KR102324673B1 - Skin external composition for reducing skin irritation and inflammation containing extracts of Glycyrrhiza uralensis and Paeonia lactiflora - Google Patents

Abstract

The present invention relates to a composition for external application to the skin for relieving skin irritation and inflammation containing an extract of a mixture of licorice and peony as an active ingredient, and more particularly, to licorice and peony extract, peony, angelica, rhubarb, poison ivy, white paper, taekran It relates to a composition having an effect of alleviating skin irritation caused by various external toxic substances and inhibiting inflammation by further containing any one or more herbal extracts selected from hyeonggae and hwangbaek.
The composition for external application for skin according to the present invention uses a natural extract to minimize side effects, and detoxifies and discharges toxins by increasing the expression of NQO1, an enzyme that detoxifies toxins introduced from the outside, and GSTT1, an enzyme that releases toxins, and , protects the skin by increasing the expression of hBD3, a natural immune factor. In addition, it suppresses the cytokines IL-6, IL-8, TNF-α, etc., which are generated by ultraviolet rays, and has an excellent effect of suppressing NO generated by yellow dust or heavy metals contained in yellow sand, and air pollutants. , skin irritation and inflammatory reactions are relieved. In addition, the composition for external application for skin according to the present invention has excellent antioxidant effect.
Due to these effects, the composition for external application for skin according to the present invention can be variously applied to the cosmetic industry.

Description

TECHNICAL FIELD [0002] Skin external composition for reducing skin irritation and inflammation containing extracts of Glycyrrhiza uralensis and Paeonia lactiflora, containing extracts of licorice and peony mixture as active ingredients

The present invention relates to a composition for external application to the skin for relieving skin irritation and inflammation containing an extract of a mixture of licorice and peony as an active ingredient, and more particularly, to licorice and peony extract, peony, angelica, rhubarb, poison ivy, white paper, taekran It relates to a composition having an effect of alleviating skin irritation caused by various external toxic substances and inhibiting inflammation by further containing any one or more herbal extracts selected from hyeonggae and hwangbaek.

The skin is a part of the body that is directly exposed to the external environment, and acts as a protective film to protect the body from external stimuli. However, when the skin is exposed to external stimuli such as excessive ultraviolet rays or air pollutants such as heavy metals of yellow dust, skin irritation such as erythema and itching and an inflammatory reaction are induced. Skin troubles caused by these causes are not only a cosmetic problem, but also substances generated in the inflammatory reaction process incidentally cause skin pigmentation, promote the breakdown of skin elastic fibers, and even increase skin wrinkles. is known

In particular, when ultraviolet rays are directly and excessively irradiated to the skin, it promotes the formation of erythema or the production of melanin in the skin cells, which can cause spots and blemishes. It can cause trouble and, in severe cases, can cause skin cancer. Ultraviolet rays are divided into UV-A (320-400 nm), UV-B (280-320 nm), and UV-C (200-280 nm) depending on the wavelength. is known as UV-A and UV-B.

In addition, UV exposure increases the formation of free radicals and the generation of reactive oxygen species (ROS) in the skin, and induces oxidative stress on cellular components such as DNA, cell membranes and proteins. It is known to cause skin aging.

In addition, yellow sand is a phenomenon in which small sand or loess floats in deserts located inland such as China and Mongolia and is transported far away by the upper wind and falls close to the ground. In Korea, yellow sand occurs periodically every spring. Yellow sand is a complex of organic and inorganic substances, and its physical properties and components vary greatly depending on the time and place of occurrence, and it also contains metal components that can have a biological effect. Large particles of yellow dust mainly stay at the source or in the vicinity, and those that enter Korea are usually particulate matter 10 (PM10) with a diameter of 10 μm or less. It has been reported that inhalation of such fine dust can cause damage to the respiratory system. Clinically, when the yellow dust wind directly touches the skin, it causes an allergic reaction, which is easy to cause contact dermatitis, dry skin, keratin, and itching. , yellow sand, acne, atopy, etc. are known to cause.

In addition to the skin irritants mentioned above, natural substances that are always stimulated by atmospheric soot, cigarette smoke, and pollutants, which protects the skin from causing an inflammatory reaction and relieves skin irritation and inflammation by reducing toxic substances introduced from the outside The demand for development is gradually increasing.

In the traditional medicine book, it is explained that inflammation is caused by blockage of qi and blood, and the prescriptions for drinking decoction for the symptoms of ongjeo and ongjeo thrombosis include Gamisipgisan, Baenongnaebosan, and Gubohwan, and a prescription for washing the affected area with herbal decoction Myoseungsan and Detox soup are included.

Accordingly, the present inventors have made diligent efforts to find a method for obtaining the best effect by combining natural substances, particularly herbal ingredients, for alleviating skin inflammation caused by external stimuli, licorice, peony, angelica, rhubarb, poison ivy, white paper, taekran , Hyeonggae, Hwangbaek, etc., found that while reducing the toxicity of toxic substances introduced from the outside, it relieves skin irritation and inflammation caused by various factors such as ultraviolet rays, heavy metals from yellow dust and other pollutants, and the present invention was completed.

An object of the present invention is to contain an extract of a mixture of licorice and peony as an active ingredient, preferably by further containing one or more herbal extracts selected from the group consisting of Angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae, and hwangbaek. An object of the present invention is to provide a composition for external application for skin that exhibits an effect of alleviating skin irritation and inflammation.

Another object of the present invention is to contain an extract of a mixture of licorice and peony as an active ingredient, and preferably, one or more herbal extracts selected from the group consisting of angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae, and hwangbaek. It is to provide a composition for external application to the skin exhibiting an antioxidant effect.

In order to achieve the above object, the present invention provides a composition for external application for skin for relieving skin irritation and inflammation containing an extract of a mixture of licorice and peony as an active ingredient.

In addition, the present invention provides an antioxidant composition for external application for skin containing an extract of a mixture of licorice and peony as an active ingredient.

The composition for external application for skin according to the present invention uses a natural extract to minimize side effects, and detoxifies and discharges toxins by increasing the expression of NQO1, an enzyme that detoxifies toxins introduced from the outside, and GSTT1, an enzyme that releases toxins, and , protects the skin by increasing the expression of hBD3, a natural immune factor. In addition, it suppresses the cytokines IL-6, IL-8, TNF-α, etc., which are generated by ultraviolet rays, and has an excellent effect of suppressing NO generated by yellow dust or heavy metals contained in yellow sand, and air pollutants. , skin irritation and inflammatory reactions are relieved. In addition, the composition for external application for skin according to the present invention has excellent antioxidant effect.

Due to these effects, the composition for external application for skin according to the present invention can be variously applied to the cosmetic industry.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

Among the herbal medicines used in the present invention, "licorice" is a plant of the legume family, and its scientific name is Glycyrrhiza uralensis or Glycyrrhiza glabra . Terpene compounds such as Glycyrrhetinic acid and Glycyrrhizin and flavonoids such as Liquiritigenin and Liquiritin are known. differences are reported. When the active ingredient, Glycyrrhizin, induced toxicity in the neuronal cell line PC12 cell, research results were reported on its activity for alleviating toxicity. Studies have shown that it reduces toxicity.

"Peony" used in the present invention is a plant of the Peony family, whose scientific name is Paeonia lactiflora . Terpene compounds such as Albiflorin, Paeniflorin, and Oxypaeoniflorin are known as the ingredients contained therein. It has been reported that the active ingredient, Peaoniflorin, reduces mortality by detoxifying in an animal experiment administered with the toxic substance Aconitine.

"Angi" used in the present invention is a plant of the family Hemiaceae, and the scientific name is Angelica gigas . Coumarin ingredients such as nodakenin, decursin, and decursingangelate are known as the ingredients contained.

"Rhubarb" used in the present invention is a plant of the family Rheumaceae and the scientific name is Rheum palmatum , Rheum tanguticum or Rheum officinale . Anthraquinones such as Sennoside A, Aloe emodin, Rehin, Emodin, Chrisophanol, and Physcion are known. have.

As used in the present invention, "poisonous" is a plant of the Araliaceae family, and its scientific name is Aralia continentalis . Terpene compounds such as kaurenoic acid and continentalic acid are known as the ingredients contained.

"Taekran" used in the present invention is a plant of the Lamiaceae family, and its scientific name is Lycopus lucidus . Flavonoids such as Quercimertrin and Astragalin are known as ingredients.

The "white paper" used in the present invention is a plant of the Umbrella family, and its scientific name is Angelica dahurica . Coumaring compounds such as oxypeucedanin, Imperatorin, and Isoimperatorin are known as ingredients included.

"Hyunggae" used in the present invention is a plant of the Lamiaceae family, and the scientific name is Schizonepeta tenuifolia . Known ingredients include pulegone, hesperidine, and ursolic acid.

"Hwangbaek" used in the present invention is a plant of the rutaceae family, and the scientific name is Phellodendron amurense or Phellodendron chinense . Alkaloids such as Berberine, Palmatin, and Magnoflorine are known as ingredients.

Hereinafter, the present invention will be described in more detail.

In Examples and Test Examples of the present invention, in order to confirm the skin irritation and inflammation relief efficacy of a composition containing a mixed extract of licorice, peony, Angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae, and hwangbaek according to the present invention. Efficacy experiments were conducted. As a result, the composition according to the present invention has an excellent antioxidant effect, reduces the toxicity of toxic substances introduced from the outside, and at the same time relieves skin irritation and inflammation caused by various factors such as ultraviolet rays, heavy metals from yellow dust and other pollutants. could check

Accordingly, in one aspect, the present invention relates to a composition for external application for skin for relieving skin irritation and inflammation, comprising an extract of a mixture of licorice and peony as an active ingredient.

In another aspect, the present invention relates to an antioxidant composition for external application for skin containing an extract of a mixture of licorice and peony as an active ingredient.

In the present invention, the composition may further contain one or more herbal extracts selected from the group consisting of angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae, and hwangbaek. Most preferably, it may contain the extract of a mixture of all of the above licorice, peony, angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae, and hwangbaek.

In addition, in the present invention, the licorice, peony, angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae, and hwangbaek are 1-5: 1-5: 1-5: 1-5: 1-5: 1-5 : 1-5: 1-5: A weight ratio of 1-5, preferably 4:4:4:3:4:3:2:2:4 can be characterized in that it is mixed and extracted.

In addition, the composition includes an extract of the licorice and peony mixture, and one or more herbal extracts selected from the group consisting of Angelica asiatica, rhubarb, poison ivy, white paper, taekran, hyeonggae, and hwangbaek, in a dry state, 0.01 based on the total weight of the composition. It may be characterized in that it contains an amount of from 10.0% by weight to 10.0% by weight. If the content of the extract is less than 0.01% by weight, skin irritation and inflammation relief effect is hardly observed, and if it exceeds 10% by weight, the formulation may be affected.

In addition, the extracts contained in the composition for external application for skin according to the present invention may be obtained by extraction and separation from nature by a method commonly used to extract an active ingredient from a plant. In addition, the 'extract' as defined in the present invention is extracted from a prescription drug using an appropriate solvent, and may include all of a hot water extract, a polar solvent soluble extract, or a non-polar solvent soluble extract.

As a suitable solvent for preparing the extract, any solvent acceptable in the art may be used, and water or an organic solvent may be used. For example, purified water, methanol (methanol), ethanol (ethanol), propanol (propanol), isopropanol (isopropanol), alcohols having 1 to 4 carbon atoms including butanol (butanol), acetone (acetone), ether (ether) , benzene (benzene), chloroform (chloroform), ethyl acetate (ethyl acetate), methylene chloride (methylene chloride), hexane (hexane) and various solvents such as cyclohexane (cyclohexane) can be used alone or in combination, but Not limited. Preferably, extraction can be performed using 70% ethanol or 80% methanol.

As the extraction method, any one of methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method, compression method, etc. can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process, and may be purified using a conventional purification method. There is no limitation on the method for preparing the extract of the present invention, and any known method may be used.

For example, the extract included in the composition of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying the primary extract extracted by the hot water extraction or solvent extraction method described above. In addition, the first extract was further purified using various chromatography methods such as silica gel column chromatography, thin layer chromatography, and high performance liquid chromatography. you may get

Therefore, in the present invention, the herbal medicine extract constituting the traditional prescription is a concept including all extracts, fractions and purified substances, their dilutions, concentrates, or dried substances obtained in each step of extraction, fractionation or purification, and the extract is a separate extract of each herbal medicine. It is also possible to use a mixture of extracts, or it is also possible to use the one prepared by extracting the herbs at once after mixing. In addition, the extract according to the present invention can be dissolved in 1,3-butylene glycol and blended into the composition.

In addition, in the present invention, the extract relieves skin irritation and inflammation by detoxifying toxins introduced from the outside and inducing discharge, and alleviating skin irritation and inflammation caused by ultraviolet rays and yellow dust or heavy metals contained in yellow dust can be done with

NQO1 (NAD(P)H Quinone Oxidoreductase 1) is a reductase that is composed of amino acids and exists in the cytoplasm to detoxify toxins, and GSTT1 (Glutathion S-transferase theta-1) is the It catalyzes conjugation to the compound to release the toxin. Hman Beta Defensin 3 (hBD3) induces migration and activation of antigen presenting cells and functions as an immune enhancer. In addition, when an inflammatory substance is introduced, NO (Nitric acid) is produced in the immune system. When present in a small amount, NO plays an important role in neurotransmission and host defense mechanisms, but when excessively produced, it causes chronic inflammation. Cytokines produced during an inflammatory response include TNF-α, IL-1α/β, IL-6, and IL-8. Examples and test examples show that the composition of the present invention is very effective in detoxifying and excreting toxins through the above mechanisms, functioning as an immune enhancer, and alleviating inflammation by inhibiting NO and these cytokines generated due to external stimuli. could be verified through

The composition for external application for skin according to the present invention is a concept encompassing the overall composition used for external application to the skin, and may be, for example, a cosmetic composition or a pharmaceutical composition, but preferably a cosmetic composition.

The cosmetic composition may include, for example, basic cosmetics, makeup cosmetics, hair cosmetics, body cosmetics, and the like, and the formulation is not particularly limited, and may be appropriately selected according to the purpose.

For example, the cosmetic composition may be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray. However, the present invention is not limited thereto. More specifically, softening lotion, nourishing lotion, lotion, body lotion, nourishing cream, massage cream, moisture cream, hand cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, gel, patch, oil in water Basic cosmetics such as (O/W) type and water-in-oil (W/O) type cosmetics, color cosmetics such as lipstick, makeup base or foundation, shampoo, conditioner, body cleanser, cleaning agent such as toothpaste or mouthwash, hair tonic, It may be formulated as a cosmetic composition for hair, such as a hair dressing agent such as a gel or mousse, a hair agent or a hair dye agent.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

[Example 1] Preparation of hot water extract

200 g of licorice, 200 g of peony, 200 g of angelicae, 150 g of rhubarb, 200 g of poison ivy, 150 g of white paper, 100 g of taekran, 100 g of hyeonggae, and 200 g of hwangbaek, add 10-20 times the weight of oriental medicine in purified water It was extracted while boiling for 10 to 15 hours. After completion of the extraction, the extract from which the raw material was removed was concentrated using a rotary vacuum concentrator and freeze-dried to obtain a solid extract. Purified water was added thereto, so that the extract was contained at a concentration of 5% by weight based on the total weight of the composition.

[Example 2] Preparation of ethanol extract

200 g of licorice, 200 g of peony, 200 g of angelicae, 150 g of rhubarb, 200 g of poison ivy, 150 g of white paper, 100 g of taekran, 100 g of hyeonggae, and 200 g of hwangbaek were each taken and 100% ethanol of 10-20 times the weight of herbal medicine was added, followed by the same process as in Example 1. A solid extract was obtained through Purified water and DMSO (dimethyl sulfoxide) were added thereto, so that the extract was contained at a concentration of 5% by weight based on the total weight of the composition.

[Example 3] Preparation of ethanol mixed solvent extract

200 g of licorice, 200 g of peony, 200 g of Angelica rhubarb, 150 g of rhubarb, 200 g of poison ivy, 150 g of white paper, 100 g of taekran, 100 g of hyeonggae, and 200 g of hwangbaek were each taken and 70% ethanol of 10-20 times the weight of herbal medicine was added, followed by the same process as in Example 1. to obtain an extract in a solid state. Purified water and DMSO (dimethyl sulfoxide) were added thereto, so that the extract was contained at a concentration of 5% by weight based on the total weight of the composition.

[Example 4] Preparation of methanol extract

200 g of licorice, 200 g of peony, 200 g of Angelica rhubarb, 150 g of rhubarb, 200 g of poison ivy, 150 g of white paper, 100 g of taekran, 100 g of hyeonggae, and 200 g of hwangbaek were each taken and 100% methanol 10-20 times the weight of herbal medicine was added, followed by the same process as in Example 1. A solid extract was obtained through Purified water and DMSO (dimethyl sulfoxide) were added thereto, so that the extract was contained at a concentration of 5% by weight based on the total weight of the composition.

[Example 5] Preparation of methanol mixed solvent extract

200g of licorice, 200g of peony, 200g of angelicae, 150g of rhubarb, 200g of poison ivy, 150g of white paper, 100g of taekran, 100g of hyeonggae, and 200g of hwangbaek, respectively, and after adding 80% methanol 10-20 times the weight of herbal medicine, the same process as in Example 1 A solid extract was obtained through Purified water and DMSO (dimethyl sulfoxide) were added thereto, so that the extract was contained at a concentration of 5% by weight based on the total weight of the composition.

[Test Example 1] Measurement of antioxidant efficacy

In order to confirm the antioxidant effect, free radical scavenging test and active oxygen scavenging test were performed. The sample used for the test was the sample obtained in Examples 1-5, and in order to compare the antioxidant effect, ascorbic acid (L-ascorbic acid) having excellent antioxidant effect was used as a positive control.

The free radical scavenging test uses that the stable absorbance of DPPH shows the maximum absorbance at 517 nm, and the more the antioxidant compound reacts with DPPH, the more it reacts with DPPH, and the more the free radical scavenging rate increases, the less the absorbance at the 540 nm wavelength. Application was carried out by the assay test method.

In 1 ml of 200 μM DPPH (1,1-diphenyl-2-picryl-hydrazyl radical, Sigma) solution, 1 ml of the composition obtained in Example 1-5 was diluted to an appropriate concentration in methanol, respectively, and mixed, followed by 15 at 37 ° C. After leaving for a minute, the absorbance was measured at a wavelength of 520 nm using a microplate reader (UVT-06685, Thermo max, USA).

In the free radical scavenging test, 1ml of DPPH and 1ml of methanol were added to the control group and measured in the same manner, and 1ml of methanol and 1ml of the sample were added to obtain color correction values for the sample and the control. The free radical scavenging rate was calculated using Equation 1 below, IC ₅₀ is the concentration of the sample required to remove 50% of free radicals, and the smaller the value, the higher the antioxidant activity.

[Equation 1]

Free radical scavenging rate (%) = (control absorbance - absorbance of the sample) / control absorbance X 100

The active oxygen scavenging test uses the generation of active oxygen by the enzymatic reaction of xanthine/xanthine oxidase (Sigma) to measure the change in absorbance using the oxidation of nitroblue tetrazolium (NBT) by active oxygen. By measuring, the scavenging ability of active oxygen can be known.

Sodium carbonate (Na ₂ CO ₃ ) 2.4ml, xanthine (Sigma) 0.1ml, ethylenediamine tetraacetic acid (EDTA) 0.1ml, bovine serum albumin (BSA, Sigma) 0.1ml, NBT 0.1 ml and 0.1ml of the sample, vortexed and left at 25℃ for 10 minutes, then 0.1ml of xanthine oxidase was added and reacted at 25℃ for 20 minutes, and then 6mM CuCl ₂ was added to stop the reaction. , the absorbance was measured at a wavelength of 540 nm using a microplate reader.

The control group in the active oxygen scavenging activity test was measured in the same way by adding tertiary distilled water instead of the sample solution, and adding tertiary distilled water instead of the xanthine enzyme solution to obtain each color correction value for the extracted sample and the control was set. .

The active oxygen scavenging rate was calculated using Equation 2 below, and IC ₅₀ is the concentration of the sample required to remove 50% of active oxygen, and the smaller the value, the higher the antioxidant activity.

[Equation 2]

Reactive oxygen scavenging rate (%) = 100-((absorbance of sample / absorbance of control group) × 100)

Sample name Free radical scavenging rate IC ₅₀
(μg/mL) Reactive oxygen scavenging rate IC ₅₀
(μg/mL) L-ascorbic acid 4.85 6.02 Example 1 7.25 9.08 Example 2 6.54 7.36 Example 3 4.96 6.13 Example 4 7.30 6.69 Example 5 5.14 6.50

Through the antioxidant efficacy measurement experiment, it was confirmed that the antioxidant efficacy of the ethanol-purified water mixed solvent extract of Example 3 and the methanol-purified water mixed solvent extract of Example 5 was relatively excellent.

[Test Example 2] Confirmation of gene expression of toxins detoxification and excretion factors, natural immune factors

In order to confirm the toxin detoxification and excretion efficacy of the composition according to the present invention and the gene expression of natural immune factors, human keratinocytes (HaCaT cells) were cultured in an incubator maintained at 95% or more humidity at _{5% CO 2 and 37 ° C.} It was cultured in DMEM (Dulbeccos modified Eagle's medium) medium containing % fetal calf serum, 100 μg/ml streptomycin and 100 unit/ml penicillin.

Examples 1-5 were added to the human keratinocyte culture medium at a concentration of 10 ppm, respectively, and incubated with the cells for 24 hours. Then, the cells were washed twice with 10 ml of phosphate buffer, and Trizol (Invitrogen) was used to Intracellular total RNA was isolated. The isolated RNA was re-purified using an RNA kit of Qiagen, and the quality of the RNA was checked using a TECAN instrument. cDNA was synthesized from the isolated RNA using Invitrogen's Superscript Reverse Transcriptase (RT) II kit, and this was followed by a real time-reverse transcription polymerase chain reaction (Q-RT-). PCR) was quantitatively analyzed. Changes in the expression patterns of NQO1 (Hs01045994_m1) and hBD3 (hs00218678_m1) were evaluated using SYBR Green, and the results are shown in Table 2 below.

Sample name
Expression (%) GSTT1 NQO1 hBD3 Example 1 25.5 26.6 15.0 Example 2 19.0 28.7 16.9 Example 3 41.4 36.8 33.0 Example 4 23.9 27.5 25.7 Example 5 36.7 32.1 37.6

Through the above experiment, Examples 1-5 all tend to increase the gene expression amount of GSTT1, an enzyme that discharges toxins from human normal keratinocytes, NQO1 that detoxifies toxic substances, and skin antibacterial peptide hBD3, which is a natural immune factor. was confirmed, and it was found that there was an effect of increasing the detoxification and excretion of toxins and the expression of natural immune factors.

[Test Example 3] Efficacy of inhibiting NO production by endotoxin

In order to confirm the anti-inflammatory effect of the composition according to the present invention, RAW264.7 cells (ATCC), which are murine macrophages, were used. DMEM (Dulbecco's modified Eagle's medium), fetal bovine serum (FBS), penicillin-streptomycin and insulin used for cell culture were purchased from Welgene. The RAW264.7 cells were cultured using 10% FBS and 1% penicillin streptomycin in DMEM medium, and cultured in a humidified CO₂ incubator (5% CO₂/95% air) at 37°C. After the cells were cultured in about 80% of the culture dish cold, they were subcultured by treatment with 0.25% trypsin and 2.56 mmol/L EDTA.

A cell number of 5×10 ⁴ was suspended in 10% FBS-DMEM medium, inoculated in a 96-well plate, and cultured at _{37° C., 5% CO 2 in an incubator for 24 hours.} Thereafter, each concentration of Examples 1-5 was treated and reacted for 12 hours, followed by treatment with LPS in the wells. After completion of the reaction, the culture medium was collected and the amount of NO secretion was measured using the Griess reagent system (Promega, USA), and the results are shown in Table 3 below. The measurement method using the Griess reagent system was, in more detail, according to the protocol provided by Promega, the manufacturer of the system. Sulfanilamide solution was first added to the collected cell culture medium, and then the NED solution was put into the microplate. Measurement was performed at 540 nm using a reader.

Sample name treatment concentration NO inhibition rate (%) Example 1 50ppm 21.6 25ppm 16.4 Example 2 50ppm 59.4 25ppm 41.5 Example 3 50ppm 89.1 25ppm 55.0 Example 4
50ppm 55.7 25ppm 42.2 Example 5 50ppm 85.9 25ppm 61.7

Through the above experiment, it was confirmed that there was an NO inhibitory effect in all of Examples 1-5, especially in the case of Example 3 using an ethanol-purified water mixed solvent extract and Example 5 using a methanol-purified water mixed solvent extract, NO inhibition It was found that the effect was very good.

[Test Example 4] Efficacy of inhibiting inflammatory cytokine production by endotoxin

In order to confirm the cytokine production inhibitory effect of the composition according to the present invention, RAW264.7 cell (ATCC), a mouse macrophage, was used. Cells were cultured in the same manner as in Test Example 3, suspended in 10% FBS-DMEM medium with a cell number of ^{5×10 4} and cultured in 96 plates, and cultured in an incubator at _{37° C., 5% CO 2 for 24 hours.} Examples 1-5 were treated for each concentration, and LPS was treated in the wells after 12 hours of reaction. After completion of the reaction, the culture medium was collected.

IL-1β, IL-6 and TNF-α were tested using an ELISA kit from R&D system, and according to the company's manual. The measurement results are shown in Table 4 below.

Sample name
treatment concentration
Expression inhibition rate (%) TNF-α IL-1β IL-6 Example 1 50ppm 16.2 10.1 19.2 25ppm 9.5 8.0 11.3 Example 2 50ppm 23.6 15.6 24.4 25ppm 18.8 11.5 20.7 Example 3 50ppm 30.1 27.4 32.1 25ppm 21.5 15.5 22.7 Example 4 50ppm 26.6 19.1 22.5 25ppm 19.8 14.1 17.3 Example 5 50ppm 32.7 25.8 35.5 25ppm 25.1 19.9 22.7

As a result of the measurement, it was observed that each Example decreased the production of the stimulation mediators, IL-1β, IL-6 and TNF-α, in particular, in the case of Examples 3 and 5, IL-1β, IL-6 And it was confirmed that the production amount of TNF-α was reduced compared to each extract.

[Test Example 5] Skin cell protection effect by UV rays

In order to confirm the skin cell protection efficacy of the composition according to the present invention by ultraviolet rays, human keratinocytes (HaCaT cells) were dispensed in a 96-well plate to 1×10 ⁵ cells/ml, 37° C., 5% CO ₂ Incubator After culturing for 16 hours, 100 μg/ml of Example 1-5 of the present invention was added. After 24 hours, UV-B was irradiated with 24 mJ/cm and then incubated at 37° C. for 48 hours. For MTT assay, 50 μL of MTT stock solution (2 mg/mL) was added to each well so that the total reaction volume was 200 μL. After the addition, the cells were further cultured for 4 hours to reduce MTT. The plate was centrifuged at 800 × g for 5 minutes, the supernatant was removed, 150 μl DMSO (dimethyl sulfoxide) was added to each well to dissolve formazan crystal, and absorbance was measured at 540 nm with a scanning multi-well spectrophotometer. This absorbance represents the amount of MTT reduced by the cells and is therefore proportional to the number of viable cells present in each well. As a control, PBS was used instead of the extract of the present invention. The results are shown in Table 5 below.

Sample name treatment concentration Cell viability (%) Example 1 50ppm 20.1 25ppm 16.5 Example 2 50ppm 29.9 25ppm 20.5 Example 3 50ppm 41.8 25ppm 30.6 Example 4
50ppm 32.1 25ppm 22.0 Example 5 50ppm 45.7 25ppm 33.0

Through the above experiment, in particular, in Example 3 using the ethanol-purified water mixed solvent extract and Example 5 using the methanol-purified water mixed solvent extract, the cell viability was excellent, and it was found that the skin protection effect by ultraviolet rays was excellent.

[Test Example 6] Efficacy of inhibiting inflammatory cytokine production by ultraviolet rays

In order to confirm the efficacy of the composition according to the present invention to inhibit inflammatory cytokine production by ultraviolet rays, human keratinocytes (HaCaT cells) were dispensed in a 96-well plate at 1×10 ⁵ cells/ml at 37° C., 5% CO _{2 After} culturing in an incubator for 16 hours, each of Examples 1-5 of the present invention was added at 100 μg/ml. After 24 hours, UV-B was irradiated with 24 mJ/cm, and then incubated at 37° C. for 48 hours, the culture medium was collected and ELISA analysis was performed.

PGE2, IL-1β, IL-6 and TNF-α were tested using an ELISA kit from R&D system, and according to the company's manual. The measurement results are shown in Table 6 below.

Sample name
treatment concentration
Expression inhibition rate (%) PGE2 IL-1β IL-6 Example 1 50ppm 12.0 13.6 10.1 25ppm 7.2 9.7 8.0 Example 2 50ppm 18.8 11.2 15.6 25ppm 11.5 7.7 11.5 Example 3 50ppm 21.4 16.3 27.4 25ppm 15.8 9.5 15.5 Example 4 50ppm 15.2 10.7 19.1 25ppm 10.1 6.1 14.1 Example 5 50ppm 19.0 15.8 25.8 25ppm 10.7 11.0 19.9

Through the above experiment, especially in the case of Example 3 using the ethanol-purified water mixed solvent extract and the methanol-purified water mixed solvent extract and Example 5 using the mixed solvent extract, the cytokine PGE2, IL-1β, IL-6 expression inhibition rate of all of the excellent, UV It was found that the inhibitory effect of inflammatory cytokines by

[Test Example 7] Skin cell protection effect by heavy metals

In order to confirm the skin cell protection effect of the composition according to the present invention, human fibroblasts (KTCC) were ^{inoculated to 1×10 4} /well in a 24-well plate, and cultured at 37° C. in DMEM medium for 24 hours. . Then, after removing the cell culture medium, Example 1-5, 50ppm lead, 50ppm mercury, 10ppm chromium, 10ppm copper 10ppm, respectively or after adding a mixture of DMEM medium without serum, and further cultured for 24 hours.

For MTT assay, 50 μL of MTT stock solution (2 mg/mL) was added to each well so that the total reaction volume was 200 μL, and then the cells were further cultured for 4 hours to reduce MTT. The plate was centrifuged at 800 × g for 5 minutes, the supernatant was removed, 150 μl of dimethylsulfoxide was added to each well to dissolve formazan crystal, and absorbance was measured at 540 nm with a scanning multi-well spectrophotometer. This absorbance represents the amount of MTT reduced by the cells and is therefore proportional to the number of viable cells present in each well. As a control, PBS was used instead of the extract of the present invention. The results are shown in Table 7 below.

Sample name treatment concentration Cell viability (%) Example 1 50ppm 21.9 25ppm 17.0 Example 2 50ppm 28.9 25ppm 21.5 Example 3 50ppm 41.8 25ppm 30.1 Example 4
50ppm 30.7 25ppm 22.0 Example 5 50ppm 45.7 25ppm 35.4

Through the above experiment, in particular, in Example 3 using the ethanol-purified water mixed solvent extract and Example 5 using the methanol-purified water mixed solvent extract, the cell viability was excellent, and it was found that the skin cell protection effect by heavy metals was excellent.

[Test Example 8] NO production inhibitory effect on air pollutants (soot)

In order to check the NO production inhibitory effect of the composition according to the present invention on air pollutants, particularly soot, a cotton swab was used to scrape off soot residues on the exhaust tub of a diesel vehicle, then DMSO was added and extracted for 24 hours. After vortexing was performed, the extract was obtained using a filter to obtain a sample of automobile soot.

The experiment was carried out by culturing RAW264.7 cells (ATCC), which are macrophages of mice. Cells were cultured in the same manner as in Test Example 3, suspended in 10% FBS-DMEM medium with a cell number of ^{5×10 4} and inoculated in a 96-well plate, and cultured in an incubator at _{37° C., 5% CO 2 for 24 hours.} Thereafter, the soot sample was treated in the wells and cultured for 24 hours, and Examples 1-5 were treated for each concentration and reacted for 48 hours. After completion of the reaction, the culture medium was collected and the amount of NO secretion was measured using the Griess reagent system (Promega, USA), and the results are shown in Table 8 below.

Sample name treatment concentration NO inhibition rate (%) Example 1 50ppm 20.3 25ppm 15.0 Example 2 50ppm 29.2 25ppm 11.8 Example 3 50ppm 32.1 25ppm 24.0 Example 4
50ppm 25.5 25ppm 19.7 Example 5 50ppm 33.9 25ppm 11.7

Through the above experiment, especially in Example 3 using the ethanol-purified water mixed solvent extract and Example 5 using the methanol-purified water mixed solvent extract, the NO inhibition rate was excellent, and the NO production inhibitory effect on air pollutants, especially soot, was excellence was confirmed.

[Test Example 9] NO production inhibitory effect on other pollutants (cigarette smoke)

In order to confirm the NO production inhibitory effect of the composition according to the present invention on cigarette smoke, after smoking, the cigarette butts were collected and the filter part was separated. 1ml of DMSO per one cleaned filter was added and extracted for 5 hours using a shaker. After shaking the sample for 10 minutes using a vortexer, the filter was removed and the precipitate was removed by rotating the sample in a centrifuge at 300 rpm for 5 minutes to obtain a tobacco sample.

The experiment was carried out by culturing RAW264.7 cells (ATCC), which are macrophages of mice. Cells were cultured in the same manner as in Test Example 3, suspended in 10% FBS-DMEM medium with a cell number of ^{5×10 4} and inoculated in a 96-well plate, and cultured in an incubator at _{37° C., 5% CO 2 for 24 hours.} Thereafter, the tobacco samples were treated in the wells and cultured for 24 hours, and Examples 1-5 were treated for each concentration and reacted for 48 hours. After completion of the reaction, the culture medium was collected and the amount of NO secretion was measured using the Griess reagent system (Promega, USA), and the results are shown in Table 9 below.

Sample name treatment concentration NO inhibition rate (%) Example 1 50ppm 18.8 25ppm 15.0 Example 2 50ppm 17.6 25ppm 12.2 Example 3 50ppm 27.3 25ppm 21.7 Example 4
50ppm 20.5 25ppm 13.1 Example 5 50ppm 29.4 25ppm 18.0

Through the above experiment, in particular, in Example 3 using the ethanol-purified water mixed solvent extract and the methanol-purified water mixed solvent extract in Example 5 using the mixed solvent extract, the NO inhibition rate was excellent, and the NO production inhibitory effect on air pollutants, especially tobacco smoke was found to be excellent.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (13)

A cosmetic composition for relieving skin irritation and inflammation, comprising, as an active ingredient, an extract of a plant mixture consisting of licorice, peony, angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae and hwangbaek. A cosmetic composition for skin antioxidant use comprising, as an active ingredient, an extract of a plant mixture consisting of licorice, peony, angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae and hwangbaek. A cosmetic composition for enhancing skin immunity comprising, as an active ingredient, an extract of a plant mixture consisting of licorice, peony, angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae and hwangbaek. A cosmetic composition for skin protection comprising, as an active ingredient, an extract of a plant mixture consisting of licorice, peony, angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae and hwangbaek. The method according to any one of claims 1 to 4, wherein the plant mixture is licorice, peony, angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae and hwangbaek 1-5: 1-5: 1-5: 1- 5: 1-5: 1-5: 1-5: 1-5: A cosmetic composition, characterized in that it is mixed in a weight ratio of 1-5. The cosmetic composition according to any one of claims 1 to 4, wherein the content of the extract is 0.01 to 10.0% by weight based on the total weight of the composition. The cosmetic composition according to any one of claims 1 to 4, wherein the extract is obtained by adding an alcohol mixed solvent consisting of alcohol and water having 1 to 4 carbon atoms to the plant mixture and then extracting. The cosmetic composition according to claim 7, wherein the alcohol having 1 to 4 carbon atoms is at least one selected from the group consisting of ethanol, methanol, propanol and butanol. The cosmetic composition according to claim 1, wherein the extract detoxifies toxins introduced from the outside and relieves skin irritation and inflammation by inducing discharge. The cosmetic composition according to claim 1, wherein the cause of skin irritation or inflammation is at least one selected from the group consisting of ultraviolet rays, heavy metals, soot, yellow dust, cigarette smoke and air pollutants. According to claim 5, wherein the plant mixture is licorice, peony, Angelica, rhubarb, poison ivy, white paper, taekran, hyeonggae and hwangbaek 4-5: 4-5: 4-5: 3-5: 4-5: 3~ 5: 2-5: 2-5: A cosmetic composition, characterized in that it is mixed in a weight ratio of 4-5. The method according to claim 7, wherein the extract is obtained by adding water and dimethyl sulfoxide to the extracted solid material after adding an alcohol mixed solvent consisting of alcohol and water having 1 to 4 carbon atoms in the plant mixture, cosmetic composition. The cosmetic composition according to claim 4, wherein the skin protection is achieved through the effect of increasing the survival rate of skin cells.