KR101973950B1 - Composition comprising mixture of a citrus unshiu peel extract and chamaecyparis obtuse extract for alleviating skin inflammation or skin hyperpigmentation by yellow sand or fine dust - Google Patents

Abstract

The present invention relates to a composition containing a mixture of a Citrus unshiu peel extract and a Chamaecyparis obtusa extract. The mixture inhibits the expression of NO production and inflammatory cytokines induced by yellow dust or fine dust, and also inhibits tyrosinase activity and melanin production induced by yellow dust or fine dust. Therefore, the composition of the present invention has excellent effects of relieving skin inflammation, caused by yellow dust or fine dust, as well as alleviating skin pigmentation.

Description

[0001] The present invention relates to a composition for alleviating skin irritation or improving skin pigmentation induced by yellow dust or fine dust including dermis and flounder mixed extracts, or a composition for improving skin pigmentation, or fine dust}

The present invention relates to a composition for alleviating skin inflammation induced from yellow dust or fine dust, including a mixture of a dermis extract and a cottonseed extract. The present invention also relates to a composition for improving skin pigmentation induced by yellow dust or fine dust, including a mixture of a dermis extract and a cottonseed extract.

Yellow sand is a phenomenon that small sand, loess or dust of desert and loess in the center of Asian continent such as China and Mongolia float in the sky and fly away in the upper wind. It can be moved to Jeju Island by long-distance mobile air pollutant. Do. The fine dust is collectively referred to as PM10 (Particulate Matter 10) and is classified into PM2.5 (Particulate Matter 2.5) which is an ultra fine dust of 2.5 μm or less. It mainly occurs when fossil fuels are burned, and China's micro dust, which is 70% dependent on coal, accounts for a large part.

It is estimated that about 25-33% of current industrialized diseases are caused by environmental hazards, and in Europe, about 310,000 people die early due to contaminated air, and the American Cancer Society estimates that 10% Ug increases the overall mortality rate by 7%. According to a joint study by the Ministry of Environment in 2013 (Journal of Allergy and Clinical Immunology, 132 (2), 2013, 495-498), symptoms of dermatitis become worse when the concentration of fine dust or air pollutants increases . When fine dust (PM10) is 1 占 퐂 / m3 The average symptom of dermatitis increased by 0.4% in each increase and the symptoms of dermatitis increased by 2.59% and 2.74%, respectively, as the total volatile organic compounds (TVOC) and benzene increased by 0.1 ppb.

In addition, when dusts or fine dusts are generated, the air pollutes the atmosphere with a yellowish brown color, and the dust amount on the air is increased by a factor of four on average, so that the skin tends to easily wear and lose vitality. It can deeply enter into the skin, causing skin troubles, aggravating various symptoms such as irritant dermatitis, allergic dermatitis, skin pigmentation, acne, spots, freckles, dry skin.

Although steroids are mainly used for treating skin inflammation caused by dusts or fine dusts, there are various side effects such as skin atrophy and possibility of delayed growth at the time of long-term administration, and recently, use of non-steroids is increasing. However, non-steroids also show side effects such as erythema, itching, and weakening of immunity, making it difficult to treat skin inflammation.

In addition, dust or fine dust penetrates deep into the skin with melanocytes through hair follicles, and harmful substances (polycyclic aromatic hydrocarbons (PAH), phthalates, dioxins ) Cause proliferation of melanocytes to cause skin pigmentation and darken skin tone.

Currently, many studies have been conducted to find a novel substance having a high therapeutic effect and low side effect from a safety-proven natural material-derived material. Typically, there are many kinds of researches such as Raspberry Extract (registered Patent No. 10-0563548), Raspberry Extract (Patent No. 10-1309172) , Extract of Spirulina (Patent No. 10-2009-0079468), extract of Deerfly (registered patent No. 10-0919852), extract of Mulberry bark extract and extract of licorice (registered patent No. 10-0700352), but the skin inflammation induced by yellow dust or fine dust No extracts have been developed that have excellent effects on alleviating or improving skin pigmentation.

The inventors of the present invention confirmed that the skin irritation alleviating effect and the skin pigment deposition improving effect are enhanced by mixing the dermis extract and the whitening extract, and completed the present invention.

Accordingly, it is an object of the present invention to provide a composition for alleviating skin irritation induced from yellow dust or fine dust containing a mixture of a dermis extract and a cottonseed extract as an active ingredient.

In addition, a problem to be solved by the present invention is to provide a composition for improving skin pigmentation induced from yellow dust or fine dust containing a mixture of a dermis extract and a cottonseed extract as an active ingredient.

Hereinafter, the present invention will be described more specifically.

In order to solve the above problems, the present invention provides a composition for alleviating skin irritation induced from yellow dust or fine dust comprising a mixture of a dermis extract and a cottonseed extract as an active ingredient.

The present invention also provides a composition for improving skin pigmentation induced from yellow dust or fine dust comprising a mixture of a dermis extract and a cottonseed extract as an active ingredient.

The dermis refers to the mature peel of citrus unshiu Markovich or Citrus reticulata Blanco, Citrus Sunki, etc. in Korea. In Japan, Citrus unshiu Markovich and Citrus reticulata Blanco (柑) are designated as the dermis, and in China, Citrus reticulata Blanco (柑) and its cultivated varieties as the dermis.

The dermis is said to be better in the long term, and the redder the better, so the name of Hongpie and Dermis is formed. The fruit is called tangerine (Tachibana), the leaf is tachibana (橘 葉), the yellow part where the inside of the fruit bark is removed is called 柑 紅 (橘), the seed is called 橘 核 (橘 核) . It has a peculiar odor, is slightly irritating, has a spicy flavor, and is warm.

Chamaecyparis obtusa is mostly distributed in Asia, and is also called cypress tree or conifers. It is an evergreen scaly tree belonging to Cuperssaceae and originates from Japan. The whitening is effective for nourishment, branching, hemostasis, rheumatism, neuralgia, hemorrhage, intestinal hemorrhage, and tonic, and phytoncide is contained in a large amount in the whitish leaves, thereby enhancing psychological stability, cardiopulmonary function, and strengthening of the heart.

The dermis extract and the flatulence extract may be obtained by extracting with water, a C ₁ to C ₄ lower alcohol or a mixed solvent thereof, and the lower alcohol may be ethanol or methanol, more specifically, ethanol.

Since the dermis extract and the sclerotin extract have a relative effect in inhibiting the expression of the respective inflammation-causing substances, a mixture of the dermis extract and the cottonseed extract may be used to relieve skin inflammation caused by yellow dust or fine dust, A complementary effect can be given to the improvement.

Preferably, the mixture of the dermis extract and the whitening extract is mixed at a weight ratio of 4: 1 to 1: 2, and more preferably 3: 1 to 1: 1. When the weight ratio of the dandruff extract to the mixed extract was more than 4, the activity of the dandelion extract was decreased by 20% or more when the dandelion extract was used alone. When the weight ratio of the dandelion extract was more than 2, It was 30% more active than single use.

These dusts are long-haul migratory air pollutants and are caused by small sand, loess or dust floating in the sky in the desert and loess areas in the center of Asian continent such as China and Mongolia, It says.

The fine dust may include one or more fine dusts selected from the group consisting of PM10, PM2.5, and TSP.

The fine dust is collectively referred to as PM10 (Particulate Matter 10) and is classified into PM2.5 (Particulate Matter 2.5) which is an ultra fine dust of 2.5 μm or less. It mainly occurs when fossil fuels are burned, and China's micro dust, which is 70% dependent on coal, accounts for a large part. Also, total suspended dust (generally all suspended particles (TSP) in the air below 50 μm) are also widely contained in the fine dust.

In addition, skin inflammation caused by yellow dust or fine dust is a kind of contact dermatitis. It is a kind of contact dermatitis which is caused by general skin inflammation (atopic dermatitis due to antigen contact, dermatitis which is caused by skin ingestion by internal substances, Inflammatory cytokines, which specifically increase expression levels, are present, unlike inflammatory diseases (e.g.

That is, as shown in Experimental Example 1, in the present invention, the yellow dust or fine dusts are interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), tumor necrosis factor- wherein the expression level of at least one inflammatory cytokine selected from the group consisting of Tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony stimulating factor (GM-CSF) .

Thus, the mixture of the dermis extract and the cottonseed extract of the present invention can inhibit yellow dust or fine dust-induced NO production and inflammatory cytokines (IL-1 alpha, IL-2, TNF-a and GM-CSF) have.

In addition, the mixture of the dermis extract and the albumen extract of the present invention can inhibit tyrosinase activity induced by yellow dust or fine dusts and inhibit melanin production.

The mixture of the dermis extract and the whitening extract has an excellent effect on the improvement of skin irritation caused by yellow dust or fine dust, and thus can be used as a cosmetic composition for alleviating skin irritation.

In addition, the mixture of the dermis extract and the whitening extract has an excellent effect for improving skin pigmentation caused by yellow dust or fine dust, and thus can be used as a cosmetic composition for skin pigmentation improvement.

The mixture may be included in an amount of 0.001 to 10% by weight based on the total weight of the composition. When the amount of the mixture is less than 0.001% by weight based on the total weight of the composition, the effect of alleviating skin irritation or skin pigmentation is not exhibited. When the amount of the mixture is more than 10% by weight, the degree of skin irritation mitigation or skin pigmentation improving effect The stability of the shape is problematic, and it is not economical.

The cosmetic composition may contain components conventionally used in cosmetic compositions, and includes, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and perfumes, and carriers.

The cosmetic composition may be prepared in any form conventionally produced in the art. In one embodiment, it may be any one selected from the group consisting of a high-viscosity emulsifier type, a low-viscosity emulsifier type, and a solubilized formulation, but the present invention is not limited thereto. And may contain components of the cosmetic composition commonly used in the technical field to which the invention belongs, depending on the formulation to be produced. For example, as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, packs, massage creams and sprays But is not limited thereto. More specifically, the present invention relates to a cosmetic lotion composition comprising: (A) at least one cosmetic composition selected from the group consisting of a lotion, a nutritional lotion, a nutritive cream, a massage cream, an essence, an eye cream, a sun lotion, a sunscreen, a makeup base, a bib cream, a cleansing cream, a cleansing foam, a cleansing water, Balm type product, spray or powder. Depending on the formulation to be prepared, the water phase component, the oil component, the surfactant, the moisturizer, the lower alcohol, the thickening agent, the chelating agent, the preservative, and the fragrance may be selected and added in addition.

The mixture of the dermis extract and the cottonseed extract may be manufactured by a manufacturing method including, but not limited to, the following steps.

1) extracting the dermis or leek leaf with an extraction solvent;

2) filtering the extract of step 1), respectively;

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

4) drying the concentrated extract of step 3) to powder;

5) mixing the powdered extract of step 4) at a constant ratio.

In this method, the dermis or filamentous foliage of step 1) may be used without limitation, such as cultivated or marketed.

In the above method, conventional methods such as filtration, hot water extraction, immersion extraction, reflux extraction, ultrasonic extraction, supercritical extraction, and the like can be used. Lt; / RTI >

Further, the extraction solvent may be obtained by extracting with water, a C ₁ to C ₄ lower alcohol, or a mixed solvent thereof, and the lower alcohol may be ethanol or methanol. The extraction solvent may be added in an amount of 1 to 20 times the amount of the dermis or lincepin, and may be added in an amount of 5 to 15 times, but is not limited thereto. The extraction temperature may be 20 to 70 캜, specifically 25 to 65 캜, and more specifically, 30 to 60 캜, but is not limited thereto. In addition, the extraction time may be 1 to 40 hours, and may be 10 to 30 hours, but is not limited thereto.

In this method, the extract of step 2) may be filtered 1 to 5 times. As the filtration method, conventional methods in the art such as sieving, suction filtration, ultrafiltration, and filter press can be used, but the present invention is not limited thereto.

In the above method, the vacuum concentration of the step 3) may be performed using a vacuum decompression concentrator or a vacuum rotary evaporator, but the present invention is not limited thereto.

In the above method, the drying method of step 4) may be a conventional method such as hot air drying or freeze drying, but is not limited thereto.

In the above method, the mixing method of step 5) may be carried out by mixing each of the powdered extract ingredients in a weight ratio and dissolving them in a conventional solvent used in the art such as water, propanediol, and ethanol, It does not.

The mixture of the dermabrasion extract and the whiteness extract of the present invention has an excellent effect on the improvement of skin irritation by inhibiting the increase of NO production and inflammatory cytokine induced by yellow sand or fine dusts.

In addition, the mixture of the dermis extract and the whiteness extract of the present invention inhibits tyrosinase activity induced by yellow dust or fine dusts, and inhibits melanin production, thereby improving skin pigmentation.

FIG. 1 is a graph showing the effect of inhibiting NO production induced by yellow dust or fine dusts in the mixture of the dermis extract and the white flour extract of the present invention.
2 is a graph showing the effect of the mixture of the dermis extract and the leish extract of the present invention on IL-1? Production induced by yellow dust or fine dust.
FIG. 3 is a graph showing the effect of the mixture of the dermis extract and the leish extract of the present invention on IL-2 production induced by yellow dust or fine dust.
FIG. 4 is a graph showing the effect of the mixture of the dermis extract and the leish extract of the present invention on TNF-α production induced by yellow dust or fine dust.
FIG. 5 is a graph showing the effect of the mixture of the dermis extract and the fine linseed extract of the present invention on GM-CSF production induced by yellow dust or fine dust.
FIG. 6 is a graph showing the inhibitory effect of tyrosinase activity induced by yellow dust or fine dusts on the mixture of the dermis extract and the white flour extract of the present invention.
FIG. 7 is a graph showing the melanin production inhibitory effect induced by yellow dust or fine dusts of the mixture of the dermis extract and the white flour extract of the present invention.
8 is a graph showing the effect of the mixture of the dermis extract and the albumen extract of the present invention on tyrosinase activity induced by melanocyte stimulating hormone.
FIG. 9 is a graph showing the melanin production inhibitory effect induced by the melanocyte stimulating hormone in the mixture of the dermis extract and the albumen extract of the present invention.

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

However, the following Production Examples, Examples, Formulation Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by Production Examples, Examples, Formulation Examples and Experimental Examples.

Production Example 1. Preparation of Dermal Ethanol Extract

To 50 g of dried pulverized dermis (Citrus unshiu Markovich peel) was added 500 ml of 70% ethanol mixed at a volume ratio of purified water to ethanol of 30:70 and extracted with shaking at room temperature for 20 hours. The extract was filtered, concentrated under reduced pressure to remove ethanol, and then dried to obtain a powdered dermis ethanol extract at a yield of 18.7% by weight.

Production Example 2: Production of elongated hot water extract

The dried Chamaecyparis obtusa leaf was pulverized and then subjected to hot water extraction at 100 ° C for 5 hours. The obtained extract was filtered and concentrated under reduced pressure at 60 ° C to 1/5 of the initial weight. The ethanol precipitation reaction was carried out by slowly dropping ethanol of 5 times the volume of the concentrate. The precipitate was collected, washed with ethanol, and then filtered to dryness. This gave a powdered hydrocolloid extract at a yield of 4% by weight.

Production Example 3. Preparation of yellow dust or fine dust extract

1) Sampling of dust or fine dust: A sample was taken from a sampler equipped with a 47 mm Teflon filter at a flow rate of 16.7 L / min for 24 hours in a low volume air sampler installed in Bucheon city, Gyeonggi Province in March 2013. The sample could be collected with Total Suspended Particles (TSP), which is all the dust floating in the air below 50 μm.

2) Preparation of total suspended dust (TSP) extract: Ethanol was added to a filter cut into a size of about 1 ㎡ and extracted with stirring for 12 hours. The extract was separated and extracted with extraction solvent. The extract was again extracted with ultrasonic for 4 hours. The extracts were combined and concentrated under reduced pressure to a constant volume. At this time, the extracted sample used ethanol as an extraction solvent.

Examples 1. to 5. Preparation of a mixture of a dermis extract and a cottonseed extract

The dermal extract powder and the monoaxial extract powder obtained in Preparation Examples 1 and 2 were mixed in a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3) 4) and 1: 2 (Example 5), and the mixture was dissolved in a mixed solvent of water, propanediol and ethanol to prepare a mixture of the dermis extract and the whitening extract.

EXPERIMENTAL EXAMPLE 1 Evaluation of Inflammation Inducing Ability of Yellow Dust or Fine Dust Extract

There are reports that when yellow dust or fine dusts come into contact with the skin, various adverse reactions may occur. The following experiment was carried out to investigate the specific effect of dust or fine dust on skin. In order to confirm that the TSP that was captured in the air was extracted to induce inflammation in the cells, experiments were carried out on the anti - inflammatory effect.

(1) NO production induction evaluation

Raw cells were seeded at 6 × 10 ⁴ cells / well in a 96-well plate and cultured at 37 ° C and 5% CO 2 for 24 hours. TSP extract was treated and cultured again for 24 hours. Then, the cell culture was mixed with Griess reagent 1: 1 and the NO production was measured by measuring the absorbance at 540 nm with a micro-plate reader. The results are shown in Table 1. A negative control (control) for NO production and a positive control (lipopolysaccharide (LPS) 1 占 퐂 / ml) were treated together. At this time, the cytotoxicity of the sample raw cells to induce NO production was not found.

(2) Evaluation of inflammatory cytokine production induction

HaCaT was dispensed into 96-well plates at 2.5 × 10 ⁴ cells / well and cultured at 37 ° C and 5% CO 2 for 24 hours. IL-2, TNF-α, and GM-CSF were measured by ELISA kit for IL-1α, IL-2, TNF-α and GM-CSF, respectively. The results are shown in Table 1. At this time, there was no cytotoxicity of HaCaT cells in the sample to induce the production of IL-1 alpha, IL-2, TNF-alpha and GM-CSF.

Sample NO
production
(%) IL-1?
Production
(%) IL-2
production
(%) TNF-a
production
(%) GM-CSF
production
(%) Control 100.0 + - 3.4 100.0 ± 2.2 100.0 ± 3.3 100.0 0.7 100.0 ± 1.6 LPS 1 ug / ml 196.7 ± 3.9 186.0 + - 4.7 192.3 ± 2.4 227.8 ± 5.4 229.7 ± 2.1 TSP extract 248.9 ± 1.5 160.5 ± 3.5 201.5 ± 2.6 157.7 ± 2.1 235.5 ± 0.6

As shown in Table 1, it was confirmed that TSP extract induced NO production and inflammatory cytokine production.

Experimental Example 2 Evaluation of inhibitory effect of TSP-induced NO production

(Preparation Example 1) and the whitening extract (Preparation Example 2) were added at concentrations of 100 ug / ml and 50 ug / ml without cytotoxicity, respectively, after treatment with TSP (Total Suspended Particles) (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), and 1: 1 (Example 3) : 1 (Example 4), and 1: 2 (Example 5). The NO measurement method is as follows.

Raw cells were seeded at 6 × 10 ⁴ cells / well in a 96-well plate. Cells were cultured in a cell incubator at 37 ° C and 5% CO 2 for 24 hours. After 24 hours, the samples were treated by concentration, cultured, and the cell culture solution and Griess reagent were reacted at a ratio of 1: 1. The absorbance was measured at 540 nm using an ELISA reader.

The NO concentration was determined using a sodium nitrite standard curve, and the final amount of NO was compared with the negative control (control) in terms of NO amount per constant protein. For protein quantification, after cell culture and sample treatment, the medium was recovered and each well was washed with PBS. The cells were treated with 1 N NaOH and protein was quantitated by the Lowry assay, one of the protein assay methods. To quantify the protein, Bio-Rad's DC Protein Assay (500-0116) was used. Cells were treated with Reagent A: Reagent S = 50: 1, the reagent contained in the Bio-Rad's DC Protein Assay, and treated with Reagent B at room temperature. The amount of protein was determined by measuring the absorbance at 750 nm with an ELISA reader, and the amount of final protein was determined using a standard curve of bovine serum albumin.

Sample Concentration (ug / ml) NO production (%) Control 100.0 + - 3.4 TSP extract 248.9 ± 1.5

Sole extract
Production Example 1 100 98.7 ± 2.1 50 120.6 ± 4.0 25 159.8 ± 3.1
Production Example 2 100 139.8 ± 1.3 50 177.9 ± 2.8 25 200.1 ± 1.6





Mixed extract
Example 1 100 106.1 ± 3.3 50 133.7 ± 2.7 25 170.1 ± 4.3
Example 2 100 75.8 ± 1.2 50 101.6 ± 2.0 25 120.3 + 1.7
Example 3 100 105.3 ± 2.3 50 130.4 ± 1.5 25 165.8 ± 2.4
Example 4 100 135.8 ± 3.1 50 172.4 ± 2.3 25 209.9 ± 2.1
Example 5 100 157.4 ± 2.5 50 195.6 ± 3.8 25 242.5 ± 3.5

As shown in the above Table 2, the NO production amount (248.9%) increased by TSP was reduced to 98.7% by the dermis extract of 100 ug / ml (Preparation Example 1) It was confirmed that the extract was reduced to 139.8% by the extract of Fagaceae (Preparation Example 2).

In addition, the dermis extract and the whitening extract were mixed at a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: Ex. 5) decreased to 106.1%, 75.8%, 105.3%, 135.8% and 157.4%, respectively, at the concentration of 100 ug / ml compared to the amount of NO produced by TSP.

That is, the NO production inhibition effect was most excellent in Example 2, and the NO production amount was reduced by 22.9% and 64.0%, respectively, compared with the dermis extract (Preparation Example 1) and the whitening extract (Preparation Example 2).

When the amount of NO increased by TSP was taken as 100%, 100.9% inhibition of the dermis extract (Preparation Example 1) and 73.3% inhibition of the whitening extract (Preparation Example 2) were observed at a concentration of 100 ug / ml. In Example 2, which showed the most excellent effect, 116.3% of the inhibitory activity was observed, and it was confirmed that the inhibitory effect on NO production was 15.4% higher than that of the dermis extract (Preparation Example 1) and 43.0% I could.

Experimental Example 3. Assessment of TSP-induced inhibition of cytokine production

After treatment with TSP, which is a fine dust promoting cytokine production, the dermis extract (Preparation Example 1) and the whitening extract (Preparation Example 2) were treated at a concentration of 100 ug / ml, 50 ug / ml and 25 ug / ml (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: 1 (Example 4), 1: 2 (Example 5), and the cells were treated. IL-1α, IL-2, TNF-α and GM-CSF were measured as cytokines.

HaCaT was dispensed into 96-well plates at 2.5 × 10 ⁴ cells / well. Cells were cultured in a cell culture incubator at 37 ° C and 5% CO 2 for 24 h. After incubation, the samples were treated by concentration, and the next day, experiments were carried out using an ELISA kit. Absorbance was measured at 450 nm with an ELISA reader.

The amount of final cytokine was compared with the negative control (Control) in terms of the amount of cytokine per constant protein.

Sample Concentration (ug / ml) IL-1? Production (%) Control 100.0 ± 2.2 TSP extract 160.5 ± 3.5

Sole extract
Production Example 1 100 99.8 ± 2.2 50 115.9 ± 1.1 25 134.4 ± 3.7
Production Example 2 100 120.1 ± 2.4 50 139.8 ± 3.9 25 155.5 ± 1.5





Mixed extract
Example 1 100 105.1 ± 0.8 50 120.0 ± 3.3 25 131.5 ± 0.9
Example 2 100 70.7 ± 2.3 50 85.1 ± 3.5 25 101.6 ± 2.9
Example 3 100 95.1 ± 1.1 50 113.7 ± 4.2 25 129.6 ± 3.9
Example 4 100 122.9 ± 2.8 50 144.3 ± 2.5 25 160.7 ± 5.0
Example 5 100 128.9 ± 1.0 50 142.9 ± 2.6 25 157.8 ± 1.4

As shown in Table 3, the amount of IL-1α production (160.5%) increased by TSP was reduced to 99.8% by 100 μg / ml of dermis extract (Preparation Example 1) / ml < / RTI > (Preparation Example 2).

In addition, the dermis extract and the whitening extract were mixed at a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: Example 5), the concentration of IL-1α was decreased to 105.1%, 70.7%, 95.1%, 122.9%, and 128.9%, respectively, when the concentration of IL-1α was increased by 100 μg / ml.

That is, the IL-1α production inhibitory effect was the most excellent in Example 2, and the IL-1α production was reduced by 29.1% and 49.4% more than the dermis extract (Preparation Example 1) and the whitening extract (Preparation Example 2), respectively.

In addition, when the amount of IL-1α increased by TSP was 100%, the dermis extract (Preparation Example 1) was 100.3% and the whitening extract (Preparation Example 2) was 66.8% at a concentration of 100 ug / ml. In Example 2, which had the best effect, 148.4% inhibitory effect was obtained and 48.1% inhibition of IL-1α production was obtained than dermis extract (Preparation Example 1) and 81.7% .

Sample Concentration (ug / ml) IL-2 production (%) Control 100.0 ± 3.3 TSP extract 201.5 ± 2.6

Sole extract
Production Example 1 100 120.8 ± 1.9 50 139.8 ± 3.4 25 150.1 ± 1.4
Production Example 2 100 143.8 ± 1.8 50 167.2 ± 1.7 25 188.3 ± 1.2





Mixed extract
Example 1 100 135.4 ± 1.2 50 155.5 ± 2.8 25 167.9 ± 3.8
Example 2 100 92.5 ± 1.6 50 108.8 ± 3.1 25 121.1 ± 1.6
Example 3 100 120.5 ± 3.1 50 131.9 ± 2.0 25 149.8 ± 3.7
Example 4 100 147.3 ± 2.9 50 173.2 ± 1.2 25 192.4 + - 5.4
Example 5 100 150.2 ± 4.0 50 174.7 ± 1.8 25 191.4 ± 3.6

As shown in Table 4, the IL-2 production (201.5%) increased by TSP decreased to 120.8% by the dermis extract of 100 ug / ml (Preparation Example 1) / ml < / RTI > (Preparation Example 2).

The dermis extract and the cotton-white extract were mixed in a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: ) Decreased to 135.4%, 92.5%, 120.5%, 147.3% and 150.2%, respectively, at the concentration of 100 ug / ml relative to the amount of IL-2 produced by TSP.

That is, the IL-2 production inhibitory effect was most excellent in Example 2, and the IL-2 production was 28.3% lower than that of the dermis extract (Preparation Example 1) and 51.3% lower than that of the white flour extract (Preparation Example 2).

In addition, when the amount of IL-2 increased by TSP was taken as 100%, the dermis extract (Preparation Example 1) was inhibited by 70.5% and the whitening extract (Preparation Example 2) by 37.9% at a concentration of 100 ug / ml. In Example 2, which had the best effect, the inhibitory effect was 110.6%, and the inhibitory effect on IL-2 production was 40.1% higher than that of dermis extract (Preparation Example 1) and 72.8% .

Sample Concentration (ug / ml) TNF-α production (%) Control 100.0 0.7 TSP extract 157.7 ± 2.1

Sole extract
Production Example 1 100 103.8 ± 2.8 50 117.7 ± 1.2 25 135.4 ± 0.8
Production Example 2 100 120.8 + 1.7 50 136.8 ± 1.1 25 149.0 ± 2.6





Mixed extract
Example 1 100 105.7 ± 2.1 50 126.2 ± 0.6 25 139.8 ± 1.7
Example 2 100 78.1 ± 2.4 50 90.5 ± 2.6 25 103.6 ± 1.1
Example 3 100 98.7 ± 1.9 50 117.8 ± 0.6 25 130.1 ± 2.6
Example 4 100 117.3 ± 1.3 50 135.5 ± 1.8 25 153.8 ± 3.8
Example 5 100 123.5 ± 1.8 50 145.7 ± 1.9 25 156.6 ± 1.3

As shown in Table 5, the amount of TNF-α production (157.7%) increased by TSP was reduced to 103.8% by 100 μg / ml of dermis extract (Preparation Example 1) / ml of the cottonseed extract (Preparation Example 2), it was confirmed to be reduced to 120.8%.

In addition, the dermis extract and the whitening extract were mixed at a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: Example 5), the amount of TNF-α produced by TSP decreased to 105.7%, 78.1%, 98.7%, 117.3% and 123.5% at a concentration of 100 μg / ml, respectively.

That is, the TNF-α production inhibitory effect was most excellent in Example 2, and the TNF-α production was 25.7% and 42.7% lower than that of the dermis extract (Preparation Example 1) and the whitening extract (Preparation Example 2), respectively.

In addition, when the amount of TNF-? Increased by TSP was 100%, the dermis extract (Preparation Example 1) and the whitening extract (Preparation Example 2) were inhibited by 93.4% and 64.0%, respectively, at a concentration of 100 ug / ml. Example 2, which showed the most excellent effect, showed a suppressive effect of 138.0% and a TNF-α production inhibitory ability which is 44.5% higher than the dermis extract (Preparation Example 1) and 74.0% superior to the whiteness extract (Preparation Example 2) .

Sample Concentration (ug / ml) GM-CSF production (%) Control 100.0 ± 1.6 TSP extract 235.5 ± 0.6

Sole extract
Production Example 1 100 100.7 ± 4.0 50 129.7 ± 0.4 25 147.6 ± 2.5
Production Example 2 100 201.7 ± 3.1 50 226.1 ± 3.6 25 233.9 ± 4.8





Mixed extract
Example 1 100 115.8 ± 1.8 50 159.9 ± 2.1 25 170.5 ± 2.0
Example 2 100 67.9 ± 3.0 50 80.0 ± 0.6 25 97.8 ± 0.9
Example 3 100 94.8 ± 2.4 50 121.7 ± 2.0 25 130.9 ± 1.4
Example 4 100 134.8 ± 1.5 50 167.4 ± 1.2 25 183.1 ± 1.1
Example 5 100 161.2 ± 2.7 50 194.2 ± 3.3 25 211.6 ± 1.8

As shown in Table 6, GM-CSF measurement showed that the GM-CSF production amount (235.5%) increased by TSP was reduced to 100.7% by the dermis extract of 100 ug / ml (Preparation Example 1) / ml of the leishmanial extract (Preparation Example 2), it was confirmed that it decreased to 201.7%.

In addition, the dermis extract and the whitening extract were mixed at a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: Example 5), the concentration of GM-CSF decreased by 115.8%, 67.9%, 94.8%, 134.8% and 161.2%, respectively, when the concentration of GM-CSF was increased by 100 μg / ml.

That is, the GM-CSF production inhibitory effect was most excellent in Example 2, and the GM-CSF production was reduced by 32.8% and 133.8%, respectively, compared with the dermis extract (Preparation Example 1) and the whitening extract (Preparation Example 2).

In addition, when the amount of GM-CSF increased by TSP was taken as 100%, 99.5% of the dermis extract (Preparation Example 1) and 24.9% of the extract of the whiteness extract (Preparation Example 2) were inhibited at a concentration of 100 ug / ml. In Example 2, which showed the best effect, 123.7% of the inhibitory effect was shown, and it had a GM-CSF production inhibitory ability which is 24.2% higher than the dermis extract (Preparation Example 1) and 98.7% superior to the whiteness extract (Preparation Example 2) .

Experimental Example 4 Evaluation of Tyrosinase Activity and Melanin Production Induced by TSP

After treatment of the cells with TSP, the fine dust, the dermis extract (Preparation Example 1) and the whitening extract (Preparation Example 2) were treated at a concentration of 100 ug / ml, 50 ug / ml and 25 ug / ml, The dermis extract and the cotton-white extract were mixed in a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: ), And the cells were treated. Sample treatment, tyrosinase activity and melanin content measurement method are as follows.

After 2.5 x 10 ⁵ cells / well of human epidermal keratinocytes in a 6-well plate, the cells were incubated in a cell culture incubator at 37 ° C and 5% CO 2 for 24 hours. After 24 hours, TSP and samples were treated and cultured for 24 hours.

Human epidermal keratinocytes were plated at 1.5 × 10 ⁵ cells / well in a 6-well plate and cultured in a cell incubator at 37 ° C and 5% CO 2 for 24 hours to allow the cells to adhere well to the plate.

On the next day, the culture of human epidermal keratinocytes treated with TSP and the sample was treated for 72 hours, and the tyrosinase activity in the keratinocytes of the human epidermis was measured. Cells were lysed with lysis buffer (50 mM sodium phosphate buffer, 1% triton X-100, 0.1 mM phenylmethyl sulfonyfluoride) and centrifuged at 13,000 rpm for 20 minutes. After centrifugation, the obtained supernatant was reacted with 10 mM L-DOPA in 50 mM sodium phosphate buffer (pH 6.8) as a tyrosinase enzyme solution at 37 ° C for 60 minutes. The tyrosinase activity was measured at 490 nm using an ELISA reader.

The content of melanin was determined by dissolving the pellet of melanocytes remaining after centrifugation in 1 N NaOH by measuring the absorbance at 405 nm with an ELISA reader.

The final tyrosinase activity and the content of melanin were compared with the negative control (control) in terms of tyrosinase activity and melanin content per a certain protein.

Sample Concentration (ug / ml) Tyrosinase activity (%) Control 100.0 ± 2.6 TSP extract 163.6 ± 1.8

Sole extract
Production Example 1 100 111.8 ± 0.1 50 129.7 ± 0.1 25 140.7 ± 1.1
Production Example 2 100 135.4 ± 3.9 50 150.1 ± 2.3 25 158.0 + - 3.0





Mixed extract
Example 1 100 117.0 ± 0.1 50 135.5 ± 0.2 25 150.8 ± 4.1
Example 2 100 80.7 ± 2.2 50 99.1 ± 0.6 25 117.8 ± 0.1
Example 3 100 105.4 ± 0.1 50 124.7 ± 1.0 25 138.8 ± 3.0
Example 4 100 121.1 ± 1.4 50 141.0 ± 4.0 25 152.5 ± 0.1
Example 5 100 133.6 ± 1.9 50 153.8 ± 1.4 25 165.1 ± 1.5

As shown in Table 7, the tyrosinase activity (163.6%) increased by TSP was decreased to 111.8% by the dermis extract of 100 ug / ml (Preparation Example 1), and 100 ug / ml And it was confirmed to be reduced to 135.4% by the extract of the white flour (Preparation Example 2).

In addition, the dermis extract and the whitening extract were mixed at a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: Example 5), when they were mixed at a concentration of 100 ug / ml against the tyrosinase activity increased by TSP, they decreased to 117.0%, 80.7%, 105.4%, 121.1% and 133.6%, respectively.

That is, the tyrosinase activity inhibitory effect was the most excellent in Example 2, and the tyrosinase activity was reduced by 31.1% from the dermis extract (Preparation Example 1) and by 54.7% from the whitening extract (Preparation Example 2).

In addition, when the activity of tyrosinase increased by TSP was 100%, the dermis extract (preparation 1) and the whitening extract (preparation 2) were inhibited by 81.4% and 44.3%, respectively, at a concentration of 100 ug / ml. In Example 2, where the effect was most excellent, 130.3% of the inhibitory effect was shown, which was 48.9% more effective than the dermis extract (Preparation Example 1) and 86.0% superior to the whitening extract (Preparation Example 2) I could confirm.

Sample Concentration (ug / ml) Melanin content (%) Control 100.0 ± 0.2 TSP extract 155.7 ± 1.0

Sole extract
Production Example 1 100 111.8 ± 2.5 50 123.9 ± 0.4 25 138.1 ± 0.7
Production Example 2 100 130.1 ± 2.5 50 139.8 ± 3.2 25 146.7 ± 0.3





Mixed extract
Example 1 100 107.2 ± 1.2 50 120.7 ± 0.2 25 133.9 ± 3.3
Example 2 100 83.7 ± 0.7 50 100.1 ± 1.2 25 107.8 ± 0.3
Example 3 100 103.4 ± 3.5 50 116.8 ± 1.5 25 122.3 ± 1.7
Example 4 100 114.4 ± 2.8 50 129.7 ± 2.3 25 141.6 ± 0.1
Example 5 100 125.7 ± 3.0 50 135.7 ± 1.0 25 148.0 ± 1.4

As shown in Table 8, the melanin content (155.7%) increased by TSP was decreased to 111.8% by the dermis extract of 100 ug / ml (Preparation Example 1) And it was confirmed that the extract was reduced by 130.1% by the extract of the white cotton (Preparation Example 2).

In addition, the dermis extract and the whitening extract were mixed at a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: Example 5), the amount of melanin was decreased to 107.2%, 83.7%, 103.4%, 114.4%, and 125.7%, respectively, at a concentration of 100 ug / ml relative to the amount of melanin produced by TSP.

That is, the melanin production inhibitory effect was the highest in Example 2, and the melanin production was 28.1% lower than that of the dermis extract (Preparation Example 1) and 46.4% more than that of the whitening extract (Preparation Example 2).

In addition, when the amount of melanin increased by TSP was 100%, the dermis extract (Preparation Example 1) was inhibited by 78.8% and the whitening extract (Preparation Example 2) by 46.0% at a concentration of 100 ug / ml. In Example 2, which had the best effect, the inhibitory effect was 129.3%, which is 50.4% higher than that of the dermis extract (Preparation Example 1) and 83.3% superior to that of the whiteness extract (Preparation Example 2) I could confirm.

Experimental Example 5 Evaluation of tyrosinase activity and melanin production induced by melanocyte-stimulating hormone (a-MSH)

After treatment with a-MSH promoting melanin production, the dermis extract (Preparation Example 1) and the whitening extract (Preparation Example 2) were treated at a concentration of 100 ug / ml, 50 ug / ml and 25 ug / ml, (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: 1 (Example 4) and 1: 2 Example 5), and the cells were treated. Sample treatment, tyrosinase activity and melanin content measurement method are as follows.

Human epidermal keratinocytes were plated at 1.5 × 10 ⁵ cells / well in a 6-well plate and cultured in a cell incubator at 37 ° C and 5% CO 2 for 24 hours to allow the cells to adhere well to the plate.

The following day, the a-MSH and the sample were treated and cultured for 72 hours, and the tyrosinase activity in the keratinocytes of the human epidermis was measured. Cells were lysed with lysis buffer (50 mM sodium phosphate buffer (pH 6.8), 1% triton X-100, 0.1 mM phenylmethyl sulfonyfluoride) and centrifuged at 13,000 rpm for 20 minutes. After centrifugation, the obtained supernatant was reacted with 10 mM L-DOPA in 50 mM sodium phosphate buffer (pH 6.8) as a tyrosinase enzyme solution at 37 ° C for 60 minutes. The tyrosinase activity was measured at 490 nm using an ELISA reader.

The content of melanin was determined by dissolving the pellet of melanocytes remaining after centrifugation in 1 N NaOH by measuring the absorbance at 405 nm with an ELISA reader.

The final tyrosinase activity and the content of melanin were compared with the negative control (control) in terms of tyrosinase activity and melanin content per a certain protein.

Sample Concentration (ug / ml) Tyrosinase activity (%) Control 100.0 + - 0.4 a-MSH 207.8 ± 3.2

Sole extract
Production Example 1 100 120.8 ± 0.8 50 137.8 ± 1.2 25 158.8 ± 1.2
Production Example 2 100 150.6 ± 1.4 50 169.8 ± 1.6 25 180.8 ± 2.0





Mixed extract
Example 1 100 133.8 ± 1.2 50 140.8 ± 0.6 25 155.8 ± 0.7
Example 2 100 90.1 ± 0.9 50 108.9 ± 0.6 25 129.1 ± 0.6
Example 3 100 123.0 ± 1.3 50 135.8 ± 0.3 25 150.5 ± 2.7
Example 4 100 135.9 ± 0.9 50 158.0 + - 0.4 25 177.5 ± 0.7
Example 5 100 146.8 ± 0.8 50 168.2 ± 1.5 25 189.8 ± 0.6

As shown in Table 9, tyrosinase activity (207.8%) increased by a-MSH was decreased to 120.8% by 100 μg / ml of dermis extract (Preparation Example 1) ml of the extract of Clostridium perfringens (Preparation Example 2).

In addition, the dermis extract and the whitening extract were mixed at a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: Example 5), 133.8%, 90.1%, 123.0%, 135.9%, and 146.8% decrease, respectively, at the concentration of 100 ug / ml compared to the tyrosinase activity increased by TSP.

That is, the tyrosinase activity inhibitory effect was the most excellent in Example 2, and the tyrosinase activity was reduced by 30.7% and 60.5% more than the dermis extract (Preparation Example 1) and the whitening extract (Preparation Example 2), respectively.

In addition, when the activity of tyrosinase increased by a-MSH was 100%, the dermis extract (Preparation Example 1) was inhibited by 80.7% and the whitening extract (Preparation Example 2) by 53.1% at a concentration of 100 ug / ml. In Example 2, which showed the most excellent effect, 109.2% of the inhibitory effect was observed, and it was confirmed that the inhibitory activity against tyrosinase activity was 28.5% higher than that of the dermis extract (Preparation Example 1) and 56.1% I could.

Sample Concentration (ug / ml) Melanin content (%) Control 100.0 ± 0.5 a-MSH 198.8 ± 0.9

Sole extract
Production Example 1 100 130.0 ± 1.1 50 142.2 ± 0.9 25 159.0 ± 0.8
Production Example 2 100 157.3 ± 1.7 50 166.7 ± 1.3 25 185.6 ± 1.6





Mixed extract
Example 1 100 130.8 ± 2.1 50 147.6 ± 0.9 25 176.1 ± 2.2
Example 2 100 103.4 ± 0.7 50 119.7 ± 2.6 25 130.5 ± 1.3
Example 3 100 120.7 ± 1.8 50 137.7 ± 0.4 25 155.4 ± 0.7
Example 4 100 148.1 ± 2.2 50 161.3 ± 2.0 25 183.1 ± 2.3
Example 5 100 166.9 ± 1.3 50 180.5 + 1.7 25 201.4 ± 0.6

As shown in Table 10, the amount of melanin (198.8%) increased by a-MSH was reduced to 130.0% by 100 μg / ml of dermis extract (Preparation Example 1) lt; / RTI > of the extract (Preparation Example 2).

In addition, the dermis extract and the whitening extract were mixed at a ratio of 4: 1 (Example 1), 3: 1 (Example 2), 2: 1 (Example 3), 1: Example 5) decreased to 130.8%, 103.4%, 120.7%, 148.1% and 166.9%, respectively, at a concentration of 100 ug / ml relative to the amount of melanin produced by TSP.

That is, the melanin production inhibitory effect was the highest in Example 2, and the melanin production was 26.6% lower than that of the dermis extract (Preparation Example 1) and 53.9% more than the whitening extract (Preparation Example 2).

In addition, when the amount of melanin increased by a-MSH was 100%, the dermal extract (Preparation Example 1) showed a inhibitory effect of 69.9% and the whitening extract (Preparation Example 2) had a inhibitory effect of 42.0% at a concentration of 100 ug / ml. In Example 2, which showed the best effect, 96.6% inhibitory effect was confirmed, and it was confirmed that the inhibitory effect on melanin formation was 26.9% higher than that of dermis extract (Preparation Example 1) and 68.8% I could.

Formulation Examples 1. to Formulation 8. Preparation of O / W cream formulations containing dermis and flour extracts

Formulations 1 to 8 of an O / W cream formulation having the composition shown in the following Table 11 were prepared. Extracts were dried powders, and other ingredients were generally used for cosmetics.

(Unit: wt%) Prize ingredient Formulation Example One 2 3 4 5 6 7 8 Number
Prize Purified water To 100 glycerin 20 Betaine 20 Sodium hyaluronate 20 Incrementer Suitable amount Metal ion sequestrant Suitable amount U
Prize FAGE -100 stearate One Glyceryl stearate One Polysorbate 60 0.8 Stearic acid 0.5 Cetearyl alcohol 2 Capric caprylic triglyceride 15 Tocopheryl acetate 0.5 The
end
Ⅰ Production Example 1 - 0.67 - - - - - - Production Example 2 - - 0.67 - - - - - Example 1 - - - 0.67 - - - - Example 2 - - - - 0.67 - - - Example 3 - - - - - 0.67 - - Example 4 - - - - - - 0.67 - Example 5 - - - - - - - 0.67 The
end
Ⅱ incense Suitable amount Preservative Suitable amount Other additives Suitable amount

<Manufacturing Method>

(1) The water phase and the oil phase were warmed and homogeneously mixed and dissolved respectively.

(2) The oil phase was added to the aqueous phase at 75 DEG C, and the mixture was emulsified

(3) After the step (2), the additive I was added and mixed at 45 캜, and then the additive II was added and mixed.

EXPERIMENTAL EXAMPLE 6. Evaluation of whitening effect according to the mixing ratio of dermis and leish extract

Twenty healthy adults (20 males and 2 females) adhered with opaque tape with six holes (1.5 cm in diameter) to the subject's skin. The subjects were exposed to ultraviolet light (UVB) of 1.5-2 times the minimum erythema dose The darkening of the skin was induced by irradiation, the test materials were applied, and after two months, the darkness and darkness of the skin were measured using a chroma meter. Formulation Examples 1 to 8 were used as test substances, and each test substance was applied twice a day (morning and evening) daily.

The determination of the whitening effect was determined by determining the "L" value representing the shade of the skin (the non-burned Korean skin color generally has a value of 50 to 70). When the test substance is applied, the L value gradually increases when the test substance is effective. The comparison between the test substances is expressed by a ΔL value (final L value - L value before application of the test substance). The larger the value of? L, the greater the whitening effect.

The mean value of? L for the 20 subjects according to the experimental results is shown in Table 12 below.

Test substance Formulation Example One 2 3 4 5 6 7 8 Whitening effect
(DELTA L) 0.796 1.751 1.315 2.432 2.986 2.337 2.152 1.952

As shown in Table 12, although Formulation Examples 2 and 3 each containing the dermis extract and the whitening extract of the present invention exhibited a whitening effect higher than that of Formulation 1 which did not contain both the dermis extract and the whitening extract, The results were lower than those of Examples 4 ~ 8. Among Formulation Examples 4 to 8 in which the dermis extract and the cottonseed extract were mixed, Formulation Example 5 (dermis extract: cottonseed extract = 3: 1 weight ratio) showed the best skin whitening effect.

Claims (9)

For improving skin inflammation and pigmentation induced by yellow dust or fine dust comprising a 3: 1 by weight mixture of dermis ( citrus unshiu Markovich peel) extract and chamaecyparis obtuse extract as active ingredients, comprising the steps of: Method of preparing composition:
(a) adding 70% ethanol to the dried pulverized dermis and shaking extracting at room temperature for 20 hours;
(b) filtering and concentrating the extract of step (a);
(c) drying the extract of step (b) to obtain a powdered dermis ethanol extract;
(d) pulverizing the dried soft cotton leaves and subjecting them to hot water extraction at 100 ° C for 5 hours; And
(e) filtering the extract of step (d) and concentrating it under reduced pressure to 1/5 of the initial weight to obtain a mono-hydrolyzed extract.

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