Topical skin preparation

Abstract

To provide a topical skin preparation with both the effect of inhibiting the expression of aging-related factors and the effect of promoting the expression of cell growth factors.SOLUTION: A topical skin preparation contains a mixture of an extract derived from Viola mandshurica flowers and a Lactococcus lactis culture derived from Japanese cherry flowers as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an external skin preparation containing an extract of violet flowers and a culture broth of lactococcus as active ingredients. [Background technology]

[0002] In recent years, research has been conducted to elucidate skin aging at the cellular level, and it has been discovered that, for example, factors (inflammatory cytokines, etc.) that induce aging in surrounding cells from aged cells and factors (cell growth factors) that are involved in the proliferation of epidermal or dermal cells exist. The phenomenon in which inflammatory cytokines etc. induce aging in surrounding cells is called the senescence-associated secretory phenotype (SASP), and research into preventing and improving skin aging by suppressing the secretion of these SASP factors or promoting the growth factors of the above-mentioned epidermal or dermal cells has attracted attention (Non-Patent Documents 1, 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Naoko Otani, Cellular senescence and the secretory phenomenon associated with cellular senescence, SASP: its induction mechanism and role in the body. Journal of the Osaka City Medical Association. 2017, 66, 1-6. [Non-Patent Document 2] Naoko Otani, Induction mechanism of cellular senescence and its role in the body: Cellular senescence of hepatic stellate cells and promotion of liver cancer by intestinal bacterial metabolites, Journal of the Japanese Association of Women Scientists / Vol. 15 (2015) No. 1 DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0004] In view of the above-mentioned conventional techniques, an object of the present invention is to provide an external skin preparation having an effect of suppressing the expression of inflammatory cytokines and an effect of promoting the expression of cell growth factors. [Means for solving the problem]

[0005] The present invention is an external skin preparation containing as active ingredients a mixture of an extract of violet (Viola mandshurica) flowers and a Lactococcus lactis culture derived from cherry blossoms. Effect of the Invention

[0006] The present invention can provide an external skin preparation that has the effect of suppressing the expression of inflammatory cytokine factors and the effect of promoting the expression of cell growth factors by using an extract of violet (Viola mandshurica) flowers and a culture of Lactococcus lactis derived from cherry blossom flowers as active ingredients. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 shows the results of a test evaluating the inhibition of SA-β-galactosidase (senescence-associated β-galactosidase) activity between epidermal keratinocytes and fibroblasts. BEST MODE FOR CARRYING OUT THEINVENTION

[0008] The topical skin preparation of the present invention is characterized in that it contains as an active ingredient a mixture of an extract of Viola mandshurica flowers and a culture of Lactococcus lactis derived from cherry blossoms.

[0009] The "violet" used in the present invention is Viola mandshurica, which belongs to the Viola genus of the Violaceae family.

[0010] The violet flowers to be used for extraction may be used as they are, but may also be subjected to processing such as washing, drying, or chopping before extraction.

[0011] As the extraction solvent, any solvent generally used for plant extraction can be used appropriately, for example, water, lower alcohols (methanol, ethanol, propyl alcohol, butanol, isobutanol) or polyhydric alcohols (propylene glycol, 1,3-butylene glycol, glycerin). The above extraction solvent may be used alone or in a mixture of two or more kinds. When a mixture is used, for example, a mixture of water and ethanol or water and 1,3-butylene glycol is preferred.

[0012] In preparing the extract, the pH is not particularly limited, but is generally preferably in the range of 3 to 9. In this sense, if necessary, an alkalinity regulator such as sodium hydroxide, sodium carbonate, or potassium hydroxide, or an acidity regulator such as citric acid, hydrochloric acid, phosphoric acid, or sulfuric acid may be added to the extraction solvent to adjust the pH to a desired level.

[0013] The extraction conditions such as extraction temperature and extraction time vary depending on the type and pH of the solvent used. For example, when the solvent is water, a mixture of water and ethanol, or a mixture of water and 1,3-butylene glycol, the extraction temperature is generally in the range of 0°C to 80°C, and the extraction time is in the range of 1 to 168 hours (1 hour to 1 week).

[0014] Next, the lactic acid bacterium used in the present invention is "Lactococcus lactis" derived from cherry blossoms.

[0015] The carbon source used in culturing lactic acid bacteria is not particularly limited, and examples of the carbon source include glucose, fructose, lactose, raffinose, etc. In addition to the carbon source, a nitrogen source may be added, and examples of the nitrogen source include amino acids and peptones.

[0016] The culture temperature for lactic acid bacteria is generally in the range of 20° C. to 40° C., preferably in the range of 25° C. to 37° C., and the pH is generally in the range of 3.0 to 8.5, preferably in the range of 3.5 to 7.5, and the culture time is generally in the range of 12 hours to 10 days, preferably in the range of 1 day to 7 days.

[0017] In the present invention, the lactic acid bacteria culture solution cultured under the above conditions is filtered after the cultivation is completed, and the culture supernatant is used as an active ingredient.

[0018] The present invention uses as an active ingredient a mixture of the above-mentioned violet flower extract and the above-mentioned lactic acid coccus culture solution, where the blending ratio of the violet flower extract to the lactic acid coccus culture solution is in the range of 7:3 to 3:7, and more preferably 1:1.

[0019] The above mixture, which is an active ingredient of the present invention, can be incorporated into skin external preparations (cosmetics, quasi-drugs, external medicines). Examples of skin external preparations include milky lotions, creams, lotions, essences, packs, lipsticks, foundations, liquid foundations, makeup press powders, blushers, face powders, face washes, body shampoos, scalp and hair shampoos, hair conditioners, shampoos or tonics for hair growth and nourishment, cleansing cosmetics such as soaps, and bath agents, but the present invention is not limited to these.

[0020] When the mixture according to the present invention is blended into a skin external preparation (such as a cosmetic, a quasi-drug, or a medicine for external use), ingredients that are usually used in skin external preparations, such as oily ingredients, surfactants (synthetic or natural), thickeners, emulsifiers or emulsifying aids, preservatives / bactericides, powder ingredients, ultraviolet absorbers, antioxidants, sequestering agents, pigments, and fragrances, can be blended in. In addition, moisturizers, anti-inflammatory agents, pigmentation prevention agents, anti-wrinkle agents, and other physiologically active ingredients can also be blended in as needed.

[0021] Examples of moisturizing agents include mucopolysaccharides (hyaluronic acid and its derivatives, hyaluronic acid fermentation liquid, chondroitin and its derivatives, or heparin and its derivatives, etc.), elastin or its derivatives, collagen or its derivatives, collagen peptides, and amino acids or their derivatives.

[0022] Anti-inflammatory agents include allantoin, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, β-glycyrrhetinic acid, stearyl glycyrrhetinate, ε-aminocaproic acid, bactiol, tranexamic acid, pantothenyl alcohol, retinol palmitate, vitamin E or a derivative thereof (tocopherol acetate, etc.), and the like.

[0023] Examples of pigmentation preventive agents include ascorbic acid or its derivatives, hydroquinone or its derivatives, ellagic acid or its derivatives, resorcinol derivatives, 4-methoxysalicylic acid potassium salt, nicotinic acid or its derivatives, etc. Examples of ascorbic acid derivatives include sodium L-ascorbic acid-2-phosphate, magnesium L-ascorbic acid-2-phosphate, sodium L-ascorbic acid-2-sulfate, magnesium L-ascorbic acid-2-sulfate, L-ascorbic acid-2-glucoside, L-ascorbic acid-5-glucoside, ascorbyl tetrahexyldecanoate, ascorbyl palmitate phosphate 3Na, 3-O-ethyl ascorbic acid, etc. Examples of hydroquinone derivatives include arbutin (hydroquinone-β-D-glucopyranoside), α-arbutin (hydroquinone-α-D-glucopyranoside), etc.

[0024] Anti-wrinkle agents include niacinamide, vitamin A and its derivatives.

[0025] Next, the present invention will be described in more detail with reference to Production Examples and Test Examples, but the present invention is not limited thereto.

[0026] Preparation Example 1. Preparation of the mixture according to the present invention [Preparation of Violet Flower Extract] 2800g of purified water was added to 56g of violet (Viola mandshurica) flowers and stirred at 4°C for 18 hours. This was filtered to obtain 2640g of a pale purple crude extract of violet flowers (solid concentration 0.11%). The obtained crude extract of violet flowers was concentrated to obtain 363g of a concentrate. 1,3-butylene glycol was added to the 363g concentrate and filtered to obtain 510g of a yellow transparent violet flower extract (solid concentration 0.40%). [Preparation of Lactococcus culture medium] A liquid medium containing glucose and peptone was prepared and sterilized, and 18 g of a preculture solution of Lactococcus lactis derived from cherry blossoms, which had been cultured in GYP liquid medium, was added to 900 g of the sterilized liquid medium and cultured at 30° C. for 6 days. This lactococcus culture solution was heat sterilized and filtered to obtain 749 g of lactococcus culture solution (solid concentration 1.10%). [Preparation of the mixture] 500 g of the above violet flower extract was mixed with 500 g of the above lactococcus culture solution and filtered to obtain 970 g of a pale yellow-brown, transparent mixture of violet flower extract and lactococcus culture solution (solid concentration 0.63%).

[0027] Test Example 1. Evaluation of the promoting effect of cell growth factor expression Human normal epidermal keratinocytes (NHEK) were cultured at 6 × 10 in a 24-well plate (IWAKI) using HuMedia-KG2 (Kurabo). 4The cells were seeded at 100 cells / well and cultured at 37°C for one day in the presence of 5% CO2. The medium was then replaced with HuMedia-KB2 (Kurabo), and the same medium was further replaced with a sample solution in which the mixture of Production Example 1 was added to the same medium so that the final concentration was 2.0%, and a control solution in which 30% 1,3-butylene glycol was added to the same concentration instead of the sample solution (the mixture of Production Example 1) was prepared, and these were added to each well and cultured for another 24 hours. The culture supernatant was used for analysis using Human Growth Factor Antibody Array - Membrane (41 Targets), ab134002 (Abcam). The expression level of cell growth factors was expressed as a relative amount when the value of the 30% 1,3-butylene glycol-added group was set as 100 as the control.

[0028] The results of Test Example 1 are shown in Table 1. [Table 1] JPEG2024169304000002.jpg14289

[0029] As shown in Table 1, it was confirmed that the mixture of the present invention significantly promotes the expression of cell growth factors.

[0030] Since "VEGF," "VEGF-D," "VEGFR2," and "VEGFR3" shown in Table 1 are vascular endothelial cell growth factors, it is suggested that the mixture of the present invention exerts a hair growth effect through angiogenesis by promoting the expression of these factors.

[0031] Furthermore, "PDGF-AA," "PDGF-AB," "PDGF-BB," and "PIGF" shown in Table 1 are platelet-derived growth factors known as growth factors for fibroblasts, and "PDGFRα" and "PDGERβ" are receptor factors for platelet-derived growth factors. This suggests that the mixture of the present invention promotes the proliferation of fibroblasts, thereby promoting the production of extracellular matrix such as collagen in fibroblasts, thereby exerting the effect of improving wrinkles and sagging skin.

[0032] Since "TGF-α," "TGF-β," "TGF-β2," and "TGF-β3" shown in Table 1 are transforming growth factors known as factors that initiate collagen synthesis, it is suggested that the mixture of the present invention has the effect of promoting collagen synthesis and improving wrinkles and sagging skin.

[0033] In addition, since "IGF-1" shown in Table 1 is insulin-like growth factor-1 that promotes the proliferation of fibroblasts, and "IGF-ISR" is an insulin receptor substrate factor, it is suggested that the mixture of the present invention promotes the proliferation of fibroblasts, promotes the production of extracellular matrices such as collagen, and exerts the effect of improving wrinkles and sagging skin.

[0034] Furthermore, since "FGF-7" shown in Table 1 is a fibroblast growth factor, it is suggested that the mixture of the present invention promotes the proliferation of fibroblasts, promotes the production of extracellular matrix such as collagen, and exerts the effect of improving wrinkles and sagging skin.

[0035] Furthermore, since "HGF" shown in Table 1, a hepatocyte growth factor, exhibits a proliferation-promoting effect on various epithelial cells, it is suggested that the mixture according to the present invention has an effect of improving skin turnover.

[0036] Test Example 2. Evaluation test of cytokine secretion suppression effect Human normal epidermal keratinocytes (NHEK) were cultured at 6 × 10 in a 24-well plate (IWAKI) using HuMedia-KG2 (Kurabo). 4Cells were seeded at 100 cells / well and cultured at 37°C for one day in the presence of 5% CO2. The medium was then replaced with HuMedia-KB2 (Kurabo Industries, Ltd.), and a sample solution was prepared by adding the mixture of Production Example 1 to the same medium so that the final concentration was 2.0%, and a control solution was prepared by adding 30% 1,3-butylene glycol to the same concentration instead of the sample solution (the mixture of Production Example 1). These were then added to each well and cultured for a further 24 hours. After that, an ultraviolet lamp (TL20W / 12RS, Phillips Industries, Ltd.) was used to illuminate the bottom of the incubator with approximately 50 mJ / cm2. 2 The cells were irradiated with ultraviolet B (UV-B) rays for 100 h. The cells were further cultured for 24 h, and the supernatant was collected. This supernatant was used for analysis using Human Cytokine Antibody Array (42 Target 8 memb) ab133997 (Abcam). The amount of cytokines was expressed as a relative amount to the control group, which was not irradiated with ultraviolet (UV-B) rays and had 30% 1,3-butylene glycol added, and the value was set at 100.

[0037] The results of Test Example 2 are shown in Table 2. [Table 2] JPEG2024169304000003.jpg68152

[0038] As shown in Table 2, it was first confirmed that UV-B irradiation induces cytokine secretion from epidermal cells, and that the mixture of Production Example 1 suppresses the secretion of inflammatory cytokine factors.

[0039] "IL-6," "MCP-1," and "IL-1α" shown in Table 2 are SASP factors that induce aging of surrounding cells, suggesting that the mixture of the present invention has the effect of suppressing skin aging by inhibiting the secretion of these factors.

[0040] Furthermore, "IL-7" shown in Table 2 is a type of cytokine that has the function of enhancing immune responsiveness. Therefore, the mixture of the present invention is suggested to have the effect of suppressing excessive immune responses by inhibiting the secretion of this factor, thereby keeping the skin healthy, and inhibiting skin aging.

[0041] Furthermore, since "IL-10" and "IL-12p40 / 70" shown in Table 2 are inflammatory signal factors such as cytokines, the mixture of the present invention is suggested to have the effect of suppressing inflammation by inhibiting the secretion of these factors, thereby keeping the skin healthy, and inhibiting skin aging.

[0042] Furthermore, since "MCP-2" and "MCP-3" shown in Table 2 are chemokines derived from monocytes and macrophages, respectively, the mixture of the present invention inhibits the secretion of these factors, thereby suggesting an effect of inhibiting the migration of leukocytes and thus inhibiting skin aging.

[0043] Test Example 3: Evaluation test of the inhibitory effect on aging-related factors Normal human epidermal cells (NHEK) were cultured in a 24-well plate at 6 × 10 4 The cells were seeded at 100 cells / well and cultured at 37°C under 5% CO2 and saturated water vapor. After 24 hours of culture, the mixture of Production Example 1 was added as a sample solution to a final concentration of 2.0%, and cultured for another 24 hours. Next, an ultraviolet lamp (TL20W / 12RS, Philips) was used to irradiate the cells from the bottom of the culture vessel with approximately 50 mJ / cm2. 2 After that, the medium was replaced with one not containing the sample solution and cultured for 48 hours, after which the supernatant was collected (supernatant A). On the other hand, normal human fibroblasts (neonatal fibroblasts and adult fibroblasts were used) were cultured at 6 × 10 4The cells were prepared at a concentration of 100 / mL, seeded at 1 mL per well on a 24-well plate, and cultured at 37°C under 5% CO2 and saturated steam. After 24 hours of culture, the above-mentioned supernatant A was added and cultured. As a control, a test group was set up in which the culture supernatant containing only 30% 1,3-butylene glycol aqueous solution of the same concentration was added to epidermal cells instead of the sample solution (control group). After 72 hours of culture, the cells in each test group were harvested with 0.5 mL of ISOGEN II reagent (Nippon Gene Co., Ltd.). 200 μL of RNase-free water was added to the harvested cells, which were then stirred and mixed, left at room temperature for 15 minutes, and then centrifuged for 15 minutes at 15,000 rpm and 4°C in a centrifuge (TOMY Co., Ltd. / MX-160), and 500 μL of the supernatant was taken. 500μL of isopropanol (Wako Pure Chemical Industries, Ltd.) was added to the collected supernatant, stirred and mixed, left at room temperature for 10 minutes, and then centrifuged at 15,000 rpm and 4℃ for 10 minutes to obtain a total RNA precipitate. 0.5mL of 75% ethanol was added to the total RNA, and the precipitate was collected by centrifugation at 15,000 rpm and 4℃ for 3 minutes. The supernatant was discarded, and this process was repeated twice. After completely removing the supernatant, the mixture was air-dried and dissolved in RNase-free water. The collected total RNA was reverse transcribed using a specified kit [PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio)] to synthesize cDNA. Using the synthesized cDNA as a sample, the expression of various genes and the expression of the internal standard ACTB gene were detected using Thermal Cycler Dice (registered trademark) Real Time System Single (manufactured by Takara Bio Inc.) and SYBR (registered trademark) Premix Ex TaqTM II (Perfect Real Time) (manufactured by Takara Bio Inc.). Here, ACTB (β-actin) is one of the housekeeping genes (a gene that is expressed at a constant amount in common in many tissues and cells, is constantly expressed, and is essential for the maintenance and proliferation of cells), and since its expression level is always constant, it is used as an internal standard in PCR experiments.The test results were obtained by comparing the expression levels of each gene in each test group when the expression level of the ACTB gene was held constant. In this test system, the expression level of the gene in the control group, where epidermal cells were irradiated with ultraviolet light (UV-B), was set at 100, and the relative values ​​of the expression levels of the genes in the other test groups were calculated.

[0044] The evaluation results of Test Example 3 for neonatal fibroblasts are shown in Table 3, and the evaluation results of Test Example 3 for adult fibroblasts are shown in Table 4. [Table 3] JPEG2024169304000004.jpg57114

[0045] [Table 4] JPEG2024169304000005.jpg56130

[0046] As a result of Test Example 3, it was confirmed that the expression of matrix metalloproteinase-3 (MMP-3), known as a SASP factor, was induced by ultraviolet light (UV-B) irradiation in both neonatal and adult fibroblasts, and that the induction rate was particularly high in adult fibroblasts. Furthermore, it was confirmed that the mixture of Production Example 1 according to the present invention suppressed the expression of "MMP-3" in both neonatal and adult fibroblasts. Furthermore, it was confirmed that the mixture of Production Example 1 according to the present invention suppressed the expression of "IL-6" and "IL-8", which are known as other SASP factors, in adult fibroblasts. Therefore, according to the present invention, the effect of suppressing the chain phenomenon of suppressing the secretion of SASP factors and inducing aging of other cells, and suppressing skin aging such as wrinkles or age spots, is suggested.

[0047] Test Example 4. Evaluation test for inhibition of SA-β-galactosidase activity between epidermal keratinocytes and fibroblasts In order to evaluate the effect of the mixture of Preparation Example 1 according to the present invention on indirect cellular senescence between epidermal keratinocytes and fibroblasts, the following test was carried out. First, normal human epidermal keratinocyte cell line (NHEK, Kurabo) was placed on a 24-well plate at 6.0 × 10 4 The wells were seeded with HuMedia-KG2 (Kurabo Industries, Ltd.) to give 1.5 cells / well, and cultured at 37°C under 5% CO2 for 24 hours. After replacing with HuMedia-KB2 (Kurabo Industries, Ltd.), a sample solution was prepared by adding the mixture of Production Example 1 as a solution to a final concentration of twice 2.0%, and a control solution was prepared by similarly adding 30% 1,3-butylene glycol instead of the mixture of Production Example 1. These were then added to each well and cultured for a further 24 hours. To prevent carryover of the mixture of Production Example 1, the culture medium was replaced again with HuMedia-KB2, and UV-B was irradiated at 50 mJ / cm2. 2 The cells were irradiated so that the temperature reached 100°C and cultured for 48 hours, allowing the secretions of epidermal keratinocytes under each condition to accumulate in the culture medium. At the same time, a test group not irradiated with UV-B was prepared as a control for comparison with the irradiated group. The culture supernatants of each group were collected, flash frozen in liquid nitrogen, and stored at 80°C. Normal human fibroblasts (NHDF) derived from adults were cultured at 2.0 x 10 4 The cells were seeded at 1000 x g / well and cultured at 37°C for 1 day under 5% CO2. The culture supernatant collected above was then rapidly thawed in a 37°C bath, and the supernatant centrifuged at approximately 3000 x g at room temperature was added and cultured for 6 days. The cells were washed twice with PBS, replaced with Eagle MEM medium containing 0.5% NCS containing 1 μM SPiDER-βGal (Dojindo Laboratories), and incubated at 37°C for 30 minutes. After the culture was completed, the cells were washed twice with PBS and then photographed using a GFP filter on a fluorescent microscope BZ-X810 (Keyence). The white arrows indicate cells with high activity of SA-β-galactosidase, which is widely used as an indicator of aging. In fact, cells have a limited number of divisions, and repeated division reduces their division ability, eventually causing them to stop dividing. This phenomenon is called cellular senescence, and SA-β-galactosidase is used as an indicator of cellular senescence because its activity increases as cellular senescence progresses.

[0048] The results of Test Example 4 are shown in Figure 1. As shown in Figure 1, it was shown that the supernatant of epidermal keratinocytes irradiated with UV-B and cultured with the addition of a control solution promotes the activation of SA-β-galactosidase in fibroblasts. On the other hand, it was confirmed that the activation of SA-β-galactosidase was suppressed even when the supernatant of epidermal cells irradiated with UV-B and cultured with the addition of the mixture of the present invention was added to fibroblasts and cultured. This suggests that the mixture of the present invention has the effect of suppressing the secretion of factors that cause fibroblast aging in epidermal cells irradiated with ultraviolet rays.

Claims

[Claim 1] A skin topical agent containing as active ingredients a mixture of an extract of violet (Viola mandshurica) flowers and a culture of Lactococcus lactis derived from cherry blossoms.

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