using 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / ml penicillin, and 100 µg / ml. Cultured in Dulbeco's Modified Eagle's Medium (DMEM) containing streptomycin. All subsequent cell cultures were cultured and used in the above manner. The Pleurotus eryngii extract of the present invention was dissolved in DMSO and treated with cells, and the control was treated with only DMSO. DMSO treated in the cells was treated at a concentration of less than 0.1%. The WST-1 assay results are shown in FIG. 1.

1, it can be confirmed that the Pleurotus eryngii mushroom extract (PEE) of the present invention is not cytotoxic.

<Example 3. Confirmation of the expression of chemokines and proteins related to atopic dermatitis>

3-1. Confirmation of TARC / CCL17 Expression-Using Immunoassay

In order to confirm the expression of cytokine TARC / CCL17, HaCaT cells were treated with the Pleurotus eryngii extract of the present invention at a concentration of 10-50 μg / ml for 30 minutes, and then TNF-α (20 ng / ml) / IFN-γ (20 ng). / Ml) was treated for 24 hours. After 24 hours, only the supernatant of the cell treatment solution was collected, and the expression of TARC was confirmed by immunoassay (immunoassay, R & D Systems), and the results are shown in FIG. 2.

Also, under the same conditions, Pleurotus eryngii extract (PEW) was also treated with HaCaT cells, and the effects thereof were compared with that of Pleurotus eryngii extract of the present invention, and the results are shown in FIG. 3.

The results of FIG. 2 indicate that TARC / CCL17, which was increased due to the treatment of TNF-α / IFN-γ, was suppressed concentration-dependently due to the treatment of the Pleurotus eryngii extract (PEE) of the present invention, whereby the extract was It can be seen that there is an inhibitory effect of atopic dermatitis.

3 shows that the Pleurotus eryngii water extract (PEW) has little inhibitory effect on TARC / CCL17 that was increased due to the treatment of TNF-α / IFN-γ, unlike the Pleurotus eryngii extract (PEE) of the present invention. Therefore, the inhibitory effect on TARC / CCL17 is confirmed to be characteristic of the ethanol extract of Pleurotus eryngii.

3-2. Check MDC / CCL22 and CTACK / CCL27-using RT-PCR

RT-PCR was performed to confirm expression of cytokines MDC / CCL22 and CTACK / CCL27.

To this end, HaCaT cells were pretreated with the Pleurotus eryngii mushroom extract of the present invention at a concentration of 10-50 μg / ml for 30 minutes, and then TNF-α (20 ng / ml) / IFN-γ (20 ng / ml) for 24 hours. Treated.

Total RNA was extracted using RNAiso Reagent (Takara). Primer conditions for RT-PCR were as follows, and the S16r gene (S16 ribosomal protein) was a housekeeping gene, and was used as a comparison group to compare the increase and decrease of all genes.

MDC / CCL22 forward: 5’-AGG ACA GAG CAT GGC TCG CCT ACA GA-3 ’

MDC / CCL22 reverse: 5’-TAA TGG CAG GGA GGT AGG GCT CCT GA-3 ’

CTACK / CCL27 forward: 5’-CTC AGC TCT ACC GAA AGC C-3 ’

CTACK / CCL27 reverse: 5’-GCC CAT TTT CCT TAG CAT CC-3 ’

S16 ribosomal protein (S16r) genes forward: 5’-TCC AAG GGT CCG CTG CAG TC-3 ’

S16 ribosomal protein (S16r) genes reverse: 5′-CGT TCA CCT TGA TGA GCC CAT T-3 ’

PCR was performed for 35 cycles under the following conditions, and the amplified DNA was identified using the SYBRSafeDNA gel staining kit, and the results are shown in FIG. 4.

PCR conditions: [denaturation at 98 ° C for 10 sec, annealing at 60 ° C for 30 sec, elongation at 72 ° C for 60 sec]

The results of FIG. 4 show that MDC / CCL22 and CTACK / CCL27, which were increased due to the treatment of TNF-α / IFN-γ, are suppressed concentration-dependently due to the treatment of the Pleurotus eryngii extract (PEE) of the present invention. It can be seen that the extract has an inhibitory effect on atopic dermatitis.

3-3. Confirmation of Expression of ICAM-1-Using Western Blotting

Western blotting was performed to confirm the expression of ICAM-1, a receptor protein for lymphocyte surface antigen.

HaCaT cells were pretreated with Pleurotus eryngii mushroom extract of the present invention at 10-50 μg / ml for 30 minutes, and treated with TNF-α (20 ng / ml) / IFN-γ (20 ng / ml) for 24 hours, each of 80 μg. Proteins were transferred to 10% SDS-PAGE and polyvinylidene difluoride (PVDF) membranes. After membrane blocking, the primary antibody was reacted, and a secondary antibody (horseradish peroxidase-conjugated anti-IgG secondary antibody) was reacted. Final expression comparison was performed using ECL reagent, β-actin (house keeping gene) as a comparison group, the results are shown in FIG.

5 shows that ICAM-1, which has been increased due to the treatment of TNF-α / IFN-γ, is suppressed concentration-dependently due to the treatment of the Pleurotus eryngii extract (PEE) of the present invention, whereby the extract is It can be seen that there is an inhibitory effect of atopic dermatitis.

< Example  4: Toxicity Test>

4-1: Oral administration

ICR-based mice (25 g) were divided into 5 groups of 10 animals, and the worm mushroom extract according to the present invention was orally administered at doses of 0, 2, 10, 20 and 50 ㎍ / kg, respectively, and observed for 2 weeks. In all cases, all of them survived.

4-2: intraperitoneal administration

ICR-based mice (25 g) were divided into 5 groups of 10 rats, followed by intraperitoneal administration of the Pleurotus eryngii mushroom extracts according to the present invention at doses of 0, 2, 10, 20 and 50 ㎍ / kg, respectively, for 24 hours. Results All five groups survived.

From the above results, it was confirmed that the Pleurotus eryngii mushroom extract of the present invention has little acute toxicity.

< Examples  1: Pharmaceutical Formulation example >

1-1: Preparation of Tablet

20 g of Pleurotus eryngii extract of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

1-2: Preparation of Injection Solution

2 g of Pleurotus eryngii extract of the present invention was dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

< Examples  2: Food Manufacturing example >

2-1: Preparation of Cooking Seasonings

Fresh pine mushroom extract of the present invention to 0.2 to 10% by weight to prepare a cooking seasoning for health promotion.

2-2: Preparation of Wheat Flour Food

The Pleurotus eryngii mushroom extract of the present invention is added to flour at 0.1 to 5.0% by weight, and bread, cake, cookies, crackers and noodles are prepared using the mixture to prepare foods for health promotion.

2-3: soup  And juicy ( gravies Manufacturing

The Pleurotus eryngii mushroom extract of the present invention was added to the soup and gravy by 0.1 to 1.0% by weight to prepare meat products for health promotion, soup of noodles and gravy.

2-4: Dairy Products ( dairy products Manufacturing

The Pleurotus eryngii mushroom extract of the present invention is added to milk at 0.1 to 1.0 wt%, and various dairy products such as butter and ice cream are prepared using the milk.

< Examples  3: Drink Manufacturing example >

3-1: Vegetable juice  Produce

0.5 g of the Pleurotus eryngii extract of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

3-2: Fruit juice  Produce

0.1 g of Pleurotus eryngii extract of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

Claims (4)

A composition for the prevention or treatment of atopic dermatitis, comprising ethanol extract of Pleurotus eryngii as an active ingredient. delete Health functional food for the prevention or improvement of atopic dermatitis, comprising ethanol extract of Pleurotus eryngii as an active ingredient. delete

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