KR101738469B1 - A composition comprising ethyl acetate or n-butanol fraction of Crataegi Fructus for preventing or treating inflammatory disease - Google Patents

Abstract

The present invention relates to a composition comprising an ethyl acetate or n-butanol fraction of Crataegi fructus for preventing or treating inflammatory diseases. The fraction of the present invention has an excellent activities for inhibiting the generation of IL-6 and NO in macrophagocytes and for inhibiting the generation of IP-10, RANTES, and MDC, thereby being able to be usefully utilized as a composition or health functional food for preventing or treating inflammatory diseases, or a cosmetic composition for preventing or treating skin inflammation.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for preventing or treating an inflammatory disease comprising an ethyl acetate fraction or an n-butanol fraction,

The present invention relates to a composition for preventing or treating an inflammatory disease comprising an ethyl acetate fraction or an n-butanol fraction of a marine sperm.

Inflammation (inflammation) is caused by damage to tissues (cells) or infection with external infectious agents (bacteria, fungi, viruses, various allergens), various inflammatory mediators and immune cells are involved, Complex physiological responses such as substance secretion, fluid infiltration, cell migration, and tissue destruction, and external symptoms such as erythema, edema, fever, and pain. In the normal case, the inflammatory reaction removes the external infectious agent and regenerates the damaged tissue to regenerate the organism's function. However, when the antigen is not removed or the internal substance causes the inflammatory reaction to occur excessively or continuously, , And some of them lead to rheumatoid arthritis, osteoporosis, sepsis, vascular diseases, cancer and the like. In other words, the inflammatory response is the most important pathological mechanism of the host defense mechanism against the wound, microbial infection, but persistent and excessive inflammation damages the tissue.

Macrophages are the most representative immune cells that regulate these inflammatory responses. They play an important role in host defense mechanisms in harmful environments and are involved in the progress of various diseases such as autoimmune diseases. In particular, stimulation of cytokines such as lipopolysaccharide (LPS), IFN-y (interferon-y), TNF-alpha (tumor necrosis factor-a), IL-6 (interleukin- The inducible nitric oxide synthase (iNOS), which is expressed by LPS, produces a large amount of NO over a long period of time. NO is a physiologically important homeostatic regulator and plays a variety of roles, such as eliminating intestinal bacteria and tumors, regulating blood pressure, or mediating neurotransmission. However, when inflammation occurs, the amount of iNOS expressed in the inflammation-related cells is increased to produce a large amount of NO. Excessively generated NO causes tissue damage, genetic mutation, nerve damage, and increases vascular permeability and induces edema . In addition, when an inflammatory reaction occurs, PGE ₂ is produced by macrophage COX-2, which is an inflammatory factor mainly involved in pain and fever (Wang, MT et al., 2007). Therefore, the anti-inflammatory effect can be confirmed by confirming the inhibition of the production of NO, COX-2, etc. produced in the inflammatory reaction.

Since the skin of the human body covers the surface of the body, there are many opportunities for external stimulation such as ultraviolet rays, direct contact with various pathogens, and strong influence from the body. In addition, diseases are caused by various causes because they are affected by pathological changes such as genetic, inflammatory, benign and malignant tumors, hormones, trauma, degenerative degeneration. Dermatitis is an inflammation of the skin that can be divided into atopic dermatitis, contact dermatitis and seborrhoic dermatitis. Atopic dermatitis is a recurrent chronic dermatitis caused by environmental, genetic, immunological reaction and skin barrier abnormality. Contact dermatitis is dermatitis caused by contact with external substances, and seborrheic dermatitis is caused by sebum secretion It refers to dermatitis that occurs well in the area. In particular, atopic dermatitis is recurrent chronic dermatitis with severe itching, with about 10 to 15% of children having atopic dermatitis, of which 90% are reported to heal spontaneously within 5 years. It is generally known that about 30-40% of adult patients do not recur dermatitis. However, it is known that it is difficult for adult to become skin irritation such as skin irritation, skin irritation caused by irritant substance, and housewife eczema even when becoming adult. In addition, in the case of sensitive skin, there is a high possibility that the skin becomes atopic skin because of low water retention and recovery ability, skin keratinization and itching.

Although the exact pathophysiology of atopic dermatitis has not yet been fully understood, it is believed that immunologic and nonphysiologic mechanisms are involved with genetic predisposition. It has been reported that exogenous atopic dermatitis, which accounts for the majority of atopic dermatitis, is caused by an immunologic mechanism associated with IgE (immunoglobulin E), which implies a delayed immune response due to T cell abnormality rather than an immediate immune response to a specific allergen There are many. In addition, nowadays, Th2-related cytokines such as IL-4, which induces the production of IgE from B cells, have been reported to be the cause of atopy (Kang, J. S. et al., 2008).

Although steroids are widely used as the most common therapeutic agents for the treatment of inflammatory diseases as described above, they can exhibit various side effects such as decreased immune function, skin thinning, atrophy and flushing, and when they are taken for a long time, steroid acne, Serious side effects have been reported, including skin (skin atrophy, capillary vasodilation, purpura), steroid injection, dermatitis in situ, hair follicle, pigment desilyl, ancestral, bulbous dermatitis, skin bleeding. In addition, nonsteroidal agents are used as therapeutic agents for other inflammatory diseases. In addition to anti-inflammatory effects, they cause severe adverse effects such as gastrointestinal disorders, hepatic disorders, renal diseases and the like, and thus they are restricted in long-term use (Rajakariar R et al. 2006) . Therefore, there is a demand for the development of therapeutic agents derived from natural products, which have few side effects and are stable even after long-term administration.

Crataegi Fructus refers to the fruit of crataegus pinnatifida, and the flowers of the hawthorn tree bloom in May and the fruit is round and round with white spots. In Korea, it has been known to have effects such as dryness, digestion, convergence, and pain. In Europe, the fruit of European hawthorn is also called crataegus, and it is also used as a cardiotonic agent. Examples of the pharmacological ingredient include caffeic acid, citric acid, corosolic acid, crataegolic acid, euscaphic acid, hyperin, Hyperoside, procatechuic acid, pyrogallol, oleanolic acid, ursolic acid, and vitexin have been reported. The antioxidant activity of antioxidants such as antioxidants (Ryu, HY et al., 2007), and the effect of the antimicrobial agent (Korean Patent No. 10-0570120), antibacterial effect (Kim Jung Hyun, 2010)

Korean Patent Laid-Open No. 10-2005-0079256 discloses a composition for preventing and treating allergic diseases in which a mixture containing a mixture of Horseshoe, Bokbunja, [Li, C. et al., 2011] discloses that a water fraction of a mountain sage has an anti-inflammatory effect, but the fact that a fraction obtained by fractionating the sasa extract with an organic solvent can be used as a preventive or therapeutic agent for an inflammatory disease .

Korean Patent Laid-Open No. 10-2005-0079256, a composition for the prevention and treatment of an allergic disease containing a mixed extract of Bacillus subtilis and Bokbunja as an active ingredient, published on Aug. 10, 2005. Korean Registered Patent No. 10-0570120, cosmetics for free radical scavenging containing horseradish extract, registered on Apr. 05, 2006.

Kim, JH, et al., Antimicrobial effect of methanol extract of mountain sodas, Dongduk Pharma Research, 14, 33-38, 2010. Kang, J. S. et al., Inhibition of atopic dermatitis by topical application of silymarin in NC / Nga mice, Int Immunopharmacol., 8 (10), 1475-1480, 2008. Li, C. et al., Anti-inflammatory effect of the water fraction from hawthorn fruit on LPS-stimulated RAW 264.7 cells, Nutr Res Pract., 5 (2), 101-106, 2011. Ryu, H. Y. et al., Thrombin Inhibition Activity of Fructus Extract of Crataggus pinnatifida Bunge, Journal of life sciences, 17 (4), 535-539, 2007. Wang, M. T. et al., Cyclooxygenases, prostanoids, and tumor progression, Cancer Metastasis Rev., 26 (3-4), 525-534, 2007.

The object of the present invention is to provide a composition for preventing or treating an inflammatory disease comprising an ethyl acetate fraction or an n-butanol fraction of a marine spermatozoa. The present invention also provides a composition for preventing or treating an inflammatory disease, Or a cosmetic composition for preventing or ameliorating skin inflammation.

The present invention relates to a method for treating a crustacean (Crataegi fructus) which is obtained by extracting and concentrating Crataegi fructus with a C1 to C4 lower alcohol or a lower alcohol aqueous solution, suspending it in water, and subsequently fractionating and concentrating it with ethyl acetate and n-butanol Ethyl acetate fraction or n-butanol fraction, wherein the C1 to C4 lower alcohols are methanol or ethanol.

The inflammatory disease is selected from the group consisting of allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, A disease selected from the group consisting of rheumatoid arthritis, periarthritis, tendonitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases.

In addition, the present invention relates to a method for producing a sansapae extract, which comprises suspending a sansapae extract obtained by extracting and concentrating a sansa with a lower alcohol of C1 to C4 or a lower alcohol aqueous solution and concentrating it with ethyl acetate and n-butanol sequentially, Or a fraction thereof or an n-butanol fraction thereof. The present invention also relates to a health functional food for preventing or ameliorating an inflammatory disease.

In another aspect, the present invention relates to a method for preparing a plant extract obtained by suspending a marine extract obtained by extracting and concentrating a marinade from a C1 to C4 lower alcohol or a lower alcohol aqueous solution, and subsequently fractionating and concentrating the mixture with ethyl acetate and n-butanol Which comprises an ethylacetate fraction or an n-butanol fraction of sansapae, and a cosmetic composition for preventing or improving skin inflammation.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for preparing a herbal extract, which comprises suspending a marine extract obtained by extracting and concentrating a marinade with a lower alcohol or a lower alcohol aqueous solution of C1 to C4, followed by successive fractionation and concentration with ethyl acetate and n-butanol, n-butanol fraction. The present invention also relates to a pharmaceutical composition for preventing or treating an inflammatory disease.

The lower alcohol or the lower alcohol aqueous solution of the C1 to C4 is preferably a 50 to 100% lower alcohol or a lower alcohol aqueous solution, and the lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, Methanol or ethanol may be used.

The inflammatory disease may be selected from the group consisting of allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, But are not limited to, those selected from the group consisting of osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay fever, periuritis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases .

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising an ethyl acetate fraction or an n-butanol fraction of a marine spermatozoa, wherein the pharmaceutical composition comprising the marine fraction comprises , Tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterilized injection solutions. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, 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 formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . 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 the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the pharmaceutical composition comprising the fragrance fraction of the present invention will vary depending on the age, sex, body weight, the particular disease or condition to be treated, the severity of the disease or condition, 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 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition comprising the fragile fraction of the present invention can be administered to mammals such as rats, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the fraction has little toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

The present invention also provides a health functional food for the prevention or amelioration of an inflammatory disease comprising an ethyl acetate fraction or an n-butanol fraction of a marinade and a pharmaceutically acceptable food-aid additive, By weight to 0.001% by weight to 100% by weight. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids, and examples of foods to which the compound of the present invention can be added include various foods, beverages, gums, tea, vitamins .

In another aspect, the ethyl acetate fraction or the n-butanol fraction of a marinade of the present invention can be used as a cosmetic composition having an effect of preventing or ameliorating skin inflammation, and the skin inflammation can be classified into allergic dermatitis, Dermatitis, seborrheic dermatitis, seborrheic dermatitis, urticaria, psoriasis, dry skin, eczema, and the like.

In addition, the cosmetic composition may contain all components commonly used in cosmetics, in addition to the fragrance fractions. For example, it may contain common auxiliary components such as emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers and perfumes. The above-mentioned components can be selected within a range that does not impair the effect inherent in the cosmetic, depending on the purpose of formulation or use. In addition, the cosmetic composition may be prepared in any form conventionally produced in the art, and examples thereof include solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More preferably, it may have one type of formulation selected from softening longevity (skin), astringent lotion, nutritional lotion, nutritional cream, massage cream, essence, pack, skin sticking patch and skin sticking gel.

In addition, the cosmetic composition containing the fragrance of the mountain can be used every day, and can be used for an unspecified period of time. Preferably, the cosmetic composition can be used for the user's age, skin condition or skin type, And the period can be adjusted.

The present invention relates to a composition for the prophylaxis or treatment of an inflammatory disease comprising an ethyl acetate fraction or an n-butanol fraction of spermatozoa, wherein the fraction is an activity of inhibiting IL-6 and NO production in macrophages, A composition for preventing or treating an inflammatory disease or a composition for preventing or ameliorating skin inflammation or a composition for preventing or ameliorating an inflammatory disease or a composition for preventing or ameliorating skin inflammation, . ≪ / RTI >

1 is a result of analyzing IL-6 and NO produced after applying the niger-butanol fraction of the present invention to LPS-treated macrophages.
FIG. 2 shows the results of analysis of the amount of IP-10, Rantes, and MDC produced after applying the nasal butanol fraction of the present invention to human keratinocytes treated with TNF-α and 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, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1 >

70% ethanol aqueous solution (3 L × 2 times) was taken in 600 g of a hot-air dried mountain sampler collected from Geumsan-myeon, Geumsan-gun, and refluxed for 5 hours. The mixture was cooled to room temperature, filtered and concentrated to obtain 115 g of ethanol extract. Thereafter, 2 L of distilled water was added to the ethanol extract, dissolved in an ultrasonic washing machine, and then sequentially fractionated with ethyl acetate (500 mL 2 times) and n-butanol (500 mL 2 times) to obtain the ethyl acetate fraction 4.2 g) and the n-butanol fraction (16.8 g) of Example 2 were obtained.

Prepared in the same manner as in Examples 1 and 2 except that 50% ethanol aqueous solution, 100% ethanol, 70% methanol aqueous solution, 50% methanol aqueous solution and 100% methanol were added instead of 70% ethanol aqueous solution Examples 3 to 12 were prepared.

Condition Sansa extract solvent Solvent conditions of re-fractionation of Sansa extract Re-fractionating solvent Total amount of fraction obtained Example 3 50% aqueous ethanol solution Ethyl acetate 3.6 g Example 4 n-butanol 13.8g Example 5 100% ethanol Ethyl acetate 4.2 g Example 6 n-butanol 16.2 g Example 7 70% aqueous methanol solution Ethyl acetate 3.6 g Example 8 n-butanol 15g Example 9 50% aqueous methanol solution Ethyl acetate 3g Example 10 n-butanol 13.8g Example 11 100% methanol Ethyl acetate 4.2 g Example 12 n-butanol 15.6 g

< Comparative Example  1. Preparation of ethanol extract of sansapae>

70% ethanol aqueous solution (3 L × 2 times) was added to 600 g of hot-air dried fish samarium collected from Geumwon-myeon, Geumsan-gun, and refluxed for 5 hours. After cooling to room temperature, filtration and concentration were performed to produce 114.6 g of ethanol extract of mountain sainfoin.

&Lt; Comparative Example 2 > Preparation of water-for-hull water extract &

600 g of hot sauce, which was collected from Geumwon-myeon, Geumsan-gun and mixed with 6 liters of water, was extracted at 90 ℃ for 10 hours.

&Lt; Comparative Example 3 >

Prepared in the same manner as in Example 1, fractionated with ethylacetate and n-butanol, and the remaining water layer was filtered and concentrated to obtain 73.8 g of a fragrant water fraction.

<Experimental Example 1> Cell culture>

The cell culture conditions used to measure the anti-inflammatory activity of the fern fractions of the present invention are as follows.

Mouse macrophage RAW 264.7 cells and human keratinocyte HaCaT cells were purchased from Korean Cell Line Bank. The cells were incubated with 10% FBS (fetal bovine serum), 10% T-stim (USA), 0.05 mM 2-mercaptoethanol, 2 mM L-glutamine (Sigma, USA) Were cultured in a DMEM culture medium containing streptomycin at 37 ° C under 5% CO ₂ (CO ₂ incubator (Forma Sci, USA)).

EXPERIMENTAL EXAMPLE 2 Measurement of Cell Survival Rate [

(3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed to measure the cell viability of the mountainous flesh fraction of the present invention.

RAW 264.7 cells and HaCaT cells cultured under the conditions of Experimental Example 1 were dispensed in a 96-well plate at a concentration of 1 × 10 ⁶ cells / ml, respectively, and then cultured for 24 hours. Then, the fungus fractions of Example 2 were prepared at a concentration of 12.5, 25, 50, 100, 200, 400 and 800 μg / μl in the DMEM culture medium to which no FBS and antibiotics were added and cultured for 24 hours Cells and cultured for an additional 24 hours. At this time, the cell survival rate was confirmed by treating Comparative Example 1 to 3 or control group (untreated group) at a concentration of 800 μg / μl instead of Example 2.

At the end of the incubation, MTT solution (0.5 mg / ml) corresponding to one tenth of DMEM was added, followed by further reaction for 4 hours to reduce the MTT. The supernatant was removed and dried in the dark for 30 minutes. The formazan crystal was dissolved in 100 μl of DMSO and the absorbance at 540 nm was measured with an absorbance meter (ELISA reader, molecular device, USA). The cell viability was then calculated by the following equation and is shown in Table 2 below.

[Equation]

RAW 264.7 cells  HaCaT cells Control group (untreated group) 100.00 100.00 Example 2 12.5 / / ㎕ 98.41 105.74 25 / / ㎕ 99.68 91.15 50 / / ㎕ 95.27 94.09 100 mu g / mu l 97.59 99.87 200 [mu] g / 97.34 108.51 400 μg / μl 106.82 94.13 800 / / ㎕ 116.60 100.60 Comparative Example 1 800 / / ㎕ 108.88 110.92 Comparative Example 2 800 / / ㎕ 110.42 100.60 Comparative Example 3 800 / / ㎕ 101.84 107.66

As shown in Table 2, in the case of treating the RAW 264.7 cells and the HaCaT cells with the butanol fraction of the present invention (Example 2), the cell survival rate similar to that of the control group (untreated group) was observed regardless of the concentration of the spermine butanol fraction, And no cytotoxicity was observed. In addition, although not shown in Table 2, it was confirmed that Example 1 and Examples 3 to 12 were free from cytotoxicity.

<Experimental Example 3> Measurement of IL-6 and NO production amount>

In order to measure the inflammation-inhibiting activity of the mountainous flesh fraction of the present invention, the amount of IL-6 and NO produced was measured.

For this, the first, the RAW 264.7 cell cultures under the conditions of Experimental Example 1 in the 24-well plate at 2 × 10 ⁵ cells / well were inoculated in a concentration of, 37 ℃, 5% CO ₂ and 95% O ₂ in the condition 24 Lt; / RTI &gt; Thereafter, the supernatant was removed, and 2 μl of the positive control 10 μg / ml of cyclosporin A or 400 μg / ml of the samples 1 to 12 or 400 μg / ml of the comparison In the case of Example 2, 2 ml of each concentration was added to each concentration (25, 50, 100, 200 and 400 / / ml), followed by further incubation for 24 hours After the supernatant was separated and stored in the freezer, it was used to measure IL-6 and NO production.

ELISA kit (Mouse IL-6 ELISA kit, eBioscience, USA) was used to measure IL-6 (Mouse IL-6) first in inflammatory cytokines. In accordance with the manufacturer's instructions, the antibody was dispensed at 100 μl / well into the culture broth of the present invention or the positive control group treated at this time, and reacted at 4 ° C. for 16 hours. Then, each well was washed with wash buffer, , 200 μl of assay diluent was added to each well, the wells were incubated for 1 hour, and then cultured at room temperature. The standard was diluted, the supernatant was diluted 20-fold, the plate was washed, 100 μl of each standard and the supernatant were added, the wells were incubated for 2 hours and then cultured at room temperature. Plates were washed and a working detector was prepared. 100 μl of each well was added to each well. After incubation for 1 hour, the wells were incubated at room temperature. The plate was washed and substrate solution was prepared. And incubated at room temperature in the dark for 30 minutes. The stop solution was added to each well in an amount of 50 μl, and the absorbance at 450 nm was measured by a microplate spectrophotometer. The results are shown in Table 3 and FIG. 1A.

Next, to measure NO production amount, a grease reagent was used, which was prepared by dissolving solution A (0.2% Naphthylethylene diamine dihydrochloride in DW) and solution B (2% sulfonylamide in 5% H ₃ PO ₄ ) Two solutions were mixed 1: 1 immediately before the next use. In the above procedure, 100 쨉 l of the supernatant stored in the freezer was dispensed into a 96-well plate, and 100 쨉 l of the same amount of a grease reagent was mixed. Absorbance was measured at 540 nm using an absorbance meter. And FIG. 1B.

Condition IL-6 (pg / ml) NO (μM) Example 1 2589 22.3 Example 2 2432 20.0 Example 3 2686 23.1 Example 4 2554 22.8 Example 5 2557 23.7 Example 6 2654 23.0 Example 7 2797 23.6 Example 8 2703 24.7 Example 9 2861 24.7 Example 10 2875 24.3 Example 11 2842 25.5 Example 12 2953 25.9 Comparative Example 1 6824 43.5 Comparative Example 2 7034 45.4 Comparative Example 3 7208 46.3

In the case of the above Table 3, the results of treatment of Examples 1 to 12 and Comparative Examples 1 to 3 at a concentration of 400 μg / ml show the results of the production of IL-6 and NO. Examples 1 to 12 of the present invention It was confirmed that the effect of decreasing the amount of IL-6 and NO production was superior to those of Examples 1 to 3.

In addition, referring to FIG. 1, when Example 2 of the present invention was treated with macrophages treated with LPS, IL-6 and NO production were decreased in a concentration-dependent manner. Especially, at a concentration of 400 μg / And the fractions of the present invention were found to be IL-6, an inflammatory cytokine, and a substance exhibiting NO production inhibitory activity.

<Experimental Example 4> Confirmation of skin inflammation and damage inhibitory effect>

The treatment of TNF-α and IFN-γ (10 ng / ml), which induce an inflammatory response in human keratinocyte HaCaT cells, resulted in the production of IPA-10, Rantes, and MDC The effect was confirmed.

First, the HaCaT cell cultures under the conditions of Experimental Example 1 in the 24-well plate at 1 × 10 ⁶ cells / well and inoculated, 24 hours at 37 ℃, 5% CO ₂ and 95% O ₂ conditions (standard incubator conditions) And then the supernatant was removed. Thereafter, TNF- [alpha] and IFN- [gamma] were treated at a concentration of 10 [mu] g / ml and then treated with 2 ml of 10 [micro] g / ml of cyclosporin A as a positive control or 200 [ 2 ml of each of Comparative Examples 1 to 3 was added, followed by further incubation for 24 hours. Then, the supernatant was separated and stored in a freezer to measure IP-10, Rantes and MDC production. Particularly, in the case of Example 2, IP-10, Rantes and MDC production amounts were also measured by treatment at concentrations of 25, 50, 100, 200 and 400 μg / ml, respectively.

Using the IP-10, Rantes, and MDC ELISA kits (eBioscience, USA), according to the manufacturer's instructions, in the above procedure, the culture of the Example or Comparative Example or the positive control group The antibody was dispensed at 100 μl / well and reacted at 4 ° C. for 16 hours. Each well was washed with the washing solution, 200 μl of the assay dilution was added, and the well was blocked for 1 hour and then cultured at room temperature. The standard was diluted, the supernatant was diluted 20-fold, the plate was washed, 100 μl of each standard and the supernatant were added, the wells were incubated for 2 hours and then cultured at room temperature. 100 μl of each well was added to each well. After incubating for 1 hour, the wells were incubated at room temperature. The plates were washed, and a substrate solution was prepared. 100 μl of each solution was added to each well. . Then, 50 μl of a stop solution was added to each well, and the absorbance at 450 nm was measured with a microplate spectrophotometer, and the results are shown in Table 4 and FIG.

IP-10 (pg / ml) Rantes (pg / ml) MDC (pg / ml) Example 1 4625.87 21.875 41.362 Example 2 4578.66 20.151 39.632 Example 3 4883.77 22.110 42.265 Example 4 4842.62 21.253 41.052 Example 5 5025.51 23.488 42.580 Example 6 5125.14 23.962 42.562 Example 7 5339.98 25.002 44.351 Example 8 5235.25 25.859 43.352 Example 9 5541.27 26.152 45.365 Example 10 5454.58 25.821 46.358 Example 11 5621.02 27.696 44.521 Example 12 5492.22 26.368 45.214 Comparative Example 1 11058.81 40.414 112.125 Comparative Example 2 11525.58 43.553 132.363 Comparative Example 3 12158.39 44.041 148.255

In the case of Table 4 above, the results of IP-10, Rantes and MDC production in the treatment of all of Examples 1 to 12 and Comparative Examples 1 to 3 at a concentration of 200 μg / ml are shown in Examples 1 to 12 It was confirmed that the effect of reducing IP-10, Rantes and MDC production was better than those of Comparative Examples 1 to 3.

2, when Example 2 of the present invention is applied to keratinocytes of skin treated with TNF-α and IFN-γ which cause an inflammation reaction, atopy-related factors IP-10, Rantes and MDC production was decreased in a concentration dependent manner. In addition, when the above Example 2 was treated at a concentration of 200 μg / ml or more, the expression level of cyclosporin A was lower than that of the positive control group. Thus, it was confirmed that the fragrant mountainous fraction of the present invention exhibited dermatitis inhibiting activity.

<Experimental Example 5> Toxicity test>

Experimental Example 5-1. Acute toxicity

The present experiment was conducted to investigate toxicity to the animal body in an acute (within 24 hours) when an excessive amount of Example 2 (northeastern n-butanol fraction) of the present invention was consumed in a short period and determine the mortality rate. Twenty ICR mice were prepared, and 10 mice were assigned to each group. In the control group, 30% PEG-400 alone was administered, and the experimental group was orally administered at a concentration of 1.0 g / kg in Example 2, respectively. After 24 hours of administration, the respective mortality rates were examined. As a result, both the control group and the experimental group administered with the above example survived.

Experimental Example 5-2. Organ organs toxicity test in experimental group and control group

The long-term toxicity test was carried out to examine the effects of C57BL / 6J mice on the organs (tissues) of the animals, the experimental group of Example 2 (nosanthus n-butanol fraction) administered at a concentration of 1.0 g / After 8 weeks from the control group, the blood was collected and the concentration of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) in the blood was measured using Select E (vital scientific NV, Netherland) . As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal and histological observation was performed with an optical microscope through a conventional tissue section production process, but no abnormal abnormalities were observed.

EXPERIMENTAL EXAMPLE 5-3. Human body skin irritation confirmation experiment

The creams of the following formulation 2-3, containing Example 2 of the present invention (northeastern n-butanol fraction), were subjected to a patch test on 10 volunteers. As a result, no skin irritation was observed.

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200 g of Example 2 (northeastern n-butanol fraction) 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.

Formulation Example 1-2. Injection preparation

1 g of the Example 2 (northeastern n-butanol fraction) of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were 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.

Formulation Example 1-3. Manufacture of Ointment Agents

10 mg of Example 2 (northeastern n-butanol fraction) of the present invention, 250 mg of PEG-4000, 650 mg of PEG-400, 10 mg of white petrolatum, 1.44 mg of methoxybenzoic acid methyl, 0.18 mg of p- And then an ointment agent was prepared according to a conventional ointment preparation method.

&Lt; Preparation Example 2: Preparation of cosmetic &

Formulation Example 2-1. Production of flexible lotion

Using the Example 2 (northeastern n-butanol fraction) of the present invention, a flexible lotion was produced according to the composition shown in the following Table 5 by a conventional method.

Raw material name Content (% by weight) Example 2 (Sansa n-Butanol fraction) 4.0 Butylene glycol 3.5 glycerin 2.5 Polyoxyethylene hardened castor oil 0.1 ethanol 2.5 Betaine 1.0 Citric acid 0.01 Sodium citrate 0.03 antiseptic Suitable amount Spices Suitable amount Purified water to 100

Formulation Example 2-2. Manufacture of nutrition essences

Nutritional essence was prepared by the usual method according to the composition described in Table 6 below using Example 2 (northeastern n-butanol fraction) of the present invention.

Raw material name Content (% by weight) Example 2 (Sansa n-Butanol fraction) 7.0 Cetostearyl alcohol 1.0 Self emulsifying monostearic acid 1.0 Wax 0.5 Squalane 5.0 Isocetyl octanoate 3.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 8.0 glycerin 4.0 Propylene glycol 0.2 Carboxy polymer 0.22 Triethanolamine 0.25 antiseptic Suitable amount Spices Suitable amount flash Suitable amount Purified water to 100

Preparation Example 2-3. Manufacture of cream

Creams were prepared in the usual manner according to the composition shown in Table 7 below using Example 2 (northeastern n-butanol fraction) of the present invention.

Raw material name Weight% (w / w) Example 2 (Sansa n-Butanol fraction) 7.0 Cetostearyl alcohol 3.0 Self emulsifying monostearic acid 1.5 Chin type monostearate 1.5 Wax 0.5 Liquid paraffin 8.0 Squalane 7.0 Isocetyl octanoate 4.0 Refined jojoba oil 4.0 Dimethylsiloxane 0.3 Sorbitan monostearate 1.0 Polyethylene glycol monostearate 1.2 glycerin 6.0 Propylene glycol 4.0 Betaine 4.0 Xanthan gum 0.06 Triethanolamine 0.10 antiseptic 0.25 Spices Suitable amount flash Suitable amount Purified water to 100

Formulation Example 2-4. Manufacture of packs

A pack was prepared by the usual method according to the composition shown in the following Table 8 by using Example 2 (northeastern n-butanol fraction) of the present invention.

Raw material name Content (% by weight) Example 2 (Sansa n-Butanol fraction) 8.0 glycerin 7.0 1,3-butylene glycol 3.0 Squalene 3.0 Dimethicone 3.0 Sodium hydrulonate 0.1 Glycine 2.0 Polyacrylamide 5.0 Triethanolamine 0.1 EDTA 0.1 Purified water to 100

&Lt; Formulation example 3. Food production >

Formulation Example 3-1. Manufacture of cooking seasonings

Example 2 (marine n-butanol fraction) of the present invention was added to the cooking sauce at 1 wt% to prepare a cooking sauce for health promotion.

Formulation Example 3-2. Manufacture of flour food products

Example 2 (marine n-butanol fraction) of the present invention was added to wheat flour at 0.1% by weight, and bread, cake, cookies, crackers and noodles were prepared using this mixture to prepare foods for health promotion.

Formulation Example 3-3. Manufacture of soups and gravies

Example 2 (marine n-butanol fraction) of the present invention was added to soup and juice at 0.1 wt% to prepare soup for health promotion and juice.

Formulation Example 3-4. Manufacture of dairy products

Example 2 (marine n-butanol fraction) of the present invention was added to milk in an amount of 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 3-5. Vegetable juice manufacturing

0.5 g of Example 2 (northeastern n-butanol fraction) of the present invention was added to 1,000 ml of tomato juice or carrot juice to prepare vegetable juice for health promotion.

Formulation example  3-6. Manufacture of fruit juice

0.1 g of Example 2 (northeastern n-butanol fraction) of the present invention was added to 1,000 ml of apple juice or grape juice to prepare health promotion fruit juice.

Claims (8)

The Crataegi Fructus was extracted and concentrated with an ethanol or ethanol aqueous solution and suspended in water and then fractionated and concentrated with ethyl acetate and n-butanol to obtain ethyl acetate fraction or n-butanol fraction A pharmaceutical composition for preventing or treating skin inflammation, which is selected from the group consisting of allergic dermatitis, atopic dermatitis, contact dermatitis, seborrheic dermatitis, urticaria, psoriasis, dry skin and eczema. delete delete The Crataegi Fructus was extracted and concentrated with an ethanol or ethanol aqueous solution and suspended in water and then fractionated and concentrated with ethyl acetate and n-butanol to obtain ethyl acetate fraction or n-butanol fraction A health functional food for preventing or ameliorating skin inflammation, which is selected from the group consisting of allergic dermatitis, atopic dermatitis, contact dermatitis, seborrheic dermatitis, urticaria, psoriasis, dry skin and eczema. delete delete The Crataegi Fructus was extracted and concentrated with an ethanol or ethanol aqueous solution and suspended in water and then fractionated and concentrated with ethyl acetate and n-butanol to obtain ethyl acetate fraction or n-butanol fraction A cosmetic composition for prevention or improvement of skin inflammation, which is selected from the group consisting of allergic dermatitis, atopic dermatitis, contact dermatitis, seborrheic dermatitis, urticaria, psoriasis, dry skin and eczema. delete

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