KR20200114291A - Composition for preventing or improving skin photoaging comprising unsaponifiable matter from Perilla frutescens by-product as effective component - Google Patents

Abstract

The present invention relates to a cosmetic composition for preventing or improving skin photoaging containing unsaponifiable matter derived from a perilla seed as an active ingredient, a sunscreen containing the cosmetic composition, and a skin external use composition for preventing or improving skin photoaging containing unsaponifiable matter derived from a perilla seed as an active ingredient.

Description

A cosmetic composition for preventing or improving skin photoaging comprising unsaponifiable matter from Perilla frutescens by-product as effective component}

The present invention relates to a cosmetic composition for preventing or improving skin photoaging, containing an unsaponifiable product derived from perilla seeds as an active ingredient.

Photoaging refers to a phenomenon in which skin damage occurs, such as decreased elasticity and moisture of the skin and wrinkles formed due to repeated or prolonged exposure to ultraviolet radiation (UVR) of sunlight. As the desire to maintain beautiful and healthy skin increases due to the improvement of the standard of living, and as interest in preventing skin damage caused by exposure to sunlight increases as outdoor activities increase, research on skin aging control is being actively conducted. .

UV rays are classified into UVA (ultraviolet A, 320-400nm), UVB (ultraviolet B, 280-320nm), and UVC (ultraviolet C, 200-280nm) according to their wavelength. Among them, UVB is very harmful rays that are involved in epidermal damage or cause dryness, skin erythema, pigmentation, and epidermal thickening. In addition, it causes an imbalance of antioxidant mechanisms and damages lipids, proteins, and enzymes that make up the skin, thereby intensifying inflammatory reactions, skin cancer and photoaging. On the other hand, UVA has little power in a short term compared to UVB, but its long wavelength penetrates deep into the skin and damages the dermis of the skin, blackening the skin and promoting skin aging. The standard of efficacy of materials and products that can inhibit UVA and UVB is used as the UV blocking index, and to develop natural functional materials and products that can exhibit efficacy without side effects even when used for a long time for skin damage due to skin photoaging. Research is being actively conducted.

ROS (reactive oxygen species) produced by UVB stimulation is one of the causes of photoaging and stimulates the intracellular signaling system, causing oxidative stress on biomolecules such as DNA, lipids and proteins, causing skin tissue damage. ROS stimulates epidermal keratinocytes and dermal fibroblasts to activate transcription factors of AP-1 and nuclear factor kappa-light-chain-enhancer of activator B cells (NF-κB) and secrete inflammation-related cytokines. Thus, it increases the expression of MMP (matrix metalloproteinase) and promotes the decomposition of collagen and elastic fibers constituting the skin tissue to form wrinkles. In vivo, antioxidant enzymes such as CAT (catalase), SOD (superoxide dismutase) and GST (glutathione-s-transferase) act to protect against lipid peroxides and oxidative enzymes produced and activated from ROS stimulation. In vitro, vitamin C, It protects against damage caused by ROS through bioactive substances such as E and polyphenols and flavonoids.

Perilla frutescens ) is an annual plant of the labiatae, mint family, a dicotyledonous plant, and metabolic phenotypes in a reddish pigmented form and a greenish pigmented form depending on the effectiveness of anthocyanins. There are two chemovarietal forms. Perilla seeds are one of the traditional medicinal crops and have been used to treat various diseases such as depression, tumors, cough, antibacterial, anti-fungal, and allergies.

On the other hand, Korean Patent No. 1934793 discloses'a composition for preventing or improving skin photoaging that contains an extract of Goyom trees as an active ingredient', and Korean Patent No. 0614970 discloses a recognition containing perilla seed extract as an active ingredient. A composition for preventing and improving dysfunction is disclosed, but there is no description of a cosmetic composition for preventing or improving skin photoaging containing the non-saponifiable product derived from perilla seeds of the present invention as an active ingredient.

The present invention was derived from the above requirements, and the present inventors treated human fibroblast cell line Hs68 cells with UVB to induce photoaging and then treated unsaponifiable products derived from perilla seeds, resulting in reduced Hs68 cells by UVB irradiation. It was confirmed that the survival rate of was increased and the amount of ROS production increased by UVB decreased. In addition, the unsaponifiable product derived from perilla seeds of the present invention inhibits the phosphorylation of MAPKs (JNK, ERK and p38) and AP-1 (c-fos and c-jun) induced by UVB irradiation, thereby inducing skin aging. The present invention was completed by confirming that the expression of the transforming growth factor (TGF)-β1 and Smad2/3, which decreases protein expression and promotes collagen production, is increased.

In order to solve the above problems, the present invention provides a cosmetic composition for preventing or improving skin photoaging, containing an unsaponifiable matter derived from perilla seed as an active ingredient.

In addition, the present invention provides a sunscreen comprising the cosmetic composition.

In addition, the present invention provides a composition for external application for skin for preventing or improving skin photoaging, containing unsaponifiable matter derived from perilla seeds as an active ingredient.

The unsaponifiable material derived from perilla seeds of the present invention not only reduces the activity of proteins that induce skin aging, but also has an excellent effect of increasing the activity of factors that promote collagen production, so it is a functional material for preventing and improving photoaging caused by ultraviolet rays. And it can be usefully used as a cosmetic composition.

1 is a graph (A) showing the survival rate of cells by irradiation with UVB after pretreatment of perilla seed meal unsaponifiable (PSU) or perilla seed meal methanol extract (1, 2.5 or 5 μg/ml) to human fibroblast cell line Hs68 cells. A graph showing cell viability (B and C) and intracellular collagen production (D and E) by UVB irradiation after PSU pretreatment on Hs68 cells.
FIG. 2 is a graph showing the ROS (reactive oxygen species) generated in the cells after pretreatment with PSU (1, 2.5 or 5 μg/ml) on Hs68 cells and then irradiation with UVB, measured with a fluorescence spectrophotometer.
3 shows the production amount (A and B) and mRNA expression level (C) of MMP (matrix metalloproteinase)-1 and MMP-3 proteins by UVB irradiation after pretreatment of PSU (1, 2.5 or 5 μg/ml) in Hs68 cells. And D) are measured and shown.
4 is a result of measuring the phosphorylation levels of JNK(A), ERK(B) and p38(C) by pretreatment with PSU (1, 2.5 or 5 μg/ml) in Hs68 cells and irradiation with UVB.
5 is a result of measuring the phosphorylation activity of c-jun (A) and c-fos (B) by pretreatment with PSU (1, 2.5 or 5 μg/ml) in Hs68 cells and irradiation with UVB.
Figure 6 shows by measuring the expression levels of TGF-β1 (A), Smad2/3 (B) and Smad7 (C) by pretreatment with PSU (1, 2.5 or 5 μg/ml) in Hs68 cells and irradiation with UVB. It is a graph.

In order to achieve the object of the present invention, the present invention provides a cosmetic composition for preventing or improving skin photoaging, containing as an active ingredient unsaponifiable matter derived from perilla.

In the present specification, the term'unsaponifiable' is also referred to as unsaponifiable, and generally refers to a substrate that is not hydrolyzed by alkali, and when compared to saponified in lipids, hydrocarbons such as sterols, vitamins such as fat-soluble pigments, etc. It means a material containing.

In the cosmetic composition of the present invention, the unsaponifiable product derived from perilla seed can be prepared using a conventional method known in the art, and preferably, a saponified product is treated with an alkali to the perilla by-product obtained after preparing perilla oil. Can be obtained by decomposition. At this time, the remaining residue except for the decomposed saponified product may be extracted using an organic solvent, but is not limited thereto. The organic solvent may be hexane, ethyl acetate, or a mixed solution thereof, more preferably a mixed solution of hexane and ethyl acetate, but is not limited thereto.

In the present specification, the term'photoaging' refers to a phenomenon in which skin damage, pigmentation, wrinkle formation, or skin elasticity decreases upon repeated or prolonged exposure to ultraviolet rays of sunlight.

In the cosmetic composition of the present invention, the unsaponifiable product derived from perilla seeds has an excellent effect of increasing collagen synthesis and reducing the expression of collagen-degrading enzymes MMP (matrix metalloproteinase)-1 or MMP-3. Inhibiting the activity of MMP-1 protein, type I collagen degrading enzyme and MMP-3 protein, type IV collagen degrading enzyme using unsaponifiable perilla, can reduce collagen degradation, thus maintaining elasticity of skin tissue. It can prevent wrinkles.

In addition, the unsaponifiable product derived from perilla seeds reduces the phosphorylation reaction of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, c-fos or c-jun in cells, and TGF-β1 And it is excellent in the effect of preventing skin aging by increasing the expression of Smad2/3 to decrease the expression of the MMPs protein. Activation of the mitogen activated protein kinase (MAPK; ERK, JNK, and p38) pathway can increase the expression of MMPs by inducing the continuous activity of AP-1 (a transcription factor consisting of Fos and Jun family proteins). For example, phosphorylation of ERK increases the expression and phosphorylation of c-fos constituting AP-1, and phosphorylation of JNK or p38 increases the expression and phosphorylation of c-jun constituting AP-1 to express MMPs. Increase Therefore, by inhibiting the activity of MAPK and AP-1 using perilla extract, it is possible to prevent wrinkles by inhibiting the expression of MMPs. In addition, if the perilla extract is used to induce the expression and phosphorylation of TGF (transforming growth factor)-β1 and Smad2/3 to increase the activity, it is possible to promote collagen production through activation of transcription factors in the nucleus.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion It can be formulated as a foundation, wax foundation and spray, and more specifically, skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, Moisture cream, sunscreen, hand cream, essence, nutritional essence, pack, cleansing foam, cleansing water, cleansing cream, body lotion, body cleanser, soap, and powder can be prepared in any one formulation selected from, preferably UV It may be prepared in the form of a blocking agent, but is not limited thereto. The cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.

In addition to the above active ingredients, the cosmetic composition of the present invention includes ingredients commonly used in cosmetic compositions, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents ( foaming agents), fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives , Conventional adjuvants such as lipid vesicles, and carriers.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide as carrier components Etc. may be used.

When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydro Propellants such as carbon, propane-butane or dimethyl ether.

When the formulation of the cosmetic composition 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, propylene Glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the cosmetic composition 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 cosmetic composition of the present invention is a surfactant containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acetionate, imidazolinium derivative, methyltaurate, sarcosinate , Fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty diethanolamide, vegetable oil, linoline derivative or ethoxylated glycerol fatty acid ester, and the like may be used.

The cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrophobic substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreen agents, vitamins, plant extracts, etc. .

In addition to the active ingredient, the cosmetic composition of the present invention may further contain one or more ingredients that exhibit the same or similar functions or enhance the effect. For example, as an anti-aging and wrinkle-improving component known in the art, one or more components selected from retinoic acid, protein derived from animal placenta, betulinic acid and chlorella extract may be further included. The added component may be included in an amount of 0.0001 to 10 parts by weight, based on 100 parts by weight of the total composition, and the content range may be adjusted according to requirements such as collagen synthesis promoting activity, skin safety, and ease of formulation. .

The present invention also provides a composition for external application for skin for preventing or improving skin photoaging, containing an unsaponifiable product derived from perilla seed as an active ingredient.

In the composition for external application for skin for preventing or improving skin photoaging of the present invention, the non-sample derived from perilla seeds not only increases collagen synthesis and decreases the expression of collagenase, matrix metalloproteinase (MMP)-1 or MMP-3, By reducing the phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), p38, c-fos or c-jun in cells, and increasing the expression of TGF-β1 and Smad2/3 Since it can reduce the activity of MMPs, which are collagenases, it is excellent in preventing skin damage, pigmentation, wrinkle formation, or reduction of skin elasticity and related photoaging caused by repeated or prolonged exposure to UV rays.

The external preparation for skin of the present invention may be applied to the human body in various ways or forms of the composition of the present invention, but may be preferably prepared in a formulation of a skin external preparation applicable by being applied topically to the skin. For example, a powder, an ointment, a gel, a cream, a liniment, a lotion, a liquid, or an aerosol may be used, but the present invention is not limited thereto.

When formulated for the external application for skin, it can be prepared by appropriately blending additives in addition to the composition of the present invention. The additives are commonly used in the art and can be blended within a range that does not impair the objects and effects of the present invention.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

One. Perilla origin Unsaponifiable Produce

Perilla oil was prepared in a production container, and 3 g of the remaining perilla seed (perilla by-product) was taken, 20 ml of 6% pyrogallol was added, followed by nitrogen filling, ultrasonic treatment for 5 minutes, and potassium hydroxide (KOH) 8 ml. After filling with nitrogen again, the lid was replaced with a cooling tube, and the mixture was cooled after reacting for 50 minutes in a thermostat at 75°C. After cooling, 30 ml of 2% sodium chloride (NaCl) was added, and extraction was repeated 3 times with 30 ml of an extraction solvent (hexane:ethyl acetate=85:15, without BHT). The extract was concentrated under reduced pressure to remove the solvent and redissolved in dimethyl sulfoxide (DMSO) to treat cells.

2. Vitamin E content measurement

The prepared perilla meal unsaponifiable was redissolved in hexane, filtered, and analyzed by NPHPLC (normal phase HPLC). LiChrosphere ^® Diol 100 column (250 x 4 mm id, 5 um, Merck, Germany) was used, and the mobile phase was 1.0 ml/ of hexane/isopropanol (98.9:1.1, v/v) It flowed at the speed of minutes. The concentrations of unsaponifiable tocopherol and tocotrienol were calculated using the average peak area compared to the standard.

3. Phytosterol ( phytosterol ), Policosanol ( policosanol ), squalene ( squalene) analysis

The concentrations of phytosterol, policosanol, and squalene in the unsaponifiable perilla meal were measured using 5α-cholestane as an internal standard. Gas chromatography (Varian 3800; Varian Inc., USA) was equipped with a SAC-5 fused-silica capillary column (30m×0.32mm i.d.; Supelco, USA) and a flame ionization detector. The transport gas was helium, and the temperatures of the injector and detector were 310°C and 320°C, respectively, and phytosterol, policosanol, and squalene were distinguished through retention time and standard materials.

4. Cell culture and cytotoxicity test

Human fibroblast line Hs68 cells (American Type Culture Collection, Manassas, USA) are DMEM containing FBS (fetal bovine serum, 10%), penicillin (100 units/ml), and streptomycin (50µg/ml). The medium was cultured in an incubator controlled by 37° C., 5% CO ₂ , and 95% humid air. Hs68 cells were dispensed in a 96-well incubator at a concentration of 1.0×10 ⁴ cells/well and cultured for 24 hours, and then the samples were diluted in a medium containing no FBS and cultured for 24 hours. 20 µl of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, 5 mg/ml) solution was added to each well and reacted for 2 hours, and the blue formazan ( formazan) crystals were dissolved in DMSO and absorbance was measured.

5. UVB Cellular protective effect on irradiation

Hs68 cells were dispensed in a 96-well incubator at a concentration of 1.0 × 10 ⁴ cells/well, cultured for 24 hours, and then diluted in a medium containing no FBS and cultured for 24 hours. After the culture was completed, the medium was removed, washed once with PBS (phosphate buffered saline), and then irradiated with UVB (30mJ/cm ² ) with a UVB lamp, and a UV LIGHT METER (UV-340A, LT Lutron, Taiwan) was used. UV intensity was monitored. All UVB irradiation was performed after applying a thin layer of PBS to the cells attached to the 96-well incubator, and after irradiation, the sample was diluted in a medium containing no FBS and irradiated to the cells for 24 hours. The control was carried out under the same conditions as above without UVB irradiation, and the cell protective effect against UVB was confirmed through the same method as in the cytotoxicity experiment using the MTT.

6. ROS Measurement of production

Samples were pretreated to Hs68 cells for 24 hours, washed with PBS, and then UVB (30mJ/cm ² ) was irradiated on the cells. The sample was treated on UVB-irradiated cells for 30 minutes, stained with 25 μM of DCFH-DA (dichlorodihydro-fluorescein diacetate), and then excitation wavelength of 485 nm and emission wavelength of 530 nm using a fluorescent spectrophotometer. ) To measure the amount of ROS (reactive oxygen species) produced in cells.

7. Water-soluble collagen and MMPs Measure

The amount of MMP-1 and MMP-3 produced was determined by measuring the content of MMP-1 and MMP-3 present in the medium using a human MMP-1 and MMP-3 ELISA kit (Merck & Co. Inc., USA). The content of collagen was measured using a Sircol ^TM soluble collagen assay kit.

8. qRT - PCR Using mRNA Expression level measurement

Total RNA was extracted using TRIzol reagent (SolGent), and cDNA was synthesized using (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific, Inc.). QRT-PCR (quantitative real-time polymerase chain reaction) was performed using a reaction mixture in which the synthesized cDNA, primer, RNase-free water, etc. were mixed, and TaqMan primers used for PCR were purchased and used from Thermo Fisher Scientific. (MMP1 Assay ID; Hs00899658_m1, MMP3 Assay ID; Hs00968305_m1).

9. Measurement of protein expression level using Western blot

After loading the sample onto a 10% SDS-polyacrylamide gel, it was transferred to a PVDF membrane. The membrane was blocked in a TBST (Tris-Buffered Saline+Tween) solution containing 5% skim milk for 1 hour, and then reacted overnight at 4°C using a primary antibody diluted with a TBST solution containing 5% skim milk. After the completion of the primary antibody reaction, it was washed with a TBST solution for 30 minutes, reacted with a secondary antibody, and then treated with an ECL solution in a dark room to sensitize the X-ray film.

Example One. Perilla origin Unsaponifiable Vitamin C within, Phytosterol , Policosanol And measuring the content of squalene

As a result of measuring the content of vitamin C, phytosterol, policosanol, and squalene in the unsaponifiable perilla seed using high-performance liquid chromatography (HPLC) and gas chromatography (GC), The most abundant vitamin E was γ-tocotrienol (330.67mg/100g USM), and the main policosanol was C24 (tetracosanol; 857.72mg/100g USM), C26 (hexacosanol; 252.88mg/100g USM), and C28 ( octacosanol; 1802.98mg/100g USM), C30 (triacontanol; 847.77mg/100g USM), and it was confirmed that β-sitosterol (sitosterol; 23016.25mg/100g USM) occupies the most content among phytosterols ( Table 1).

Example 2. Perilla origin Unsaponifiable Cell protection effect and collagen synthesis

Hs68 cells, a human fibroblast cell line, were treated with an unsaponifiable product (hereinafter, referred to as PSU) derived from perilla and were irradiated with UVB to measure cell viability and intracellular collagen production.

First, as a result of comparing the cytoprotective effect of the non-saponifiable perilla seed and methanol extract of perilla seed, the UVB irradiation group (no PSU treatment) decreased the cell viability to about 75% compared to the UVB/PSU-free control group, but PSU ( 1, 2.5 or 5 ㎍ / ㎖) or perilla seed meal methanol extract (1, 2.5 or 5 ㎍ / ㎖) was pretreated and irradiated with UVB, it was confirmed that the cell viability increased in a concentration-dependent manner. In particular, the PSU treatment group at a concentration of 1㎍/㎖ significantly increased cell viability compared to the UVB irradiation group (no PSU treatment), but it was confirmed that there was no significant difference in the methanol extract treatment group at the concentration of 1㎍/mL perilla seeds. I did. Through this, it was found that the non-saponifiable perilla perilla extract has excellent cell protection effect against UVB in the proliferation of normal fibroblasts compared to the methanol extract of perilla perilla, and has excellent cell protection effect even at a low concentration (FIG.

In addition, in Hs68 cells not irradiated with UVB, the cell viability between the PSU-treated group (1, 2.5, or 5 µg/ml) and the PSU-free control group was similar, indicating that PSU had no cytotoxicity (Fig. 1B). . In addition, the UVB irradiation group (no PSU treatment) decreased the cell viability to about 74% level compared to the UVB/PSU untreated control group, but it was confirmed that the cell viability increased depending on the PSU concentration when UVB was irradiated after pretreatment of PSU. (Fig. 1C), through this, it was found that the PSU has a cytoprotective effect against UVB in the proliferation of normal fibroblasts.

In addition, as a result of measuring intracellular collagen production by treating PSU at different concentrations in Hs68 cells not irradiated with UVB, it was confirmed that the PSU 5 ㎍ / ㎖ treatment group significantly increased the collagen production amount compared to the PSU untreated group ( 1D), the amount of collagen production in the UVB-irradiated group decreased to a remarkable level compared to the non-UVB-irradiated group, but it was confirmed that the amount of collagen production increased depending on the PSU concentration when UVB was irradiated after pretreatment of the PSU (FIG. 1E ).

Example 3. Perilla origin Unsaponifiable ROS Analysis of production inhibition effect

As a result of analyzing the effect of inhibiting the amount of ROS production of the unsaponifiable (PSU) derived from perilla seeds, the amount of ROS produced in the UVB irradiated group (no PSU treatment) increased by about two times compared to the control group without UVB/PSU, but after pretreatment of the PSU When irradiated with UVB, it was confirmed that the amount of ROS production increased by UVB irradiation significantly decreased (FIG. 2).

Example 4. Perilla origin Unsaponifiable MMP Protein production inhibition effect analysis

MMPs (matrix metalloproteinases) proteins are enzymes that break down the proteins that make up the skin's extracellular matrix (ECM), and the production of MMPs in keratinocytes and dermal fibroblasts is caused by various stresses such as aging, UV or heat. When it increases, the amount of collagen and elastin, which are the main components of ECM, decreases, causing the skin to lose elasticity and wrinkles. There are 19 types of MMPs in human skin, of which only MMP-1, MMP-3 and MMP-9 increase their expression by UV irradiation. In particular, MMP-1 is a major collagen-degrading enzyme that decomposes type-I and Ⅲ, the main collagen types in the skin. Since type-1 accounts for 80-90%, MMP-1 has the greatest influence, and type-1 collagen Is known to be synthesized and secreted in the form of procollagen, a precursor of collagen, from dermal fibroblasts. Collagen degraded by MMP-1 is then further degraded by MMP-3, and MMP-3 is known to activate pro MMP-1. Accordingly, the amount of production of MMP-1 and MMP-3 and the amount of mRNA expression in cells were measured to determine the effect of the non-saponifiable perilla (PSU) on the MMP protein increased by UVB stimulation.

As a result, the UVB irradiation group significantly increased the amount of MMP-1 and MMP-3 production compared to the UVB/PSU untreated control group, but when UVB was irradiated after pretreatment of the PSU, compared to the UVB irradiation group (no PSU treatment). It was confirmed that the amount of production of MMP-1 and MMP-3 decreased depending on the PSU concentration (FIGS. 3A and B). In addition, it was confirmed that the measurement results of the mRNA expression levels of MMP-1 and MMP-3 also appear very similar to the results of the production of MMP-1 and MMP-3 (FIGS. 3C and D).

Through this, it was thought that the PSU of the present invention could effectively prevent skin photoaging caused by ultraviolet rays by inhibiting collagen degradation by inhibiting both the expression and production of MMP-1 and MMP-3.

Example 5. Perilla origin Unsaponifiable MAPK Analysis of inhibitory effect on phosphorylation

The effect of unsaponifiable (PSU) derived from perilla seeds on the phosphorylation of ERK, JNK and p38, which are mitogen activated protein kinase (MAPK), was analyzed. JNK and ERK phosphorylation were expressed as phosphorylated MAPK expression/MAPK expression.

As a result, the level of JNK phosphorylation was significantly increased in the UVB irradiation group (no PSU treatment) compared to the UVB/PSU non-treatment control group, but UVB irradiation after pretreatment of PSU at each concentration (1, 2.5 or 5㎍/㎖) When compared to the UVB irradiation group (no PSU treatment), it was confirmed that the phosphorylation level of JNK decreased to 61%, 30.9%, and 19.7% depending on the PSU concentration (FIG. 4A).

In addition, ERK and p-38 phosphorylation levels were also very similar to those of the JNK phosphorylation level measurement results, and when UVB irradiation after pretreatment of PSU concentrations, ERK phosphorylation levels were 78.9%, 45.3%, depending on PSU concentration, It was confirmed that it decreased to 12.8% (FIG. 4B), and the p-38 phosphorylation level decreased to 60.9%, 27.8%, and 15.3% depending on the PSU concentration (FIG. 4C). Through this, it was found that the PSU of the present invention can inhibit the expression of collagen-degrading enzyme by inhibiting the phosphorylation of MAPK.

Example 6. Perilla origin Unsaponifiable Analysis of AP-1 transcription factor activity inhibition effect

The transcription factor AP-1 is composed of c-jun and c-fos proteins and is activated by UVB irradiation, and the amount of MMPs produced is increased by the activated AP-1. In the above experiment, it was confirmed that the unsaponifiable (PSU) derived from perilla seed reduced both the expression and production of the MMP protein, and it was investigated whether PSU also reduced the activity of AP-1, the upper regulator of the MMP protein. AP-1 activity was expressed by the expression level of p-c-jun or p-c-fos/c-jun or c-fos expression level.

As a result, the c-fos phosphorylation level was significantly increased in the UVB irradiation group (no PSU treatment) compared to the UVB/PSU non-treatment control group, but after pretreatment of PSU at each concentration (1, 2.5 or 5㎍/㎖), UVB When investigated, it was confirmed that the c-fos phosphorylation level decreased to 68.1%, 40.1%, and 23.3% depending on the PSU concentration (FIG. 5A).

In addition, the c-jun phosphorylation level was also very similar to the c-fos phosphorylation level measurement result, and when UVB was irradiated after pretreatment of the PSU by concentration, the c-jun phosphorylation level was 88.6%, 80.0% depending on the PSU concentration. , It was confirmed that it decreased to 63.8% (FIG. 5B). Through this, it was found that the PSU of the present invention can inhibit the expression of collagen-degrading enzyme by inhibiting the phosphorylation of AP-1 transcription factor.

Example 7. Perilla origin Unsaponifiable TGF -β pathway activity enhancement effect analysis

The extracellular matrix of the skin is gradually divided during aging, and genes related to the extracellular matrix of the dermis in aged human skin are reduced through the TGF-β pathway. TGF-β promotes phosphorylation of Smad2/3 and, in the case of Smad7, competes with Smad2 and Smad3 to bind to activated TGFR1. It is known that ROS increased by UVB inhibits the action of TGF-β in dermal fibroblasts and activates AP-1, an intracellular transcription factor. Accordingly, the effect of perilla seed-derived unsaponifiable (PSU) on the TGFβ/smad signaling pathway, a major regulator of Type 1 procollagen synthesis, was analyzed.

As a result, the expression levels of TGF-β1 and Smad2/3 significantly decreased in the UVB irradiation group (no PSU treatment) compared to the UVB/PSU non-treatment control group, but the PSU concentration by concentration (1, 2.5 or 5㎍/㎖) After pretreatment with UVB, it was confirmed that the expression levels of TGF-β1 and Smad2/3 increased in a PSU concentration-dependent manner compared to the UVB irradiation group (no PSU treatment) (Figs. 6A and B).

On the other hand, the expression level of Smad7, which inhibits the activity of TGF-β1 and Smad2/3, was significantly increased in the UVB irradiation group (no PSU treatment) compared to the UVB/PSU non-treatment control group. When irradiated, it was confirmed that the amount of Smad7 expression decreased in a PSU concentration-dependent manner compared to the UVB irradiation group (no PSU treatment) (FIG. 6C ).

Claims (8)

A cosmetic composition for preventing or improving skin photoaging containing unsaponifiable matter derived from perilla seeds as an active ingredient. The cosmetic composition for preventing or improving skin photoaging according to claim 1, wherein the photoaging is skin damage, pigmentation, wrinkle formation, or reduction of skin elasticity due to ultraviolet irradiation. The cosmetic composition for preventing or improving skin photoaging according to claim 1, wherein the unsaponifiable product derived from perilla seed meal is obtained by decomposing the saponified product by treating perilla seed meal with alkali. The prevention or improvement of skin photoaging according to claim 1, wherein the unsaponifiable product derived from perilla seeds increases the synthesis of collagen and decreases the expression of the collagenase MMP (matrix metalloproteinase)-1 or MMP-3. Cosmetic composition. The method of claim 1, wherein the unsaponifiable product derived from perilla seeds reduces the phosphorylation reaction of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), p38, c-fos or c-jun, and TGF -A cosmetic composition for preventing or improving skin photoaging, characterized in that increasing the expression of β1 or Smad2/3. A sunscreen comprising the cosmetic composition according to any one of claims 1 to 5. A composition for external application for skin for preventing or improving skin photoaging, containing unsaponifiable matter derived from perilla seeds as an active ingredient. [8] The composition for external application for skin photoaging for preventing or improving skin according to claim 7, wherein the photoaging is skin damage, pigmentation, wrinkle formation, or reduction of skin elasticity caused by UV irradiation.

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