WO2023128728A1

WO2023128728A1 - Composition for skin function improvement

Info

Publication number: WO2023128728A1
Application number: PCT/KR2023/000070
Authority: WO
Inventors: 박한오; 김명희; 연승우; 유원백; 이솔
Original assignee: (주)에이스바이옴
Priority date: 2022-01-03
Filing date: 2023-01-03
Publication date: 2023-07-06
Also published as: KR20230105165A

Abstract

The present invention relates to a composition for skin function improvement and, particularly, to a composition for skin function improvement comprising a Lactobacillus gasseri BNR17 culture medium, and a cosmetic including the composition.

Description

Composition for improving skin function

The present invention relates to a composition for improving skin function, and specifically relates to a composition for improving skin function comprising a Lactobacillus gasseri BNR17 culture medium and cosmetics comprising the composition.

Cosmetics are basically for the purpose of beauty, but in addition, functional cosmetics such as whitening, wrinkle improvement, and antioxidant are showing remarkable growth. There is an analysis that the general public's interest in beauty and makeup is rapidly increasing as interest in appearance increases as the quality of life improves. Accordingly, studies are being conducted to discover new materials for the development of functional cosmetics, and recently, cosmetics using microorganisms such as enzymes and lactic acid bacteria have been developed. In particular, lactic acid bacteria used as major microorganisms include Lactobacillus acidophilus, Lactobacillus casei , Lactobacillus gasseri , and Bifidobacterium longum . The strains have been reported to have antibacterial activity, antioxidant activity and anti-inflammatory activity.

What is closely related to the functional study of cosmetics is the melanin synthesizing cell. Melanin plays a major role in determining the color of the animal's skin, hair, and eyes, and also plays a role in protecting the skin from UV damage. However, excessive melanin accumulation causes excessive pigmentation, resulting in skin damage such as freckles and melasma. In the melanin synthesis process, reactive oxygen species (ROS), including hydrogen peroxide, are generated to place melanin-synthesizing cells in an oxidative stress environment, and melanin biosynthesis is continuously induced. In addition, the excess active oxygen produced is one of the representative causes of skin aging, and also causes wrinkles of the skin by increasing collagen degrading enzymes. There is type I collagen, which is the most abundant in skin connective tissue, and there are other types such as elastin, fibronectin, integrin, fibrillin, and proteoglycan. The newly synthesized procollagen is secreted into the extracellular space of skin cells and used as a raw material for forming collagen fibers. Collagen basically plays an important role in providing skin binding and elasticity. Therefore, inhibition of reactive oxygen species has been suggested as a way to prevent melanin biosynthesis from melanin-synthesizing cells and at the same time prevent skin aging ( Biol Chem. 2019 Apr 24;400(5):589-612, Aging Res Rev. 2015 May;21:16-29, Antioxidants (Basel. 2020 Jan 10;9(1):63 ).

Melanin production is mainly caused by the activity of an enzyme called tyrosinase, which is a metalloprotein containing copper ions. After converting the amino acid L-tyrosine into L-DOPA (3,4-dihydroxyphenylalanine) as a substrate, It is converted to L-DOPA-quinone by oxidation again. L-DOPA-quinone converted in this way is synthesized into melanin by other proteins such as phospholipase C (PLC), tyrosinase related protein-1 (TRP-1), and tyrosinase related protein-2 (TRP-2) ( Science. 1982 Sep 17;217(4565):1163-5, Materials (Basel). 2012 Sep;5(9): 1661-1685 ). Therefore, inhibition of melanin production by inhibiting tyrosinase enzyme activity results in skin whitening effect ( Materials (Basel). 2012 Sep; 5(9): 1661??1685, J Enzyme Inhib Med Chem. 2017 Dec;32(1) :403-425 ).

Substances currently used as whitening agents include arbutin, kojic acid, and L-tyrosine competitive inhibitors. In addition to this, there are substances developed as other whitening effects, but they also show side effects such as weak inhibitory activity of tyrosinase or cytotoxicity. Therefore, there is a demand for the development of whitening materials from materials that can effectively inhibit tyrosinase while having low cytotoxicity.

Based on these studies, studies to find substances for whitening and wrinkle improvement using natural materials with antioxidant effects are still being actively conducted. In line with this, while the present inventors were seeking ways to discover new materials for the development of functional cosmetics, Lactobacillus gasseri BNR17 ( Lactobacillus gasseri BNR17) culture medium, including antioxidant activity, confirmed the effect that can help whitening and wrinkle improvement By doing so, the present invention was completed.

발명의 요약Summary of Invention

An object of the present invention is to provide a skin function improvement use of Lactobacillus gasseri BNR17 strain culture medium.

In order to achieve the above object, the present invention provides a composition for improving skin function selected from the group consisting of increasing antioxidant activity, whitening and wrinkle improvement, including Lactobacillus gasseri BNR17 culture medium. The present invention also provides a method for improving skin function selected from the group consisting of activity increase, whitening and wrinkle improvement, which includes treating Lactobacillus gasseri BNR17 culture medium. The present invention further provides a use of the Lactobacillus gasseri BNR17 culture medium for preparing a composition for improving skin function selected from the group consisting of increasing antioxidant activity, whitening, and improving wrinkles.

The present invention also provides a cosmetic comprising the composition.

1 is a result of analyzing the viability of mouse-derived melanoma cells B16-F10 cells by Lactobacillus gasseri BNR17 culture supernatant.

2 is a result of evaluating the effect of the Lactobacillus gasseri BNR17 culture supernatant on the secretion of melanin synthesized in B16-F10 cells into the extracellular space.

Figure 3 is a result of evaluating the effect of Lactobacillus gasseri BNR17 culture supernatant on melanin synthesis in B16-F10 cells.

Figure 4 is a result of measuring the whitening activity to the extent that the culture supernatant of Lactobacillus gasseri ( Lactobacillus gasseri ) BNR17 inhibits the activity of tyrosinase enzyme in B16-F10 cells.

Figure 5 shows the results of confirming the expression of MITF, tyrosinase, TRP-1 and TRP-2 in B16-F10 cells by Lactobacillus gasseri BNR17 culture supernatant at the mRNA level.

6 is a result showing cell viability in the case of treatment with Lactobacillus gasseri BNR17 culture supernatant in HaCaT cells, which are human skin cells.

Figure 7 shows the results of performing real-time polymerase chain reaction (qRT-PCR) after treating HaCaT cells with Lactobacillus gasseri BNR17 culture supernatant.

Figure 8 is a result of confirming the expression of HO-1, catalase, collagen and MMP1 mRNA by performing real-time polymerase chain reaction (qRT-PCR) after treating HaCaT cells with Lactobacillus gasseri BNR17 culture supernatant is shown.

Figure 9 shows the results of confirming the antioxidant effect of the culture supernatant of Lactobacillus gasseri ( Lactobacillus gasseri ) BNR17.

발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In the present invention, when treating a composition containing a Lactobacillus gasseri BNR 17 culture medium derived from human milk, particularly a cell-free culture supernatant, the expression of mRNAs related to melanin synthesis, tyrosinase activity and related signaling systems this was suppressed Additionally, it was confirmed that collagen mRNA increased and MMP1 mRNA decreased, and a cytoprotective effect according to an increase in HO-1 mRNA was observed.

Based on this, the present invention, in one aspect, relates to a composition for improving skin function selected from the group consisting of increasing antioxidant activity, whitening, and improving wrinkles, including Lactobacillus gasseri BNR17 culture medium.

The present invention also relates to a method for improving skin function selected from the group consisting of activity increase, whitening and wrinkle improvement, which includes treating Lactobacillus gasseri BNR17 culture medium. The present invention further relates to the use of a culture medium of Lactobacillus gasseri BNR17 for preparing a composition for improving skin function selected from the group consisting of increasing antioxidant activity, whitening and improving wrinkles.

In one embodiment, the Lactobacillus gasseri BNR17 may include a strain having an accession number of KCTC 10902BP.

The culture medium of the Lactobacillus gasseri BNR17 may refer to a medium containing by-products produced by culturing the Lactobacillus gasseri BNR17 strain in a medium so that the strain consumes nutrients and is metabolized. . In addition, a concentrated or diluted solution of the culture solution, a dried product obtained by drying the culture solution, a crude or purified product of the culture solution, or a mixture thereof may also be included as an active ingredient in the composition of the present invention.

Specifically, in the present invention, a culture supernatant of Lactobacillus gasseri BNR17 having an accession number of KCTC 10902BP may be included.

The culture supernatant is Lactobacillus gasseri BNR17 prepared in Korean Patent Publication No. 10-2008-0110447 '2. It means that the strain, especially the cell-free state, is obtained by centrifuging the culture solution obtained after culturing using the 'method of producing lactic acid bacteria powder'.

The culture supernatant is specifically, not limited to the MRS liquid medium, but may also be produced in an MRS liquid medium in which some components have been changed, and basically, Lactobacillus gasseri ^BNR17 is added to the MRS liquid medium at a It can be obtained by inoculation at 37 ° C. for 18-24 hours with 30% NaOH by volume as a neutralizing agent and pH-control fermentation to pH 5.7 ± 0.2, followed by centrifugation at 4 ° C. at 10,000xg. .

The present invention relates to the use of the Lactobacillus gasseri BNR17 culture medium, in particular, the use of improving skin function selected from the group consisting of increasing antioxidant activity, whitening and improving wrinkles of the culture supernatant.

Skin is composed of epidermis, dermis and subcutaneous tissue. The epidermis, the outer layer of the skin, is composed of stratified squamous epithelium and acts as the body's protective barrier against the external environment. The epidermis is divided from the outside into the stratum corneum, the stratum lucidum, the stratum granulosum, the stratum spinosum, and the stratum basale. The dermis is a layer between the epidermis and subcutaneous tissue and is divided into papillary dermis and reticular dermis. Sensory nerves, fibroblasts, and macrophages are present and occupy the largest part of the skin.

The composition may be a cosmetic composition, and may be prepared in formulations such as lotion, skin, toner, essence, ampoule, emulsion, gel, water, solid, suspension, foam, aerosol, foam, pack, patch, or cream. It is not limited.

In some cases, it may further include one or more cosmetically acceptable carriers, for example, oil and fat components, humectants, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, Bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavors, blood circulation promoters, cooling agents, and the like may be appropriately incorporated, but are not limited thereto.

In another aspect, the present invention relates to cosmetics comprising the composition. In particular, the cosmetic according to the present invention may be a functional cosmetic for improving one or more skin functions selected from the group consisting of increased antioxidant activity, whitening, and wrinkle improvement.

The cosmetics may be applied and blended with general ingredients used in the manufacture of general skin cosmetics, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., as necessary. In addition, the cosmetics are mist, serum, nutrient lotion, softening lotion, softening water, emulsion, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, Hand Cream, Foundation, Powder, Makeup Base, Essence, Nutritional Essence, Pack, Soap, Cleansing Foam, Cleansing Lotion, Cleansing Cream, Body Lotion, Body Cleanser, Face Wash, Treatment, Beauty Serum, Beauty Pack, Ointment, Gel, Linement , It can be prepared as a formulation selected from the group consisting of liquids, patches, sprays, bath additives, sunscreens, sun oil and hair products, and various conventional carriers and additives suitable for each formulation and well known in the art can include

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Example

Example 1. Experimental method and preparation

1. Evaluation of whitening efficacy

1-1. cell culture

In this experiment, mouse-derived melanoma cells, B16-F10 cells, were purchased from ATCC and cultured. Basic B16-F10 cell culture was performed in DMEM (Hyclon, Logan, UT, USA) medium containing 10% FBS (Hyclon, Logan, UT, USA) and 1% Antibiotic/Antimycotic Solution (Hyclon, Logan, UT, USA). was subcultured by adapting to a 37°C 5% CO ₂ incubator, and phenol red free DMEM (Hyclon, Logan, UT, USA) was used for the experimental conditions for measuring extracellular melanin.

1-2. Cell viability evaluation

In order to determine the treatment concentration of Lactobacillus gasseri BNR17 culture supernatant (hereinafter referred to as BNR17 culture supernatant) having accession number of KCTC 10902BP, B16-F10 cells were treated with various concentrations, and cell viability was measured by WST (AbFrontier, Seoul, Korea) was confirmed using a cell viability measurement kit. To confirm cell viability, B16-F10 was inoculated in a 96-well plate at 1X104 cells/well, and cultured for 24 hours at 5% CO ₂ and 37°C. After changing the culture medium, BNR17 culture supernatant was treated with 0, 0.1, 0.5, 1, 2.5 and 5 (v / v %) in each well, and after 24 hours at 37 ° C, the WST kit was measured according to the method of the instruction manual. In summary, 10 μl of WST reagent was added to each well, incubated at 37 ° C for 1 and a half hours, and the resulting formazan was measured for absorbance at 450 nm using a microplate reader. measured. The cell viability was shown with the group not treated with the BNR17 culture supernatant as 100%.

1-3. Measurement of extracellular melanin concentration

For the whitening activity assay, B16-F10 cells were suspended in 3ml of phenol red free DMEM medium containing 10% FBS and inoculated at 1X10 ⁵ cells/well in a 6-well plate, followed by 5% CO ₂ , 37 Incubated for 24 hours at ℃. Thereafter, α-MSH (Sigma, CA, USA) was treated at a concentration of 200 nM and the BNR17 culture supernatant was treated at each concentration (0.5 v/v % and 1 v/v %) and cultured for 48 hours at 37 ° C. production was induced. As a control, 200 μM of arbutin (Sigma, CA, USA) was treated in the same experimental manner as the BNR17 culture supernatant treatment. After further culturing, only the culture medium from which the cells were removed was taken and the absorbance was measured at 492 nm with a microplate reader, and the sample was compared with the untreated control at 100% and relative values.

1-4. Measurement of intracellular melanin concentration

As for the whitening activity assay, the intracellular melanin content was measured using the method of the Effectiveness Evaluation Guidelines for Functional Cosmetics Helping in Skin Whitening (2020. 9). After processing the cells under the same conditions as in Example 1-3, measuring the extracellular melanin concentration, the cells were collected and counted, and the same number of cells was collected and centrifuged to obtain only the cells. The recovered cells were dissolved in 100 μl of 1 N NaOH (Sigma, CA, USA) solution containing 10% DMSO at 60 ° C for 2 hours to obtain a solution containing intracellular melanin, and the absorbance was measured at 492 nm with a microplate reader. It was measured, and the sample was compared with the relative value with the untreated control set as 100%.

1-5. Measurement of intracellular tyrosinase activity

In this experimental example, in order to confirm the whitening activity of the samples identified in Examples 1-4, a manual method using L-DOPA (Sigma, CA, USA) as a substrate and a Tyrosinase Activity Assay Kit (Abcam, Cambridge, UK) ) was used to test tyrosinase inhibition as follows. B16-F10 cells were inoculated into a 60 mm plate and cultured for 24 hours at 5% CO ₂ and 37 °C. Thereafter, α-MSH (Sigma, CA, USA) was treated at a concentration of 200 nM and the BNR17 culture supernatant was treated at each concentration (0.5 v/v % and 1 v/v %), incubated at 37 ° C. for 40 hours, and melanin production was induced. As a control, 200 μM of arbutin (Sigma, CA, USA) was treated in the same experimental manner as the BNR17 culture supernatant treatment. After 40 hours, the cells were washed with PBS (phosphate buffered saline) and recovered using centrifugation. The obtained cells were dissolved in a buffer in the Tyrosinase Activity Assay Kit (Abcam), and then centrifuged at 13,000 rpm, 15 minutes, 4 ℃, and the cell culture medium from which the cells were removed was used. After quantifying the amount of protein using BCA protein assay kit (Thermo Scientific, USA), the protein sample (20 μg/well) was treated with 80 μl of 2 mg/ml L-DOPA, reacted at 37 ° C for 1 hour, and microplate Absorbance was measured at 492 nm using a reader. The same protein sample (5 μg/well) was mixed with the Tyrosinase Activity Assay Kit reaction mix in the Tyrosinase Activity Assay Kit (Abcam), reacted at 37° C. for 10 minutes, and the absorbance was measured at 510 nm with a microplate reader. After performing the same experiment as above using arbutin as a control group, the degree of tyrosinase inhibition of the BNR17 culture supernatant treatment group was compared.

1-6. Intracellular melanin synthesis effect

Changes in mRNA levels related to melanin synthesis during BNR17 culture supernatant treatment were measured using a quantitative real time polymerase chain reaction (qRT-PCR) method. The B16-F10 cells were suspended in 1ml of DMEM medium containing 10% FBS, inoculated into a 12-well plate at 5X10 ⁴ cells/well, and cultured for 24 hours at 5% CO ₂ and 37 °C. After incubation, α-MSH was treated at a concentration of 200 nM and BNR17 culture supernatant was treated with 1 (v/v %) to induce melanin production by culturing at 37° C. for 6 hours and 40 hours. Arbutin 200 μM was used as a control. After additional incubation for the corresponding time, the cells were washed with Dulbecco's phosphate buffered saline (DPBS, WELGENE), and recovered with 1 mL Trizol (Thermo Fisher Scientific, Waltham, MA, USA). Then, 0.2 mL of chloroform (Chloroform, Sigma, CA, USA) corresponding to a ratio of 1/5 of Trizol was added, stirred, and centrifuged at 4° C. at 14000 rpm for 15 minutes to obtain a supernatant. 0.5 mL isopropanol (Isopropanol, Darmstadt, Germany) was added to the supernatant, reacted for 10 minutes, and centrifuged at 14000 rpm for 15 minutes to obtain a precipitate. The supernatant was removed and washed twice using 1mL 75% ethanol (Ethanol, Darmstadt, Germany). After completely drying the washed precipitate at room temperature, it was dissolved in 20 μL of water. After quantification of the melted RNA, cDNA was synthesized using the same amount of RNA for each group. Real-time polymerase chain reaction (qRT-PCR) was performed on the synthesized cDNA using cyanine dye 2X GreenStar qPCR Master Mix (Bioneer, Daejeon, Korea) as a target. Through real-time polymerase chain reaction, finally, the expression level of MITF, tyrosinase, tyrosinase related protein-1, and tyrosinase related protein-2 mRNA was evaluated.

2. Measurement of anti-wrinkle and antioxidant effects using human skin cells

2-1. cell culture

HaCaT cells, human skin cells used in this experiment, were purchased from Accegen Biotechnology (Fairfield, NJ, USA) and cultured. HaCaT cells were cultured in DMEM (Hyclon, Logan, UT, USA) medium containing 10% FBS (Hyclon, Logan, UT, USA) and 1% Antibiotic/Antimycotic Solution (Hyclon, Logan, UT, USA). ℃, 5% CO ₂ It was subcultured by adapting to an incubator.

2-1. Cell viability evaluation

Samples were treated according to concentration, and the cell viability changed for 24 hours was confirmed using a WST (AbFrontier, Seoul, Korea) cell viability measurement kit. To confirm cell viability, HaCaT cells were inoculated in a 96-well plate at 1X10 ⁴ cells/well, and cultured at 5% CO ₂ and 37 °C for 24 hours. After changing the culture medium, BNR17 culture supernatant was treated with 0, 1, 2.5 and 5 (v / v %) in each well, and after 24 hours at 37 ° C, the WST kit was measured according to the method of the instruction manual. Briefly, 10 μl of WST reagent was added to each well, followed by incubation at 37 ° C. for one and a half hours, and the absorbance of the resulting formazan was measured at 450 nm using a microplate reader. The cell viability was shown with the group not treated with the BNR17 culture supernatant as 100%.

2-2. Intracellular wrinkle improvement and antioxidant effect

HaCaT cells were suspended in 1 ml of DMEM medium containing 10% FBS, inoculated into a 12-well plate at 5X10 ⁴ cells/well, and cultured for 24 hours at 5% CO ₂ and 37 °C. Thereafter, the culture medium was replaced with a new culture medium, and samples of the BNR17 culture supernatant were treated for each concentration. After 24 hours of additional culture, cDNA was synthesized by extracting RNA from cells in the same manner as in Example 1-6, and finally heme oxygenase-1 (HO-1), catalase, glutathione peroxidase 1 through real-time polymerase chain reaction (GPx1), superoxide dismutase 1 (SOD1), collagen type 1a (Col1a1), and metalloproteinase 1 (MMP1) mRNA expression levels were evaluated. In order to analyze the function of BNR17 according to the induction of oxidative stress, the cells were inoculated in a 12-well plate at 5X10 ⁴ cells/well and cultured for 24 hours. Then, the culture medium was changed to a new one, and the BNR17 culture supernatant sample was pretreated for 30 minutes at each concentration (1 v/v % and 3 v/v %), and hydrogen peroxide (H2O2, Sigma, St Louis, MO, USA) at a concentration of 100 μM. After additional culturing for 24 hours, RNA was extracted from the cells in the same manner as in Examples 1-6 to synthesize cDNA, and finally, the levels of HO-1, catalase, Col1a1, and MMP1 mRNA expression were evaluated through real-time polymerase chain reaction.

2-3. Evaluation of superoxide anion radical scavenging ability

In order to evaluate the superoxide anion radical scavenging ability of the BNR17 culture supernatant, the OxiTec™ DPPH Antioxidant Assay Kit (BIOMAX, Seoul, Korea) was used and measured according to the method of the instruction manual. In summary, BNR17 culture supernatant was prepared by concentration, DPPH working solution and Assay buffer were added, reacted for 30 minutes, and absorbance was measured at 517 nm with a microplate reader. The superoxide anion radical scavenging ability of the sample was evaluated by substituting it into trolox standard (0, 40, 60, 80, 100 μg/mL). Superoxide anion radical scavenging ability of BNR17 culture supernatant was evaluated based on trolox 100 μg/mL.

Example 2. Results

2-1. Cell viability of B16-F10 cells

In order to evaluate the effect of the BNR17 culture supernatant according to the present invention on cell activity, cell viability was measured using B16-F10 cells. BNR17 culture supernatant samples were treated with 0, 0.1, 0.5, 1, 2.5 and 5 (v/v %) and cultured for 24 hours. The result of observing the cell viability thereafter is shown in the graph in FIG. 1 below. As shown in FIG. 1, no significant difference was observed in cell viability by the BNR17 culture supernatant until treatment with the highest 1 (v/v %).

2-2. Inhibition of melanin synthesis by BNR17 culture supernatant

The results of evaluating the effect of the BNR17 culture supernatant according to the present invention on melanin synthesis are shown in FIGS. 2 and 3 .

As shown in Figure 2, as a result of measuring the secretion of synthesized melanin in the culture supernatant in the B16-F10 cell culture medium, it did not reach the result of arbutin, which is well known as a whitening functional material as a comparison group, but as the concentration of the culture supernatant increased Accordingly, the amount of melanin secretion was suppressed by about 25% or more compared to the α-MSH treated group. In addition, as shown in FIG. 3, as a result of measuring the degree of inhibition of melanin synthesis in B16-F10 cells in the culture supernatant, as the concentration of the culture supernatant increased, the amount of melanin produced was reduced by about 30% or more compared to the α-MSH treated group. It was confirmed that the inhibitory effect was equal or greater than that of the control group, arbutin and α-MSH co-treated group. Accordingly, it can be seen that the whitening effect of the BNR17 culture supernatant according to the present invention is remarkably excellent.

2-3. Tyrosinase activity inhibitory effect

The results of measuring the whitening activity to the extent that the BNR17 culture supernatant according to the present invention inhibits the activity of tyrosinase enzyme in B16-F10 cells are shown in FIG. 4 . 'Example 1-5. As a result of measuring the activity of tyrosinase using L-DOPA as a substrate by using the intracellular protein obtained by the 'Measurement of Intracellular Tyrosinase Activity' method, the enzyme activity of the BNR17 culture supernatant-treated sample decreased at least equal to that of the arbutin-treated sample as a control group. . As a result of repeated verification of the same protein sample with a tyrosinase activation measurement kit, it was confirmed that the enzyme activity was reduced to a similar level.

2-4. Measurement of gene expression involved in intracellular melanin synthesis in B16-F10

Various melanin synthesis-related genes directly affect the mRNA expression of MITF (microphthalmia-associated transcription factor), which is known as a transcriptional regulator, and tyrosinase, TRP (tyrosinase-related protein)-1, and TRP-2, which are involved in melanin synthesis in a MITF-dependent manner. An experiment was performed to investigate the effect of BNR17 culture supernatant on the Cells were prepared in the same manner as in Examples 1-6, and expressions of MITF, tyrosinase, TRP-1, and TRP-2 were confirmed at the mRNA level.

As shown in FIG. 5 , it can be confirmed that the mRNA expression level of MITF induced by α-MSH stimulation in B16-F10 cells was inhibited by treatment with BNR17 culture supernatant at 6 hours after stimulation induction. The mRNA expression of tyrosinase, TRP-1, and TRP-2 decreased in a concentration-dependent manner at 40 hours, when α-MSH stimulation was sufficiently induced and melanin synthesis could occur sufficiently. In particular, it showed more effective results than the control group, arbutin treatment group, through which the BNR17 culture supernatant suppressed the expression of MITF, a key transcriptional regulator of melanin synthesis, and at the same time, tyrosinase, TRP-1 regulated by MITF , it was confirmed that it can be effective for skin whitening by suppressing the mRNA expression of TRP-2.

2-5. Cell viability of HaCaT cells

As shown in FIG. 6, the BNR17 culture supernatant in human skin cells showed high cell viability even when the cells were treated with the compound at a high concentration of 5 (v / v%). This indicates cell viability similar to that of the untreated group without any treatment, and that the BNR17 culture supernatant has little toxicity to human skin cells within the concentration range.

2-6. Measurement of gene expression involved in cytoprotection, antioxidant and wrinkle improvement in HaCaT cells

Heme oxygenase-1 (HO-1) is induced by ultraviolet radiation, hydrogen peroxide, cytokine, hypoxia, and glutathione (GSH) consumption in various types of cells, and in various tissues/cells, it has anti-inflammatory, antioxidant, and active apoptosis It is known to have an inhibitory effect. Enzymatic antioxidants typically involved in oxidative damage include catalase (CAT), glutathione peroxidase (GPx1), and superoxide dismutase 1 (SOD1). Their main roles are to reduce oxidative stress in cells and tissues in the human body. reduce oxidative damage.

Collagen is a major constituent protein that accounts for about 90% of the skin's dry weight among the components that make up the connective tissue of skin cells. impact can be evaluated. Among dozens of metalloproteinases (MMPs) produced in the body, MMP-1 is a proteolytic enzyme that acts specifically on collagen. By inhibiting the activity of MMP-1 to protect collagen, skin tissue elasticity is improved. It is known to be able to maintain and prevent the formation of wrinkles.

In order to evaluate the efficacy of the BNR17 culture supernatant itself, HaCaT cells were treated with BNR17 culture supernatant at concentrations of 1 and 3 (v/v%), followed by real-time polymerase chain reaction (qRT-PCR). As a result, as shown in Figure 7, the mRNA expression involved in cytoprotection (HO-1), antioxidant (Catalase, GPx1, SOD1) and wrinkle improvement (Col1a1) in HaCaT cells increased in a concentration-dependent manner by treatment with BNR17 culture supernatant. can confirm that MMP-1, a proteolytic factor that acts specifically on collagen, was not significant, but showed a tendency to decrease.

In order to observe the antioxidant phenomenon in HaCaT cells treated with the BNR17 culture supernatant, the BNR17 culture supernatant was pretreated at concentrations of 1 and 3 (v/v%) according to the method of Example 2-2, and then treated with hydrogen peroxide. Then, real-time polymerase chain reaction (qRT-PCR) was performed to confirm HO-1, catalase, collagen, and MMP1.

As a result, as shown in FIG. 8, HO-1 mRNA expression significantly increased in the group treated with the BNR17 culture supernatant, and the mRNA expression of MMP1, which was increased by hydrogen peroxide, decreased in a BNR17 culture supernatant concentration-dependent manner.

As described above, it was confirmed that the BNR17 culture supernatant can be effective not only in itself, but also in cell protective effect and anti-oxidation against oxidative stress induced by hydrogen peroxide, and wrinkle improvement.

2-7. Measurement of superoxide anion radical scavenging ability

DPPH assay was performed and confirmed the superoxide anion radical scavenging ability of the BNR17 culture supernatant obtained in the above example. As shown in FIG. 9, in the case of the BNR17 culture supernatant, the superoxide anion radical scavenging ability was shown from 0.625 (v / v %), and as the capacity of the BNR17 culture supernatant increased, it was confirmed that it increased to 68% compared to Trolox, the control group. It was confirmed that there is an antioxidant effect according to the oxide anion radical scavenging.

The Lactobacillus gasseri BNR17 culture medium according to the present invention is a breast milk lactic acid bacterium whose safety has been proven by the Ministry of Food and Drug Safety, and is effective in whitening and wrinkle improvement, including antioxidant activity, and can be usefully used as a cosmetic composition There will be.

As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC10902BP

Entrusted date: 20060123

Claims

A composition for improving skin function selected from the group consisting of increasing antioxidant activity, whitening, and improving wrinkles, including a Lactobacillus gasseri BNR17 culture medium.
The composition according to claim 1, wherein the Lactobacillus gasseri BNR17 has an accession number of KCTC 10902BP.
The composition according to claim 1, wherein the culture medium is a culture supernatant.
A cosmetic comprising the composition of any one of claims 1 to 3.