KR20120118188A - Compostion for reducing irritation and inflammaion in skin comprising extracts of astragalus membranaceus - Google Patents

Abstract

PURPOSE: A skin irritation or dermatitis alleviating composition including Astragali Radix extracts is provided to Promote proliferation of skin dermis cells and to have protective effects on UVB. CONSTITUTION: A skin irritation or dermatitis alleviating composition includes Astragali Radix extracts as active ingredients. The Astragali Radix extract is extracted by water, C1-2 lower alcohol or a mixed solvent thereof. The C1-2 lower alcohol is methanol or ethanol. The skin stimulant is itching, erythema, edema, or skin irritation. The skin inflammation is atopic dermatitis, contact dermatitis, seborrhea or pimple. The Astragali Radix extract suppresses the expression or activation of the NF- κ B. The cosmetic composition improves or prevents wrinkles or aging caused by the dermatitis. An external for preventing or improving skin irritation or dermatitis includes the Astragali Radix extract as the active ingredient.

Description

Compensation for reducing irritation and inflammaion in skin comprising extracts of Astragalus membranaceus}

The present invention relates to a composition for alleviating skin irritation or skin inflammation, and more specifically, Astragalus membranaceus ) relates to a composition containing the extract as an active ingredient.

 Skin is a part of the body that is directly exposed to the external environment. Skin irritation, atopic dermatitis, contact dermatitis, such as erythema, swelling, itching, and peeling when exposed to excessive ultraviolet rays, contaminants, or irritants. Inflammatory reactions such as seborrhea and acne. The skin trouble caused by the skin irritation stress is not only aesthetic problem, but also the substances produced during the inflammatory reaction incidentally cause pigmentation of the skin and promote the breakdown of the skin elastic fibers to affect the increase of skin wrinkles. Known (Toxicol In Vitro. 2004; 18: 231-43, Irritant dermatitis. 1996; pp. 87-111).

 Until now, mainly antihistamines, vitamin ointments or corticosteroids have been used to treat such inflammatory skin diseases. However, these drugs are often temporary and often have severe side effects.

Various biochemical phenomena are involved as a cause of inflammation in living bodies. When a specific stimulus is applied to a cell, the cell receives the stimulus as a signal and adjusts the DNA design to pick up the necessary protein. NF-κB is a protein complex in the cytoplasm that penetrates the nuclear membrane and increases the gene expression rate by binding to and stimulating specific sites (specific sites of the gene) of DNA specific sequences. When NF-κB binds to specific gene DNA in the nucleus, it overprints specific enzymes or proteins. NF-κB plays an important role in the development and progression of tumors as well as immune activity and inflammatory response systems. NF-κB also regulates the biological clock of aging. Aging tends to diminish the ability to regulate inflammatory and immune responses and inhibit tumor growth. This is associated with an increase in NF-κB production. Stimulation of NF-κB increases the rate of expression of certain genes, leading to an increase in the production of related enzymes and proteins, which abnormally increases inflammatory prostaglandins and other eicosanoids production. In addition, increased NF-κBB activity causes the immune system to be overactivated, exacerbating various autoimmune diseases and inflammatory responses. Therefore, limiting NF-κB production and activity can inhibit the early pathways and processes of various biological inflammatory response systems. NF-κB is also closely associated with the nitric oxide system, which promotes the overproduction of nitric oxide. When cells receive harmful signals (stress, insulin resistance, free radicals, cytokines, ultraviolet rays, viral infections, etc.), NF-κB increases, and activated NF-κB migrates to the nucleus, iNOS, COX-2 and IL-1β or By promoting the expression of genes that induce inflammatory reactions, such as various types of cytokines such as TNF-α, cancer-cellifying proteins and inflammation-enzyme proteins increase, causing cancer, autoimmune diseases, viral infections, septic shock and the like.

Inflammation of the skin is caused by UV rays, dryness, contaminants, and the like, and these factors cause collagen fibers and elastic fibers to break down, resulting in dry skin or deepening of wrinkles.

Hwanggi is a perennial plant of the dicotyledonous rosewood legume, which grows in the cracks of the mountains. It is 40 ~ 70cm high and has white soft fine hairs. Stem is gross and leaf is odd one-time quill leaf consisting of 6-11 pairs of small leaves. Small leaf is about 1 ~ 2 cm long, oval-shaped oval, and leaf is flat. Chin leaves are lanceolate with long tips. A long flower spike comes from the axilla as a gunshot, and 5 to 10 flowers run. Yellow-white flowers bloom in July-August, about 2cm long, small peduncle is about 3mm long. Calyx is about 5 mm long, dark brown hairs, and divided into 5 pieces. There are 10 stamens and fruits are fruited in November. The pod is long oval, pointed at both ends, 2 ~ 3cm long, and contains 5 ~ 7 seeds. It is distributed in Korea, Japan, Manchuria, northeastern China and eastern Siberia. It is often cultivated as a herb, and it is harvested in the fall of oriental medicine to remove the outcrops and small roots, and dried in the sun, called the yellow spice of herbal medicine, tonic, jihan, diuresis, small seed, etc. Because of the efficacy of physical weakness, fatigue boredom (혈), blood loss (氣血 虛脫), hangang (脫肛), uterine decay, visceral sewage, cold sweat, peripheral nerves are prescribed.

For this reason, in order to reduce skin irritation or skin inflammation caused by various stimulants, it is necessary to develop substances or compositions having anti-inflammatory or skin irritation efficacy.

Therefore, the present inventors have studied to develop natural plant extracts for skin irritation or skin inflammation, and as a result, Astragalus extract promotes the proliferation of dermal dermal cells, protects against UVB, expresses NF-κB and the nucleus. By confirming that there is a translocation inhibitory effect and a procollagen (procollagen) increase effect, the present invention was completed by revealing that the Astragalus extract can be usefully used in a composition for skin irritation or skin inflammation alleviation.

It is an object of the present invention to provide a cosmetic composition for skin irritation or skin inflammation alleviation.

Another object of the present invention is to provide an external preparation for skin for the prevention and improvement of skin irritation or skin inflammation.

Another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of skin irritation or skin inflammation.

Another object of the present invention is to provide a health food for preventing and improving skin irritation or skin inflammation.

In order to achieve the above object, the present invention Astragalus membranaceus ) provides a cosmetic composition for skin irritation or skin inflammation alleviation containing the extract as an active ingredient.

In addition, the present invention Astragalus membranaceus ) provides an external skin preparation for the prevention and improvement of skin irritation or skin inflammation containing the extract as an active ingredient.

In addition, the present invention Astragalus membranaceus ) provides a pharmaceutical composition for the prevention and treatment of skin irritation or skin inflammation containing the extract as an active ingredient.

In addition, the present invention Astragalus membranaceus ) provides a health food for the prevention and improvement of skin irritation or skin inflammation containing the extract as an active ingredient.

Astragalus of the present invention membranaceus extract stimulates skin dermal cell proliferation, protects against UVB, inhibits the expression of NF-κB and translocation to the nucleus, and increases procollagen, so that skin irritation or skin It can be usefully used as a composition for alleviating inflammation.

1 is a diagram showing the appearance of Astragalus membranaceus .
Figure 2 is a diagram showing the cell proliferation effect by Astragalus extract in Hs68 fibroblasts. Data are presented as percentages relative to the control and averaged over three replicates. * Significant difference between controls (*: p <0.05, ***: p <0.001)
Figure 3 is a diagram showing the effect of Astragalus extract on UVB-induced activity of NF-κB P65 in Hs68 fibroblasts. Astragalus extracts were irradiated with UVB (40 mJ / cm 2) in cultured cells with or without pretreatment (10 μg / ml, 100 μg / ml and 300 μg / ml) for 24 hours. Cells were harvested 5 hours after UVB irradiation and cell lysates were prepared for NF-κB activity assay using Western blot analysis.
4 is an immunocytochemical staining assay that inhibits the relocation of NF-κB P65 into the nucleus in Astragalus-treated (100 μg / ml) and 40 mJ / cm 2 UVB exposed human skin fibroblasts. NF-κB P65 localization from the cytoplasm to the nucleus is shown to be stained darker than the UVB control. The white arrows indicate that NF-κB P65 is relocated to the nucleus. Cultured cells were incubated with anti-p65- antibody at 4 ° C. overnight as described in the Examples of the present invention. Representative fluorescence images were obtained with a fluorescence microscope. Magnification 400 ×.
Figure 5 shows the effect of Astragalus on UVB-induced activation of procollagen in fibroblasts. Astragalus extracts were irradiated with UVB (40 mJ / cm 2) in cultured cells with or without pretreatment (10 μg / ml, 100 μg / ml and 300 μg / ml) for 24 hours. Cells were harvested 5 hours after UVB irradiation and cell lysates were prepared. Total protein levels of procollagen were detected with specific antibodies using Western blot analysis as described in the Examples.

Hereinafter, the present invention will be described in detail.

The present invention is Astragalus membranaceus ) provides a cosmetic composition for skin irritation or skin inflammation alleviation containing the extract as an active ingredient.

The Astragalus extract is preferably prepared by a manufacturing method including the following steps, but is not limited thereto.

1) extracting by adding an extraction solvent to the Astragalus;

2) cooling the extract of step 1) and filtering;

3) concentrating the filtered extract of step 2) under reduced pressure; And

4) dry powdering the concentrated extract of step 3).

In the above method, the sulfur group of step 1) can be used without limitation, such as those grown or commercially available.

The extraction solvent is preferably water, alcohol or a mixture thereof, but is not limited thereto. As the alcohol, it is preferable to use C ₁ to C ₂ lower alcohols, and as the lower alcohol, ethanol or methanol is preferably used, but is not limited thereto. As the extraction method, it is preferable to use distillation extraction, shaking extraction or reflux extraction, but not always limited thereto. Preferably, the extraction solvent is extracted by adding 2 to 10 times the amount of high water to the extract. Extraction temperature is preferably from room temperature to 100 ℃ but is not limited thereto. In addition, the extraction time is preferably 2 to 12 hours, but is not limited thereto. The number of times of extraction is preferably 1 to 3 times, but is not limited thereto. In a specific embodiment of the present invention, the lyophilized extract obtained 13.9 g, the yield was 13.9%.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. In addition, the drying of step 4) is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but not always limited thereto.

The skin irritation is preferably any one selected from the group consisting of erythema, edema, itching and peeling, but is not limited thereto.

The skin inflammation is preferably any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea and acne, but is not limited thereto.

The Astragalus extract preferably inhibits the expression or activity of NF-κB, but is not limited thereto.

The cosmetic composition is preferred to improve or prevent wrinkles or aging resulting from skin inflammation, but is not limited thereto.

In a specific embodiment of the present invention, the survival rate of Hs68 fibroblasts in the experiments not irradiated with UV light was increased to 89.93%, 105.41% and 113.93% compared to the control at the concentrations of Astragalus extract 10, 100 and 300 μg / ml, respectively. . In addition, UVB 40 mJ irradiation significantly increased the concentration of 111.76%, 141.17% and 139.70% compared to the control at each concentration. The survival rate of Hs68 cells at the concentration of 100 μg / ml of Astragalus was the highest as 141.17% compared to the control group (FIG. 2).

In a specific embodiment of the invention, Western blot analysis shows normal and UVB 40 mJ / cm 2. The irradiated control (control) was measured in the range of the drug concentration 10 ㎍ / ㎖, 100 ㎍ / ㎖, 300 ㎍ / ㎖ as a control. As a result, the amount of expression of NF-κB P65 in the control group was increased compared to the normal group (normal) and can be seen to decrease compared to the drug concentration (Fig. 3).

In a specific embodiment of the present invention, immunocytochemistry was performed to determine how Astragalus affects the NF-κB pathway expressed by UVB. As NF-κB P65 was translocated by UVB, it was confirmed that the concentration was higher in the nucleus than in the cytoplasm. After NF-κB P65 is activated, it moves to the nucleus and functions as a gene that causes transcription. In the cells treated with 100 ㎍ / ㎖ of the Astragalus concentration, the inhibitory effect appeared, it can be seen that the translocation is reduced (Fig. 4).

In a specific embodiment of the present invention, in order to determine the effect of Astragalus extract on procollagen synthesis that affects the matrix of skin cells in Hs68 fibroblasts, the degree of protein synthesis was performed at astragalus concentration of 100 μg / ml. saw. Compared to the control (control) showed a relatively high increase in procollagen synthesis (Fig. 5). Procollagen inhibited by UVB irradiation showed a protective effect against UVB by inhibiting the translocation of NF-κB P65 to a certain concentration of sulfur.

Therefore, Astragalus extract promotes proliferation of dermal dermal cells, protects against UVB, inhibits expression of NF-κB and translocation to the nucleus, and increases procollagen. It can be usefully used as a cosmetic composition for alleviating inflammation.

The cosmetic composition may be in the form of a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic (liposome) A cream, a skin, a lotion, a powder, an ointment, a spray, or a cone stick. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the cosmetic composition, in addition to the organic extract of the present invention, fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, Commonly used in water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics And any other ingredients that may be used, such as auxiliaries commonly used in the cosmetic field.

In the cosmetic composition containing the Astragalus extract of the present invention, the extract of the present invention may be added to the cosmetic composition usually contained in an amount of 1 to 15% by weight, preferably 2 to 10% by weight.

In addition, the present invention Astragalus membranaceus ) provides an external skin preparation for the prevention and improvement of skin irritation or skin inflammation containing the extract as an active ingredient.

The skin irritation is preferably any one selected from the group consisting of erythema, edema, itching and peeling, but is not limited thereto.

The skin inflammation is preferably any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea and acne, but is not limited thereto.

The Astragalus extract preferably inhibits the expression or activity of NF-κB, but is not limited thereto.

The external preparation for skin is preferred to improve or prevent wrinkles or aging resulting from skin inflammation, but is not limited thereto.

When using the extract of the present invention as an external preparation for skin, it is additionally used for fatty substances, organic solvents, solubilizers, thickening and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants. Common in water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or external preparations for the skin It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients used as. In addition, the ingredients may be introduced in amounts generally used in the field of dermatology.

In addition, the present invention Astragalus membranaceus ) provides a pharmaceutical composition for the prevention and treatment of skin irritation or skin inflammation containing the extract as an active ingredient.

The skin irritation is preferably any one selected from the group consisting of erythema, edema, itching and peeling, but is not limited thereto.

The skin inflammation is preferably any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea and acne, but is not limited thereto.

The Astragalus extract preferably inhibits the expression or activity of NF-κB, but is not limited thereto.

The pharmaceutical composition is preferred to improve or prevent wrinkles or aging resulting from skin inflammation, but is not limited thereto.

The pharmaceutical composition of the present invention may further contain commonly used excipients, disintegrants, sweeteners, lubricants, flavors and the like, and may be formulated into tablets, capsules, powders, granules, suspensions, Syrups, and other liquid preparations.

Specifically, the pharmaceutical compositions of the present invention can be formulated for oral administration, for example, as tablets, troches, lozenges, aqueous or aqueous suspensions, prepared powders or granules, emulsions, hard or soft capsules, It is formulated into elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, celluloses or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, and disintegrants such as stearic acid Magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally, and it is preferable to select subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection injection method for parenteral administration. To formulate into parenteral formulations, the coriander extract of the present invention is mixed in water with a stabilizer or buffer to prepare a suspension, which is formulated in unit dosage forms of ampoules or vials.

The dosage of the active ingredient according to the present invention is appropriately selected according to the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, but in the case of oral administration in general Adults can be administered once to several times in an amount of 0.0001 to 500 mg of the extract of the present invention per kg of body weight per day, it is preferable to administer in an amount of 0.001 to 100 mg.

In addition, the present invention Astragalus membranaceus ) provides a health food for the prevention and improvement of skin irritation or skin inflammation containing the extract as an active ingredient.

The skin irritation is preferably any one selected from the group consisting of erythema, edema, itching and peeling, but is not limited thereto.

The skin inflammation is preferably any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea and acne, but is not limited thereto.

The Astragalus extract preferably inhibits the expression or activity of NF-κB, but is not limited thereto.

The health food is preferably to improve or prevent wrinkles or aging resulting from skin inflammation, but is not limited thereto.

The health food of the present invention can be used as it is, or can be used together with other food or food ingredients, or can be appropriately used in accordance with conventional methods.

There is no particular limitation on the kind of the food. Examples of foods to which the Astragalus extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, teas, and drinks. , Alcoholic beverages and vitamin complexes, and includes all of the health food in the usual sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, maltose, disaccharides such as sugar, and polysaccharides such as dextrin, cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as tautin and stevia extract, or synthetic sweeteners such as saccharin or aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the Astragalus extract of the present invention is various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols And carbonation agents used in carbonated beverages. In addition, the Astragalus extract of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples.

However, the following Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Preparation Examples.

Hwanggi ( Astragalus membranaceus ) Preparation of extract

Hwanggi used in this experiment was purchased through Wonkwang Pharmaceutical Co., Ltd., Gyeonggi Yangnyeong Market, Jegi-dong, Jegi-dong, Dongdaemun-gu, Seoul, Korea. Stored.

Astragalus membranaceus ) 100 g of precisely weighed and placed in a reflux extractor with 2000 ml of primary distilled water, the temperature was raised to near 100 ℃, extracted by heating for 2 hours from the boiling point of the liquid, and then decompressed with filter paper The filtered filtrate was concentrated using a rotary vacuum evaporator (Eyela, Japan). This concentrated solution was used as a sample using a dry powder using a freeze dryer (Eyela, Japan). Lyophilized extract obtained 13.9 g, yield 13.9%.

Hwanggi  Viability of Skin Dermal Cells of Extracts viability ) Impact analysis

<2-1> cell culture

The cell line used in the experiment was purchased from the American Type Culture Collection (ATCC, USA) as Hs68 (dermal fibroblast cells, human). This cell line is derived from human foreskin and belongs to dermal cells.

Hs68 fibroblast cell line was 10% FBS (fetal bovine serum, GIBCO BRL, USA), penicillin (100 μg / ml), streptomycin (37%) at 37 ° C. and 5% CO _2. 100 μg / ml) was incubated with DMEM medium (Dulbecco's Modified Eagle Medium, GIBCO BRL, USA). Hs68 cells were fully grown in 75 cm 2 flasks (Falcon, USA) and then washed at 3 days of culture with phosphate buffered saline (PBS) solution, followed by 1 ml of 0.25% trypsin per 50 ml flask. A trypsin) -EDTA (GIBCO BRL, USA) solution was added and treated for 1 minute at room temperature. The trypsin solution was discarded and stored at 37 ° C. for 5 minutes to detach and passage cells. The detached cells were suspended in 10 ml of DMEM medium added with 10% FBS and then transferred to a new culture vessel (50 ml culture flask) in a CO ₂ incubator (Sanyo, Japan) (37 ° C.) at a split ratio of 1:20. , 5% CO ₂ ).

<2-2> Viability of skin dermal cells

Mosmann (1983), Kotnik (1990) and others were applied to investigate the effects of Astragalus extract on the proliferation of Hs68 fibroblasts. 100 μl of 2 × 10 ⁶ cells / ml cells were added to a 96 well plate, incubated for 24 hours at 37 ° C. in a 5% CO ₂ incubator, and then the medium was discarded and the cultured cell surface was washed with PBS solution. 10, 100 and 300 [mu] g / ml of samples dissolved in the same amount of medium and PBS were treated in each well and incubated for 24 hours. 20 μl of 5 mg / ml MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazoliumbromide; MTT, Sigma, USA) dissolved in PBS 4 hours before the end of culture After shading with aluminum foil and incubated at the same conditions for the rest of the time. After removing all of the culture medium and treated with 200 μl of DMSO (dimethyl sulfoxide; DMSO, Sigma, USA) and standing at 37 ° C. for 2 hours, absorbance was measured at 570 nm using a microplate reader (Molecular Devices, USA). .

Cell viability was calculated by the following formula.

The experimental results were expressed as mean ± standard error (Mean ± SE), and the difference between the control group and the experimental group was determined by the Student's t-test to be statistically significant when p <0.05.

As a result, the survival rate of Hs68 fibroblasts in the experiments not irradiated with ultraviolet rays increased to 89.93%, 105.41% and 113.93% compared to the control at the concentration of 10, 100 and 300 ㎍ / ㎖ of Astragalus extract. In addition, UVB 40 mJ irradiation significantly increased the concentration of 111.76%, 141.17% and 139.70% compared to the control at each concentration. The survival rate of Hs68 cells at the concentration of 100 μg / ml of Astragalus was highest as 141.17% compared to the control group (FIG. 2).

Hwanggi  Extract NF Effect on -κB Expression

Western blot analysis was performed to determine how Astragalus affects the NF-κB signaling pathway expressed by UVB.

<3-1> UVB  Protein Extraction After Irradiation

Hs68 cells were incubated with medium in a 100 mm Petri dish (Corning, USA) at a density of 2 × 10 ⁶ cells / ml, incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator, then discarded and the surface of the culture cells was PBS. After washing with the solution, all PBS was removed and the same amount of medium and 100 ㎍ / ㎖ sample dissolved in PBS was treated in a dish and incubated for 24 hours. After incubation, the medium was discarded, washed with PBS, 4 ml of PBS was added, irradiated with UVB 40 mJ / cm 2, washed with PBS solution again, and treated with DMEM without FBS in each dish and incubated for 5 hours. The cells from which the culture solution was removed were washed twice with PBS, and then 300 µl of lysis buffer was added and scraped off using a scraper. The supernatant was taken by centrifugation at 12,000 rpm for 10 minutes in 1.5 ml Eppendorf tubes, and 1 μl of the supernatant was taken to quantitate the protein by Bradford's method.

<3-2> Western Blot  analysis

Western blot analysis showed normal and UVB 40 mJ / ㎠ The irradiated control (control) was measured in the range of the drug concentration 10 ㎍ / ㎖, 100 ㎍ / ㎖, 300 ㎍ / ㎖ as a control.

15 μg of sample protein was isolated by electrophoresis on 10% SDS-PAGE Tris-glycine gel and then transferred to PVDF membrane. Protein-transferred PVDF membrane was blocked for 4 to 12 hours using PBS containing 0.1% tween20 and 5% BSA (Bovine serum albumin) and then incubated with NF-κB antibody (1: 1000) for 2 hours. Thereafter, the remaining residual antibodies were removed by stirring three times for 10 minutes with 0.1% Tween20's T-PBS solution, incubated at room temperature for 2 hours using rabbit secondary antibody (1: 2000), and then again 0.1% T- The remaining residual antibodies were removed by stirring 6 times with PBS solution for 10 minutes. Thereafter, the developer was buried in a film to show a film.

As a result, the amount of expression of NF-κB P65 in the control group was increased compared to the normal group (normal) and can be seen to decrease compared to the drug concentration (Fig. 3).

Hwanggi  By extract NF -κB P65  Inhibition

Immunocytochemistry was performed to determine how Astragalus affects the NF-κB pathway expressed by UVB.

Immunocytochemical staining was incubated at ⁵ × 10 ⁵ cells / ml on a cover slide coated with 0.1% gelatin and fixed with 95% alcohol. In order to inhibit the endogenous peroxidase activity, it was treated with 3% hydrogen peroxide for 10 minutes and washed with phosphate buffered saline (PBS). To remove nonspecific reactions of immunocytochemical staining, the cells were treated with goat serum (Jackson Immno Research, USA, 1:10) for 60 minutes, the excess solution was removed and the anti-nfkb antibody (Santa Cruz, USA, rabbit polyclonal, 1: 100) is reacted overnight at 4 ℃. After completion of the reaction, the cells were washed three times with PBS for 5 minutes and left for 2 hours in a secondary antibody to which FITC (Jackson Immno Research, USA, 1:50) was attached. Thereafter, it was embedded with glycerol gelatin and subjected to control staining with hematoxylin, and observed with an optical microscope.

Immunocytochemical staining results showed that NF-κB P65 was translocated by UVB and observed at a higher concentration in the nucleus than the cytoplasm. After NF-κB P65 is activated, it moves to the nucleus and functions as a gene that causes transcription. In the cells treated with 100 ㎍ / ㎖ of the Astragalus concentration, the inhibitory effect appeared, it can be seen that the translocation is reduced (Fig. 4).

Hwanggi  Procollagen Expression by Extracts

In order to examine the effect of Astragalus extract on procollagen synthesis in Hs68 fibroblasts, which affects the matrix of skin cells, the degree of protein synthesis was performed at Astragalus concentration of 100 ㎍ / ml.

<5-1> UVB  Protein Extraction After Irradiation

The protein was extracted according to Example <3-1>.

<5-2> Western Blot  analysis

15 μg of sample protein was isolated by electrophoresis on 10% SDS-PAGE Tris-glycine gel and then transferred to PVDF membrane. Protein-transferred PVDF membranes were blocked for 4 to 12 hours using PBS containing 0.1% tween20 and 5% Bovine serum albumin (BSA), followed by 2 hours with pro-collagen antibody (1: 1000). Incubated for Thereafter, the remaining residual antibodies were removed by stirring three times for 10 minutes with 0.1% Tween20's T-PBS solution, incubated at room temperature for 2 hours using rabbit secondary antibody (1: 2000), and then again 0.1% T- The remaining residual antibodies were removed by stirring 6 times with PBS solution for 10 minutes. Thereafter, the developer was buried in a film to show a film.

As a result, the procollagen synthesis increase was relatively higher than that of the control (FIG. 5). Procollagen inhibited by UVB irradiation showed a protective effect against UVB by inhibiting the translocation of NF-κB P65 to a certain concentration of sulfur.

< Manufacturing example  1> Manufacture of cosmetics

Cosmetics for the prevention and improvement of inflammatory diseases containing the astragalus extract or fractions thereof of the present invention as an active ingredient can be prepared. The present inventors prepared the cosmetic of solubilized formulations such as cosmetics and soft cosmetics of the emulsion formulations such as nutrient cosmetics, creams, essences and the like as the cosmetics.

<1-1> Cosmetic Preparation of Emulsifying Formulations

To the cosmetic composition of the emulsifier type was prepared in the composition shown in Table 1. The manufacturing method is as follows.

1) The mixture which mixed the raw materials of 1-9 was heated at 65-70 degreeC.

2) 10 was added to the mixture of step 1).

3) The mixture of the raw materials of 11-13 was heated to 65-70 degreeC, and completely dissolved.

4) Through step 3), the mixture of 2) was slowly added to emulsify for 2-3 minutes at 6,000 rpm.

5) The raw material of 14 was dissolved in a small amount of water, and then added to the mixture of step 4) and emulsified for 2 minutes.

6) After weighing 15 to 17 raw materials, the mixture of step 5) was further emulsified at 40 ° C. for 30 seconds.

7) After emulsifying the mixture of step 6) through a degassing process to prepare a cosmetic of emulsion type by cooling to 25 ~ 35 ℃.

Composition of Emulsifying Formulations 1, 2, 3 Furtherance Emulsifier 1 Emulsifier 2 Emulsifier 3 One Stearic acid 0.3 0.3 0.3 2 Stealy alcohol 0.2 0.2 0.2 3 Glyceryl Monostearate 1.2 1.2 1.2 4 Wax 0.4 0.4 0.4 5 Polyoxyethylene sorbitan
Monolauric acid ester 2.2 2.2 2.2 6 Methyl paraoxybenzoate 0.1 0.1 0.1 7 P-hydroxybenzoic acid profile 0.05 0.05 0.05 8 Cetylethylhexanoate 5 5 5 9 Triglyceride 2 2 2 10 Cyclomethicone 3 3 3 11 Purified water To 100 To 100 To 100 12 Concentrated glycerin 5 5 5 13 Triethanolamine 0.15 0.15 0.15 14 Polyacrylic acid polymer 0.12 0.12 0.12 15 Pigment 0.0001 0.0001 0.0001 16 incense 0.10 0.10 0.10 17 Ethanol Extract of Example 1 0.0001 One 10

<1-2> Solubilization  Cosmetic preparation of the formulation

A cosmetic of a solubilized formulation was prepared with the composition shown in Table 2 below. The manufacturing method is as follows.

1) Raw materials 2 to 6 were placed in raw material 1 (purified water) and dissolved using a still mixer.

2) The raw materials of 8 to 11 were put into the raw material of 7 (alcohol) and completely dissolved.

3) The mixture of step 2) was solubilized with slow addition to the mixture of step 1).

Composition of Solubilized Formulations 1, 2, 3 Furtherance Solubilized Formulation 1 Solubilized Formulation 2 Solubilized Formulation 3 One Purified water To 100 To 100 To 100 2 Concentrated glycerin 3 3 3 3 1,3-butylene glycol 2 2 2 4 EDTA-2Na 0.01 0.01 0.01 5 Pigment 0.0001 0.0002 0.0002 6 Normal-hexane fraction of Example 2 0.1 5 5 7 Alcohol (95%) 8 8 8 8 Methyl paraoxybenzoate 0.1 0.1 0.1 9 Polyoxyethylene
Hydrogenated Ester 0.3 0.3 0.3 10 incense 0.15 0.15 0.15 11 Cyclomethicone - - 0.2

< Manufacturing example  2> Preparation of Pharmaceutical Formulations

<2-1> Sanje  Produce

2 g of ethyl acetate fraction of Example 2 of the invention

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<2-2> Preparation of Tablet

100 mg of the normal-butanol fraction of Example 2 of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<2-3> Preparation of Capsule

100 mg of the normal-hexane fraction of Example 2 of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

<2-4> Preparation of the ring

1 g of normal-butanol fraction of Example 2 of the invention

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

<2-5> Preparation of Granules

150 mg of ethyl acetate fraction of Example 2 of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

< Manufacturing example  3> Manufacture of food

Food containing the Astragalus extract or fractions thereof of the present invention was prepared as follows.

<3-1> Production of flour food

0.5-5.0 parts by weight of Astragalus extract of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<3-2> soup  And juicy ( gravies Manufacturing

0.1-5.0 parts by weight of the normal-hexane fraction of the Astragalus extract of the present invention was added to soups and broths to prepare meat products for health promotion, soups and noodles for noodles.

<3-3> Ground Beef  Produce

10 parts by weight of the normal-butanol fraction of the Astragalus extract of the present invention was added to the ground beef to prepare a ground beef for health promotion.

<3-4> Dairy products ( dairy products Manufacturing

5-10 parts by weight of the ethyl acetate fraction of the Astragalus extract of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<3-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The ethyl acetate fraction of the extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by spraying and drying with a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds, and coarse ethyl acetate fractions prepared above were prepared by combining the following ratios.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Astragalus ethyl acetate fraction (3 parts by weight),

(0.5 part by weight),

(0.5 parts by weight)

< Manufacturing example  4> Manufacturing of beverages

<4-1> Health drink  Produce

Instant sterilization was carried out by homogeneously combining 5 g of the Astragalus extract of the present invention with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). Then it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<4-2> Production of vegetable juice

Vegetable juice was prepared by adding 5 g of ethyl acetate fraction of the Astragalus extract of the present invention to 1,000 ml of tomato or carrot juice.

<4-3> Preparation of fruit juice

1 g of a normal-hexane fraction of the Astragalus extract of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

Claims (13)

Astragalus membranaceus ) Cosmetic composition for skin irritation or skin inflammation alleviation containing the extract as an active ingredient.
The cosmetic composition according to claim 1, wherein the extract is extracted with water, C ₁ to C ₂ lower alcohols, or a mixed solvent thereof.
The method of claim 2, wherein C ₁ To The lower alcohol of C ₂ is a cosmetic composition, characterized in that methanol or ethanol.
According to claim 1, wherein the skin irritation is a cosmetic composition, characterized in that any one selected from the group consisting of erythema, edema, itching and peeling.
The cosmetic composition of claim 1, wherein the skin inflammation is any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea, and acne.
The cosmetic composition according to claim 1, wherein the Astragalus extract inhibits the expression or activity of NF-κB.
The cosmetic composition according to claim 1, wherein the cosmetic composition improves or prevents wrinkles or aging resulting from skin inflammation.
Astragalus membranaceus ) Skin external preparation for the prevention and improvement of skin irritation or skin inflammation containing extract extract as an active ingredient.
9. The external preparation for skin according to claim 8, wherein the skin inflammation is any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea and acne.
Astragalus membranaceus ) Pharmaceutical composition for the prevention and treatment of skin irritation or skin inflammation containing the extract as an active ingredient.
The pharmaceutical composition of claim 10, wherein the skin inflammation is any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea and acne.
Astragalus membranaceus ) Health food for the prevention and improvement of skin irritation or skin inflammation containing the extract as an active ingredient.
The health food of claim 12, wherein the inflammatory skin disease is any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea, and acne.

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