KR101661203B1 - Cosmetic composition for prevention or amelioration of skin photodamage containing extracts of Cornus walteri leaves as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating photodamage of skin caused by ultraviolet rays, which comprises an extract of horse chestnut leaf. The cosmetic composition for preventing or ameliorating the photodamage of skin according to the present invention shows a photoprotective effect against cell damage induced by ultraviolet rays and an elimination activity against intracellular reactive oxygen species induced by hydrogen peroxide and ultraviolet rays, Reduces death and restores cell viability.

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition for prevention or amelioration of photodamage of skin,

TECHNICAL FIELD The present invention relates to a composition for preventing, ameliorating or treating skin photodamage caused by ultraviolet rays, which contains an extract of Liliaceae leaf as an active ingredient.

The skin is the largest organ of our body and is exposed to the outside, protecting the body from external stimuli. Among the various external stimuli, ultraviolet light is known to be the main cause of photoaging. Ultraviolet rays are classified into UVA (320-400 nm), UVB (280-320 nm) and UVC (200-280 nm) depending on wavelength. Ultraviolet rays reaching the earth surface and affecting the human body are known as UVA and UVB have. Part of the UVB, in particular, penetrates the epidermis and reaches the papillary layer of the dermis. Excessive irradiation of the skin with ultraviolet rays causes erythema reaction, sunburn, pigmentation, photoaging, skin cancer, and the like. Ultraviolet rays are directly absorbed by the DNA bases in the cell, and when the DNA bases absorb directly, the pyrimidine bases existing in close proximity due to the DNA photodamage are cross-linked cyclobutane-type cyclobutane-type cyclobutane- -pyrimidine dimers (CPD), and pyrimidine- (6-4) -pyrimidone photo-products (pyrimidine- (6-4) -pyrimidone photoproduct, 6-4PP). Such DNA damage is restored by the temporary arrest of cell cycle by the expression of p53 protein. However, when the cells fail to recover from damage, apoptosis is induced by expression of proteins such as caspase-3 involved in apoptosis and fragmentation of DNA (Lo HL, et al. BMC Cancer, 5: 135 (2005)); Elmore S., Toxicol. Pathol. 35 (4), 495 (2007); Haupt S., et al., J. Cell Science, 116, 4077 (2003)). Thus, effects such as DNA damage recovery by ultraviolet rays, inhibition of p-p53 and caspase-3 expression can be presented as biomarkers for treatment of skin photodamage.

In addition to direct damage by ultraviolet light, cells indirectly absorb ultraviolet light through free radical scavengers (porphyrin, riboflavin, etc.) and generate free radicals. The resulting free radicals oxidize skin cells, proteins, lipids, etc., or cause DNA damage, leading to apoptosis or skin cancer (Kang, S., et al., Am. J. Pathol., 166, 1691 2005); Matsumura, Y., et al., Toxicol. Appl. Pharmacol., 195 (3), 298 (2004)).

In order to protect skin cells from ultraviolet rays, it is important to protect cells from reactive oxygen and DNA damage. In recent years, researches for protecting skin cells from ultraviolet rays have been actively conducted by using various herbal medicines and edible plant extracts.

Cornus walteri Wanger is a deciduous broad-leaved arboreous tree with a height of 10 m. Its bark is divided like a net and is dark brown. Leaves are alive, broad ovate or elliptical, flowers bloom in June, fruits are round, ripen in black from September to October (Lee Changbok, Korean Plants Book, Dongmunsa Temple, Seoul (1989) , Seoul 249 (1996)). It is used to stop diarrhea and to treat lacquer and stop the diarrhea. In private, it is called 'fresh tree', 'liquor tree' or ' It has been used in the name of 'sniper'. Recently, Choi et al. Have studied gallic acid, (+) - gallocatechin, (+) - catechin, (+) - catechin and quercetin quercetin, quercitrin, rutin, isoquercitrin, and hyperoside (Choi WH, et al., Kor. J., Pharmacogn., 29, 217 (1998)). Among them, gallic acid and (+) - gallocatechin were reported as 0.054% and 0.022%, respectively. Lim et al. (Bacchus et al., 2002), and that the body weight of the subjects was significantly lower than that of the control group (2005)). In addition, Park and Cha showed that lipid-lowering effect in the sand-leaf-treated group was more effective than lipid-lowering treatment in the sand-leaf-treated group. (Park, WH, et al., J. Oriental Rehab. Med., 19, 59 (2009)), which has been shown to be effective in the inflammatory activity and in the hyperlipidemia due to obesity and related metabolic diseases.

Korean Patent Registration No. 10-0478136 discloses an antioxidant active composition containing an extract of Aspergillus oryzae, and Korean Patent Publication No. 10-2013-0097033 discloses an antioxidant composition for improving obesity containing a extract of Aspergillus oryzae .

However, the treatment and prevention of photodamage has not been known.

Accordingly, it is an object of the present invention to provide a composition for preventing, ameliorating or treating skin photodamage caused by ultraviolet rays.

The present invention provides a composition for preventing, ameliorating or treating skin photodamage comprising an extract of Liliaceae leaf as an active ingredient. The present invention also provides a method for preventing, ameliorating, or treating skin photodamage by applying a composition comprising an extract of Liliaceae leaf as an active ingredient to skin.

Preferably, the Liliaceae leaf extract can be extracted using an alcohol having 1 to 4 carbon atoms, water, or a mixture thereof as an extraction solvent.

More preferably, the Lilium leaf extract comprises the steps of: (s1) extracting the Lilium leaves with an alcohol having 1 to 4 carbon atoms, water, or a mixture thereof; (s2) adding an enzyme to the extract of step (s1) and shaking / culturing; And (s3) extracting the culture of step (s2) by adding an organic solvent to the culture.

For example, the alcohol may be at least one selected from the group consisting of ethanol, methanol, isopropanol, and butanol, and preferably ethanol may be used.

The inventors of the present invention have discovered the surprising fact that the survival rate of cells is remarkably excellent when the extract of P. falciparum is treated with light-damaged cells by ultraviolet light. The present invention has been completed by applying this fact to a composition for preventing, improving or treating photodamage of the skin.

The reason why the extract of the present invention has excellent photodamage prevention, improvement or therapeutic effect is presumed to be due to the synergistic action between the components contained in the extract of Liliaceae, and the excellent effect of Liliaceae leaf extract The reason why it can be obtained is presumed to be attributable to the selection of the seedling as the raw material, the use of only the stem leaf, the specific production method, and the like, but the present invention is not limited thereto.

In the present invention, symptoms caused by "skin photodamage" include erythema, sunburn, pigmentation, photoaging, skin cancer, skin cell DNA damage, increased reactive oxygen species, increased lipid peroxidation, cellular apoptosis, caspase- ) Activity, increase of? -H2A.X, increase of cyclobutane pyrimidine dimer (DPD) in DNA, increase of p-p53 expression level, and the like. It is not.

The caspase-3 is a protein involved in apoptosis, which induces apoptosis by DNA fragmentation. The above-mentioned γ-H2A.X shows DNA damage in the form of DNA double strand separation in H2A.X, which is one of the histone H2Ab families, and phosphorylation of Ser 139 existing in the C-terminal tail. The cyclobutanepyrimidine dimer is formed when thymine is irradiated with ultraviolet light in a continuous DNA sequence, which indicates DNA damage. p-p53 is a protein involved in the repair of damaged DNA by temporarily stopping the cell cycle.

In the present invention, " prevention " means any action that suppresses or delays skin photodamage. &Quot; Improvement " also refers to any action that alleviates the state of photodamage of the skin or reduces the severity of the symptoms. The term " treatment " means all the actions that the composition of the present invention is applied to the skin damaged by the ultraviolet rays to improve or improve the skin photodamage symptoms.

It is preferable that the safflower leaves contained in the composition of the present invention are dried and crushed to improve the extraction yield. In addition, the composition of the present invention may further include an extract extracted from stem, skin, fruit, seed, root,

In the composition of the present invention, the Lilium leaf extract (s1) comprises the steps of: (s1) extracting the Liliaceae leaves with an alcohol having 1 to 4 carbon atoms, water or a mixture thereof; (s2) adding an enzyme to the extract of step (s1) and shaking / culturing; And (s3) fractionating the culture of step (s2) by adding an organic solvent to the culture.

The alcohol in step (s1) may be selected from the group consisting of ethanol, methanol, isopropanol, and butanol. For the purpose of the present invention, it is preferable to use ethanol as the alcohol in consideration of the enzymatic treatment or toxicity of the subsequent step (s2). The concentration of alcohol in the mixture of alcohol and water used in step (s1) is 10 to 99 wt%, preferably 25 to 90 wt%, more preferably 35 to 80 wt%, and most preferably 50 to 75 wt% %. ≪ / RTI >

The enzyme of step (s2) may be selected from the group consisting of carbohydrate hydrolase, pectin hydrolase and cellulose hydrolase. For the purpose of the present invention, the enzyme is most effective in preventing, ameliorating or treating photodamage when pectin hydrolase is used.

As the organic solvent for fractionation in the step (s3), at least one selected from the group consisting of ethyl acetate, diethyl ether, trichloromethane, butanol, and dichloromethane can be used. For the purpose of the present invention, the use of ethyl acetate as the organic solvent is most effective in preventing, improving or treating photodamage of the skin of a composition comprising an extract of Liliaceae leaf.

The present invention also relates to a method for producing a plant, comprising the steps of: (s1) extracting a leaf of a Chinese persimmon tree using an alcohol having 1 to 4 carbon atoms or water; (s2) adding an enzyme to the extract of step (s1) and shaking / culturing; And (s3) a step of fractionating the culture of step (s2) by adding an organic solvent to the culture, and a method for preparing a composition for preventing, improving or treating photodamage of skin . For the purpose of the present invention, the composition comprising the extract of Liliaceae leaf which is prepared through the above-mentioned production method is remarkably excellent in prevention, improvement or treatment of photodamage of the skin.

The alcohol in step (s1) may be at least one selected from the group consisting of ethanol, methanol, isopropanol, and butanol. For the purpose of the present invention, it is preferable to use ethanol as the extraction solvent in consideration of the enzymatic treatment or toxicity of the subsequent step (s2). The concentration of alcohol in the mixture of alcohol and water may be from 10 to 99% by weight, preferably from 25 to 90% by weight, more preferably from 35 to 80% by weight, most preferably from 50 to 75% by weight.

The step (s1) in the above production process is preferably carried out at an extraction temperature of 60 to 90 ° C. If the extraction temperature is lower than 60 ° C, the extraction yield is lowered and the extraction time becomes longer. When the extraction temperature is higher than 90 ° C, the components contained in the extract may be denatured.

The enzyme of step (s2) may be selected from the group consisting of carbohydrate hydrolase, pectin hydrolase, and cellulose hydrolase. For the purpose of the present invention, the enzyme is most effective in preventing, ameliorating or treating photodamage when pectin hydrolase is used.

In the above production method, the step (s2) is preferably carried out at a temperature of 40 to 70 ° C. If the temperature is lower than 40 ° C, the incubation rate is decreased. If the temperature is higher than 70 ° C, the components contained in the extract may be denatured.

Preferably, the method further comprises the step (s1) between the steps (s1) and (s2) to further purify the extract of step (s1) and increase the efficiency of shaking and culturing using the enzyme of step (s2) And concentrating the extract and re-dissolving the concentrate in an aqueous alcohol solution. The alcohol may be at least one selected from the group consisting of ethanol, methanol, isopropanol, and butanol, and preferably ethanol may be used. The alcohol aqueous solution may have a concentration of 5 to 30% by weight, preferably 6 to 20% by weight, more preferably 7 to 15% by weight, most preferably 10% by weight, .

As the organic solvent for fractionation in the step (s3), at least one selected from the group consisting of ethyl acetate, diethyl ether, trichloromethane, butanol, and dichloromethane can be used. For the purpose of the present invention, the use of ethyl acetate as the organic solvent is most effective in preventing, improving or treating photodamage of the skin of a composition comprising an extract of Liliaceae leaf.

The composition comprising the extract of Liliaceae leaf extract according to the present invention as an active ingredient can be used for various purposes, for example, as a cosmetic composition, a pharmaceutical composition, a health functional food composition and the like.

Accordingly, the present invention provides a cosmetic composition for prevention or improvement of photodamage, comprising an extract of Liliaceae leaf as an active ingredient. In addition, the present invention provides a method for preventing or improving photodamage by applying a cosmetic composition for preventing or improving photodamage, which comprises as an active ingredient, an extract of a moss-free leaf extract, to the skin.

The cosmetic composition comprising an extract of Liliaceae leaf extract according to the present invention as an active ingredient can be prepared in various formulations of basic products such as lotion, essence, cream, pack, foundation or make-up base, . Specifically, it can be applied to various properties such as liquid, cream, paste, or solid phase, and can be applied to a conventional cosmetic preparation method.

The cosmetic composition according to the present invention may further comprise at least one cosmetically acceptable carrier mixed with a cosmetic composition for general skin. Examples of the cosmetic composition include oil, water, a surfactant, a moisturizer, a lower alcohol, , A chelating agent, a coloring agent, a preservative, a perfume, and the like may be appropriately compounded, but the present invention is not limited thereto.

In the cosmetic composition according to the present invention, the extract of Liliaceae leaf is 0.01 to 50% by weight, preferably 0.1 to 40% by weight, more preferably 1 to 30% by weight, most preferably 5 to 20% by weight, ≪ / RTI > If the extract is less than 0.01% by weight, the effect of preventing or improving photodamage is insignificant. When the extract is more than 50% by weight, there is a problem in stability of the cosmetic formulation.

In addition, the present invention provides a pharmaceutical composition for preventing or treating photodamage of skin comprising an extract of Liliaceae leaf as an active ingredient.

The pharmaceutical composition according to the present invention may further comprise an appropriate carrier, excipient, diluent or the like conventionally used in the production of a pharmaceutical composition. The pharmaceutically acceptable carriers, excipients or diluents that may be included in the pharmaceutical compositions according to the extractive embodiments of the present invention include lactose, dextrose, sucrose, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, But is not limited to, at least one selected from the group consisting of water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The pharmaceutical composition according to the present invention can be formulated into various formulations such as ointments, creams, gels, tablets, capsules, granules, suspensions, or syrups according to conventional methods known in the art.

The pharmaceutically effective amount and the effective dose of the pharmaceutical composition according to the present invention may vary depending on the formulation method, administration method, administration time and / or administration route of the pharmaceutical composition. The type and degree of the reaction to be achieved by the administration of the pharmaceutical composition, the kind of the subject to be administered, age, weight, general health condition, symptom or degree of disease, sex, diet, excretion, The components of the drug or other composition used in conjunction therewith, and the like, and similar factors well known in the medical arts. One of ordinary skill in the art can readily determine and prescribe dosages effective for the desired treatment. Administration of the pharmaceutical composition according to the present invention can be administered once a day, or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention. A preferred dose of the pharmaceutical composition according to the present invention may be from 1 mg / kg to 1, OOO mg / kg per day.

The composition for preventing, ameliorating or treating photodamage of the skin comprising the extract of Liliaceae according to the present invention not only exhibits cytotoxic activity against intracellular reactive oxygen species but also restores cell damage caused by ultraviolet rays, And the like.

Figure 1 shows the formation of CPD and 6-4 PPs by ultraviolet light (Sinha RP, HD, P., Sinha P., Photochem. Photobiol. 1, 225 (2002)).
Figure 2 shows the cell survival rate for each of the plant extracts at 250 mJ UBV (*: significant results according to the students test ( p <0.05), **: significant results according to the Student's test ( p < 0.01), * p < 0.05, ** p < 0.01 compared to UVB treated groups, respectively).
FIG. 3 shows the cytotoxicity of extracts of Fusarium oxysporum when treated with fibroblasts (*: significant results according to the Student's test ( p <0.05)).
Fig. 4 shows the effect of inhibiting hydrogen peroxide induced intracellular ROS formation of the extract of A. fumigatus (*: significant results according to the students test ( p <0.05), **: significant results according to the Student's test ( p < 0.01), * p < 0.05, ** p <
Figure 5 shows the effect of inhibiting intracellular ROS production induced by UVB irradiation of the leaves of the L. stigma (*: significant results according to the students test ( p <0.05), **: significant Results ( p <0.01), * p <0.05, ** p <0.01 compared to UVB treated groups, respectively).
FIG. 6 shows the inhibitory effect of 8-isoprostane induced by UVB irradiation on the extract of the leaves of the Liliaceae (*: significant results according to the students test ( p <0.05) **: Significant results ( p <0.01), * p <0.05, ** p <0.01, respectively, compared with the group treated with UVB).
Figure 7 shows the effect on the apoptosis of fibroblasts in UVB-irradiated fibroblasts (*: significant results according to students test ( p <0.05), **: significant results according to the Student's test ( p < 0.01), * p < 0.05, ** p <
Figure 8 shows the activity of caspase-3 involved in apoptosis when treated with UVB-irradiated fibroblasts (**: significant results ( p <0.01) according to the Student's test, ** p &Lt; 0.01 UVB treated group).
Figure 9 shows the recovery of y-H2A.X as a result of DNA damage when treated with UVB-irradiated fibroblasts (**: significant results according to the Student's test ( p < 0.01), ** p < 0.01 UVB treated group).
Figure 10 shows the residual amount of CPD when treated with UVB-irradiated fibroblasts (*: significant results according to the students test ( p <0.05), **: according to the Student's test Significant results ( p <0.01), * p <0.05, ** p <0.01 compared to UVB treated groups, respectively).
Figure 11 shows the expression level of p-p53 protein when treated with UVB irradiated fibroblasts (*: significant results ( p <0.01) according to the Student's test, * p &lt; Compared to the group).

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1. Preparation of plant extracts

Weigh each of dried date, leaf, oriental leaf, oak, cercophera, papaver, paulownia, and japonks, and weigh 100 times the weight of 100% methanol And the mixture was heated at 60-90 ° C for extraction and filtered. The extraction procedure was repeated twice, and the obtained crude extract solution was concentrated under reduced pressure to prepare an extract. The extract was dissolved in DMSO (dimethyl sulfoxide) at 100 mg / mL to prepare a stock solution.

Example 2. Free radical scavenging measurement

The free radical scavenging activity was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH). For the experiment, 100 μL of 0.2 mM DPPH solution in methanol was added to a 96-well plate, and ethanol and a sample were added to a final concentration of 10 μg / mL, followed by incubation at room temperature for 20 minutes. After incubation, absorbance was measured at 515 nm using a spectrophotometer (Synergy ^HT , Bio-tek, USA). The DPPH scavenging activity (%) of each plant extract is shown in Table 1 below.

Botanical name DPPH scavenging ability (%) Control group 0.0 Jujube leaves 89.3 Leaves 88.5 Biplane leaf 94.9 Oak leaf 92.7 Hemlock 94.0 Island wilt leaves 92.7 Tung leaves 90.6 Poppy leaf 91.0

As shown in Table 1, the DPPH scavenging ability of the plant extract according to Example 2 of the present invention was 80% or more. Therefore, a cytotoxicity test was conducted on these materials (data not shown), and the cell viability against UVB was measured at a concentration where no toxicity was observed.

Example 3. Measurement of cell survival rate against ultraviolet light

Cell viability for ultraviolet light was measured by Moseman's method (Moseman T., J. Immuno. Methods, 65, 55 (1983)) and Skaper et al. (Skaper SD, et al., Cell Culture, (3-4,5-dimethylthiazol-2yl) -2,5-diphenyl (3-4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide -2H-tetrazolium bromide) measurement method. Human fibroblast HS68 was cultured in a 96-well plate. The cultured cells were irradiated with 250 mJ UVB, treated with the plant extract prepared according to the method of the present invention, and cultured in a 5% CO ₂ incubator at 37 ° C. for 12 hours. MTT 5 mg / mL solution was added to each well After the treatment, the reaction was carried out at 37 DEG C for 4 hours. After the medium was removed, DMSO was added to the resulting solution to dissolve the formed crystals, and the resultant was measured at 595 nm using an ELISA reader. [Figure 2] shows the cell viability (%) of each plant extract against ultraviolet light.

[Figure 2] shows the cell survival rate for 250 mJ UVB as a result of treating the plant extract according to Example 1 of the present invention. Cell viability was 69% when treated with UVB alone and cell survival rate was lower when treated with plant extracts than with UVB only treatment except for the extract of L. var. As shown in the above Table 1, it was confirmed that the plant extract did not have a cytoprotective effect against ultraviolet rays because of its antioxidative effect. On the other hand, the survival rate of UVB on the extracts of Liliaceae was 94%, which was significantly different from other plant extracts.

Example 4. Preparation of Leaf Extract

50 g of dried leafy leaves were pulverized, and then 500 mL of 75% ethanol aqueous solution was added thereto, followed by heating at 60-90 DEG C for 2 hours and filtering. This extraction procedure was repeated twice and concentrated to obtain crude extract. This crude extract was redissolved in 10% aqueous ethanol solution and incubated at 47 [deg.] C for 20 hours with Novozym 33095; Pectin lyase (Novozymes, Denmark) was added. The enzyme-treated culture broth was filtered, concentrated, and dispersed in water at a ratio of 1:10 with respect to the solid content. Then, the same amount of ethyl acetate was added thereto, followed by fractionation and concentration to obtain a final extract. The extract was dissolved in DMSO (dimethyl sulfoxide) at 100 mg / mL to prepare a stock solution.

sample yield Macaroni leaf extract 2.44%

As shown in [Table 2], the yield of the extract of Liliaceae according to Example 1 of the present invention was 2.44%.

Example 5. Cytotoxicity of Leaf Extracts

Cell cytotoxicity was assessed by Moseman's method (Moseman T., J. Immuno. Methods, 65, 55 (1983)) and Skaper et al. (Skaper SD, et al., Cell Culture, 2, 17-33 (3-4,5-dimethylthiazol-2yl) -2,5-diphenyl-2,7-diphenyl-2H-tetrazolium bromide) 2H-tetrazolium bromide) measurement method. Human fibroblast HS68 was cultured in a 96-well plate. Twelve hours after the treatment of the leaf tea leaf extract, MTT 5 mg / mL solution was added to each well, followed by reaction at 37 ° C for 4 hours. After removing the medium, DMSO was added to the resulting solution to dissolve the formed crystals, and the resultant was measured at 595 nm using an ELISA reader. Toxicity to cells was expressed as a percentage of the average absorbance value of each control.

In order to investigate the cytotoxicity of the extracts of Fusarium oxysporum, the extracts of 1, 20 and 50 μg / mL of L. japonica leaf extracts were compared with those treated with control DMSO alone Cytotoxicity was investigated. The results are shown in Fig.

FIG. 3 shows the cytotoxicity of the extracts of A. cinerea on the fibroblasts according to the present invention. The cell survival rate was found to be 95% or more at 50 μg / mL. Therefore, it was confirmed that there was no cytotoxicity at the concentration of less than 50 μg /

Example 6. Production of Reactive Oxygen Species (ROS) in Cells

To measure ROS in H ₂ O ₂ -treated fibroblasts, cells were inoculated in 24-well plates at a concentration of 4 × 10 ⁴ cells / well, stabilized, replaced with serum-free DMEM medium, and incubated with 500 μM H ₂ O ₂ The extracts were incubated in a 5% CO ₂ incubator at 37 ° C for 12 hours. The cultured cells were cultured in DMEM medium supplemented with 10 μM DCFH-DA for 30 min at 37 ° C in a 5% CO ₂ incubator, washed with DCFH-DA 3 times with HBSS, and then stained with a fluorescence microplate reader ( Fluorescence was measured at excitation 485 nm and emission 530 nm using a Bio-Tek instrument, USA.

In order to measure ROS in UVB-irradiated skin cells, the extract was treated with 250 mJ UVB in the same manner as described above. Then, the extracts were incubated in a 37 ° C, 5% CO ₂ incubator for 12 hours, DMF medium supplemented with 10 μM DCF-DA was added and DCF was measured 30 minutes later. FIG. 4 shows H ₂ O ₂ -induced inhibitory effect on ROS formation in the cells of A. cinerea leaf extract according to an embodiment of the present invention.

DCFH-DA assay was performed to observe the effect of Horseradish Leaf Extract on the increased intracellular ROS by H ₂ O ₂ . The fibroblasts were treated with 500 μM H ₂ O _2, and then the fluorescence absorbance of the extract was measured by treating the extract of L. leucocyte with concentration. As shown in FIG. 4, the treatment of H ₂ O ₂ alone significantly increased intracellular ROS production, and the production of ROS was inhibited by 1, 20, and 50 μg / mL And significantly suppressed intracellular ROS. Thus, the antioxidant activity of the extracts of Liliaceae leaves suggest that there is a therapeutic and preventive effect of photodamage of the skin by removing active oxygen generated by stress such as ultraviolet rays. FIG. 5 shows the inhibitory effect of UVB irradiation on the production of intracellular ROS by the extract of A. cinerea according to an embodiment of the present invention.

As shown in FIG. 5, intracellular ROS by ultraviolet light was significantly inhibited by 20, 50 μg / Therefore, it is suggested that the extract of Rootstock leaf extract has a therapeutic and preventive effect on photodamage of skin by removing ROS generated by ultraviolet rays.

Experimental Example 7. Lipid Peroxidation Assay

The amount of 8-isoprostane secreted by the fibroblasts into lipid peroxidation was measured using a commercially available enzyme immunoassay (Cayman Chemical, Ann Arbor, MI, USA) (Colorimetric determination).

Whether the extract of Liliaceae leaf can inhibit the membrane lipid peroxidation reaction in UVB irradiated cells was measured 12 hours after UVB irradiation. The lipid peroxidation reaction was confirmed by measuring the amount of 8-isoprostane in the skin cell culture medium. As a result, as shown in Fig. 6, the increase in 8-isoprostanes in UVB- It was confirmed that the level of stannan was significantly and dose - dependently reduced by treatment of A. fumigatus leaf extract. As a result, as shown in [Figure 4] and [Figure 5], the extract of Liliaceae showed reduced intracellular reactive oxygen, thereby inhibiting lipid peroxidation as shown in [Figure 6]. Therefore, it is suggested that the extracts of A. falciparum have a therapeutic and preventive effect on skin photodamage caused by ultraviolet rays.

Example 8. Measurement of Apoptosis of Extract of Lentinus edodes

Cellular death was measured using a cell death detection ELISA plus kit (Poche Diagnostics, Mannheim, Germany). The experimental method will be briefly described as follows. After incubation for 12 hours, the cells were separated from the cultured fibroblasts by 250 mJ UVB irradiation, washed, washed, and transferred to a 1.5-ml tube. Each tube was allowed to have 10 ⁴ cells, followed by centrifugation for 10 minutes. After carefully removing the supernatant, 200 μL of lysis buffer was added to each well, followed by reaction at 15 to 25 ° C for 30 minutes. The tube was again centrifuged for 10 minutes, and then 20 μL of each supernatant was transferred to a 96-well plate coated with streptavidin. After adding 80 μL of the immune reaction solution to each well, the plate was wrapped with a cover foil and reacted at a temperature of 15 to 25 ° C for 2 hours at 300 rpm. After removing the solution and washing three times with the reaction solution, 100 μL of ABTS solution was added to each well, and the mixture was reacted at 250 rpm for about 10 to 20 minutes until a sufficient coloring reaction was observed. Absorbance was measured with an ELISA reader at 405 nm.

FIG. 7 shows the presence or absence of cellular apoptosis when treated with UVB-irradiated fibroblasts according to the embodiment of the present invention. It was confirmed that the extracts of Lilium japonica leaf extract decreased at 20 and 50 μg / mL, respectively. These results show that the extracts of A. fumigatus prevent cell death from photodamage caused by UVB.

Example 9. Measurement of Caspase-3 Activity of Leaf Extract

Caspase-3 activity was measured using a Caspase-3 assay kit supplied by Abcam. Briefly, 250 ml of UVB-irradiated fibroblasts were treated for 12 hours with PBS, washed with PBS, lysed at 4 ° C for 10 minutes, and centrifuged at 10,000 g to remove supernatant &Lt; / RTI &gt; The obtained supernatant was subjected to the method provided by abcam using a caspase-3 activity assay kit and was performed using a microplate reader (Bio-Tek instrument, USA) And the absorbance at 405 nm was measured.

FIG. 8 shows the activity of caspase-3, which is involved in cellular apoptosis, upon treatment of UVB-irradiated fibroblasts according to the embodiment of the present invention. The activity of caspase-3 was negligible in the group not irradiated with UVB, whereas the activity of caspase-3 was significantly increased in the group irradiated with UVB. The activity of caspase-3 was significantly decreased at 20, 50 μg / mL when treated with 1, 20, and 50 μg / mL extracts. These results suggest that the extracts of A. fumigatus inhibit cell death caused by UVB-induced photodamage of skin cells.

Example 10. Measurement of H2A.X (pSer139) of Leaf leaf extract

The amount of H2A.X (pSER139) was measured using the H2A.X (pSer139) assay kit supplied by Abcam. Briefly, 250 mJ UVB-irradiated fibroblasts were treated with 4% paraformaldehyde in PBS for 12 hours, followed by washing with PBS. Immobilized cells were washed with PBS, and then permeabilized with Triton-X 100 for 10 minutes. Then, the cells were immersed in a blocking solution at room temperature for 2 hours, and then reacted with a primary antibody (anti-H2A.X (pSer139)) diluted with blocking solution overnight at 4 ° C. Subsequently, the cells were washed with a washing buffer and allowed to react with fluorescent secondary antibody for 2 hours at room temperature. After washing with PBS, washing with PBS, Janus Green Stain was added and the reaction was allowed to proceed at room temperature for 5 minutes. After washing with deionized water, 0.5 mM HCl was added and absorbance was measured at 595 nm using a microplate reader (Bio-Tek instrument, USA).

FIG. 9 shows the recovery of γ-H2A.X according to DNA damage when treated with UVB-irradiated fibroblasts according to the embodiment of the present invention. In order to observe the effect of the extracts of Liliaceae on UVB - damaged DNA, the amount of H2A.X was measured by intracellular ELISA analysis. H2A.X is one of the histone H2Ab families. When DNA double strand separation occurs, Ser 139 present in the C-terminal tail of H2A.X is phosphorylated within 1 to 3 minutes, resulting in γ-H2A.X . Thus, it is possible to determine whether DNA damage has occurred. As shown in [Figure 9], the extract of the Lycoris spp. Recovered intracellular DNA damage due to UVB stimulation in a concentration-dependent manner.

Example 11: CPD measurement of Leaf leaf extract

When ultraviolet light is applied to a continuous DNA sequence of thymine, a cyclobutane pyrimidine dimer (DPD) is formed. In order to confirm the effect on DNA damage, a commercially available OxiSelect ( ^TM ) Cell UV Induced DNA Damage Staining Kit (CPD) (Cell Biolabs Inc, USA) was used. The residual amount of CPD was measured and analyzed in order to confirm the effect of the leaf extract on the recovery of the DNA injury reaction (FIG. 10).

As a result, as shown in FIG. 10, when the extract was treated with UV light, CPD amount was significantly decreased in a concentration dependent manner. It was confirmed that the extracts of Liliaceae leaf not only protect against UV-induced apoptosis but also recover DNA damage reaction.

Example 12. Expression of phospho-p53 (phospho-p53, p-p53) expression in the extract of Liliaceae

In order to measure the amount of p53 expressed by UV-induced cell damage, 250 mJ UVB was irradiated on fibroblasts, and then treated with 1, 20, 50, or 100 μg / The cultured cells were homogenized with PRO-PREP protein extraction solution (iNtRON Bio Technology Co., Ltd), vortexed for 10 minutes, centrifuged at 4 ° C for 12 minutes at 12,000 rpm for 30 minutes, Respectively. The expression of p-p53 was measured using the same amount of protein using a sandwich ELISA kit (Cell Signaling). In order to examine the effect of the extract of the leafy leaf on the protein expression of the phospho-p53 involved in repairing the damaged DNA, ELISA method was used to measure the result, and the results are shown in FIG.

As shown in Fig. 11, when the ultraviolet light was not treated, the amount of expression was small, but when the ultraviolet light was irradiated, the expression of p-p53 remarkably increased. When UV light was irradiated, the expression of p-p53 protein was decreased compared with UVB-treated group. These results suggest that the expression of p-p53 is decreased due to the recovery of DNA damage according to the results shown in [Fig. 9] and [Fig. 10]. Therefore, it is suggested that the extracts of A. falciparum can be recovered from the photodamage of skin cells by ultraviolet rays.

In view of the above results, the intracellular antioxidant activity, lipid peroxidation effect, apoptosis, caspase-3 activity, p-p53 expression, CDP, and H2A.X inhibitory effect of the extract of A. cinerea were confirmed. Therefore, it could be confirmed that the Korean leaf tea leaf is an excellent material for treating and preventing skin photodamage caused by ultraviolet light.

As described above, the present invention is very useful in the life, bio, food, or pharmaceutical industries because it provides a natural composition having an excellent effect of preventing, improving, or treating skin photodamage containing an extract of Lilac leaves.

Claims (12)

A cosmetic composition for preventing or ameliorating the photodamage of the skin, comprising an extract of Liliaceae leaf as an active ingredient. [Claim 2] The cosmetic composition according to claim 1, wherein the horse leaf leaf extract is extracted using an alcohol having 1 to 4 carbon atoms, water, or a mixture thereof as an extraction solvent. The method according to claim 1,
(s1) Extracting the leaves of a Chinese persimmon tree with an alcohol having 1 to 4 carbon atoms, water or a mixture thereof;
(s2) adding an enzyme to the extract of step (s1) and shaking / culturing; And
(s3) adding an organic solvent to the culture of step (s2)
Wherein the cosmetic composition is extracted. The cosmetic composition according to claim 3, wherein the enzyme is a pectin hydrolyzing enzyme. The cosmetic composition according to claim 3, wherein the organic solvent is at least one selected from the group consisting of ethyl acetate, diethyl ether, trichloromethane, butanol, and dichloromethane. (s1) Extracting the leaves of a Chinese persimmon tree with an alcohol having 1 to 4 carbon atoms, water or a mixture thereof;
(s2) adding an enzyme to the extract of step (s1) and shaking / culturing; And
(s3) adding an organic solvent to the culture of step (s2)
Wherein the extract is a mixture of at least two kinds of cosmetic ingredients. The method according to claim 6, wherein the enzyme is a pectin hydrolase. The method according to claim 6, wherein the organic solvent is at least one selected from the group consisting of ethyl acetate, diethyl ether, trichloromethane, butanol, and dichloromethane. 7. The method according to claim 6, further comprising, between step (s1) and step (s2), further concentrating the extract of step (s1) and re-dissolving the concentrate in an aqueous alcohol solution. The method according to claim 9, wherein the concentration of the alcohol aqueous solution is 5 to 30% by weight. A pharmaceutical composition for preventing or treating photodamage of skin, comprising an extract of Liliaceae leaf as an active ingredient. delete

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