KR20110075070A - Composition for preventing or treating colitis containing (2s)-2'-methoxykurarinone from sophora flavescens extracts - Google Patents

Abstract

PURPOSE: A composition containing Sophora flavescens Aiton extract or (2S)-2 prime-methoxykurarinone derived from the extract are provided to effectively express HO-1 and to suppress Th2 chemokine. CONSTITUTION: A composition for preventing and treating atopic dermatitis contains Sophora flavescens Aiton extract prepared by mixing dried Sophora flavescens Aiton with a solvent, maturing, and filtering, and concentrating. A composition for preventing and treating atopic dermatitis also contains (2S)-2 prime-methoxykurarinone derived from the extract as an active ingredient.

Description

Composition for preventing or treating colitis containing (2S) -2'-methoxykurarinone from Sophora flavescens extracts (2S) -2prime-methocycurinone

The present invention relates to a composition for preventing and treating atopic dermatitis, and more particularly, to a composition for preventing and treating atopic dermatitis using a ginseng extract containing (2S) -2 prime-methocycurinone.

Atopic dermatitis is a type of skin eczema caused by poly-factors such as immunological, genetic, pharmacological and physiological, and environmental (Leung DY & Bieber T, Lancet . 2003: 361 (9352): 151-160). Atopic dermatitis is a chronic relapsing inflammatory skin disease with repeated improvement and deterioration caused by infections, stress, seasonal and climate changes, irritation and allergens, and is widespread in infants and adults. Two types: exrinsic form (70-80%) by IgE-mediated sensitization and intrinsic form (20-30%) by non-IgE-mediated sensitization In the form of leung DY et al . , J Clin Invest. 2004: 113 (5): 651-657; Mohrenschlager M et al., J Eur Acad Dermatol Venereol . 2006: 20 (5): 503-513; Novak N & Bieber T. J Allergy Clin Immunol . 2003: 112 (2): 252-262).

Atopic dermatitis is a skin disease with a complex characteristic that activates immune and inflammatory responses through various pathways. It is a complex interaction between sensitive genes, a patient's environment, defects in skin barrier function, and a systemic or local immune response. Cause. In general, clinical features of atopic dermatitis include disruption of the skin barrier due to severe itch, inflammation, increased serum IgE, infiltration of eosinophils in the inflammatory site, and biased development of Th2 cells.

Chemokines represent chemoattractants known as low molecular weight proteins and are known as the superfamily. In other words, according to the structure and function of the protein can be divided into four subfamily (C, CC, CXC and CX3C subfamily), these chemokines infiltrate the white blood cells in the inflammatory site to induce an inflammatory response (Power CA) & Proudfoot AE.Curr Opin Pharmacol . 2001: 1 (4): 417-424.).

Thymus & activation-regulated chemokine (TARC / CCL17), macrophage-derived chemokine (MDC / CCL22), and cutaneous T cell-attracting chemokine (CTACK / CCL27) are representative Th2 chemokines that induce the migration and invasion of Th2 cells to the inflammatory site. It is known. TARC / CCL17 and MDC / CCL22 act via CC chemokine receptor 4 (CCR4), while CTACK / CCL27 exert its effect via CCR10 (Reiss Y et al. al ., J Exp Med . 2001: 194 (10): 1541-1547. In particular, the serum of TARC / CCL17, MDC / CCL22 and CTACK / CCL27 are detected in the serum of patients with atopic dermatitis, which is known to be closely related to atopic dermatitis (Nakazato J et. al ., Pediatr Allergy Immunol . 2008: 19 (7): 605-613). These Th2 chemokines are mainly produced by exogenous antigens or cytokines generated in vivo in keratinocytes and infiltrate into the wound of immune cells causing inflammation.

Oxidative stress, on the other hand, is known to cause tissue damage by causing various inflammatory diseases including atopic dermatitis. Antioxidants are useful agents to alleviate or treat inflammatory diseases. Heme oxygenase-1 (HO-1) is known as a major antioxidant enzyme in vivo. HO-1 is an inducible enzyme that shows anti-inflammatory, anti-cell proliferation and anti-apoptosis. It is an important antioxidant that modulates the body's molecular response and has a pharmacological action to treat various inflammatory diseases (Kirino M et al ., J Allergy Clin Immunol . 2008: 122: 290-297; Clark JE et al ., Biochem J. 2000: 348: 615-619).

NC / Nga is a DBA / 2 family mouse with immunophysiological characteristics such as high sensitivity to X-ray irradiation and high sensitivity to anaphylactic shock caused by egg white albumin injection, and chemical antigen and house dust. Biological antigens such as house dust mite can induce skin diseases similar to human atopic dermatitis, and are widely used for the evaluation of etiology and therapeutic drugs of atopic dermatitis (Matsuda H et al., Int Immunol . 1997: 9: 461-466; Suto H et al., Int Arch Allergy Immunol . 1999: 120: 70-75)

Meanwhile, Sophora flavescens Aiton is a perennial herb that belongs to the legume and is widely distributed in Korea, Japan and China. Ginseng is a medicinal plant that has been used for diarrhea, gastrointestinal hemorrhage, and eczema. Recently, major pharmacological agents such as quinolizidine alkaloids and flavonoids have been isolated and studied. However, there have been no studies of atopic dermatitis using Ginseng extract containing these ingredients (Ding PL et al., J Asian Nat Prod Res . 2005: 7 (3): 237-243; Jung HJ et al., Arch Pharm Res . 2005: 28 (12): 1333-1336)

Therefore, the present invention isolates and identifies (2S) -2 prime-methosycurinone derived from ginseng, and its pharmacological effect is to induce HO-1 effectively against Haketa cells (HaCaT), a human-derived keratinocyte cell line. CTACK / CCL27 chemokine, the key cause of the action yesterday, and dust mites ( Dermatophagoides) farinae , Df ) induces atopic dermatitis similar to humans in NC / Nga mice using oral administration of (2S) -2prime-methocycurinone-containing ginseng extract, and dermalitis scores. ), Skin histology, serum IgE and Th2 chemokines (TARC / CCL17, MDC / CCL22, CTACK / CCL27) were very effective.

It is an object of the present invention to develop and provide a novel atopic dermatitis prevention and treatment technology by discovering natural products-derived Th2 chemokine inhibitors using high ginseng extract containing (2S) -2 prime-methocycurinone.

In order to achieve the above object, the present invention relates to a composition for the prevention and treatment of atopic dermatitis using a ginseng extract containing metoshicurinone, the dried ginseng is mixed with an extraction solvent, aged, and then the active substance (2 S) -2-prime-mail Toshio Tokura leverage rinon says that the material that can effectively induce HO-1 in human-derived HaCaT cells, an extract of Sophora based on this targeting NC / Nga mice Df Oral administration to induced NC / Nga mice is characterized by the inhibition of Th2 chemokine, a major cause of atopic dermatitis.

According to the present invention, (2S) -2prime-methocycurinone derived from Ginseng ginseng effectively prevents new atopic dermatitis based on a mechanism of inhibiting Th2 chemokines such as CTACK / CCL27 produced in HaCaT cells by effectively expressing HO-1. It offers the possibility of developing therapeutics.

In particular, the present invention dust ginseng extract house dust ( Dermatophagoides farinae , Df ) antigen-induced atopic dermatitis similar to humans in NC / Nga mice, followed by oral administration of Ginseng extracts for skin dermatitis scores, skin histology, serum IgE and Th2 chemokine (TARC / CCL17, MDC / CCL22, CTACK / CCL27) was found to have a very good effect, according to (2S) -2 prime-methosicuranone-containing ginseng extract was prepared for atopic dermatitis release and treatment It can be used effectively as.

Hereinafter, a composition for preventing and treating atopic dermatitis using (2S) -2prime-methocycurinone-containing Gosam extract prepared according to the present invention will be described in detail.

According to one embodiment of the present invention, the atopic dermatitis treatment agent according to the present invention is mixed with 20 to 40 parts by weight of dried ginseng with respect to 100 parts by weight of the extraction solvent, and then sealed and left for 5 to 14 days to mature, the aged The mixture was obtained by fractions of organic solvents, and was isolated and identified as an active substance (2S) -2prime-methocycurinone, followed by a verification step of effectively inducing HO-1 to suppress Th2 chemokines, and filtering using a filter. It refers to all the steps effective to improve and treat atopic dermatitis in the atopic dermatitis-like NC / Nga mice prepared composition comprising the step, concentrating the filtered mixture with a concentrator, drying the concentrate.

Ginseng used in the present invention means that obtained by extracting from various organs or parts (for example, leaves, flowers, roots, stems, shells, fruits, etc.), it is preferable to use the root of the ginseng. Ginseng roots are washed and dried, broken into small pieces, and then put into extractant.

As the extraction solvent used in the present invention, water, methanol, ethanol, ethyl acetate and the like may be used, but it is preferable to use ethanol and methanol to contain methosycurinone.

In addition, it is preferable that 20-40 weight part of ginseng roots is added with respect to 100 weight part of ethanol, and, as for the mixing ratio of a ginseng root and ethanol, More preferably, 30 weight part is mixed.

After putting ginseng root in ethanol, seal the container and leave it for about 5 ~ 14 days to mature.

The aged mixture is filtered using a 0.45 μm filter and then concentrated in a concentrator. The concentrated concentrate is lyophilized at low temperature to obtain a ginseng extract.

Although the formulation of the composition according to the present invention is not particularly limited, cosmetic ingredients such as moisturizers and ointments may be used as external preparations, oral or injectable medicines may be used for oral administration, and may also be used as food additives.

The compositions of the present invention may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of dry powders which are dissolved in sterile, pyrogen-free water prior to use to form cosmetics, oral formulations, subcutaneous injections, and veins. It may be formulated as a parenteral formulation such as injection or intramuscular injection.

For external formulations such as cosmetics and ointments, such as moisturizers and lotions, the compositions of the present invention may be used in a known manner using pharmaceutically acceptable carriers and excipients, for example, tablets, troches, sugar-containing tablets, aqueous or oily suspensions. , Dispersible powders or particles, in the form of emulsions, which can be prepared in unit dosage form and in multi-dose containers to produce the finished product.

In the case of oral formulations, the compositions of the present invention may be used in known manner using pharmaceutically acceptable carriers and excipients, for example tablets, troches, sugar-containing tablets, aqueous or oily suspensions, dispersible flours or particles. , In the form of emulsions, soft or hard capsules, syrups or elixirs, which can be prepared in unit dosage form and in multi-dose containers to produce the finished product.

In oral formulations, tablets include inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate and sodium phosphate; Granulating agents such as corn, starch and alginic acid; Disintegrants; Binders such as starch, gelatin and acacia; And lubricants such as magnesium stearate, stearic acid and talc, in admixture with excipients usable for the manufacture of tablets. Tablets may be used uncoated or coated to inhibit absorption and degradation of the tablets in the gastrointestinal tract. For example, time-inhibiting substances, such as glyceryl monostearate and glyceryl distearate, can also be applied. Hard capsules are a compound of the present invention mixed with an inert solid diluent such as calcium carbonate, calcium phosphate and kaolin, and soft capsules are solvents such as polypropylene glycol, PEGs (polyethylene glycol) and ethanol that can be mixed with water. And the active ingredient in oil solvents such as peanut oil, liquid paraffin and olive oil.

The water-soluble suspending agent is a mixture of excipients and active ingredients suitable for the preparation of water-soluble suspending agents, and excipients include, for example, sodium carboxymethyl cellulose, methyl cellulose, hydroxy-propyl methyl cellulose, sodium alginate, polyvinyl Suspending agents such as pyrrolidone, gum tragacanth and gum acacia; Compounds condensing fatty acids such as polyoxyethylene stearate and alkylene oxides; Compounds in which alkylene oxides are condensed with long fatty acids such as heptadecaethyleneoxycetanol; Compounds which condensate partial esters derived from anhydrous hexitol anhydride and fatty acids, such as polyoxyethylene sorbitol monooleate, with ethylene oxide; Wetting agents; Or dispersing agents. The water-soluble suspending agent may further contain preservatives, colorants, spices and sweeteners.

The oily suspension is a suspension of an active ingredient in a vegetable oil such as olive oil, sesami oil or a mineral oil such as liquid paraffin. For example, a thickening agent such as beeswax, hardened paraffin and cetyl alcohol may be used. It may contain. In addition, it may further contain preservatives, colorants, spices and sweeteners, such composition can be preserved by the addition of antioxidants such as vitamins C and E.

Dispersible powders and particles have an active ingredient in a state of mixing together a dispersing agent, wetting agent, suspending agent and preservative. Suitable dispersing, wetting or suspending agents can be mentioned by way of example already mentioned above. Additional excipients include, for example, nanoparticles, essential oils and colorants.

The water-in-oil emulsion is made from an evening primrose seed oil, vegetable oils such as olive oil, or mineral oils such as liquid paraffin, and is used in esters of natural phospholipids such as soy bean lecithin, anhydrous hecitol or fatty acids such as sorbitan monooleate. The compound derived from hexitol anhydride and a fatty acid such as hydroxyoxysorbitol monooleate, and a partial ester derived from fatty acid are emulsified with an ethylene oxide as an emulsifier to emulsify the active ingredient.

Syrups and elixirs can be prepared by mixing the active ingredients with sweeteners such as glycerol, propylene glycol, sorbitol and sucrose.

Parenteral formulations may be injected as sterile injectable solutions or as suspensions in which the active ingredient is suspended in a non-toxic usable diluent or solvent such as 1,3-butanediol. Among the excipients or solvents that can be used are water, Ringer's solution and isotonic saline solution. Cosolvents such as ethanol, polyethylene glycol, and polypropylene glycol can also be used. In addition, sterile, nonvolatile oils can be customarily used as solvents or suspending solvents. Mixed, non-stimulating, bland fixed oils for this purpose include synthetic mono- and diglycerides. In addition, fatty acids such as oleic acid can be used to prepare the injections. The suppository form is formulated by mixing the drug with a suitable non-irritating excipient, e.g. cocoa butter, polyethylene glycol, which is solid at room temperature but liquid at the rectal temperature and will melt in the rectum to release the drug. May be administered.

Food additives may include beverages, baking, drinks, fats, ice cream, sweets, rice cakes, snacks, baby food, alcoholic beverages, seasonings, retort or canned food.

Cosmetic additives may include a variety of cosmetics such as moisturizing cream, moisturizing lotion, moisturizing soap, lip balm, skin, lotion, whitening agent, wrinkle cream, sunscreen.

When treating atopic dermatitis using the composition of the present invention, the dose of ginseng extract containing the active ingredient methoxykura (2S) -2prim-methoxycurinone is determined by the age, body weight, general state of health, Depending on sex, meal, time of administration, rate of excretion, drug combination and degree of disease during treatment, preferably, up to 10-1,000 mg / kg body weight of high ginseng extract may be used on a daily basis per patient.

Hereinafter, the content of the present invention will be described in more detail through examples and test examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention. All experimental values were expressed as mean ± SE, statistical analysis was performed by ANOVA and Student's t-test, and the significance limit was set to P <0.05.

Test Example 1 Preparation of Ginseng Extract

The ginseng medicine used in the present invention was purchased in 2005 in Yangnyeong Market, Jeonbuk, Jeollabuk-do, and was used in the present invention after it was identified by Professor Hong-Joon Kim, Department of Oriental Medicine, Woosuk University. .

300 g of well-dried red ginseng was injected into a beaker containing 1,000 ml of ethanol, sealed, left to stand for one week, filtered using a 0.45 μm filter, concentrated in a concentrator (Eyela, Japan), and then in a freezer at -70 ° C. After drying to obtain 50g was used in the experiment while storing at -20 ℃.

[Test Example 2] Establishment of atopic dermatitis-like model animals

Skin diseases of NC / Nga mice have been reported to have very similar immunological characteristics as well as the apparent characteristics of human atopic dermatitis (Matsuoka et. al . , Int Immunol . 1997: 9: 461-946; Yamamoto et al . , Allergol Int . 2007: 56 (2): 139-148. Naturally over time, but atopic dermatitis is caused, 6 weeks of age in young mice but atopic dermatitis symptoms, house dust mite, when injected into the subcutaneous antigen Df of the (house dust mite, Dermatophagoides farinae, Df) or hayeoteul applied to the skin It has been known to react sensitively and accelerate atopic dermatitis. The appearance features of these NC / Nga mice include itching, erythema and hemorrhage, followed by edema of the skin, superficial erosion, and deep excoriation. It has been used to develop drugs for preventing and treating atopic dermatitis because it has serious symptoms such as scaling, drying and low growth.

The five-week-old male NC / Nga mouse used in the present invention was purchased from the Central Animal Laboratory (Seoul) and fixed for 12 hours at day and night cycles in a specific pathogen-free (SPF) state for 1 week. Laboratory) and sterilized water, and then used for the experiment.

Induction of atopic dermatitis by Df antigen immunity is described by Matsuoka et al. Immunol . 1997: 9: 461-946. And Yamamoto et al. ( Allergol Int . 2007: 56 (2): 139-148) and modified the method slightly designed and tested. In short, the 6-week-old NC / Nga mouse's dorsal hair is carefully removed with an ophthalmic scissors and then applied with a depilatory agent (Hanyang, Seoul), followed by 30 complete removal of the hair with water, followed by hair removal cream. Df antigen ointment (100 mg) per mouse was applied to the cuticle layer. In order to destroy the cuticle layer from the second time, the application site was sprayed with 4% SDS and completely dried for 2 hours, and then the same amount of antigen was applied twice a week for 8 weeks. The mice used in the control group and each experimental group were tested by assigning 5 mice each. The control group was sprayed only with 4% SDS, and saline was administered orally once a day. The experimental group was 100-400 mg / kg of ginseng ethanol extract ( Sophora flavescens Aiton ethanol extract (SFE) was administered once daily for 8 weeks. Finally Df With antigen At the same time with immunization, the drug was coagulated by taking the blood from the heart for 5 hours and then leaving it at room temperature for 1 hour. The coagulated blood was centrifuged at 3,000 rpm for 20 minutes to obtain serum. Serum was used in the experiment while stored at -70 ℃. Model animal establishment and experimental design are as follows.

[Establishment of Model Animals and Experimental Design]

Test Example 3 Effect of High Ginseng Extract (SFE) on Skin Lesions of NC / Nga Mice

As shown in Test Example 2, 100 mg of Df antigen ointment was applied twice a week to 100% of atopic dermatitis on NC / Nga (6 week old) mice for 8 weeks. Induced. The normal control group was only applied base ointment (100 mg / mol) and did not receive drug. Positive controls were only Df antigen ointment and only saline. The experimental group was applied with Df antigen ointment at the same time while administering 100-400 mg / kg SFE per horse once a day. To examine the changes in the skin lesions during the experiment, the skin condition after ointment application was examined by taking pictures for 8 weeks at intervals of 2 weeks from 0 weeks. Check the dryness, scaling, erosion, oysters, and bleeding of the skin to check for zero lesions, one for mild conditions, and one for mildness. Skin lesions were evaluated by giving a modest 2 points and a severe 3 points. As a result, it is shown in FIG.

In the result of FIG. 1, there was a slight change in the skin lesion of the normal control group, but the positive control group to which the Df antigen ointment was applied increased the skin lesion from the 4th week and increased significantly until 8 weeks, but the SFE drug was administered. In the experimental group, skin lesions were improved in a concentration-dependent manner. In particular, in the experimental group administered 200-400 mg / kg was inhibited effect from the fourth week (p <0.05, p <0.01).

These ginseng extracts have been identified as effective drugs with a pronounced effect of improving skin lesions induced by Df .

Test Example 4 Effect of Red Ginseng Extract (SFE) on Skin Tissue and Leukocyte Infiltration in NC / Nga Mice

To determine the effect of SFE on skin tissue and leukocyte infiltration of Nc / Nga mice immunized with Df antigen, skin histological examination was performed on mice administered with antigen and drug for 8 weeks. According to the experimental procedure, the control group and the experimental group were sacrificed, and skin tissue was extracted and kept flat using a clip, fixed with 4% paraformaldehyde (pH 7.4), and paraffin blocks were prepared through a series of processes. After that, the skin tissue was sectioned into longitudinal sections at 5 μm intervals. To examine histological and leukocyte infiltration, hematoxylin & eosin or Congo red stained, observed at low magnification (x40) and photographed at x200 microscopy and magnified, and examined at x400 microscopy to confirm leukocyte infiltration. (Olympus, Japan). The results are shown in AE of FIG. 2.

A and the normal control of Figure 2 did not change the skin tissue, but Df The skin tissue of the mice immunized with the antigen was very severe in acanthosis, hyperkeratosis, wound and bleeding levels (FIG. 2B). However, in the experimental group administered SFE, the skin tissue lesions were significantly improved depending on the concentration (CE of Figure 2). In particular, the effect was obvious in the experimental group administered 200-400 mg / kg (DE of Figure 2).

In addition, in order to determine the effect of SFE on the leukocyte infiltration, the tissue was stained with Congo red and verified in a 400 × microscope field of view.

There was no change in white blood cells in the normal control group, as shown in FIG. In positive control group immunized with Df antigen, mononuclear cells were 835.54 ± 120.52 / mm, mast cells were 238.67 ± 42.36 / mm, eosinophils were 315.02 ± 43.90 / mm and neutrophils were 291. Leukocyte infiltration was significantly increased to ± 32.51 / mm, but leukocyte infiltration such as monocytes, mast cells, eosinophils and neutrophils was significantly suppressed in the experimental group administered with SFE (p <0.05, p <0.01).

These ginseng extracts have been identified as effective drugs that effectively inhibit the invasion of leukocytes that cause lesions and inflammation of skin tissues induced by Df .

[Test Example 5] Effects of the SFE of Sophora flavescens extract on serum IgE production in NC / Nga mice

It is well known that high IgE in serum is detected, similar to exogenous human atopic dermatitis, which is an important immunological feature in atopic dermatitis model NC / Nga mice induced with Df antigen. In the present invention, in order to determine the effect of Ginseng extract on serum IgE of NC / Nga mice immunized with Df antigen, serum was obtained from mice administered with antigen and drug for 8 weeks and the amount of IgE antibody using ELISA assay kit. Was investigated. First, the total amount of IgE in serum was measured by diluting the serum 25 times and injecting into each well and injecting the standard sample provided by Shibayagi according to the method suggested by the manufacturer. As a result, it is shown in FIG.

As shown in FIG. 4, the serum IgE amount of the normal control group was base level, but the positive control group immunized with Df antigen was significantly increased to 3,095.57 ± 330.06 / ml. However, the experimental group treated with SFE significantly suppressed the production of serum IgE depending on the concentration. The effect was evident in the experimental group administered 200-400 mg / kg.

These ginseng extracts have been identified as effective drugs that effectively inhibit the production of IgE in serum induced by Df .

Test Example 6 Effect of Red Ginseng Extract (SFE) on Serum Th2 Chemokine Production in NC / Nga Mice

TARC / CCL17, MDC / CCL22 and CTACK / CCL27 are representative Th2 chemokines and induce the migration and invasion of Th2 cells in the inflammatory site. In particular, TARC / CCL17 and MDC / CCL22 produced by immune cells, such as peripheral blood monocytes, are critical molecules that cause chemoattractant to move to the site of inflammation by binding to CCR4 of Th2 cells. In addition to being detected in high amounts in the serum of atopic dermatitis patients, it is an important chemokine that accelerates the migration and invasion of Th2 cells (Tsunemi Y et. al ., Eur J Immunol . 2006: 36 (8): 2116-2127; Sebastiani S et al., Eur J Immunol . 2005: 35 (3): 746-756). In particular, it has been found that a larger amount is detected in patients irritated with ovalbumin, thus attracting attention as an important target molecule for etiology of atopic dermatitis (Nakazato J et. al ., Pediatr Allergy Immunol . 2008: 19 (7): 605-613). CTACK / CCL27 also shows its effect via CCR10 (Homey B et al., Nat Med . 2002: 8 (2): 157-165), an important phenomenon was found in the basal layer of the epidermis, the skin lesion of NC / Nga mice. Therefore, excessive production of CTACK / CCL27 induces a severe inflammatory response at the lesion site and plays an important role in mediating migration of cutaneous lymphocyte-associated antigen (CLA) positive lymphocytes (Morales J et. al ., Proc Natl Acad Sci USA . 1999: 96 (25): 14470-14475).

In order to determine the effect of Th2 chemokine on the movement of leukocytes in Nc / Nga mice immunized with Df antigen in high ginseng extract, TARC / CCL17, MDC Serum Th2 chemokine levels were measured using an ELISA assay kit specific for / CCL22 and CTACK / CCL27. Quantification of serum TARC / CCL17, MDC / CCL22 and CTACK / CCL27 was measured by diluting the serum 10-fold and injecting into each well and injecting standard samples provided by R & D according to the method suggested by the manufacturer. As a result, it is shown in FIG.

In the positive control group immunized with Df antigen as shown in FIG. 5, the amounts of TARC / CCL17, MDC / CCL22 and CTACK / CCL27 were measured as 3,250.80 ± 530.20 pg / ml, 2,330.32 ± 420.05 pg / ml and 930.05 ± 180.15 pg / ml, respectively. Although significantly higher than the normal control group, TARC / CCL17 (FIG. 5A) and CTACK / CCL27 (FIG. 5B) were inhibited in a concentration-dependent manner in the SFE-administered group, and the effect was remarkable in the 200-400 mg / ml experimental group. (p <0.05, p <0.01). Furthermore, as shown in FIG. 5C, MDC / CCL22 had a clear inhibitory effect up to 100-400 mg / ml (p <0.05, p <0.01).

These ginseng extracts have been identified as effective drugs that effectively inhibit Th2 chemokine production such as TARC / CCL17, MDC / CCL22 and CTACK / CCL27 in serum induced by Df .

Test Example 7 Isolation Identification of (2S) -2 Prime-Methoxycurinone Derived from Red Ginseng Extract

In the present invention, after confirming the excellent effect that the alpine extract can improve atopic dermatitis in NC / Nga model mouse, to determine what the active compound contained in the ginseng extract (2S) -2 prime-methocycurinone Separated.

(2S) -2 Prime-Methoxykurarinone ((2S) -2'-methoxykurarinone, MOK)} was prepared from the roots of dried ginseng, Kang et al. ( J Nat Prod . 2000: 63: 680-681) and Lee et al . (2001: 45: 588-590). Briefly, the sample was extracted with methanol, concentrated, dissolved in distilled water, and fractionated using CHCl ₃ , EtOA, and n- BuOH continuously. CHCl ₃ fraction (63 g) was dissolved in CHCl ₃ -EtOAc-MeOH (15: 1: 1 → 10: 1: 1) to obtain fraction 9 by silica gel column chromatography. Fraction 3 (14 g) was obtained from silica gel column chromatography by dissolving with CHCl ₃ -EtOAc-MeOH (10: 1: 1) to obtain subfraction 5 (fraction 31-35). Fraction 33 (2.7 g) was dissolved in CHCl ₃ -MeOH and subjected to column chromatography using Sephadax LH-20. Subfraction 331 was isolated by high performance liquid chromatography (HPLC, Jaigel GS320 column, MeOH, 3 mL / min and 210 nm) to obtain compound 1 (37.4 mg). The structural identification of metoshikurarinon obtained by the above process is Kang et al. ( J Nat Prod . 2000: 63: 680-681) and the like (Journal of about 2001: 45: was obtained to confirm and compare the results of the 588-590).

The chemical characteristics of (2S) -2 Prime-Methoshikurarinone are pale yellow powders. mp102-104 ° C .; ESI-MS m / z ; 453 [M + 1] ⁺ ; ¹ H-NMR (500 MHz, DMSO- d ₆ ) δ7.31 (1H, d, J = 8.3 Hz, H-6 '), 6.45 (1H, d, J = 2.1 Hz, H-3'), 6.40 ( 1H, dd, J = 2.1,8.3Hz, H-5 '), 6.14 (1H, s, H-6), 5.45 (1H, dd, J = 2.4,13.6Hz, H-2), 4.88 (1H, brt, J = 6.7 Hz, H-4 "), 4.56 (1H, brs, H _a -9"), 4.48 (1H, brs, H _β -9 "), 3.72 (3H, s, 2'-OMe) , 3.70 (3H, s, C ₅ -OMe), 3.17 (1H, dd, J = 13.6,16.4Hz, H _a -3), 2.86 (1H, dd, J = 2.5,16.4Hz, H _β -3) , 2.50 (2H, m, H-1 "), 2.49 (1H, m, H-2"), 1.92 (2H, m, H-3 "), 1.58 (3H, s, H-10"), 1.52 (3H, s, H-6 "), 1.42 (3H, s, H-7"). ¹³ C-NMR (125 MHz, DMSO- d ₆ ) δ188.7 (C-4), 162.3 (C-9 ), 162.2 (C-7), 159.5 (C-5), 158.7 (C-4 '), 157.3 (C-2'), 148.2 (C-8 "), 130.5 (C-5"), 127.3 ( C-6 '), 123.3 (C-4 "), 117.5 (C-1'), 110.6 (C-9"), 106.9 (C-8), 106.8 (C-5 '), 104.3 (C-10 ), 98.8 (C-3 '), 92.5 (C-6), 73.1 (C-2), 55.3 (C ₅ -OMe), 55.2 (C-2'-OMe), 46.7 (C-2 "), 44.1 (C-3), 30.7 (C-3 "), 26.8 (C-1"), 25.4 (C-6 "), 18.6 (C-10"), 17.8 (C-7 ").

[Chemical Formula of (2S) -2 Prime-Methoshikurarinone Derived from Ginseng]

Test Example 7 (ACK) / CCL27 Chemokine Inhibitory Effect of (2S) -2 Prime-Methoxycurinone

HaCaT cells, a human-derived keratinocyte cell line, were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). HaCaT cells use RPMI 1640 medium (GIBCO-BRL, Invitrogen Co., Grand Island, NY, USA) containing 10% heat-inactivated FBS, penicillin G (100 IU / ml) and streptomycin (100 mg / ml) And incubated. Cell cultures were incubated in 37% CO ₂ incubator at 5% CO ₂ and 95% atmosphere, and stimulated with TNF-α and IL-1β. The drug used (2S) -2 prime-methoxycurinone (MOK).

RT-PCR was performed to confirm whether the active substance inhibits the transcription of the genes encoding CTACK / CCL27. Ie 1 mM dNTPs, 1.75 U / μl RNAse inhibitor, 2.5 U / μl M-MLV reverse transcriptase, 25 U / μl oligo (dT) primer, 25 ng RNA extracted above, 5 mM MgCl ₂ And preparing a reverse transcription reaction mixture containing PCR buffer (5 mM KCl; 10 mM Tris-HCl pH 8.3) and having a total volume of 20 μl, and then preparing the reverse transcription reaction mixture for 10 minutes at room temperature, followed by thermal Reverse transcription was performed using a thermal cycler. The primers of CTACK / CCL27 used were (5'-AGCAGCCTCC CGCTGTTACTGTTG-3 '(forward), 5'-TGCTTTAT TAGTTTTGCTGTT GGG-3' (reverse) and primers of GAPDH were 5'-GCCAAGGTCATCCA TGA CAACTTTGG-3 '(forward)). , 5'-GCC TGCTTCACCACCTTCTTGA TGTC-3 '(reverse), etc. The cells were also inoculated with 1 × 10 ⁶ cells per well, pretreated with MOK and stimulated with rhIFN-γ and IL-1β for 24 hours before CTACK / Production of CCL27 was measured CTACK / CCL27 production was measured by ELISA according to the method provided by R & D.

6A and B show that stimulation of human keratinocyte line with TNF-α and IL-1β expresses and produces CTACK / CCL27. Therefore, this study confirmed that CTACK / CCL27 was expressed by stimulating HaCaT cells with TNF-α and IL-1β, and examined the effect of Gohsam-derived metoshicuranone (MOK) on CTACK / CCL27 chemokine expression. As a result, as shown in FIG. 6A, the expression of the CTACK / CCL27 gene was suppressed depending on the concentration of MOK, and as shown in FIG. 6B, the production was remarkably suppressed.

These ginseng-derived (2S) -2prime-methoxycurinone was found to effectively inhibit CTACK / CCL27 chemokine, a molecule that is an important inducer of atopic dermatitis in TNF-α and IL-1β-stimulated HaCaT cells. Could.

Test Example 8 Induction of HO-1 Expression by (2S) -2 Prime-Methoxycurinone

As in Experiment 2, the culture conditions of HaCaT cells were the same, and (2S) -2prime-methoxycurinone was treated, and then the expression of HO-1 was tested according to the concentration and time of the drug.

RT-PCR was performed to confirm whether the effective substance inhibits transcription of genes encoding HO-1. Ie 1 mM dNTPs, 1.75 U / μl RNAse inhibitor, 2.5 U / μl M-MLV reverse transcriptase, 25 U / μl oligo (dT) primer, 25 ng RNA extracted above, 5 mM MgCl ₂ And preparing a reverse transcription reaction mixture containing PCR buffer (5 mM KCl; 10 mM Tris-HCl pH 8.3) and having a total volume of 20 μl, and then preparing the reverse transcription reaction mixture for 10 minutes at room temperature, followed by thermal Reverse transcription was performed using a thermal cycler.

The drug-treated cells were harvested and injected with 1 × SDS sample buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% SDS, 2 mM beta-mercaptoethanol, 1 mM DTT, BPB, and xylene cyanol]. And the cells were eluted. Cell lysates were run on 10 or 12% SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes. After binding the primary antibody (anti-HO-1) used in this study, the secondary antibody bound to HRP was bound, and then the expression of the protein was examined using supersignal enhanced chemiluminescence (ECL) method. Production of HO-1 was measured by ELISA according to the method provided by R & D. As a result, it is shown in Figure 7 A-D.

Figure 7 A results in stimulating the human keratinocyte line with (2S) -2 prime-methoxycurranone (5-40 μM) 18 hours after the expression of HO-1 gene as a result of concentration-dependent expression It was confirmed that this increased. Therefore, when the expression of HO-1 was examined up to 6-18 hours by selecting 40 μM of methoshicurinone which showed the best effect on the expression of HO-1, it was confirmed that the highest expression was obtained at 18 hours as shown in FIG. 7B. Thus, after confirming the gene expression of HO-1, the effect of the (2S) -2 prime-methoxycurinone on the expression and production of HO-1 protein was tested. As a result, as shown in Figure 7 C and D (2S) -2 prime-methoxycurinone was confirmed to be an effective substance for inducing HO-1.

Experiment 9 Inhibitory Effect of (2S) -2 Prime-Methoxycurinone CTACK / CCL27

HaCaT cells, a human-derived keratinocyte cell line, were transfected with HO-1 siRNA, stimulated with TNF-a and IL-1β under the same conditions as in Example 2, and tested for the effect of metoshicurinone on the production of CTACK / CCL27. . CTACK / CCL27 production was measured by ELISA according to the method provided by R & D. As a result, it is shown in FIG.

As shown in FIG. 8, HO-1 siRNA was transfected into human keratinocyte line and TNF-α and IL-1β were stimulated to produce high levels of CTACK / CCL27. However, the treatment of metoshikuraniron (MOK) significantly inhibited the production of CTACK / CCL27 in normal keratinocytes ( p < 0.05), but not in keratinocytes transfected with HO-1 siRNA.

It was confirmed that the CTACK / CCL27 chemokine inhibitory effect of (2S) -2prime-methoxycurinone derived from Ginseng was achieved by inducing HO-1.

In the present invention, as a result of oral administration of high ginseng extract (SFE) to human atopic dermatitis-like NC / Nga mice, the result of testing the inhibitory effect of the occurrence of atopic dermatitis Df including skin lesion score and leukocyte infiltration There was an excellent effect of significantly inhibiting the clinical signs of induced atopic dermatitis. In addition, the administration of ginseng extract had an excellent effect of significantly inhibiting serum IgE and Th2 chemokine (TARC / CCL17, MDC / CCL22, and CTACK / CCL27) levels in a concentration-dependent manner. In addition, CTACK / CCL27 chemokine produced by keratinocytes is known to be one of the key causes of atopic dermatitis, and (2S) -2prime-methoxycurinone, an active ingredient of the ginseng extract, is an anti-stress enzyme HO-. By inducing 1, it was confirmed that there is an excellent effect of inhibiting the production of CTACK / CCL27 chemokine. Therefore, Ginseng extract containing (2S) -2prime-methoxycurinone may be used as a functional cosmetic and food or medicine for the prevention and treatment of atopic dermatitis.

1 is a graph showing the results of skin lesions of NC / Nga mice for the administration of high ginseng extract according to the test example of the present invention.

Figure 2 is an enlarged view of the skin tissue of NC / Nga mice for the administration of high ginseng extract according to the test example of the present invention.

Figure 3 is a graph of the results of the leukocyte infiltration experiment of NC / Nga mice for the administration of high ginseng extract according to the test of the present invention.

Figure 4 is a graph of the results of the serum IgE production test of NC / Nga mice for the administration of high ginseng extract according to the test example of the present invention.

Figure 5 is a graph of the results of the serum Th2 chemokine production test of NC / Nga mice for the administration of high ginseng extract according to the test example of the present invention.

FIG. 6 is the effect of metoshikuraniron (MOK) on the expression and production of CTACK / CCL27 in TNF-α and IL-1β-stimulated HaCaT according to a test example of the present invention.

7 is a graph showing the effect of MOK on HO-1 expression according to the test example of the present invention.

Figure 8 is a ginseng extract (2S) -2 prime-Methoshikura on the production of CTACK / CCL27 by HO-1 siRNA transfection in TNF-α and IL-1β stimulated HaCaT cells according to the test example of the present invention The effect of linon.

Claims (3)

After aging dried ginseng in an extraction solvent and aging, the aged mixture is filtered and concentrated to prevent and treat atopic dermatitis using a ginseng extract, characterized in that dried. A composition for preventing and treating atopic dermatitis by using a ginseng extract as an active ingredient and inhibiting serum IgE and Th2 chemokines (TARC / CCL17, MDC / CCL22, and CTACK / CCL27). The method according to claim 2, wherein the (2S) -2 prime-methoxycurinone derived from the ginseng extract induces the expression and production of Heme oxygenase-1 (HO-1), Th2 chemokine (CTACK / CCL27) composition for preventing and treating atopic dermatitis.

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