KR20140056192A - Anti-inflammation composition and an improvment composition of atopic dermatitis using an extract of immature fruit of diospyros kaki - Google Patents

Abstract

The present invention discloses an anti-inflammation composition and an atopic dermatitis alleviation composition using an extract of immature fruits of Diospyros kaki. Specifically, disclosed are an anti-inflammation composition and an atopic dermatitis alleviation composition using an extract of immature fruits of Diospyros kaki, which have activity of inhibiting production of NO, PGE2, inflammatory cytokine, etc. in macrophages stimulated with LPS and inhibiting production of MDC and TARC in HaCaT cells which is a keratinocyte cell line processed with hIFN-γ and TNF-α.

Description

[0001] The present invention relates to a composition for antiinflammation and an composition for improving atopic dermatitis using an extract and a composition for improving atopic dermatitis,

The invention persimmon (Diospyros kaki ), and compositions for improving atopic dermatitis.

Inflammation is the defensive response of a living body to external infectious agents such as external physical and chemical stimuli, bacteria, fungi, viruses and allergens.

The inflammatory response is part of the innate immune response, and as in other animals, the innate immune response of humans begins by recognizing and attacking macrophages as non-self through a pattern of cell surfaces that are specific to the pathogen. During the inflammation reaction, plasma accumulates on the inflamed area, diluting the toxicities secreted by the bacteria, increasing the blood flow, and accompanied by symptoms such as erythema, pain, edema, and fever.

These inflammatory reactions involve a variety of biochemical events, in particular, cyclooxygenase (COX), which is associated with the nitric oxide synthase (NOS) and the biosynthesis of various prostaglandins, .

There are three isomers of NOS: endotoxin of bacteria such as calcium or camodanol-dependent eNOS (endothelial NOS), nNOS (neurogenic NOS), and lipopolysaccharide (IL-1β, TNF-α, IL There are iNOS (inducible NOS) induced by various inflammatory cytokines such as IL-6, IL-8 and IL-12 and produce NO from L-arginine.

NO produced by eNOS or nNOS plays an important role in maintenance of homeostasis by performing various physiological responses related to blood pressure control, neurotransmission, learning, memory, etc. However, NO produced by iNOS is associated with arthritis, sepsis, (Moncade S. et al, Pharmacol. Rev., 1991, 43, 109, Nature Medicine, 2001, 7, 1138; Mu, S., et al., Immunoprecipitation, , MM, J. Endotoxic Res. 7, p341, 2001).

The COX enzyme synthesizes PGG ₂ and PGH ₂ from arachidonic acid with hydroperoxidase (HOX) activity together with the function of COX. These compounds include PGE ₂ , PGF ₂ , PGD ₂ , prostacyclin and create a duplex thromboxane _{a 2 (thromboxane A2, TxA2)} . Among the functions of COX, the function of PGH synthase is involved in the pain and inflammation reaction through synthesis of PGE ₂ .

There are two subtypes of COX and COX-1 is always expressed in most tissues, whereas COX-2 is rapidly induced by inflammatory cytokines and plays an important role in the inflammatory response.

Therefore, NO, PGE ₂ , Inflammatory cytokine production inhibitors can be used as therapeutic agents for inflammatory diseases.

On the other hand, atopy is derived from the Latin word 'atopia' and has the meaning of 'odd' or 'inappropriate'.

The cause of atopic dermatitis has not yet been clarified yet. However, the cause of atopic dermatitis is not clear, but the number of IgE production, CD8 + suppression / cytotoxic T lymphocyte decrease and dysfunction, Th1 (T- cell Helper type 1) Numerical increase of lymphocyte, mononuclear / macrophage infiltration, mast cell, eosinophil, dendritic cells (DCs), increase of epidermal Langerhans cell and CD4 + T lymphocyte, Increased production of cytokines (IL-10, IL-4, IL-5, TNF-α, etc.) and increased production of chemokines (TSLP, RANTES, CTACK, LARC, MDC, TARC etc.) Have been reported [J. Invest. Dermatol., 96: 523-526,1991; J. Invest. Dermatol., 97: 389-394,1991; Immunol., 11: 81-88, 1999; Curr Drug Targets Inflamm Allergy., 2: 199-120, 2003; J. Allergy Clin Immunol., 107: 871-877, 2001; Adv. Immunol. 78:57, 2001; International Immunology, 14 (7): 767-773, 2002; Pediatr Allergy Immunol 19: 605-613, 2008], a person suffering from atopic dermatitis generally exhibits an allergic reaction to foreign substances such as house dust, ticks, animal hair, food, pollen and mold, It is presumed that atopic dermatitis is caused by disturbance of the immune system in that it accompanies allergic rhinitis, asthma, conjunctivitis and intestinal inflammation.

Experiments for inhibiting degranulation using mast cells for the screening of atopic dermatitis active substances and experiments for measuring the expression levels of MDC (macrophage-derived chemokine) and TARC (thymus and activation-regulated chemokine) in keratinocytes , MDC and TARC are chemokines reported to have significantly increased serum levels in patients with atopic dermatitis (Hijnen et al., J. Allergy Clin. Immunol. 113 (2) 334-340), treatment of atopic dermatitis It has been reported that when ciclosporin A or corticosteroids are administered to patients with atopic dermatitis, the serum levels of MDC and TARC are decreased (Y. Shimada et al., J. Dermatol. Sci., 34, 201 -208, 2004), and when hIFN-γ and TNF-α were treated with human keratinocyte cell line HaCaT cells, MDC and TARC were expressed in large quantities. Substances capable of inhibiting such expression are atopic dermatitis (Horikawa et al., Int. Immunol., 14 (7) 767-773, 2002).

The present invention is based on the finding that NO, PGE ₂ , And extracts of Haemaphylococcus epidermidis having inhibitory activity against the production of MDC and TARC in hIFN-y and TNF-a treated HaCaT cells.

It is an object of the present invention to provide a composition for antiinflammation using an extract and a composition for improving atopic dermatitis.

Other objects and specific objects of the present invention will be described below.

As shown in the following Examples and Experimental Examples, the inventors of the present invention prepared a 1-year, 3-year aged product with a pressurized extract and an anti-inflammatory activity thereof in a macrophage stimulated with LPS, PGE ₂ , Inflammatory cytokine. The results showed that the anti - inflammatory activity was increased in the order of 1 - year and 3 - year aged extracts.

The present inventors also extracted extracts of 30% and 70% ethanol with distilled water at room temperature, 60 ° C, and 100 ° C, respectively, to prepare extracts for each extraction condition. The extracts of atopic dermatitis were divided into hIFN -γ, and TNF-α. The results showed that the extracts of 100 ℃ and 30% ethanol showed high activity especially on the MDC and TARC production inhibitory activities. The extracts of 100 ℃ and 30% ethanol extracts which were found to be highly active in cell experiments were incubated at room temperature (100 ℃) and 30% ethanol extract The extracts showed markedly higher activity against atopic dermatitis.

In view of the above-mentioned experimental results, the anti-inflammatory composition of the present invention is characterized in that the anti-inflammatory composition of the present invention contains an aged product of an undiluted extract and an extract, particularly an undiluted extract and a pressurized extract (an extract obtained by pressing the unrefined undiluted extract with a physical external force) In another aspect, the composition can be identified as a composition for improving atopic dermatitis, which comprises an extract of hot-water extract at 100 ° C or a 30% ethanol extract of unripe starch as an active ingredient. Where "%" is the concentration (v / v) by volume percentage and refers to a volume of solute dissolved in a volume of solution as is known in the art. For example, 30% (v / v) ethanol means that 30 ml of ethanol is dissolved in 100 ml of water.

In the present specification, the term " undamaged seed "means a sense before the harvest, more specifically, a sense that the surface is completely green.

In the present specification, the term "active ingredient" alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which is itself inactive.

As used herein, "anti-inflammatory" is meant to include the improvement, treatment, inhibition or delay of the onset of an inflammatory disease as defined below.

In the present specification, the term "inflammatory disease" means a disease accompanied by an inflammatory reaction specified by a local or systemic bio-defense reaction against an external infectious source such as external physical or chemical stimuli or bacterial, fungal, Be defined. The diseases accompanied by such an inflammatory reaction include various inflammatory mediators and activations of enzymes (e.g., iNOS, COX-2 etc.) associated with immune cells, secretion of inflammatory mediators such as NO, TNF-a, IL-6, , PGE ₂ secretion), body fluid infiltration, cell migration, and tissue destruction, and is accompanied by a series of complex physiological responses such as erythema, pain, edema, fever, deterioration or loss of specific function of the body, appear. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as it is included in the definition of inflammatory diseases as described above, it may be acute, chronic, ulcerative, allergic, Whether it is necrotic or not. Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, Inflammatory bowel syndrome, inflammatory pain, migraine headache, headache, back pain, fibromyalgia, fascia disease, viral infection (e.g., C type infection), bacterial infection, fungal infection, burn, wound due to surgical or dental surgery, These may include Prostaglandin E Overdose Syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis, eczema and multiple sclerosis.

As used herein, "atopic dermatitis" is defined as including any disease classified as atopic dermatitis in the art, regardless of the cause, either directly or indirectly, of its occurrence. In general, atopic dermatitis is classified into infantile atopic dermatitis, pediatric atopic dermatitis, adult atopic dermatitis, and maternal atopic dermatitis according to the onset period or the object of the invention. In the present specification, Of atopic dermatitis.

The composition for antiinflammation and the composition for improving atopic dermatitis of the present invention (hereinafter, referred to as "composition of the present invention") can be used for an anti-inflammatory activity, an atopic dermatitis- (Effective amount) depending on the purpose of administration, formulation, purpose of formulation, etc. A typical effective amount will be determined within the range of 0.001 wt% to 99.900 wt% based on the total weight of the composition. The term "effective amount" as used herein refers to an amount of an effective ingredient capable of inducing prevention, amelioration, treatment, or delayed onset of symptoms of an inflammatory disease or atopic dermatitis. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

The composition of the present invention can be identified as a cosmetic composition in a specific embodiment.

When the composition of the present invention is recognized as a cosmetic composition, the cosmetic composition may be prepared in various forms, for example, emulsion, lotion, cream (oil-in-water type, oil water type, multiphase), solution, suspension Water, water), anhydrous products (oil and glycol), gels, masks, packs or powders.

The composition of the present invention may contain an acceptable carrier in cosmetic preparations in addition to its active ingredients.

As used herein, the term " acceptable carrier for a cosmetic preparation "refers to a compound or composition which is already known and used in the cosmetic preparation, or which is a compound or composition to be developed in the future, and which is not toxic to the human body.

The carrier may be included in the composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from about 50% by weight to about 99% by weight of the composition, based on the total weight thereof.

However, since the ratio depends on the above-mentioned formulation of the cosmetic product and its specific application site (face or hands) or the desired amount of application thereof, the ratio is to limit the scope of the present invention in any aspect It should not be.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosifiers, emulsifiers, stabilizers, sunscreens, coloring agents and perfumes.

The compounds / compositions which can be used as the carrier and which can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosifiers, emulsions, stabilizers, sunscreens, A person skilled in the art can select and use appropriate substances / compositions.

The composition of the present invention can be identified as a soap composition in another specific embodiment.

When the composition of the present invention is identified as a soap composition, the soap composition of the present invention can be prepared by incorporating an active ingredient into a soap base, and can be prepared as an additive including a skin moisturizer, an emulsifier, a water softener and the like.

Examples of the soap base material include vegetable oils such as palm oil, palm oil, soybean oil, perm free oil, olive oil and palm kernel oil or animal fats such as tallow, lard, sunshine and fish oil. Examples of the skin moisturizers include glycerin, erythritol, polyethylene glycol , Propylene glycol, butylene glycol, pentylene glycol, hexyl glycol, isopropyl myristate, silicone derivatives, aloe vera, sorbitol and the like. Examples of the emulsifier include natural oils, wax fatty alcohols, hydrocarbons, May be used. As the water softening agent, tetrasodium ethylenediate and the like may be used.

The soap composition of the present invention may further contain, as an additive, an antibacterial agent, a dispersant, a foam inhibitor, a solvent, a scouring inhibitor, a corrosion inhibitor, a fragrance, a dye, a sequestering agent, an antioxidant and an antiseptic.

In the soap composition of the present invention, the soap base or additive may be included in a content commonly used in the art, wherein the soap base generally comprises from 99.999% to 50% by weight, based on the total weight of the soap composition And the additive may be added in an amount of 1 wt% to 20 wt%.

The composition of the present invention can be identified as a pharmaceutical composition in another specific embodiment.

The pharmaceutical composition of the present invention may be in the form of oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (including sterile injectable aqueous Or oily suspensions), stationary formulations (such as creams, lotions, ointments (semi-solid external preparations), micro loo emulsions, gels, pastes, transdermal preparations (e.g., patches, bandages, etc.).

The term "pharmaceutically acceptable" as used herein means that the application (prescribing) subject does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (e.g., corn starch, potato starch and the like), cellulose, derivatives thereof (e.g. sodium carboxymethylcellulose, ethylcellulose, etc.), malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g., TWEENS), wetting agents (For example, sodium lauryl sulfate), a coloring agent, a flavoring agent, a tableting agent, a stabilizer, an antioxidant, a preservative, water, a saline solution and a phosphate buffer solution. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, when the pharmaceutical composition of the present invention is prepared by an aqueous suspension, suitable excipients include sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose , Sodium alginate, polyvinylpyrrolidone, and the like. Suitable excipients when prepared from injection solutions include Ringer's solution, isotonic sodium chloride, and the like.

The pharmaceutical compositions of the present invention may be administered orally or parenterally, and preferably topically.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

In a specific embodiment, the composition of the present invention can be identified by a food composition such as a functional beverage.

The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof.

Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

Examples of the physiologically active substance include catechins such as catechin, epicatechin, gallocatechin and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin.

As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium and zinc can be used.

In addition, the food composition of the present invention may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners.

Such preservatives, emulsifiers and the like are preferably added in a very small amount as long as they can attain an application to which they are added. The term " trace amount " means, when expressed numerically, in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition.

Examples of the preservative which can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid).

Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin and the like.

Examples of the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.

According to another aspect of the present invention, there is provided an antimicrobial composition for Staphylococcus aureus comprising an aged product of unripe and crushed extract as an active ingredient.

Staphylococcus aureus used in the following experiment is a microorganism which is known to cause septicemia, boils, acne and the like in patients whose skin is not normally a medical problem but immunity is low. The present inventors have confirmed the antimicrobial activity against Staphylococcus aureus in the unsweetened and squeezed extract and its aged material through the minimum bacteriostatic concentration, but the minimum bacteriostatic concentration was not measured in the case of unstained and compressed extracts In the case of the aged material, the minimum bacteriostatic concentration was 2 mg / mL.

The antimicrobial composition of the present invention may be incorporated into the antimicrobial composition of the present invention in any amount as long as the active ingredient of the extract and the extract thereof exhibit antimicrobial activity. Usually, the composition is based on the total weight of the composition By weight, more preferably 0.1 to 50% by weight, and more preferably 3 to 10% by weight.

The composition for an antimicrobial of the present invention may contain a dispersing agent, a carrier and the like in addition to the active ingredient thereof, so long as it does not inhibit the active ingredient, unrefined and the antimicrobial activity of the extract.

Such dispersants include water, alcohols such as methyl alcohol, ethyl alcohol, ethylene glycol, propylene glycol, diethylene glycol, glycerin, etc., ketones such as acetone and methyl ethyl ketone, ethers such as dioxane, Aliphatic hydrocarbons such as hexane and kerosene; aromatic hydrocarbons such as benzene, toluene, xylene, naphthalene and methylnaphthalene; halogenated hydrocarbons such as chloroform, Carbon tetrachloride and the like), acid amides (e.g., dimethylformamide), esters (e.g., methyl acetate ester, ethyl acetate ester, butyl acetate ester, fatty acid glycerin ester and the like), nitrol (e.g., acetonitrile) Surfactants (higher alcohol sulfate esters, alkyl sulfonic acids, alkyl allyl sulfonic acids, quaternary ammonium salts, oxyalkylamines, fatty acid esters A polyalkylene oxide compound, and an anhydrous sorbitol compound). The dispersant may be prepared by incorporating the dispersant into the antimicrobial composition of the present invention, alone or in a mixture of two or more.

Examples of the carrier include clay (for example, kaolin, bentonite, acid clay, etc.), talc (for example, talc powder and tin powder), silica (for example, diatomaceous earth, silicic anhydride, mica powder, etc.) These carriers may be prepared by being contained in the antimicrobial composition of the present invention alone or as a mixture of two or more kinds.

In addition, the antimicrobial composition of the present invention may be prepared in any of liquid, solid and gas phases. The antimicrobial composition of the present invention may be administered by any method such as oral, parenteral, or the like, and preferably, it may be administered by a local administration method. The parenteral formulation may be in the form of an injection, a topical preparation (solution, cream, ointment, gel, lotion, patch), a suppository, (When applied to plants) and the like. Particularly, topical administration formulations include those in which the antimicrobial composition of the present invention is impregnated with a carrier made of natural fibers or synthetic fibers, those contained in cosmetics, soaps, and the like.

The antimicrobial composition of the present invention may be manufactured as a cosmetic product, a food, a medicine, or the like, and may be added to the production of these products.

As described above, according to the present invention, it is possible to provide a composition for antiinflammation and a composition for ameliorating atopic dermatitis using an extract and an extract.

The composition of the present invention can be used as a cosmetic product, medicine, food or the like.

Fig. 1 shows the results of showing the NO production inhibitory activity of the unripe and squeezed extract and its aged material.
2 to 4 are the results showing the activity of inhibiting the production of inflammatory cytokines in the unripe and squeezed extracts and aged products thereof.
Fig. 5 shows the results of showing the inhibitory activity of PGE _{2 on the} unripe and squeezed extract and its aged material.
FIG. 6 shows the results of TARC and MDC inhibitory activities of water extracts at 60 ° C and 100 ° C at room temperature, and 30% and 70% ethanol extracts, respectively.
FIGS. 7 and 8 are photographs showing the minimum bacteriostatic concentration of 1-year and 3-year aged extracts of unripe and crushed extract, respectively.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Preparation of extract and its aged product

&Lt; Example 1 >

Jeju, Jeju, was purchased in June and used as a material for this experiment. The extracts were then lyophilized and then extracted with distilled water at room temperature, 60 ℃ and 100 ℃ for more than 2 hours. The extracts were further extracted with ethanol at 30% and 70% ethanol for more than 2 hours. The obtained extracts were concentrated using a vacuum concentrator and completely free of water using a freeze dryer, and then used in the following experiments.

<Example 2> Preparation immature persimmon extract and compression and its aging water

The extracts were squeezed out and the extracts were filtered to produce liquid unsampled and compressed extracts. The aged material was prepared by immersing and immersing the liquid immature and extracts at room temperature for 12 months and 36 months, respectively.

< Experimental Example > Persimmon extract or its Aged  Anti-inflammatory activity, atopic dermatitis improvement activity and antibacterial activity test

< Experimental Example  1> Anti-inflammatory activity experiment

<Experimental Example 1-1> NO (Nitric oxide) Inhibitory evaluation

RAW 264.7 cells were adjusted to 1.5 × 10 ⁵ cells / ml using DMEM medium supplemented with 10% FBS, and then inoculated on a 24-well plate. LPS (1 μg / ml) Lt; / RTI &gt; The amount of produced NO Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid] by using the NO ₂ present in the cell culture ^- in the form of Respectively. 100 μl of the cell culture supernatant and 100 μl of Griess reagent were mixed and reacted on 96-well plates for 10 minutes, and the absorbance was measured at 540 nm. The amount of NO produced was compared with sodium nitrite (NaNO ₂ ) as standard.

In this experiment, the compressed extract of Example 2 and its aged material were used as a sample, and the results are shown in FIG.

1, it can be seen that the aged samples have higher activity of inhibiting the NO production than the squeezed extract, and that the activity of 3-year-aged water is higher than that of 1-year aged water.

<Experimental Example 1-2> Evaluation of inhibitory activity on the production of inflammatory cytokines ( TNF- α, IL- 1β and IL- 6)

RAW 264.7 cells (1.5 × 10 ⁵ cells / ml) were inoculated into a 24-well plate using DMEM medium, and cultured in 5% CO ₂ And cultured in a thermostat for 18 hours. After the medium was removed, the sample of Example and LPS (1 / / ml) were simultaneously treated and cultured under the same conditions as the pre-culture. After 24 hours, the production of inflammatory cytokines in the supernatant obtained by centrifuging the culture medium (12,000 rpm, 3 minutes) was measured. Inflammatory cytokines were quantified using the mouse enzyme-linked immnunosorbent assay (ELISA ) kit (R & D Systems Inc., Minneapolis, MN, USA) r 2 value of the standard curve for the standard was at least 0.99.

In this experiment, the crushed extract of Example 2 and its aged material were used as samples, and the production of TNF-α, IL-1β and IL-6 in the pressurized extract of Example 2 and its aged material was inhibited The activities are shown in FIG. 2 to FIG. 4, respectively.

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, α, IL-1β and IL-6, respectively.

<Experimental Example 1-3> Evaluation of inhibitory activity of PGE ₂ formation

RAW 264.7 cells were adjusted to 1.5 × 10 ⁵ cells / ml using DMEM medium, and then inoculated into a 24-well plate and cultured for 18 hours in a 5% CO ₂ incubator. After the medium was removed, the sample of the example and LPS (1 / / ml) were simultaneously treated and cultured under the same conditions as the pre-culture. After 24 hours, the supernatant was obtained by centrifuging the culture medium (12,000 rpm, 3 min) to measure PGE ₂ . PGE ₂ was quantitated using a PGE ₂ ELISA kit (R & D Systems Inc., Minneapolis, MN, USA). The r ² value of the standard curve for the standard was 0.99 or more.

In this experiment, the compressed extract of Example 2 and its aged material were used as a sample. The results are shown in Fig. 5, and similarly, the press extract, its 1-year aged material and its 3-year aged material Respectively.

<Experimental Example 1-4> Evaluation of cytotoxicity ( LDH assay )

RAW 264.7 cells (1.5 × 10 ⁵ cells / ml) were co-treated with DMEM medium and LPS (1 μg / ml) in DMEM medium for 24 hours, and then cultured for 3 minutes at 3,000 rpm for 5 minutes. The LDH (lactate dehydrogenase) assay was performed using a non-radioactive cytotoxicity assay kit (Promega). 50 μl of the culture medium obtained by centrifugation on a 96-well plate and 50 μl of the reconstituted substrate mix were added and reacted at room temperature for 30 minutes After adding 50 μl of stop solution, absorbance was measured at 490 nm using a microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA). The average absorbance values for each sample group were determined and compared with the absorbance values of the control (LDH control, 1: 5000) to evaluate cytotoxicity.

In the present experiment, the compressed extract of Example 2 and its aged material were used as a sample. The results are also shown in FIG. 1. Referring to FIG. 1, the samples of the Examples show specific cytotoxicity , Indicating that the inhibitory activity on the production of inflammatory factors is not a result of cytotoxicity.

< Experimental Example  2> Experiment to improve atopic dermatitis

<Experimental Example 2-1> Evaluation of inhibitory activity of TARC and MDC which are atopic dermatitis inducers

HaCaT cells (3.0 × 10 ⁵ cells / mL) were inoculated into a 24-well plate and DMEM culture medium. Cells were cultured in a 5% CO ₂ incubator for 18 hours. Subsequently, the medium was removed and HaCaT cells were treated with 50 μl of the extract sample of Example and 450 μl of TNF-α + IFN-γ (10 ng / ml) in a fresh medium and cultured under the same conditions as the pre-culture. After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 minutes), and the amount of atopy inducing factor produced in the supernatant was measured. Determination of TARC and MDC was quantified using the mouse enzyme-linked immnunosorbent assay (ELISA ) kit (R & D Systems Inc., Minneapolis, MN, USA) r 2 value of the standard curve for the standard was at least 0.99.

In this experiment, only the extract of each condition of Example 1 was used as a sample.

The results are shown in Fig. Referring to FIG. 6, the activity of Safflower extract, 100 ° C extract and 30% ethanol extract are particularly high.

Meanwhile, the experiment on the cytotoxicity of the extract sample according to each condition of Example 1 was performed in the same manner as in <Experimental Example 1-4>, and the results thereof are shown in FIG. 6 together. I did not see it. This suggests that the inhibitory activity of TARC and MDC, which are atopic dermatitis inducing factors, is not a result of cytotoxicity.

<Experimental Example 2-2> Clinical trial for improving atopic dermatitis

Nutritional lotions were prepared with the ingredients and contents shown in the following Table 1 for the clinical test for the improvement of atopic dermatitis.

Ingredients and quantities of nutrition lotion ingredient content
(weight%) 100% extract or 30% ethanol extract of Example 1 2.0 Stearic acid 2.0 Cetyl alcohol 2.0 Glyceryl monostearate 2.0 Polyoxyethylene sorbitan monostearate 0.5 Sorbitan sesquioleate 0.5 Glyceryl monostearate / glyceryl stearate / polyoxyethylene stearate 1.0 Wax 1.0 Liquid paraffin 4.0 Squalane 4.0 Caprylic / capric triglyceride 4.0 Carboxyvinyl polymer 0.3 Butylene glycol 5.0 glycerin 3.0 Triethanolamine 0.5 Purified water Balance

The atopic dermatitis improving activity was divided into three groups of 60 men and women having symptoms of atopic dermatitis over 5 years old and each nutrient lotion containing the extract of the above example was applied for 2 to 3 times a day for 8 weeks .

Before and after the start of the study, the SCORAD Index was developed by the European Task Force on Atopic Dermatitis, and the severity of atopic symptoms was high.

The results are shown in Table 2 below as mean ± standard deviation.

SCORAD Index division 0 parking 8 parking The nutrient lotion containing the normal temperature extract of Example 1 33.5 ± 4.7 28.4 ± 5.2 The nutrient lotion containing 100% extract of Example 1 31.2 ± 6.4 20.5 ± 6.2 * The nutrient lotion containing 30% ethanol extract of Example 1 35.6 ± 7.8 21.4 ± 4.6 * * p < 0.05

The results of the above Table 2 showed no significant difference in the case of the nutrient lotion containing the normal temperature extract of Example 1, but the 100 ° C extract and the 30% ethanol extract of Example 1 showed a significant difference And the mean value of the SCORAD index is also significantly lowered.

Statistical processing

For the analysis and analysis of the results of the clinical trial, Excel program and statistical software SPSS Window. Version 10.1 was used. For statistical significance, a significance level of 0.05 was set. Statistical methods used for analysis were analyzed by the independent sample T-test and the corresponding sample T-test.

< Example  3> Experiment of antibacterial activity  - Minimum bactericidal concentration ( Minimal bactericidal  concentration, MBC )

Staphylococcus aureus ( S. aureus KCTC 1927), a strain commonly found in the skin, was used and the medium used was Trypticase soy agar / broth (TSA) (Difco, USA) , And cultured at 37 ° C.

Measurement of the minimum bacterial killing concentration was carried out by directly spraying 100 μl of the culture solution of the strain in the solid medium containing the sample of the concentration by concentration and directly counting the number of colonies observed visually on the plate after the strain was cultured. When the colony count was confirmed to have the effect of killing 99.9% of the inoculated bacteria inoculated from the culture broth, the treatment concentration was determined as the minimum bacterial killing concentration (MBC) (Cohen MA, Huband MD, Yoder SL , Gage JW, Roland GE, 1998, Bacterial eradication by clinafloxacin, CI-990, and ciprofloxacin employing MBC test, in-vitro time-kill and in-vivo time-kill studies J Antimicrob Chemother . 41 (6): 605-14).

In this experiment, the squeezed extract of Example 2 and the aged material thereof were used as samples, and the results are shown in Table 3 below.

MBC (Minimal bactericidal concentration, mg / mL) Strain Squeezed extract 1 year ripening Aged 3 years S. aureus - 2 2

The results of the above Table 3 show that MBC was not measured for Staphylococcus aureus in the case of the squeezed extract, but all of the aged samples showed a minimum killing level of 2 mg / mL.

[Fig. 7] and [Fig. 8] are photographs showing microbial cultures of 1-year aged water and 3-year aged water, respectively.

Claims (4)

The present invention relates to a composition for improving atopic dermatitis, which comprises as an active ingredient 30% ethanol extract of Panax notoginseng and 100% hot water extract or safflower seed.
The method according to claim 1,
Wherein the composition is a cosmetic composition.
The method according to claim 1,
Wherein the composition is a pharmaceutical composition.
The method according to claim 1,
Wherein the composition is a food composition.

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