KR20210041872A - Anti-pollution cosmetic composition comprising opuntia humifusa extract - Google Patents

Abstract

An object of the present invention is to provide an anti-pollution cosmetic composition comprising, as an active component, an extract of Opuntia humifusa, which has an effect of alleviating oxidative stress on skin cells caused by fine dust. The present invention provides the anti-pollution cosmetic composition comprising, as the active component, the extract of Opuntia humifusa.

Description

Anti-pollution cosmetic composition containing Cheonnyeoncho extract as an active ingredient {ANTI-POLLUTION COSMETIC COMPOSITION COMPRISING OPUNTIA HUMIFUSA EXTRACT}

The present invention relates to an anti-pollution cosmetic composition comprising Cheonnyeoncho extract as an active ingredient, and more particularly, to prevent side effects of fine dust on skin keratinocytes, and to reduce skin inflammation and CYP1A1 expression to prevent fine dust and It relates to an anti-pollution cosmetic composition capable of preventing side effects due to environmental pollutants of dioxin.

Particulate matter refers to small dust with a diameter of less than 10 μm invisible, and is known to contain toxic substances such as sulfate and nitrate. Such fine dust is produced by photochemical reactions in the atmosphere of toxic substances or heavy metals from automobile smoke and factory chimneys, and when exposed to a high concentration of fine dust, coughing, eye irritation, skin troubles, and inflammation of lung and airway cells Can cause.

Fine dust or ultrafine dust is one of the causes of damage to the skin that has attracted attention in recent years, and refers to particulate matter (PM) with a diameter of 10 µm or less or contaminants of 2.5 µm or less. Such fine dust and ultrafine dust directly damage the skin barrier function and cause exacerbation of dermatitis. Because the particle size of fine dust is very small, it can penetrate through the hair follicles to the underside of the skin. Therefore, it can affect people who are not atopy or allergic patients or patients with sensitive skin.

When fine dust penetrates the skin, it produces free radicals and damages the mitochondria of our skin, reducing collagen synthesis, increasing decomposition, and stimulating melanocytes to induce pigmentation. The aging phenomenon can be severe. Therefore, in order to protect the skin from fine dust, it is recommended to use basic cosmetics containing antioxidants that can reduce the production of active oxygen and prevent its effects. In addition, since fine dust itself can reduce the skin barrier function, it can be helpful to drink enough moisture and make use of moisturizers to life.

Meanwhile, Opuntia humifusa is a Korean native cactus that grows naturally in Chungnam and other places. In many cases, Cheonnyeoncho is confused with Opuntia ficus-indica var saboten, which has a similar Korean name, but both have different characteristics. In other words, the centennial plant, generally known as opuntia cactus, grows to a size of 1 to 2 m and has many long and thick thorns, whereas the millennial plant has downy thorns and grows to a size of about 30 cm. Baengnyeoncho grown in Jeju Island is native to the tropical region and is difficult to cultivate in the open field because it is weak at low temperatures, whereas the Korean native cactus, Cheonnyeoncho, survives in the open field even in the dormant winter and has strong characteristics against pests and diseases. Cheonnyeoncho is known to have a higher active ingredient than 100nyeoncho, and is currently attempting to commercialize it in various ways through processing into foods and cosmetics.

The present inventors discovered that the Cheonnyeoncho extract has skin-improving effects such as inflammation relief and anti-pollution, and developed an extraction method that can increase the content of the active ingredient contained in the Cheonnyeoncho extract, containing the Cheonnyeoncho extract as an active ingredient. The anti-pollution cosmetic composition was developed.

An object of the present invention is to provide an anti-pollution cosmetic composition capable of improving oxidative stress of skin cells due to fine dust and reducing the expression of skin CYP1A1 protein.

In order to achieve the above object, the present invention provides an anti-pollution cosmetic composition comprising an extract of Opuntia humifusa as an active ingredient.

Leaves, fruits, and roots of the cheonnyeoncho can be extracted.

The viscosity of the composition may be 1.0 to 3.0 mPa.S.

The content of the cheonnyeoncho extract may be 10 to 100% by weight based on the total composition.

The composition may be any one formulation selected from the group consisting of soothing gel, lotion, cream, facial cream, lotion, essence, cleansing cream, cleansing foam, cleansing water, pack, powder, or body oil.

In addition, in order to achieve the above object, the present invention comprises the steps of lyophilizing and pulverizing the washed leaves, fruits, and roots of Opuntia humifusa; And it provides a method for preparing an anti-pollution cosmetic composition comprising the Cheonnyeoncho extract as an active ingredient comprising; and extracting the pulverized product of Cheonnyeoncho to prepare a Cheonnyeoncho extract.

According to the cosmetic composition comprising the extract of Cheonnyeoncho according to the present invention as an active ingredient, it is possible to expect an oxidative stress improvement and antioxidant effect through inhibition of the generation of active oxygen caused by fine dust.

1 is a graph showing the polyphenol content of the Cheonnyeoncho extract based on the concentration of gallic acid.
Figure 2 is a graph showing the amount of the flavonoid compound in the extract of Cheonnyeoncho based on the quercetin concentration.
3 is a graph showing the free radical scavenging ability of the Cheonnyeoncho extract based on DPPH scavenging ability (%).
4 is a graph showing the cytotoxicity test results when the extract of Cheonnyeoncho is treated with HaCaT cells for 24 hours.
5 is a graph showing the cytotoxicity test results when the Cheonnyeoncho extract was treated with HaCaT cells for 48 hours.
Figure 6 is a graph showing the effect of reducing reactive oxygen species in cells when the extract of Cheonnyeoncho is treated with HaCaT cells for 24 hours.
7 is a graph showing the effect of reducing reactive oxygen species in cells when treated with Cheonnyeoncho extract for 48 hours on HaCaT cells.
FIG. 8 is a photograph of a DNA fragment of HaCaT cells compared and measured by Comet assay when fine dust, MgAC, and Cheonnyeoncho extract were treated on HaCaT cells for 24 hours.
9 is a photograph of a DNA fragment of HaCaT cells compared and measured by a Comet assay when fine dust, MgAC (biocompatible magnesium-amino clay), and Cheonnyeoncho extract were treated on HaCaT cells for 48 hours.
10 is a graph comparing tail DNA (%) and olive tail moment (%) of HaCaT cells when fine dust, MgAC, and Cheonnyeoncho extract were treated on HaCaT cells for 24 hours and 48 hours.
11 is a graph comparing the results of apoptosis when fine dust and Cheonnyeoncho extract were treated on HaCaT cells for 24 hours.
12 is a graph comparing the results of apoptosis when fine dust and Cheonnyeoncho extract were treated on HaCaT cells for 48 hours.
13 is a graph comparing the results of ATP synthesis when fine dust and Cheonnyeoncho extract are treated with HaCaT cells.
14 is a graph comparing ATP synthesis results when a fine dust sample is treated with HaCaT cells after treatment with Cheonnyeoncho extract.
15 is a graph comparing the results of cytotoxicity experiments when fine dust and Cheonnyeoncho extract were treated on HaCaT cells for 24 hours.
16 is a graph comparing the results of cytotoxicity experiments when fine dust and Cheonnyeoncho extract were treated on HaCaT cells for 48 hours.
17 is a result of confirming the expression of the MMP-9 gene by fine dust of the Cheonnyeoncho extract according to the present invention in HaCaT cells by Western blot.
18 is a result of confirming the expression of the COX-2 gene by fine dust of the Cheonnyeoncho extract according to the present invention in HaCaT cells by Western blot.
19 is a result of confirming the expression of AhR gene by fine dust of the Cheonnyeoncho extract according to the present invention in HaCaT cells by Western blot.
20 is a result of confirming the expression of the CYP1A1 gene by fine dust of the Cheonnyeoncho extract according to the present invention in HaCaT cells by Western blot.

In the following description, it should be noted that only parts necessary for understanding the embodiments of the present invention will be described, and descriptions of other parts will be omitted without distracting the gist of the present invention.

The terms or words used in the present specification and claims described below should not be construed as being limited to a conventional or dictionary meaning, and the inventor is appropriate as a concept of terms in order to describe his own invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention on the basis of the principle that it can be defined. Therefore, the embodiments described in the present specification and the configurations shown in the drawings are only preferred embodiments of the present invention, and do not represent all the technical spirit of the present invention, and various equivalents that can replace them at the time of application It should be understood that there may be variations and variations.

The present invention provides an anti-pollution cosmetic composition comprising an extract of Opuntia humifusa as an active ingredient.

Cheonnyeoncho (Opuntiahumifusa, Eastern prickly pear) is a cactus that can winter in inland among the Opuntia sp. The cheonnyeoncho has a shape similar to the palm of the hand, so it is also called a palm cactus.

The cheonnyeoncho is a fleshy perennial plant, that is, a perennial flowering plant, and the stem is fleshy like a general cactus, and has several points of constriction like a node, and the stem has various shapes such as a cylindrical shape, a ball shape, and an oval shape, They are also classified according to their shape. The ganglia has a hetmul tube, a vascular bundle near the epidermis, and the leaves on the shoots are shaped like needles, and are fleshy, and fall after they are fully grown. Cotyledons are lanceolate or triangular, and have the same flesh, and have different flowers and bracts. In general, it is known that the cheonnyeoncho reduces its own moisture to less than half, so that wintering is possible without freezing and dying even at -20°C, thereby maintaining vitality.

Cheonnyeoncho is known to have the effect of treating constipation, diuretic effect, activating bowel movement, and increasing appetite through conventional Dongchogangmok, and has antioxidant activity, anti-obesity activity, and liver protection effect using Cheonnyeoncho stems. Is known. Studies on these cheonnyeonchos have been mainly conducted on stems and fruits, especially stems, and as a result of these studies, the flesh and stems of cheonnyeonchos are sold as food. As described above, since Cheonnyeoncho, which has been used in various ways since ancient times, has been used as a food or medicine, its safety is evaluated to be guaranteed.

The cheonnyeoncho may preferably use leaves, fruits, and roots of cheonnyeoncho.

The Cheonnyeoncho extract may be an extract prepared by a solvent extraction method of the leaves, fruits, and roots of Cheonnyeoncho. The solvent extraction may be any one selected from the group consisting of water, an organic solvent, and a mixture thereof, but may be preferably prepared using ethanol.

The organic solvent may be an alcohol having 1 to 5 carbon atoms, a polar solvent such as dilute alcohol, ethyl acetate or acetone, and a non-polar solvent of ether, chloroform, benzene, hexane, or dichloromethane, or a mixed solvent thereof. The alcohol having 1 to 5 carbon atoms may be a glycol, which is a dihydric alcohol such as ethylene glycol, propylene glycol, butylene glycol, or pinacol, or a monohydric alcohol such as methanol, ethanol, propanol, butanol, and isopropanol, but is limited thereto. no.

In addition, alcohol diluted water may be a monohydric alcohol or dihydric alcohol diluted in water at 10% (v/v) to 99.9% (v/v), but alcohol diluted to 80% (v/v) is used. It is desirable to do it.

The cheonnyeoncho extract may be prepared according to a conventional method known in the art, that is, a conventional method for preparing a plant extract. For example, it may be prepared by a method such as a cold needle extraction method, a warm needle extraction method, a heat extraction method, an ultrasonic extraction method, a steam distillation method, or a supercritical fluid extraction method, a subcritical fluid extraction method, or a microwave treatment. In order to perform the extraction method, a conventional extraction device, an ultrasonic grinding extractor, or a fractionator may be used.

The Cheonnyeoncho extract may be filtered, concentrated, or dried to remove the solvent, and filtering, concentration, and drying may be performed. Specifically, the filtration may be performed using a filter paper or a reduced pressure filter paper, the concentration may be concentrated under reduced pressure using a vacuum concentrator, for example, a rotary evaporator, and the drying may be performed by, for example, a freeze-drying method.

The cheonnyeoncho extract may be included in the cosmetic composition in an amount of 10 to 100% by weight relative to the total composition. More specifically, the content of Cheonnyeoncho extract should be at least the minimum in order to improve skin inflammation and achieve antioxidant effects due to fine dust, but it should be within the above range in consideration of skin irritation and reduction in feeling of use due to excessive addition or applicability to various formulations. Can be freely adjusted from

The cosmetic composition according to the present invention includes an extract of Cheonnyeoncho as an active ingredient, and may additionally contain a functional substance for improving antioxidant activity and skin inflammation, an excipient, and a diluent for improving physical properties. The functional material or material for improving physical properties may be added within a range maintaining the effects of the present invention.

For example, in order to improve physical properties, fragrances, pigments, disinfectants, antioxidants, preservatives, moisturizers, thickeners, inorganic salts or synthetic polymer substances may be additionally added to the cosmetic composition. In addition, a fat or oil component, an emollient agent, a surfactant, an organic pigment, an inorganic pigment, an organic powder, an ultraviolet absorber, a pH adjuster, an alcohol, a blood circulation accelerator, a cooling agent, a limiting agent, or purified water may be further included.

In addition, the cosmetic composition may take the shape of a solution, emulsion, viscous mixture, etc., and examples of any formulation commonly manufactured in the art are sprays, emulsions, creams, lotions, packs, foundations, lotions, essences, hair cosmetics It may be a formulation such as.

More specifically, the cosmetic composition according to the present invention is spray, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, skin lotion, skin softener, skin toner, lotion, milk lotion, moisture lotion, nutrition lotion, massage Cream, nutritional cream, moisture cream, hand cream, foundation, essence, nutritional essence, and may include the formulation of the pack.

For example, when the formulation of the present invention is a spray or powder, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, when the formulation of the present invention is a spray, Additionally, propellants such as chlorofluorohydrocarbon, propane/butane or dimethyl ether may be included.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

When the formulation of the present invention is a solution or emulsion, a solvent, a solvating agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3 -It may be butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable fats and oils, linoline derivatives, or ethoxylated glycerol fatty acid esters may be used.

The cheonnyeoncho extract according to the present invention contains isorhamnetin and derivatives thereof. Since isoramnetin is one of the flavonoid-based vitamins and can remove inflammation and reactive oxygen species (ROS), the Cheonnyeoncho extract according to the present invention has the effect of preventing side effects of skin inflammation due to fine dust.

In addition, in a specific embodiment, the improved antioxidant activity of the Cheonnyeoncho extract according to the present invention is characterized by an increase of about 3% or more of the total phenol content when evaluated based on the comparison of the amount of gallic acid.

In another embodiment, the improved antioxidant activity of Cheonnyeoncho extract is characterized by an increase of about 0.01% or more of the total flavonoid compounds as assessed by flavonoid content analysis.

In another embodiment, the improved antioxidant activity of the Cheonnyeoncho extract is characterized by excellent DPPH radical scavenging ability.

Example

Hereinafter, examples will be described in detail in order to describe the present invention in detail. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

1. Preparation of Cheonnyeoncho Extract

The leaves, fruits, and roots of Cheonnyeoncho were collected, thorns were removed, and then washed thoroughly with water. Then, it was finely cut into 1 cm or less, and then put into a grinder and pulverized evenly. The pulverized Cheonnyeoncho and 80% ethanol were added to the extractor at a ratio of 1:9 and extraction was performed for 90 minutes while maintaining a pressure of 0.5 atm or less at a temperature of 70°C. After the extracted liquid was filtered through a filter for 3 times, water was further evaporated in a concentrator, and 2.5% of a natural preservative was mixed as a preservation stabilizer to finally complete the Cheonnyeoncho extract.

2. Cell line and culture

HaCaT cells were distributed from the Korean Cell Line Bank, and the cell culture solution was prepared by using DMEM medium containing 1% antibiotic-antimy-cotic and 10% FBS, respectively, at 37°C and 5% CO ₂ using HaCaT cells. Incubated in a controlled CO _{2 incubator.} Subculture was carried out once every 2 days, and it was controlled so as not to exceed 10 passages.

3. Preparation of fine dust samples

The PM 2.5 particles collected at the subway station were extracted by adding ethanol to a filter cut into a size of ^{1 m 2 and stirring for 12 hours.} The extract was separated, the extraction solvent was again added to the extraction residue, and the mixture was treated with ultrasonic waves for 4 hours. The sample solution was mixed and concentrated under reduced pressure to evaporate and remove the solvent, and then re-dissolved with ethanol.

Experimental example One: Millennium Antioxidant ability of extract

During the oxidation process for energy production in vivo, a significant amount of active oxygen is produced. These active oxygens are mostly destroyed by the in vivo removal mechanism, but when active oxygen is generated in a large amount instantaneously or chronically, and the balance with the antioxidant defense system is broken, it causes various diseases. During the metabolic process of oxygen, a part of oxygen is converted into active noxious oxygen such as ^{superoxide radical (O 2} ), hydrogen peroxide (H ₂ O ₂ ), hydroxy radical (HO), and singlet oxygen (O _{2 ).} Research on antioxidants began with the discovery of SOD in 1969. A substance that prevents the aging and damage of our body due to free radicals is called an antioxidant. Antioxidants exist naturally in the human body and administered externally. It can be divided into giving. Antioxidants that naturally exist in the human body include enzymes such as SOD, glutathione, and peroxidase, uric acid, and bilirubin. Vitamins E, C, and beta-carotene are administered from the outside, and selenium is a representative of the minerals. In addition, hormones such as melatonin, phenols and flavonoids, which are abundant in natural products, are representative antioxidants.

In order to investigate the antioxidant capacity of the Cheonnyeoncho extract, a comparative experiment was performed on the total phenolic content, flavonoid content, and DPPH radical scavenging quantification.

<1-1> Millennium Determination of total phenol in extract

The quantification of phenolic compounds was analyzed by the Folin Ciocalteu reagent (FCR) color development method using gallic acid as a standard material.

The preparation of the standard was prepared by preparing a solution of gallic acid (50, 100, 200, 400, 500 μg/mL) and mixing 1 ml with 9 ml DIW.

After adding 50 µl of 2N'Folin-Ciocalteu'phenol reagent' to 10 mg/mL of Cheonnyeoncho extract to develop color, add 300 µl of 20% Na ₂ CO ₃ and let stand at room temperature for 15 minutes, and then add 1 ml of distilled water. Centrifuged for 5 minutes at 1250 rpm. Thereafter, the supernatant was measured for absorbance at 725 nm using a UV spectrophotometer, and the total polyphenol content was determined from the regression curve obtained using gallic acid as a standard. The content was calculated.

concentration Absorbance Gallic acid 50 ㎍/mL 0.0078 Gallic acid 100 ㎍/mL 0.0702 Gallic acid 200 ㎍/mL 0.2063 Gallic acid 400 ㎍/mL 0.4681 Gallic acid 500 ㎍/mL 0.5723 O.humifusa 10 mg/mL 0.3216

Table 1 is a table summarizing the absorbance values of the Cheonnyeoncho extract and the gallic acid, and FIG. 1 is a graph showing the polyphenol content of the Cheonnyeoncho extract based on the gallic acid concentration.

Referring to Table 1 and FIG. 1, as a result of the experiment, 29.51 mg of polyphenol was extracted per 1 g of the Cheonnyeoncho extract from the Cheonnyeoncho extract.

<1-2> Quantification of total flavonoids

The total flavonoid content of the Cheonnyeoncho extract was measured using a method such as the Davis method (Davis; hang CC, 2002).

To 50 mg/mL of Cheonnyeoncho extract, 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1 M potassium acetate, and 4.7 mL of ethanol were sequentially added and mixed, followed by reaction at room temperature for 40 minutes, and then 415 using an optical instrument. The absorbance was measured at nm.

Quantification of flavonoid compounds in Cheonnyeoncho extract is the same method as above by using quercetin (quercetin, Sigma-Aldrich Co., St Louis, MO, USA) so that the final concentration is 5, 10, 15, 30, 40, 50 ㎍/mL. The content of the flavonoid compound of the extract of Cheonnyeoncho was calculated from the calibration curve measured by.

concentration Absorbance Quercetin 5 μg/mL 0.1714 Quercetin 10 μg/mL 0.3599 Quercetin 15 μg/mL 0.5457 Quercetin 30 μg/mL 1.0018 Quercetin 40 μg/mL 1.2665 Quercetin 50 μg/mL 1.4711 O.humifusa 10 mg/mL 1.2670

Table 2 is a table comparing the absorbance values of Cheonnyeoncho extract and quercetin, and FIG. 2 is a graph showing the amount of flavonoid compounds in Cheonnyeoncho extract based on the quercetin concentration.

Referring to Table 2 and FIG. 2, as a result of measuring the flavonoid compound contained in the Cheonnyeoncho extract with quercetin as a standard, it was found to contain 81.84 μg of flavonoid per 1 g.

<1-3> DPPH radical scavenging quantification

1,1-diphenyl-2-picrylhydrazyl radical (DPPH, 1,1-diphenyl-2-picrylhydrazyl radical) is a relatively stable free radical and has a strong absorption (magenta) rod at a wavelength of 517 nm. , When there is a free radical remover such as GSH, the degree of discoloration to yellow has a certain ratio, so it is widely used as an antioxidant screening method.

Antioxidant activity was measured for the extract of Cheonnyeoncho and DPPH as a control. The experimental method is to take the Cheonnyeoncho extract for each concentration of 100, 200, 500, 1000, 3000, 5000 μg/ml, mix 5 µl with 80 µl 0.15 mM DPPH solution and 115 µl 70% ethanol, and the final concentration of DPPH is 0.06. It was made to be mM. DPPH solution was used as a negative control. After strongly shaking and leaving for 10 minutes in the dark, the absorbance was measured at 517 nm, and the result calculated by the following equation (= absorbance reduction rate) is shown in Table 3. A high rate of decrease in absorbance means high antioxidant efficacy.

[Equation 1]

DPPH radical scavenging rate (%) = [1-(sample absorbance ₅₁₇ nm / control absorbance ₅₁₇ nm)] x 100

Absorbance at 517 nm DPPH radical scavenging (%) DPPH 0.692 100 ㎍/ml Cheonnyeoncho extract 0.568 17.92 200 ㎍/ml Cheonnyeoncho extract 0.530 23.41 500 ㎍/ml Cheonnyeoncho extract 0.419 39.45 1000 ㎍/ml Cheonnyeoncho extract 0.273 60.55 3000 ㎍/ml Cheonnyeoncho extract 0.122 82.37 5000 ㎍/ml Cheonnyeoncho extract 0.055 92.05

Table 3 is a table showing the DPPH radical scavenging ability of the Cheonnyeoncho extract by concentration, and FIG. 3 is a graph showing the free radical scavenging ability of the Cheonnyeoncho extract based on the DPPH scavenging ability.

Referring to Table 3 and FIG. 3, it was confirmed that the higher the absorbance value after DPPH analysis, the lower the value. As a result of the experiment, it was confirmed that the scavenging ability of Cheonnyeoncho extract increased as the concentration increased, and when the concentration of Cheonnyeoncho extract was 3000 ㎍/ml or more, it was confirmed that the excellent scavenging ability increased by 80% or more.

Experimental example 2: Millennium Evaluation of the cytotoxicity of the extract ( MTT assay)

In order to evaluate the cytotoxicity of the Cheonnyeoncho extract to HaCaT keratinocytes, 5×10 ⁴ HaCaT cells were added to a 96 well plate and cultured.

Fine dust (PM) sample, magnesium-aminoclay (MgAC), and Cheonnyeoncho extract were inoculated into each well, and 20 µl of MTT solution was added to each well, 5% CO ₂ , and allowed to stand in an incubator at 37°C for 1 hour, and then the MTT solution was added. After completely removing and drying the culture plate, the formazan crystals formed in the cells were dissolved with 150 µl DMSO, and the absorbance was measured at 595 nm with an ELISA plate reader to calculate the cytotoxicity ratio.

FIG. 4 is a graph showing the cytotoxicity test results when the Cheonnyeoncho extract was treated with HaCaT cells for 24 hours, and FIG. 5 is a graph showing the cytotoxicity test results when the Cheonnyeoncho extract was treated with HaCaT cells for 48 hours.

4 and 5, as a result of performing a cytotoxicity test of the Cheonnyeoncho extract against human skin keratinocytes (HaCaT), it was confirmed that there was no cytotoxicity in the groups treated for 24 hours and 48 hours. In addition, when the treatment concentration was low, it was confirmed that the degree of cytotoxicity of the fine dust, MgAC, and Cheonnyeoncho extract was similar, but as the treatment concentration gradually increased, a significant gap in cell viability was observed.

Therefore, it was confirmed that there was no cytotoxicity in the Cheonnyeoncho extract, and in the case of treatment with other substances, it was confirmed that the cytotoxicity increased as the concentration increased.

Experimental example 3: Millennium Extract of Reactive oxygen species ( ROS ; reactive oxygen species) production inhibitory effect

In _{order to confirm the effect of Cheonnyeoncho extract on ROS (Reactive Oxygen Species) over-produced by H 2} O ₂ in HaCaT cells, fine dust (PM) samples, MgAC, Cheonnyeoncho extract were added to HaCaT cells at concentrations (5, 20, 50, 80, 120, 200 µg/mL) _{were treated with 1 mM H 2} O _2, and then 25 uM H ₂ DCF-DA was added and incubated for 48 hours, excitation 480 nm, emission ) The fluorescence intensity was measured at 530 nm and the DCF fluorescence intensity was compared.

The statistical processing of the obtained results was performed using the GraphPad Prism program, and the significance of the average value was investigated with a limit of less than 5% using a one-way analysis of vatiance (ANOVA).

FIG. 6 is a graph showing the effect of reducing active oxygen species in cells when the Cheonnyeoncho extract is treated with HaCaT cells for 24 hours, and FIG. 7 is a graph showing the effect of reducing reactive oxygen species in cells when the Cheonnyeoncho extract is treated with HaCaT cells for 48 hours. to be.

6 and 7, Cheonnyeoncho extract effectively inhibits reactive oxygen species (ROS) over-produced by _{H 2} O ₂ in proportion to the increase in concentration compared to the case of treatment with fine dust (PM) samples and MgAC. I was able to confirm that.

Experimental example 4: Millennium DNA from the extract Oxidative Measurement of the inhibitory effect of damage

A Comet assay was performed to investigate the effect of fine dust (PM) samples, MgAC, and Cheonnyeoncho extract on DNA damage. The Comet assay, also called single cell gel electrophoresis, is used in a variety of studies because it can measure DNA damage with a relatively simple method.

After culturing HaCaT cells for 24 hours, fine dust, MgAC, and Cheonnyeoncho extract were treated by concentration (50, 100 ㎍/mL), and after 12 hours, H ₂ O _{2 as a} positive control (final concentration 10 ^-3 M) After treatment, the result of staining by electrophoresis was observed with a fluorescence microscope. At this time, using an image analyzer, KOMET 3.1 (Kinetic Imaging, England), the length (um) of the tail resulting from DNA digestion in 25 cells was analyzed.

concentrations treatments Tail DNA (%) Olive tail moment (%) 24 h 48 h 24 h 48 h 50 μg/mL PM 26.17±0.49 30.72±4.38 5.25±0.81 6.75±1.07 MgAC 13.20±0.68 16.05±1.12 2.55±0.20 3.05±0.15 Millennium 5.69±0.67 8.36±0.13 1.32±0.14 1.46±0.15 100 μg/mL PM 30.84±2.51 34.96±6.14 10.68±0.75 13.25±1.34 MgAC 16.47±1.55 22.64±0.05 3.55±0.31 4.16±0.45 Millennium 10.26±1.05 12.91±1.92 2.31±0.61 2.25±0.32 positive control 52.31±3.51 63.05±9.15 11.03±1.31 21.40±2.58 negative control 4.63±1.05 6.20±1.48 1.00±0.38 1.18±0.41

Table 4 is a table summarizing the degree of DNA damage of HaCaT cells, and FIG. 8 is a photograph obtained by comparing and measuring the DNA fragments of HaCaT cells with a Comet assay when fine dust, MgAC, and Cheonnyeoncho extract were treated on HaCaT cells for 24 hours, 9 is a photograph obtained by comparing and measuring DNA fragments of HaCaT cells by Comet assay when fine dust, MgAC, and Cheonnyeoncho extract were treated on HaCaT cells for 48 hours. 10 is a graph comparing tail DNA (%) and olive tail moment (%) of HaCaT cells when fine dust, MgAC, and Cheonnyeoncho extract were treated on HaCaT cells for 24 hours and 48 hours.

Referring to Table 4 and FIGS. 8 to 10, in all of the HaCaT cells treated with fine dust (PM) samples, MgAC, and Cheonnyeoncho extract, as the treatment time and treatment concentration increased, the formation of comet tails after exposure to UVB radiation clearly increased. I could confirm.

In addition, it was confirmed that the fine dust (PM) sample had a serious effect on HaCaT cells, and MgAC caused moderate damage to HaCaT cells. On the other hand, it was confirmed that HaCaT cells treated with Cheonnyeoncho extract rarely cause damage and help cells grow better.

Experimental example 5: FACS Analysis method

In order to confirm the amount of apoptotic cells generated through UVB-induced apoptosis, it was confirmed by flow cytometry using annexin-V/PI.

HaCaT cells were cultured in 60 mm dishes, UVB irradiated, and then fine dust (PM) samples and Cheonnyeoncho extract were treated at different concentrations (100, 200 μg/mL). After 6 hours, pipetting the cell line into a ^{test tube as much as 1×10 6} cells/ml and washing with PBS, discard the supernatant and add a binding buffer (10 mM Hepes/NaOH pH 7.4, 140 mM NaCl, It was pipetted again with 2.5 mM CaCl _{2 ).} Thereafter, FITC-conjugated annexin V Apoptosis Detection Kit (BD Pharmigen, 556547, USA) and propiium Ioddide (PI) (including FITC annexin V kit) were added and incubated for 15 minutes in the dark for staining. Flow cytometer; Fluorescence-activated cell sorting, FACS calibur; Becton-Dickinson), and the percentage of apoptotic cells was measured using the software (Cell Quest software).

11 is a graph comparing apoptosis results when fine dust and Cheonnyeoncho extract were treated on HaCaT cells for 24 hours, and FIG. 12 is a graph comparing apoptosis results when fine dust and Cheonnyeoncho extract were treated on HaCaT cells for 48 hours. ) This is a graph comparing the results.

11 and 12, as a result of the experiment, it was found that the extract of Cheonnyeoncho inhibited cell death due to UVB of apoptosis cells compared to the case where the fine dust sample was treated, thereby increasing the survival rate of cells in a concentration-dependent manner. .

Experimental example 6: Millennium ATP synthesis effect of extract

<6-1>

HaCaT cells were treated with Cheonnyeoncho extract and fine dust (PM) samples, and the effect on ATP synthesis was observed in comparison with the control group. After washing the HaCaT cells twice with PBS, a lysate was obtained, and then protein was removed using 0.6 M perchloric acid, followed by centrifugation. To prevent protein pellets from being mixed, only the supernatant was rolled up, neutralized with KOH, and then lightly centrifuged to remove potassium-perchlorate. The resulting supernatant was used for ATP synthesis. ATP was measured by reduction of NADP+ in the presence of glucose, hexokinase, and glucose-6-phosphate dehydrogenase. Oligomysin was used as an inhibitor of ATPase activity.

samples synthesis ATP calculated from standard curve 24 h 48 h control 1.06±0.07 1.09±0.04 Cheonnyeoncho 100 ㎍/ml 0.67±0.01 0.82±0.01 Cheonnyeoncho 200 ㎍/ml 0.71±0.04 0.93±0.01 PM 100 ㎍/ml 0.65±0.01 0.60±0.02 PM 200 ㎍/ml 0.58±0.02 0.54±0.03

Table 5 is a table summarizing the degree of ATP synthesis when treated with Cheonnyeoncho extract and fine dust samples, and FIG. 13 is a graph comparing ATP synthesis results when fine dust and Cheonnyeoncho extract were treated with HaCaT cells.

Referring to Table 5 and FIG. 13, it was confirmed that the energy synergistic effect (ATP synthesis of HaCaT cells) was improved as the treatment concentration and treatment time of the Cheonnyeoncho extract increased. Cheonnyeoncho extract inhibited ATP synthesis at low concentrations and short-term exposure, but showed a tendency to contribute to ATP synthesis when treated at high concentrations and for a long period of time. In contrast, when the fine dust samples were treated, the degree of ATP synthesis decreased as the treatment concentration and treatment time increased. Through this, it was confirmed that fine dust is toxic to HaCaT cells by inhibiting DNA damage and ATP synthesis.

<6-2>

When treating HaCaT cells with Cheonnyeoncho extract and then treating a fine dust (PM) sample, the effect on the ATP synthesis according to the treatment time was observed in comparison with the control group.

After treatment at a concentration of 200 µg/mL of Cheonnyeoncho extract, a fine dust (PM) sample was treated to HaCaT cells at a concentration of 200 µg/mL. At this time, 200 μg/mL concentration of Cheonnyeoncho extract was treated for 3 hours, and the degree of ATP synthesis was measured every hour.

After washing the HaCaT cells treated with the Cheonnyeoncho extract and the fine dust sample twice with PBS, a lysate was obtained, and then protein was removed using 0.6 M perchloric acid, followed by centrifugation. To prevent protein pellets from being mixed, only the supernatant was rolled up, neutralized with KOH, and then lightly centrifuged to remove potassium-perchlorate. The resulting supernatant was used for ATP synthesis. ATP was measured by reduction of NADP+ in the presence of glucose, hexokinase, and glucose-6-phosphate dehydrogenase. Oligomysin was used as an inhibitor of ATPase activity.

samples synthesis ATP calculated from standard curve 24 h 48 h negative control 0.93±0.038 0.97±0.004 positive control (PM 200 ㎍/ml) 0.53±0.024 0.48±0.041 Group A (Tenyeoncho 200 ㎍/ml treated for 1 h and PM 200 ㎍/ml treated) 0.38±0.048 0.52±0.098 Group B (Tenyeoncho 200 ㎍/ml treated for 2 h and PM 200 ㎍/ml treated) 0.54±0.001 0.69±0.026 Group C (Tenyeoncho 200 ㎍/ml treated for 3 h and PM 200 ㎍/ml treated) 0.60±0.010 0.81±0.010

Table 6 is a table showing the degree of ATP synthesis of the positive control group, the negative control group, and Groups A, B, and C, and FIG. 14 is a comparison of ATP synthesis results when a fine dust sample was treated with HaCaT cells after treatment with Cheonnyeoncho extract. It is a graph.

As a result of the experiment, it was confirmed that the fine dust (PM) sample inhibited ATP synthesis compared with the case where only the Cheonnyeoncho extract was treated and the case where nothing was treated. In addition, it can be seen that the degree of ATP synthesis increases as the time for pretreatment of Cheonnyeoncho extract increases, so that the Cheonnyeoncho extract has the ability to suppress side effects caused by long-term exposure of PM samples. I did.

Experimental example 7: Cytotoxicity ( cytotoxicity ) Experiment

It was confirmed that the extract of Cheonnyeoncho reduces the cytotoxicity of HaCaT cells induced from fine dust (PM).

After treatment at concentrations of 50, 100, 150, and 200 µg/mL of Cheonnyeoncho extract, fine dust (PM) samples were treated with each of HaCaT cells at a concentration of 100 µg/mL. At this time, the Cheonnyeoncho extract was treated for 3 hours, and the cell viability was measured every hour.

HaCaT cells were inoculated into a 96 well culture plate at a concentration of 2 x 104 cells/ml. After incubation for 24 hours and 48 hours, it was washed once with DMEM medium without FBS.

Chunnyeoncho extract was dissolved in DMEM medium containing no FBS at concentrations of 50, 100, 150, and 200 μg/mL, and treated with HaCaT cells in each well washed once with DMEM medium containing no FBS. Thereafter, fine dust (PM) was treated to the HaCaT cells in each well at a concentration of 100 μg/mL. On the other hand, as a negative control, HaCaT cells were treated with nothing, and as a positive control, HaCaT cells were treated with the fine dust at a concentration of 100 µg/mL.

After culturing the Cheonnyeoncho extract and the fine dust-treated HaCaT cells for 20 to 24 hours, the CCK-8 assay solution was treated with 10 µl per well, and the absorbance was measured at 540 nm using a spectrophotometer after 1 to 4 hours. Thus, the cell viability was calculated.

15 is a graph comparing the results of a cytotoxicity experiment when fine dust and Cheonnyeoncho extract were treated on HaCaT cells for 24 hours, and FIG. 16 is a graph comparing the results of a cytotoxicity experiment when fine dust and Cheonnyeoncho extract were treated on HaCaT cells for 48 hours. cytotoxicity) is a graph comparing the experimental results.

Referring to FIGS. 15 and 16, it was confirmed that the Cheonnyeoncho extract reduced the toxicity induced from the fine dust (PM) sample, and it was confirmed that the cell viability increased as the treatment concentration and treatment time increased.

Experimental example 8: Millennium Experiment to confirm the regulation of keratinocyte differentiation of the extract

<8-1> Inhibitory Effect of Cheonnyeoncho Extract on MMP-9 Activity

The inflammatory response of the skin is regulated by several complex genes, and matrix metallopeptidase-9,2 (MMP-9,2) and cyclooxygenase-2 (COX-2) are known as major genes. In particular, it is known that MMP-9 is synthesized and secreted by various stimuli such as cytokines and growth factors, thereby affecting cell structure changes. In addition, COX-2 is _{an enzyme that generates PGE 2} , an inflammatory reaction mediator, and can cause inflammatory skin diseases. The expression of COX-2 is increased by factors such as growth factors, mitogenes, and cytokines, and plays an important role in regulating the inflammatory response.

To confirm the effect of the Cheonnyeoncho extract on the differentiation of keratinocytes (HaCaT cells), western blot was performed to confirm the protein expression and promoter activity of factors capable of confirming the degree of keratinocyte differentiation.

HaCaT cells were dispensed into a 60 mm culture dish so as to be 2Х10 ⁶ cells/well and attached, then treated with Cheonnyeoncho extract at different concentrations (50, 100, 200 ㎍/mL), and fine dust (PM) samples after 2 hours. It was treated at a concentration of 300 μg/mL. As a control, only fine dust samples were treated at different concentrations (300, 500 μg/mL).

Cheonnyeoncho extract was treated at concentrations of 50, 100, and 200 μg/mL in wells treated at a concentration of 300 μg/mL, and cultured for 24 hours. Through the above experiment, it was examined how the extract of Cheonnyeoncho affects the inflammatory response of keratinocytes due to fine dust.

에서 30 분간 단백질을 용해시켰다. 13,000 rpm으로 15 분 간 원심분리 하여 상층액을 얻은 다음 bradford assay를 이용하여 정량하였다. 단백질은 각각 30 μg씩 10 % SDS-polyacrylamide gel electrophoresis로 전기영동하고 ImmobilonP-membrane(Millipore, Billerica, USA)에 단백질을 흡착시켰다. 특이 단백질을 탐색하고자 1차 항체와 2차 항체를 반응 시킨 후 ECL kit(Amersham Pharmacia, Buckinghamshire, England)을 사용하여 X-ray film에 노출시켜 단백질 발현 정도를 확인하였다.Recover the cultured cells and add lysis buffer (50 mM tris, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 100 mM PMSF, 1 μg/mlleupeptin, 1 μg/mlaprotinin) to 4

The protein was dissolved in for 30 minutes. The supernatant was obtained by centrifugation at 13,000 rpm for 15 minutes, and then quantified using the bradford assay. Proteins were electrophoresed by 10% SDS-polyacrylamide gel electrophoresis at 30 μg each, and the proteins were adsorbed on ImmobilonP-membrane (Millipore, Billerica, USA). To search for a specific protein, the primary antibody and the secondary antibody were reacted and then exposed to an X-ray film using an ECL kit (Amersham Pharmacia, Buckinghamshire, England) to check the protein expression level.

17 is a result of confirming the expression of MMP-9 protein by fine dust of the Cheonnyeoncho extract according to the present invention in HaCaT cells by Western blot.

Referring to FIG. 17, as a result of treating only the fine dust (PM) sample, the activity of MMP-9 increased, and the higher the treatment concentration of the fine dust sample, the higher the degree of activity of MMP-9. It could be confirmed that it causes.

In addition, it was confirmed that when the Cheonnyeoncho extract was treated before processing the fine dust sample, the higher the treatment concentration of the Cheonnyeoncho extract was, the more effectively the activity of MMP-9 increased by the fine dust was reduced. Therefore, it was confirmed that the extract of Cheonnyeoncho according to the present invention is effective in inhibiting skin aging and improving skin inflammation.

<8-2> Effect of Cheonnyeoncho Extract on Inhibitory Expression of COX-2 Protein

Whether the extract of Cheonnyeoncho was related to the expression of COX-2 protein known as an inflammatory factor was investigated by Western blot by the same method as in Example 8-2.

18 is a result of confirming the expression of the COX-2 gene by fine dust of the Cheonnyeoncho extract according to the present invention in HaCaT cells by Western blot.

Referring to FIG. 18, as a result of treating only the fine dust (PM) sample, the amount of COX-2 protein expression increased, and the higher the concentration of the fine dust sample was treated, the higher the degree of expression of the COX-2 protein was found. It could be confirmed that it causes.

In addition, it was confirmed that when the Cheonnyeoncho extract was treated before processing the fine dust sample, the higher the treatment concentration of the Cheonnyeoncho extract was, the more effectively the expression level of the COX-2 protein expressed by the fine dust was reduced. In particular, it was confirmed that the COX-2 protein expression level was reduced by 2 times even at a low concentration of 50 μg/mL, so that the extract of Cheonnyeoncho was excellent in the inhibitory effect of COX-2.

Therefore, it was confirmed that the extract of Cheonnyeoncho according to the present invention is effective in inhibiting skin aging and improving skin inflammation.

<8-3> Analysis of AhR and CYP1A1 gene expression of Cheonnyeoncho extract

Skin caused by fine dust by reducing the expression of AhR (Aryl hydrocarbon receptor) and CYP1A1 (Cytochrome P450 1A1) genes related to skin damage caused by processing fine dust containing polycyclic aromatic hydrocarbons in human keratinocytes (HaCaT) It was examined whether the Cheonnyeoncho extract can prevent the damage of.

HaCaT cells were dispensed into a 60 mm culture dish so as to be 2Х10 ⁶ cells/well and attached, then treated with Cheonnyeoncho extract at different concentrations (50, 100, 200 ㎍/mL), and fine dust (PM) samples after 2 hours. It was treated at a concentration of 200 μg/mL. As a control, only fine dust samples were treated at different concentrations (100, 200, 300 μg/mL).

에서 30 분간 단백질을 용해시켰다. 13,000 rpm으로 15 분 간 원심분리 하여 상층액을 얻은 다음 Bradford assay를 이용 하여 정량 하였다. 단백질은 각각 30 μg씩 10 % SDS-polyacrylamide gel electrophoresis로 전기영동하고 ImmobilonP-membrane(Millipore, Billerica, USA)에 단백질을 흡착시켰다. 특이 단백질을 탐색하고자 1차 항체와 2차 항체를 반응 시킨 후 ECL kit(Amersham Pharmacia, Buckinghamshire, England)을 사용하여 X-ray film에 노출시켜 단백질 발현 정도를 확인하였다. Cheonnyeoncho extract was treated at concentrations of 50, 100, and 200 µg/mL in wells treated with a concentration of 200 µg/mL and cultured for 24 hours. Recover the cultured cells and add lysis buffer (50 mM tris, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 100 mM PMSF, 1 μg/mlleupeptin, 1 μg/mlaprotinin) to 4

The protein was dissolved in for 30 minutes. The supernatant was obtained by centrifugation at 13,000 rpm for 15 minutes and then quantified using Bradford assay. Proteins were electrophoresed by 10% SDS-polyacrylamide gel electrophoresis at 30 μg each, and the proteins were adsorbed on ImmobilonP-membrane (Millipore, Billerica, USA). To search for a specific protein, the primary antibody and the secondary antibody were reacted and then exposed to an X-ray film using an ECL kit (Amersham Pharmacia, Buckinghamshire, England) to check the protein expression level.

19 is a result of confirming the expression of AhR gene by fine dust of the Cheonnyeoncho extract according to the present invention in HaCaT cells by Western blot, and FIG. 20 is the expression of CYP1A1 protein by fine dust of the Cheonnyeoncho extract according to the present invention in HaCaT cells This is the result of confirming with Western blot.

Referring to FIGS. 19 and 20, on the basis of the case in which nothing was treated with human keratinocytes (HaCaT), in the control group treated with only fine dust, the gene expression of AhR (Aryl hydrocarbon receptor) was reduced by 1/3. . On the other hand, it was confirmed that when the extract of Cheonnyeoncho was treated before processing the fine dust sample, the expression of the AhR gene increased as the treatment concentration of the Cheonnyeoncho extract was increased.

In addition, the expression of CYP1A1 (Cytochrome P450 1A1) gene was increased by more than 4 times in the control group treated with only fine dust, based on the case where nothing was treated with human keratinocytes (HaCaT). When the extract was treated, it was confirmed that the CYP1A1 gene expression decreased as the treatment concentration of the Cheonnyeoncho extract increased.

Therefore, when the above experimental results are summarized, it is found that the Cheonnyeoncho extract according to the present invention has a protective effect against fine dust by regulating the expression of the toxicity-related genes AhR (Aryl hydrocarbon receptor) and CYP1A1 (Cytochrome P450 1A1) genes. I could confirm.

Until now, specific examples of the anti-pollution cosmetic composition comprising the Cheonnyeoncho extract according to an embodiment of the present invention as an active ingredient have been described, but various implementation modifications are possible within the limit not departing from the scope of the present invention. It's self-evident.

Therefore, the scope of the present invention should not be defined by being limited to the described embodiments, and should be defined by the claims and equivalents as well as the claims to be described later.

That is, it should be understood that the above-described embodiments are illustrative in all respects and not limiting, and the scope of the present invention is indicated by the claims to be described later rather than the detailed description, and the meaning and scope of the claims and All changes or modified forms derived from the equivalent concept should be construed as being included in the scope of the present invention.

Claims (6)

An anti-pollution cosmetic composition comprising an extract of Opuntia humifusa as an active ingredient. The method of claim 1,
An anti-pollution cosmetic composition comprising, as an active ingredient, an extract of Cheonnyeoncho, characterized in that the leaves, fruits, and roots of Cheonnyeoncho are extracted. The method of claim 1,
The viscosity of the composition is 1.0 to 3.0 mPa.S, characterized in that the anti-pollution cosmetic composition comprising the Cheonnyeoncho extract as an active ingredient. The method of claim 1,
The content of the Cheonnyeoncho extract is an anti-pollution cosmetic composition comprising the Cheonnyeoncho extract as an active ingredient, characterized in that 10 to 100% by weight of the total composition. The method of claim 1,
The composition is effective in any one formulation selected from the group consisting of soothing gel, lotion, cream, facial cream, lotion, essence, cleansing cream, cleansing foam, cleansing water, pack, powder, or body oil. Anti-pollution cosmetic composition containing as an ingredient. Freeze-drying and pulverizing the washed leaves, fruits, and roots of Opuntia humifusa; And
Extracting the pulverized product of Cheonnyeoncho to prepare a Cheonnyeoncho extract; Method for producing an anti-pollution cosmetic composition comprising the Cheonnyeoncho extract as an active ingredient.

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