KR20150017278A - A composition for the prevention or treatment of inflammatory skin diseases containing Astragalus sinicus L. extract or fraction thereof - Google Patents

Abstract

The present invention relates to a composition for preventing and treating inflammatory skin diseases containing Astragalus sinicus extract or fraction thererof. More specifically, the present invention relates to: a pharmaceutical composition for preventing or treating inflammatory skin diseases containing Astragalus sinicus extract or fraction thererof; and a food composition for preventing or treating inflammatory skin diseases containing Astragalus sinicus extract or fraction thererof. The Astragalus sinicus extract or fraction thererof inhibits activity of pathways of NF-kB, JAK/STAT, and PI3K/Akt promoting inflammation, inhibits expression of inflammation mediators, and thus is effective for treating inflammatory skin diseases.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating an inflammatory skin disease,

The present invention relates to a composition for the prevention or treatment of inflammatory skin diseases comprising a Cryptomeria japonica extract or a fraction thereof, and more particularly to a pharmaceutical composition for preventing or treating an inflammatory skin disease comprising a Cryptomeria japonica extract or fraction as an active ingredient, And a composition for preventing or ameliorating an inflammatory skin disease.

Inflammation induced by tissue damage and stimulation is a mechanism that is designed to protect the organ from danger, but when exposed continuously to stimuli, it is prone to chronic inflammation, such as inflammatory diseases and cancer. Many signaling steps are involved from the beginning to maintaining this state of inflammation.

During inflammatory reactions, many intracellular and intracellular signaling factors, antigen receptors, inflammatory cytokines, nuclear factor-kappa B (JF), janus kinase (JAK) / signal transducer, activator of transcription (STAT), phosphatidylinositol 3-kinases (PI3K) / Akt and activates the intracellular mitogen-activated protein (MAP) kinase signaling pathway. While these signaling pathways play an important role in regulating physiological function when in normal conditions, abnormal activation is associated with a variety of inflammation, immune disorders, cancer, and the like.

Astragalus Sinicus ( A. sinicus ) L.), also known as Chinese milk vetch, is a perennial vine herb that is traditionally cultivated in rice fields in East Asia and is found in southern China, Japan, and Korea. It is mainly used for green tea and livestock feed.

Accordingly, the present inventors have found that the extracts and fractions of Cryptomeria japonica have an excellent therapeutic effect on inflammatory skin diseases while seeking other uses of Cryptomeria japonica, thus completing the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory skin diseases, which comprises astragalus sinicus L. extract or fraction as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving an inflammatory skin disease, which comprises an extract of Cryptomeria japonica or a fraction thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory skin disease comprising Astragalus sinicus L. extract or fraction as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides a food composition for preventing or improving inflammatory skin diseases comprising an extract of Cryptomeria japonica or a fraction thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating an inflammatory skin disease comprising Astragalus sinicus L. extract or fraction as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving inflammatory skin diseases comprising an extract of Cryptomeria japonica or a fraction thereof as an active ingredient.

The Cryptomeria japonica extract or fraction of the present invention may use a cuneus outpost, but more preferably it may be a root or stem region.

Preferably, the extract of the present invention is prepared by (a) precipitating dried root or stem of Cryptomeria vulgaris in a lower alcohol having 1 to 4 carbon atoms to elute the substance 1 to 5 times, and (b) The non-polar organic solvent is added to the eluate of step a) and extracted 1 to 5 times. More preferably, (a) precipitating the dried roots or stems of the cymbals into a lower alcohol having 1 to 4 carbon atoms to elute the substance three times, and (b) adding a nonpolar organic solvent to the eluate of the step (a) It is extracted three times. The lower alcohol having 1 to 4 carbon atoms may be methanol, ethanol, propanol, butanol, and more preferably methanol. The nonpolar organic solvent may be a halogenated hydrocarbon solvent including dichloromethane, hexane, ether, chloroform, ethyl acetate, and may be used alone or as a mixture, and more preferably dichloromethane (CH ₂ Cl ₂ ).

The fragrant fraction of the present invention is obtained by fractionating the fragrant fraction of the present invention with water and n-butanol using a separating funnel, separating the fraction of n-butanol into n-hexane and a lower alcohol having 1 to 4 carbon atoms And a fraction of lower alcohol having 1 to 4 carbon atoms. More preferably, the lower alcohol fraction having 1 to 4 carbon atoms may be a fraction obtained by C ₁₈ reversed-phase vacuum flash chromatography using 20 to 30% aqueous methanol solution. The lower alcohol may be an aqueous solution thereof, and the concentration of the aqueous alcohol solution may be 1 to 50%, more preferably 5 to 30%. The lower alcohol having 1 to 4 carbon atoms may be methanol, ethanol, propanol, butanol, and more preferably methanol.

The method for producing the Cryptomeria japonica extract and fractions of the present invention is described in "Experimental Preparation 1" as one embodiment.

In the present invention, the extracts and fractions of Cryptomeria japonica extract of the present invention have anti-inflammatory activity through human-derived skin cells, mouse CD4 ⁺ T cells and animal experiments. To examine the molecular mechanism of anti-inflammatory activity, we observed the effect on the NF-κB pathway. Recently, NF-κB has caused gene expression of inflammation-promoting mediators such as cytokines, chemokines, enzymes, and adhesive molecules and has become a target for the treatment of inflammatory diseases and cancer. The inflammation-promoting mediator is known to persist for inflammation and has a molecular relationship with chronic inflammatory diseases and cancer ('A. Oeckinghaus et al., Nature Immunology; 12 (8): 695-708,2011'&Amp; M. Karin, Nature, 441 (7092): 431-436, 2006 '& RGBaker et al., Cell Metabolism; 13 (1): 11-22,2011'). The extracts and fractions rf3 and rf4 of the present invention strongly inhibited the phosphorylation and degradation of IκBα protein in human-derived skin cells stimulated with TNF-α and IFN-γ and inhibited the migration of activated NF-κB p65 to the nucleus Respectively. In addition, the transcriptional activity of NF-κB p65 was also significantly inhibited (see Example 2 and Figures 4-7).

The JAK / STAT pathway is essential for signaling by cytokines, interleukins, growth factors, and regulates the immune response. The JAK / STAT pathway plays an important physiological role in maintaining cell differentiation, proliferation, and survival, but when the pathway is abnormally regulated, it is associated with the progression of inflammatory diseases and cancer-like diseases. Thus, modulation of the JAK / STAT pathway with the NF-κB pathway has also emerged as a target for the treatment of inflammatory diseases and cancer (SS Jatiani et al., Genes &Cancer; 1 (10): 979-993, 2010 & YA Ivanenkov et al., Minireviews in Medical Chemistry; 11 (1): 55-78, 2011). The chestnut extract and fractions rf3 and rf4 of the present invention showed an activity of inhibiting phosphorylation of STAT1 and STAT3 in human-derived skin cells stimulated with TNF-α and IFN-γ. In addition, PI3K / Akt hardness inhibited by JAK / STAT was independent (see Example 3). The PI3K / Akt pathway is a pathway associated with inflammation and cancer (K. Ito et al., Journal of Pharmacology and Experimental Therapeutics; 321 (1): 1-8, 2007).

The effect of the extracts and fractions of the present invention on the cytokine-stimulated mouse immune cell, CD4 ⁺ T cell differentiation, was examined. The differentiation of CD4 + T cells, especially Th1, Th2, and Th17 cells, was suppressed, while the differentiation of Treg cells was further progressed. Along with the regulation of T cell differentiation, the expression and production of T cell specific cytokines were also regulated. The expression of IL - 4, IL-17A and IL-22, which are Th2 and Th17 cytokines, was inhibited while the expression of IL-10, a Treg cytokine, was greatly increased. This suggests that the extracts and fractions of Chamaecyparis obscata of the present invention have an effect on the regulation of quality related to the differentiation of Th2, Th17 cells such as allergies, atopy, and systemic autoimmune diseases (see Example 4).

The extracts and fractions of Cryptomeria japonica extract of the present invention are also useful as an anti-inflammatory agent such as cyclooxygenase-2 (COX-2), intercellular adhesion molecule 1 (ICAM-1) IL-10, IL-1β, IL-6, and TNF-α, and significantly increases the expression and production of IL-10, an anti-inflammatory cytokine. Thus, it can be seen that it is effective in treating inflammatory diseases caused by inappropriate NF-kB, JAK / STAT, and PI3K / Akt pathway activity (see Example 5).

IL-23 was injected intradermally into the ear of a mouse to induce psoriasis-like chronic inflammatory disease. When the fraction of the present invention was locally treated, the progression of the thickness of the ear was inhibited, (See Example 6). ≪ Desc / Clms Page number 2 >

When NF-kB, JAK / STAT, and PI3K / Akt pathways are abnormally activated, cell survival and growth are promoted by induction of anti-apoptotic and cell cycle regulatory genes. In the human-derived skin cells stimulated with TNF- [alpha] and IFN- [gamma], the chestnut extract and fractions of the present invention increased protein levels of typical apoptotic proteins, CdK inhibitor, Bax (cell death promoting gene) ).

All of the fractions (CE) and fractions (rf3, rf4) of the present invention were excellent in the treatment of inflammatory diseases. Particularly, fractions showed much better anti-inflammatory activity than crude extracts (CE). It can be seen from the results of the experiment of the present invention that rf4 among the fractions exhibits much better anti-inflammatory activity than rf3.

The pharmaceutical composition according to the present invention may contain the Cow's milk extract or fraction of the present invention, or a pharmaceutically acceptable salt thereof, alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.

The pharmaceutically acceptable salts described above are those which are physiologically acceptable and do not cause an allergic reaction or the like which are common in human administration, and the salts include salts formed by pharmaceutically acceptable free acids Acid addition salts are preferred. The free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutaric acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

 The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration.

Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like.

In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

 The pharmaceutical compositions for the prevention and treatment of inflammatory skin diseases of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration . Preferably, the pharmaceutical composition of the present invention can be transdermally administered. In the above, transdermal administration refers to administration of the pharmaceutical composition of the present invention to cells or skin to allow the active ingredient contained in the composition of the present invention to be delivered into the skin. For example, the pharmaceutical composition of the present invention can be administered in a scanning type formulation, pricking the skin lightly with a 30 gauge needle, or directly applying it to the skin.

 The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route as described above.

 In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, polysaccharides including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant.

Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

 In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

The total effective dose of the chimpanzee extract or fraction of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses have. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the preferred total volume of the cherry juice extract or fraction of the present invention is May be from about 0.01 μg to 1,000 mg, and most preferably from 0.1 μg to 100 mg, per kilogram of patient body weight per day. However, the dose of the polysaccharide fraction of the present invention is effective for the patient in consideration of various factors such as the patient's age, body weight, health condition, sex, severity of disease, diet and excretion rate as well as administration route and treatment frequency of the pharmaceutical composition In view of this, a person skilled in the art will be able to determine the appropriate effective dose according to the specific use of the polysaccharide fraction of the present invention as an immunosuppressive agent. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

Inflammatory skin diseases that can be treated by the pharmaceutical composition of the present invention include, but are not limited to, acute and chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic simplex poisoning, biliary tract deprivation dermatitis, , Sunburn dermatitis and acne.

The food composition according to the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.

For example, as a health food, the Cumin-free extract or the fraction itself of the present invention can be prepared in the form of tea, juice, and drink and then consumed, granulated, encapsulated and powdered. In addition, it can be prepared in the form of a composition by mixing it with a known substance or an active ingredient known to have an effect of improving immunity with the Cryptomeria japonica extract or fraction of the present invention.

Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled, jam, maalmalade, etc.), fish, meat and processed foods such as ham, Etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, , Retort food, frozen food, various kinds of seasonings (for example, soybean paste, soy sauce, sauces, etc.), or the like.

In the food composition of the present invention, the preferred content of the Cryptomeria japonica extract or fraction of the present invention is not particularly limited, but is preferably 0.01 to 100% by weight in the finally prepared food.

In order to use the Cryptomeria japonica extract or fraction of the present invention in the form of a food additive, it may be used in the form of a powder or a concentrated liquid.

The chestnut extract or fraction of the present invention inhibits the activity of the NF-kB, JAK / STAT, and PI3K / Akt pathways promoting inflammation and inhibits the expression of inflammatory mediators, thereby being effective for treating inflammatory skin diseases.

FIG. 1 is a diagram illustrating a method for producing the Cryptomeria japonica extract and fractions of the present invention.
Figure 2 is a graph showing the effect of H ₂ DCFDA staining on human-derived skin cells This is the result of ROS removal.
FIG. 3 shows the results of qRT-PCR measurement of the degree of expression of inflammatory mediators in human-derived skin cells.
Figure 4 shows the effect of The levels of phosphorylation of IκBα and NF-κB p56 proteins were determined by SDS-PAGE.
Figure 5 shows the effect of The degree of degradation of IκBα was observed by SDS-PAGE.
FIG. 6 shows the results of NF-κB-dependent promoter activity.
FIG. 7 shows the effect of NF-κB p65 in the cytoplasm.
Figure 8 shows that in human derived skin cells STAT1, STAT3, and Akt.
FIG. 9 shows the results of observing phosphorylation of ERK1 / 2, p38 and JNK in human-derived skin cells.
Fig. 10 shows the results of observing the phosphorylation of IFN-y-JAK-dependent Scr kinase (Src, Lck, Lyn) in human-derived skin cells.
FIG. 11 shows the results of measuring mRNA expression levels of GATA-3, T-bet, RORγt and Foxp3 by differentiating mouse immunized CD4 ⁺ T-cells.
FIG. 12 shows mRNA expression levels of TNF-a, IL-4, IL-10, IL-17A, IL-22 and IL10 in mouse immunized CD4 ⁺ Th1, Th2, Th17, and Treg cells.
FIG. 13 shows the results of measurement of the amounts of IFN-γ, IL-4, IL-17A and IL-10 produced in differentiated Th1, Th2, Th17 and Treg cells.
Fig. 14A is a graph showing that in human-derived skin cells And the production of COX-2, ICAM-1, which is an inflammatory factor.
Fig. 14B shows the effect of And the level of mRNA expression of the inflammatory factors COX-2, ICAM-1.
Fig. 15 In human-derived skin cells MRNA expression levels of IL-1α, IL-1β, IL-6, TNF-α, IL-10 and PGE ₂ were measured.
FIG. 16A is a histopathological observation of the inflammatory skin damage in the mouse ear.
FIG. 16B shows the result of measuring the thickness of the ear and observing the degree of inflammatory skin damage.
FIG. 17A shows the results of cell survival of human-derived skin cells in the case of treatment of the Cryptomeria japonica extract and fraction for 24 hours.
FIG. 17B shows the results of cell survival of human-derived skin cells in the case of treatment of Cryptomeria japonica extract and fraction for 48 hours.
18 shows the results of measurement of protein expression levels of PARP, caspase-3, 9, p21 ^{Cip / WAF1} , p27 ^Kip1 and Bax in human-derived skin cells.
FIG. 19 shows the results of observing the cytotoxicity of human-derived skin cells by FITC Annexin V staining method.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Preparation of experiment>

1. Extraction of active substance

, 9 species of beans such as Cassia obtusifolia L., Albizia julibrissin Durazz., Astragalus membranaceus Bunge, Glycyrrhiza uralensis Fisch., Astragalus sinicus L., Gleditsia japonica Miq., Pueraria lobata Ohwi, Caragana sinica Rehder, Styphnolobium japonicum Plants were purchased from Kyungdong Market, and a checklist was deposited with the National Products Research Institute of Seoul National University. Dry roots or stem extraction methods are shown in Fig. Briefly, crude extracts (CE) were prepared by repeatedly extracting dried roots or stems with methanol and dichloromethane (CH ₂ Cl ₂ ). The crude extract (CE) was fractionated with water (H ₂ O) and n-butanol, and then the n-butanol fraction was fractionated with 15% aqueous methanol solution and n-hexane. Based on the bioactivity test, the aqueous methanol fraction fraction of Astragalus sinicus L. , which exhibited the best activity, was separated into eight fractions.

The crude extract (CE) and fractions are prepared as follows.

First, 276.5 g of the sample is precipitated in 3 L of methanol in an Erlenmeyer flask, the mixture is allowed to stand at room temperature for one day to elute the material (repeated three times), and the methanol is evaporated to concentrate the sample. Next, dichloromethane is added to the sample eluted with methanol, the material is eluted in the same manner as methanol, and the dichloromethane is evaporated to concentrate the sample. Extracted samples of methanol and dichloromethane and concentrated were completely dissolved in water and butanol (2L each in water and butanol, the order of dissolution was water-butanol or butanol-water, regardless of water). When the water layer (polarity) and the butanol layer (nonpolar) are separated, the water layer is discarded and the butanol layer is separated and the butanol is evaporated to concentrate the extracted sample. The concentrated butanol layer is completely dissolved by 15% aqua methanol and nucleic acid (1.5 L each) and is shaken and left to separate into a two-layer nucleic acid layer (nonpolar) and an aqua methanol layer (polarity). The aqua methanol layer showing the physiological activity is collected and concentrated and used as a sample. At this time, after the layer separation using the butanol layer, 9.8 g of a 15% aqueous methanol layer was obtained, and 11.2 g of a nucleic acid layer was obtained. Thereafter, _C18 reversed-phase vacuum flash chromatography was performed using only 5.8 g of 9.8 g of a 15% aqueous methanol layer. Separation conditions were begun at 50% aqueous MeOH and eluted sequentially to 100% MeOH with a 10% gradient system and the residue was washed with acetone and EtOAc. The amount of each fraction thus obtained is as follows. (30% aq.MeOH, 480 mg), rf4 (20% aq.MeOH, 350 mg), rf5 (50% aq.MeOH; 1.3 g), rf2 (40% (100% acetone, 1.1 g), rf8 (EtOAc, 130 mg), rf6 (100% MeOH, 630 mg)

2. Cell and reagent preparation

HaCaT (immortalized human keratinocyte cell line) cells were purchased from CLS Cell Line Service GmbH (Eppelheim, Germany) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L- Lt; / RTI &gt; Recombinant mouse cytokines for IL-2, IL-4, IL-6 and IL-12 and mouse antibodies against anti-IL-4, anti-IFN- , USA). Recombinant human IL-23 was obtained from eBioscience (San Diego, CA, USA) and phospho-IkBa (IFN-γ and TGF-β was purchased from PROSPEC (East Brunswick, NJ, USA) STAT3 (Tyr705), phospho-Akt (Ser473), Akt, phospho-ERK1 / 2 (Thr202 / Tyr204), ERK1 / 2, phospho-p38 (Thr180 / Tyr182), p38, phospho-JNK (Thr183 / Tyr185), JNK, phospho-Src (Tyr416), Src, phospho-Lck (Tyr505) Antibodies specific for Lyn, COX-2, ICAM-1, PARP, cleaved caspase-3 (Asp175), cleaved caspase-9 (Asp330), p21, p27, Bax and GAPDH were detected by Cell Signaling Technology . Antibodies specific for IkBa, NF-kB p65 and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Horseradish peroxidase (HRP) -conjugated goat anti- rabbit and anti- USA), other chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA).

3. SDS - PAGE  And Western  Blat ( Western Blot )

Cell pellets were resuspended in lysis buffer [50 mM Tris-HCl (pH 7.4), 350 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 10% glycerol, 0.1% SDS, 1 mM EDTA, (Pall Corporation, Pensacola, FL, USA) after SDS-PAGE for all cell extracts at 4 ° C for 30 min at room temperature, 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride and phosphatase inhibitor cocktails . The membrane was treated with a blocking buffer and incubated with specific primary antibodies for the target molecule at 4 ° C for 12 hours. The signal elements were detected with an ECL detection kit (iNtRON Biotechnology, Korea), and then horseradish peroxidase-conjugated and cultured with secondary antibodies.

4. RNA  Separation and qRT ( Quantitative real - time ) - PCR  analysis

After RNA was extracted from cells or tissues using RNeasy Mini Kit (Qiagen, Valencia, CA, USA), cDNA was synthesized with QuantiTect Reverse Transcription Kit (Qiagen). qRT-PCR was performed using KAPA SYBR fast qPCR Kit (KAPA biosystems, Woburn, MA, USA) and the results were based on GAPDH gene expression. The PCR conditions were 95 ° C for 5 min, 35 cycles - 96 ° C for 20 sec, 60 ° C for 20 sec, 72 ° C for 20 sec, one cycle - 72 ° C for 5 min, and the primers were purchased from Qiagen.

5. Mouse CD4 ⁺  Isolation and differentiation of T cells

Undifferentiated CD4 ⁺ T cells were purified and differentiated in the spleen and lymph nodes of C57BL / 6 mice. Briefly, cells were concentrated in a mouse T-cell enrichment column (R & D Systems, Minneapolis, MN, USA) and separated into negative selections using a MACS column (Miltenyi Biotech Inc., Auburn, CA, USA). Cells were activated with a plate-bound anti-CD3 antibody in a 96-well plate and a soluble anti-CD28 antibody (2 mg / ml) in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine and antibiotics. Cells were treated with Th1 polarizing condition (10 ng / ml IL-12 and 10 mg / ml anti-IL-4 antibody) IL-6, 5 ng / ml TGF-b, 10 mg / ml anti-IFN-g antibody and 10 mg / ml anti-IL-4 antibody) or Treg polarizing condition (5 ng / ml TGF-b and 10 ng / ml IL-2).

6. Cytokine  And measurement of prostaglandin production

The amount of cytokines and prostaglandin E2 produced was measured by ELISA (R & D Systems, Minneapolis, MN, USA). Mouse CD4 ⁺ T cells or HaCaT cells differentiated for 4 days were stimulated by TNF-α and IFN-γ for 24 hours.

7. Trait injection and NF - kB - Dependent Reporter essay

The pRL-TK control vector containing pNF-kB-luciferase was transfected into HaCaT cells using Lipofectamine 2000 ™ (Life Technologies, Grand Island, NY, USA). After transfection for 24 hours, cells were stimulated with TNF- [alpha] and IFN- [gamma] for 24 hours, and a non-stimulated control was also prepared. The activity of NF-kB-luciferase was measured according to the manual of Dual-Luciferase Assay Kit (Promega, Madison, WI, USA) and the activity of firefly luciferase was standardized based on the activity of Renilla luciferase.

8. Intracellular ROS  Production measurement

HaCaT cells were distributed in 6-well plates at 3 × 10 ⁵ cells. Twelve hours later, stimulation with ultraviolet B (UVB, 100mJ / mL) or TNF-α and IFN-γ in serum-free DMEM medium for 4 hours was followed by treatment of the chimpanzee fraction for 1 hour. Cells were stained with Hanks 'Balanced Salt Solution (HBSS) for 30 min and stained with 2', 7'-dichlorofluorescin diacetate (H ₂ DCFDA, 10 μM) at 37 ° C for 30 min. Afterwards, intracellular ROS removal activity was analyzed with a confocal microscope (Carl Zeiss, Germany).

9. Cell activation and cell death apoptosis ) Essay

Cell activity was measured according to the manual of EZ-CyTox Enhanced Cell Viability Assay Kit (Daeil Lab Service, Korea). Briefly, the cells were distributed in a 96-well plate at a density of 10,000 cells, and then treated with DMSO (control group) and chrysanthemum fraction, respectively, for 24 hours and 48 hours, followed by incubation at 37 ° C. for 3 hours. (BioTek, Winooski, VT, USA) at a wavelength of 450 nm.

The cell death assay was performed using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA). Briefly, HaCaT cells (5 × 10 ⁵ cells / mL) were stimulated with TNF-α and IFN-γ for 24 hours and 48 hours, after which the cells were washed with PBS solution. Subsequently, the cells were incubated with binding buffer and stained with fluorescein isothiocyanate (FITC) Annexin V-propidium iodide (PI) for 15 min at room temperature and dark room. The stained cells were counted using a FACS Calibur flow cytometer (BD Biosciences, San Jose, Calif., USA).

10. Immunofluorescence ( Immunofluorescence )

Serum-starved HaCaT cells (5 × 10 ⁵ cells / well) were pre-treated for 1 hour with a chimpanzee fraction and stimulated with TNF-α and IFN-γ for 1 hour. Immunostained NF-kB p65 signal was detected using the 'Y. Kim. et al., Free Radical Biology and Medicine; 51 (11): 1985-1995,2011 '. Briefly, cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with PBS (containing 1% bovine serum albumin). For immunostaining, cells were incubated with anti-NF-kB p65 antibody for 2 hours, washed three times with blocking buffer, and incubated with Alexa Fluor 568 anti-rabbit IgG antibody (Molecular Probe, Eugene, OR, USA) And incubated together for 1 hour. Immunoreactive NF-kB p65 was stained with confocal fluorescence microscopy (Carl Zeiss, Thornwood, NY, USA) and stained with Hoechst (Molecular Probe, Eugene, OR, USA).

11. IL -23 induction psoriasis (Skin inflammation model similar)

All procedures for animal testing were approved in accordance with the guidelines provided by the Institutional Animal Care and Use Committee (IACUC) of the Catholic Medical School. C57BL / 6 mice were matched for 1 week in sterile environment and stabilized at 23 ± 2 ℃ for 12 hours at night. Control (PBS), recombinant IL-23 (500 ng / 10 uL) was injected into the skin of the ear of mice, and then 'BHKim. et al., Biochemical Pharmacology; 85 (8): 1134-1144, 2013 'for 14 days every two days. Ear thickness was measured 24 hours after the last treatment and ears were stored at -80 ° C for another experiment.

12. Histological examination

Mouse mice were fixed in PBS with 4% paraformaldehyde for 24 hours, washed with tap water, dehydrated with ethanol, and put into paraffin. The paraffin block was cut to a thickness of 4 쨉 m and then put on a glass slide. Then, the wax was removed, treated with ethanol, and hematoxylin-eosin (H & E) staining was performed. Histological analysis was performed with a fluorescence attached microscope (Olympus, Japan).

13. Statistical analysis

Data were expressed as means ± SD (SEM), and significance between the two groups was verified by two-tailed Student's t-test (p <0.05, p <0.001).

< Example  1>

The Astragalus Sinicus  L.) of the extract ROS  Removal and inhibition of inflammatory mediators

Unless otherwise stated in the present invention, the crude extract (CE) of rosewood was treated with 20 μg / mL of the crude extract and 10 μg / mL of rf3 and rf4 isolated from the crude extract. The chestnut extracts were treated 1 h before treatment with TNF-alpha (10 g / ml) and IFN-y (100 U / ml).

To verify the viability of the extracts of Cryptomeria japonica, which inhibit the inflammatory response, nine species of soybean plants were divided into polar and nonpolar layers. Of these plants, the aqueous extract of aqueous methanol in water of cuneus showed the strongest anti-inflammatory activity (tested with cytokines or ultraviolet-induced human-derived skin cells) (results not shown). Thus, eight fractions were isolated from the methanol aqueous solution extract of Cryptomeria japonica.

To test the viability of the 8 fractions isolated, we first examined the antioxidant activity of cell permeable ROS and 2 ', 7'-dichlorofluorescin diacetate (H ₂ DCFDA) reagents. When HaCaT cells, which were dormant human-derived skin cells, and the above-mentioned fractions were co-cultured, there was almost no ROS production (results not shown). ROS production was significantly increased in the experimental group treated with UV or TNF-α (10 g / mL) and IFN-γ (100 U / mL), whereas CE, rf3, rf4 Treated group showed strong ROS removal activity (see FIG. 2).

The level of anti-inflammatory activity was observed by measuring the levels of inflammatory mediators in human-derived skin cells stimulated with UV-ray by qRT-PCR. Similar to antioxidant activity, rf3 and rf4 effectively inhibited mRNA levels of inflammation-promoting factors such as IL-1α, IL-6, IL-8 and TNF-α (see FIG. As shown in FIG. 3, the anti-inflammatory activity of rf3 and rf4 fractions among the eight fractions was the most excellent.

< Example  2>

Chestnut extract NF -KB signal inhibitory effect

It is known that TNA-a binds to the TNF receptor and activates the NF-kB signal, resulting in various chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, atherosclerosis. Thus, we examined the effect of rf3 and rf4 on NF-κB signals in human-derived skin cells stimulated with TNF-α and IFN-γ. In experiments to determine the phosphorylation levels of the inhibitory proteins IκBα and NF-κB p65 proteins, the phosphorylation of the two proteins was much more inhibited by the CE, rf3, and rf4-treated groups than the untreated control, (See FIG. 4).

The degradation of the IκBα protein was consistent with the phosphorylation levels of the IκBα and NF-κB p65 proteins, indicating that CE, rf3, and rf4 blocked the degradation of IκBα (see FIG. 5).

In addition, in order to examine the NF-κB-dependent promoter activity, when human-derived skin cells transfected with NF-kB-luciferase were stimulated with TNF-α and IFN-γ, CE, rf3 and rf4 were significantly , And rf3 and rf4 showed more inhibitory activity than CE (see Fig. 6).

 To determine the location of NF-κB p65, one of the NF-kB subunit proteins, immunofluorescence staining analysis was performed. NF-κB p65 was present in the cytoplasm of dormant human-derived skin cells, but migrated to the nucleus after cytokine stimulation. The migration of NF-κB p65 to the nucleus was inhibited by CE, rf3, and rf4, and particularly the rf3 and rf4 fractions were superior (see FIG. 7).

Therefore, it can be seen from the above results that CE, rf3, and rf4 interfere with NF-kB signal transduction and have antioxidative and anti-inflammatory activities, and its activities are more stronger than rf3 and rf4 in CE.

< Example  3>

Chestnut extract JAK / STAT  And PI3K / Akt  Pathway inhibiting effect

Since CE, rf3, and rf4 stimulate JAK / STAT pathway in human-derived skin cells stimulated by TNF-α and IFN-γ, IFN-γ activates the JAK / STAT pathway, particularly JAK1 / STAT1 and JAK2 / I have tried to see if it can affect. STAT1 and STAT3 are inactivated in dormant human-derived skin cells, but stimulation with TNF-α and IFN-γ results in activation of STAT1 701 and STAT3 705 tyrosine residues. The tyrosine phosphorylation of STAT1 and STAT3 by cytokines was inhibited by CE, rf3, and rf4, and the inhibition of rf3 and rf4 was particularly strong (see Fig. 8).

In addition to the JAK / STAT pathway, IFN-γ also activates non-JAK / STAT pathways including PI3K / Akt, MAP kinases, protein kinase C, and calcium / calmodulin kinase II. These pathways are activated by IFN-y-JAK-dependent Scr-kinase. It can be seen from Fig. 8 that CE, rf3 and rf4 phosphorylate 473 serine of Akt to inhibit the PI3K / Akt pathway. However, the extracellular extract fractions did not affect MAP kinase pathways such as ERK1 / 2, p38, and JNK (see FIG. 9). In addition, the IFN-y-JAK-dependent Scr-kinase (c-Src, Lck, and Lyn) pathways were not significantly affected (see FIG. 10).

The above results are summarized as follows: CE, rf3, rf4 are human-derived skin stimulated with TNF-α and IFN-γ Inhibits the JAK / STAT pathway and the JAK / STAT-independent PI3K / Akt pathway in the cell, and the rf3 and rf4 fractions show better inhibitory effects than CE.

< Example  4>

Chestnut extract CD4 + T cell differentiation Low performance

In order to examine whether the Cryptomeria japonica Extract can regulate the immune response associated with CD4 ⁺ T cell differentiation, expression and production of T cell major transcription factors and their representative cytokines were measured in mouse CD4 ⁺ T cells. MRNA expression levels of Th2- and Th17 cell major transcription factors, GATA binding protein 3 (GATA-3) and RORγt (retinoic acid-receptor-related orphan receptor gamma) were very significant due to CE, rf3 and rf4 While T-box transcription factor (T-bet), the major transcription factor for Th-1 cells, was not affected. However, mRNA levels of Foxp3 (forkhead box P3), a major transcription factor for Treg cells, were significantly elevated (see FIG. 11).

Next, the effect of Cryptomeria japonica extract on the expression of T cell specific cytokines in the CD4 ⁺ T cell portion was examined. MRNA expression of IL-4 (Th2 cytokine) and IL-17 and IL-22 (Th17 cytokine) was effectively inhibited by CE, rf3 and rf4 and mRNA expression of TNF- Respectively. On the other hand, IL-10 (Th-2, a representative cytokine of Treg cells), an anti-inflammatory cytokine, showed an increase in mRNA expression by the chestnut extract.

Th2 and Th17 cell cytokines IL-4 and IL-17 were also significantly decreased by the extracts of CE, rf3 and f4, which is consistent with the results of the cytokine mRNA expression level. The production of IL-10 in Treg cells was greatly increased by CE, rf3, and rf4 (see FIG. 13).

From the above results, it can be seen that the Cryptomeria japonica extract of the present invention is effective for the treatment of inflammatory diseases such as allergies, asthma and autoimmune diseases induced by excessive activation of Th2 and Th17 cells.

< Example  5>

Expression and inhibition of inflammation-promoting mediator

Regulation of NF-kB and JAK / STAT pathway activity by inappropriate signals is a major tool for the treatment of inflammatory disorders, cancer, and many small molecule inhibitors have been developed to control these pathways (LA O'Neill, Nature Reviews Drug Discovery; 5 (7): 549-563, 2006 & YAIvanenkov et al., Minireviews in Medical Chemistry; 11 (1): 55-78, 2011). Since the expression of inflammatory mediators is induced by the activation of NF-kB, JAK / STAT, and PI3K / Akt pathways, the effects of chestnut extract, CE, rf3, and rf4 on the expression of inflammatory mediators were examined.

Protein production and mRNA levels of cyclooxygenase-2 (COX-2) and intercellular adhesion molecule 1 (ICAM-1), which are typical inflammatory factors regulated by NF-kB and JAK / STAT3 pathways, , and rf4 (Fig. 14).

In addition to the expression of COX-2 and ICAM-1, the chestnut extract CE, rf3 and rf4 inhibited mRNA expression and production of IL-1α, IL-1β, IL-6 and TNF-α, Significantly increased the expression and production of IL-10, an inflammatory cytokine (see FIG. 15).

Thus, it can be seen that the extracts and fractions of the present invention are effective for treating inflammatory disorders caused by inappropriate NF-kB, JAK / STAT, and PI3K / Akt pathway activity.

< Example  6>

Inhibitory effect on skin inflammation damage

Activation of the NF-kB, JAK / STAT, and PI3K / Akt pathways in human-derived skin cells is associated with inflammatory skin disease because skin cells (keratinocytes) are the major constituent cells of the skin epidermis. Since CE, rf3, and rf4 isolated from vertebrate larvae inhibit the above pathways, IL-23 was injected intradermally into mouse ears to examine whether or not inflammatory skin damage caused thereby can be controlled by the present invention. IL-23 intradermal injection into mouse skin induced psoriasis-like disease and induced epidermal hyperplasia. IL-17A and CCR6 (chemokine receptor 6) (BHKim et al., Biochemical Pharmacology; 85 (8): 1134-1144, 2013 '&amp; YSLee et al., Journal of investigative Dermatology; 133 (3): 732-741, HLRizo et al., Journal of Immunology 186 (3): 1495-1502, &lt; RTI ID = 0.0 &gt; 2011).

After H & E staining of the ear for histopathological examination, we observed progression of inflammatory skin lesions such as markedly increased ear thickness, epidermal hyperplasia (acanthosis), hyperkeratosis, and penetration of inflammatory cells into the skin Respectively. This pathological progression was significantly reduced by local treatment of rumen extract rf3, rf4 (see FIG. 16). Ear thickness measurement results and inhibition rates are shown in Table 1 below.

Group Ear thickness
(× 10 μm, mean ± SEM) Inhibition%
(mean ± SEM) PBS 41.3 ± 1.8 - IL-23 70.5 ± 3.5 - rf3 59.3 ± 2.3 38.5 ± 5.1 rf4 50.0 ± 2.0 70.1 ± 4.6

< Example  7>

Induction effect

When NF-kB, JAK / STAT, and PI3K / Akt pathways are abnormally activated, cell survival and growth are promoted by induction of anti-apoptotic and cell cycle regulatory genes. In order to test the effect of chestnut juice extract on cell proliferation, WST-1 reagent was used to confirm the effect on the proliferation and survival rate of human-derived skin cells. Treatment with rf3 and rf4, which were separated from the vagina, at various concentrations and for 24 or 48 hours, decreased cell proliferation and survival rate in a concentration / time dependent manner (see FIG. 17).

Western blot analysis of cell lysates was performed first to investigate the molecular mechanism of cell proliferation and reduction of survival rate, and the effects of Cryptomeria japonica extract on the expression of anti-apoptotic and cell cycle regulatory proteins were examined. In the cells treated with TNF-α and IFN-γ for 48 hours, CE, rf3, and rf4 were treated with poly ADP ribose polymerase (PARP), caspase-3 and 9, and p21 ^{Cip / WAF1} , Cyclin-dependent kinase (CdK) inhibitor such as p27 ^Kip1, and the protein level of the cell death-promoting gene Bax (see FIG. 18).

The same results as in FIG. 18 were obtained from the cell death assay using the FITC Annexin V staining method. In the human-derived skin cells treated with TNF- [alpha] and IFN- [gamma] for 24 hours or 48 hours, the rumen extracts rf3 and rf4 increased cell death kinetics and rf4 was stronger than CE, rf3 ).

The chestnut extract or fraction of the present invention inhibits the activity of the NF-kB, JAK / STAT, and PI3K / Akt pathways promoting inflammation and inhibits the expression of inflammatory mediators, thereby preparing a therapeutic agent for inflammatory skin diseases or an improved food And it is highly likely to be used industrially.

Claims (7)

Astragalus Sinicus L. ) extract or fraction thereof as an active ingredient for the prevention or treatment of inflammatory skin diseases.
The pharmaceutical composition according to claim 1, wherein the extract is extracted with a lower alcohol having 1 to 4 carbon atoms or dichloromethane.
The method of claim 1, wherein the extract
(a) precipitating the cymbals into a lower alcohol having 1 to 4 carbon atoms to elute the substance 1 to 5 times; And
(b) adding the nonpolar organic solvent to the eluate of step (a) and extracting the solution 1 to 5 times.

The composition according to claim 1, wherein the fraction is obtained by sequentially fractionating the Cryptomeria japonica extract with water and n-butanol, and then fractionating the n-butanol fraction with a lower alcohol having 1 to 4 carbon atoms or an aqueous solution thereof.
The pharmaceutical composition according to claim 3 or 4, wherein the lower alcohol is methanol.
The method according to claim 1, wherein the inflammatory skin disease is selected from the group consisting of acute and chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic simplex poisoning, biliary tract deprivation dermatitis, bulimia nervosa, psoriasis, &Lt; / RTI &gt;
Astragalus Sinicus L. ) extract or fraction as an active ingredient.

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