KR101348881B1 - Cosmetic Composition comprising 6-Methoxynaringenin or derivative thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising 6-methoxynalingenin or a derivative thereof as an active ingredient,
The cosmetic composition according to the present invention can be usefully used for anti-aging or anti-wrinkle use, skin whitening use, anti-inflammatory use and antibacterial use.

Description

Cosmetic composition containing 6-methoxynalingenin or derivatives thereof {Cosmetic Composition comprising 6-Methoxynaringenin or derivative thereof as an active ingredient}

The present invention relates to a cosmetic composition capable of preventing skin aging and enhancing skin whitening.

Aging of the skin can be divided into stress, disease, environmental factors, wounds, or internal aging that occurs with age, and light aging caused by exposure to light such as ultraviolet rays.

Aging of the skin is caused by the destruction of the antioxidant protective film present in the living body by the activated free radicals, thereby oxidizing lipids, proteins, polysaccharides, and nucleic acids, which are the major constituents of the skin, to destroy skin cells and tissues. In particular, when the protein is oxidized, collagen, hyaluronic acid, elastin, proteoglycan, and fibronectin, which are the connective tissues of the skin, are cleaved, which causes severe over-inflammatory reaction and skin elasticity. If this becomes more severe, mutations in the DNA can lead to mutations, cancer, and reduced immune function. Therefore, in order to prevent skin aging, it is necessary to prevent skin damage by eliminating free radicals caused by various causes, and also to restore the skin by regeneratively proliferating through active metabolism.

In addition, skin aging can be promoted by matrix metalloproteinase (MMP) (hereinafter referred to as "MMP") which is a collagen degrading enzyme. That is, in vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but as aging progresses, collagen synthesis decreases and the expression of collagen degrading enzyme MMP is promoted, resulting in decreased skin elasticity and wrinkles. . Therefore, in order to prevent skin aging and wrinkles, it is necessary to control or inhibit MMP expression in which activation is induced in cells.

On the other hand, there is a problem that the skin color is changed independently of skin aging, pigmentation is a major cause for such a change in skin color. In general, the coloring matters affecting skin color include melanin, melanoid, carotene, and hemoglobin. The most important pigment is melanin. Melanin plays an important role in preventing skin damage from ultraviolet rays by absorbing or scattering ultraviolet rays, and also excellent in removing free radical species, but sometimes melanin itself generates free radicals, and catechol in melanin structure Alternatively, quinones reduce or oxidize other substances, and melanin itself exhibits the properties of free radicals. Therefore, in order to prevent skin discoloration, it is necessary to suppress the production of melanin.

Melanin is biosynthesized into brown (pheomelanin) and black (eumelanin) polymers through a series of oxidation processes beginning with the conversion of tyrosine, an amino acid, to tyrosinase and conversion to dihydroxy phenylalanine. The biosynthesis process of melanin proceeds in a special type of brown cells in organelles called melanosomes. Through biosynthesis, melanosomes, which contain melanin granules, migrate and accumulate around the nucleus of keratinocytes.

The synthesis of melanin, the number of melanosomes, and the migration to the surrounding keratinocytes are largely genetically influenced, in part, by hormones and ultraviolet rays, and other intracellular regulators such as cytokines and copper. It is known to be affected by metal ions such as zinc, iron, and interferon, prostaglandin, histamine and the like.

Conventionally, ascorbic acid (JP-A 4-9320), hydroquinone (JP-A-192062), kojic acid (JP-A-56-7710), arbutin (JP-A 4-93150), extracts of some, etc. It is used as a whitening cosmetic because it has a tyrosinase inhibitory activity that is involved in melanin production, but its use is poor due to problems such as deterioration due to its poor stability in cosmetic formulations, discoloration, odor, and unclear efficacy at the biological level. It is limited.

It is an object of the present invention to provide a cosmetic composition comprising a new ingredient as an active ingredient that is stable and can prevent skin aging or wrinkles, and is also effective for skin whitening.

In order to achieve the above object, the present invention provides a cosmetic composition comprising 6-methoxynalingenin or derivatives thereof as an active ingredient.

The present invention provides a cosmetic composition for preventing skin aging or improving wrinkles, comprising 6-methoxynalingenin or a derivative thereof as an active ingredient.

The present invention provides a cosmetic composition for skin whitening comprising 6-methoxynalingenin or a derivative thereof as an active ingredient.

The present invention provides an anti-inflammatory cosmetic composition comprising 6-methoxynalingenin or a derivative thereof as an active ingredient.

The present invention provides an antimicrobial cosmetic composition comprising 6-methoxynalingenin or a derivative thereof as an active ingredient.

Here, the derivative of 6-methoxynalingenin may be 6-methoxynaringenin-7-O-glucoside.

The 6-methoxy naringenin or derivatives thereof may be included in an amount of 0.000001 to 30.0% by weight based on the total weight of the cosmetic composition. Alternatively, the 6-methoxynalingenin or derivatives thereof may be included in an amount of 0.0001 to 10.0 wt% based on the total weight of the cosmetic composition.

The cosmetic composition is a formulation selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, cleansing, oils, powder foundations, emulsion emulsions, wax foundations, packs, massage creams and sprays. Can be made.

The cosmetic composition according to the present invention has the following advantages by including 6-methoxynaringenin or a derivative thereof as an active ingredient.

The cosmetic composition according to the present invention may be used for preventing skin aging or anti-wrinkle because of the effects of inhibiting MMP-1 production, enhancing collagen synthesis, and improving skin wrinkles.

The cosmetic composition according to the present invention can be used for skin whitening because it has a melanin production inhibitory effect.

Since the cosmetic composition according to the present invention has an inflammatory cytokine expression suppression effect, an anti-inflammatory effect and an antimicrobial effect by ultraviolet irradiation, it can be used for anti-inflammatory use and antibacterial use.

The cosmetic composition according to the present invention has an effect of alleviating skin irritation and has a moisturizing effect.

Hereinafter, the present invention will be described in detail.

The present invention provides a cosmetic composition comprising 6-methoxynalingenin or a derivative thereof as an active ingredient.

The cosmetic composition according to the present invention can be used for anti-aging or anti-wrinkle use. Specifically, the cosmetic composition according to the present invention, as can be seen in Experimental Examples 1 and 2 to be described later exhibits an antioxidant effect, as shown in Experimental Example 3 to be described later exhibits an MMP-1 production inhibitory effect, Experimental Example 4 to be described later As can be seen from the collagen synthesis enhances the effect, as can be seen in Experimental Example 11 to be described later because it has a skin wrinkle improvement effect, it can be usefully used for anti-aging or skin wrinkle prevention.

That is, the cosmetic composition according to the present invention can prevent skin aging by preventing oxidization of skin cells and tissues by eliminating active oxygen or free radicals (see Experimental Examples 1 and 2), and also generating MMP-1. By inhibiting the breakdown of collagen to prevent skin elasticity from deteriorating and preventing wrinkles (see Experimental Example 3). Also, by enhancing collagen synthesis, skin elasticity can not be reduced and wrinkles can not be formed. (See Experimental Example 4), the skin wrinkles can be improved (see Experimental Example 11).

The cosmetic composition according to the present invention can be used for skin whitening purposes. Specifically, the cosmetic composition according to the present invention, as can be seen in Experimental Example 5 to be described later by suppressing the production of melanin pigment that can cause pigmentation can be usefully used for skin whitening use by showing a melanin production inhibitory effect.

Cosmetic compositions according to the invention can be used for anti-inflammatory applications. Specifically, the cosmetic composition according to the present invention exhibits an inhibitory effect on inflammatory cytokine expression by ultraviolet irradiation, as can be seen in Experimental Example 6, which will be described later, and as shown in Experimental Example 7, which will be described later, It can be usefully used for anti-inflammatory applications.

Cosmetic compositions according to the invention can be used for antimicrobial purposes. Specifically, since the cosmetic composition according to the present invention exhibits an antimicrobial effect as can be seen in Experimental Example 10, it can be usefully used for antibacterial purposes.

In addition, the cosmetic composition according to the present invention exhibits an effect of relieving skin irritation as can be seen in Experimental Example 8 described later, and has a moisturizing effect as can be seen in Experimental Example 9, and thus various effects in addition to the specific efficacy as described above. Because it has a can be usefully used as a cosmetic.

6-methoxynalingenin or a derivative thereof included as an active ingredient of the cosmetic composition according to the present invention is represented by the following Chemical Formula 1.

In Formula 1, R may be composed of hydroxy or glucoside. When R is OH, it is 6-methoxynaringenin, and when R is glucoside, it is 6-methoxynaringenin-7-O-glucoside. In addition, R may include various substituents other than the glucoside to correspond to various derivatives known as derivatives of 6-methoxynaringenin. That is, the derivative of 6-methoxynaringenin according to the present invention is not necessarily limited to 6-methoxynaringenin-7-O-glucoside, and may include various known derivatives.

The 6-methoxynalingenin or derivatives thereof may be included in 0.000001 to 30.0% by weight, more preferably 0.0001 to 10.0% by weight, most preferably 0.001 to 5.0% by weight based on the total cosmetic composition May be included. When the content of the 6-methoxynalingenin or its derivatives is less than 0.000001% by weight, the antioxidant effect as described above is insignificant and cannot be used for skin aging prevention purposes, and the 6-methoxynaringenin or its derivatives. If the content of more than 30.0% by weight does not show a marked increase in efficacy as the content increases may be economical.

The cosmetic composition according to the present invention contains components conventionally used in cosmetic compositions in addition to the active ingredient 6-methoxynalingenin or derivatives thereof, and for example, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and perfumes. Phosphorus aids, and carriers.

Cosmetic compositions according to the invention can be prepared in any formulations conventionally prepared in the art and include, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing It may be formulated as cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and the like, but is not limited thereto. More specifically, the cosmetic composition according to the present invention may be prepared in the form of a flexible lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder have.

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

In the case where the formulation 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. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan are used.

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

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

Hereinafter, the present invention will be described in more detail with reference to specific production examples. These preparations are merely for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these preparations.

Manufacturing example  1.6- Methoxynalingenin  And preparation of derivatives thereof

[Reaction Scheme 1]

As can be seen in Scheme 1, 1,3,5-trihydroxy-6-methoxybenzene and caffeic acid derivatives are reacted to obtain compound 1, and then heated to compound 1 under iodine and DMSO conditions for cycling. After the addition (addition) reaction, and then reduced to palladium and hydrogen to synthesize compound 2 (6-methoxynalingenin).

Thereafter, glucose was added to position 7 of compound 2 to synthesize 6-methoxynaringenin-7-O-glucoside.

6- Methoxynaringenin (1) Light yellow needles; mp 228-230 C UV (MeOH) max nm: 290, 340; ESI-MS m / z : 303 [M + H] + 1H-NMR (CD3COCD3, 400 MHz): 12.32 (1H, s, 5-OH), 7.38 (2H, d, J = 8.8 Hz, H-2 ' , 6 '), 6.88 (2H, d, J = 8.8 Hz, H-3', 5 '), 6.00 (1H, s, H-8), 5.42 (1H, dd, J = 13.2, 2.8 Hz, H -2), 3.78 (3H, s, OCH 3), 3.18 (1H, dd, J = 17.2, 13.2 Hz, H-3), 2.74 (1H, dd, J = 17.2, 2.8 Hz, H-3); 13C-NMR (CD3COCD3, 100 MHz): 79.9 (C-2), 43.5 (C-3), 198.0 (C-4), 156.2 (C-5), 130.8 (C-6), 159.7 (C-7 ), 95.7 (C-8), 159.6 (C-9), 103.3 (C-10), 129.8 (C-1 '), 129.0 (C-2'), 116.2 (C-3 '), 158.6 (C -4 '), 116.2 (C-5'), 129.0 (C-6 '), 60.8 (OCH3).

6- Methoxynaringenin- 7- O- D- glucoside (2) White powder UV (MeOH) max nm: 282, 342; ESI-MS m / z : 465 [M + H] + 1H-NMR (DMSO - d 6, 400 MHz): 11.97 (1H, s, 5-OH), 9.60 (1H, s, 4'-OH), 7.32 (2H, d, J = 8.4 Hz, H-2 ', 6'), 6.79 (2H, d, J = 8.4 Hz, H-3 ', 5'), 6.31 (1H, s, H-8) , 5.47 (1H, doublet of doublets, J = 13.2, 2.8 Hz, H-2), 4.98 (1H, d, J = 6.8 Hz, H-1``), 3.68 (3H, s, OCH3), 3.16 (1H, m, H-3), 2.72 (1H, dd, J = 17.2, 2.8 Hz , H-3) 13C-NMR (DMSO - d 6, 100 MHz): 79.4 (C-2), 42.9 (C-3), 198.5 (C-4), 155.1 (C-5), 130.8 (C- 6), 159.3 (C-7), 95.2 (C-8), 158.7 (C-9), 103.9 (C-10), 129.4 (C-1 '), 129.1 (C-2'), 115.9 (C -3 '), 158.5 (C-4'), 115.9 (C-5 '), 129.1 (C-6'), 100.5 (C-1 ''), 73.8 (C-2 ''), 77.8 (C -3 ''), 70.2 (C-4 ''), 77.3 (C-5 ''), 61.2 (C-6``), 61.0 (OCH3).

Experimental Example  One. NBT Antioxidant effect measurement

Antioxidant activity was measured by NBT method to determine the antioxidant effects of 6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside prepared according to Preparation Example 1.

In order to measure the antioxidant effect, the active oxygen produced by xanthine and xanthine oxidase was measured by NBT method, and a sample (6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside) The effect of removing the active oxygen, that is, the active oxygen scavenging effect was evaluated. The reactive oxygen scavenging rate is measured by reacting the active oxygen produced by xanthine and xanthine oxidase with Nitro Blue Tetrazolium (NBT) at the wavelength of 560 nm.

The specific measuring method is as follows.

1) To a vial bottle, add 2.4 ml of 0.05M Na ₂ CO ₃ , 0.1ml 3mM xanthine solution, 0.1ml 3mM EDTA solution, 0.1ml BSA solution, and 0.1ml 0.72mM NBT solution After adding 0.1 ml of the solution, it is left at 25 ° C. for 10 minutes. Thereafter, 0.1 ml of xanthine oxidase solution is added, stirred rapidly, and incubated at 25 ° C for 20 minutes. Thereafter, 0.1 ml of 6 mM CuCl ₂ solution is added to stop the reaction, and the absorbance St at 560 nm is measured.

2) A blank of the sample solution was measured in the same manner as in 1) except that distilled water was used instead of 0.1 ml of xanthine oxidase solution in 1) to measure the absorbance So.

3) In the blank test, absorbance Bt is measured in the same manner as in 1) except that distilled water is used instead of the sample solution in 1).

4) Blank in the blank test was measured in the same manner as in 3) except for using distilled water instead of 0.1 ml of xanthine oxidase solution in 3) to measure the absorbance Bo.

The active oxygen scavenging rate was calculated by the following Equation 1, and the results are shown in Table 1 below.

In Equation 1, St is absorbance at 560 nm after the enzymatic reaction of the sample solution, Bt is absorbance at 560 nm after the enzymatic reaction of the blank test solution, So is absorbance at 560 nm before the reaction of the sample solution without enzyme addition, Bo is the absorbance at 560 nm before the reaction in the absence of enzyme in the blank test solution.

Active oxygen scavenging rate (%) Treatment concentration (ppm) 6-methoxynalingenin (%)  6-methoxynaringenin-7-O-glucoside (%) One 63.1 62.1 5 75.1 74.4 10 86.1 83.2 25 87.5 86.2 50 90.5 89.7

As can be seen in Table 1, it can be seen that the 6-methoxynalingenin and 6-methoxynaringenin-7-O-glucoside has increased concentration-dependent antioxidant capacity.

Experimental Example  2. DPPH Antioxidant effect measurement

Antioxidant activity was measured by using the DPPH method to measure the antioxidant effects of 6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside prepared according to Preparation Example 1.

The DPPH method is to measure antioxidant activity by reducing power using a free group called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl) free radical. The free radical scavenging rate was measured at a wavelength of 560 nm by comparing the degree of DPPH reduction by the sample (6-methoxynalingenin and 6-methoxynalingenin-7-O-glucoside) to the decrease in absorbance of the blank. do.

As a reagent used, a 0.1 mM solution of 2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical (Aldrich Chem. Co., MW = 618.76) was dissolved in methanol to make 100 ml.

The specific measuring method is as follows.

1) Add 0.15 ml of 0.1 mM DPPH solution and 0.15 ml of sample solution to a 96-well plate, stir rapidly, and incubate for 10 minutes at 25 ° C. Thereafter, the absorbance St at 560 nm is measured.

2) The blank of the sample solution was measured in the same manner as in 1) except that methanol was used instead of the 0.1 mM DPPH solution in 1) to measure the absorbance So.

3) In the blank test, absorbance Bt is measured in the same manner as in 1) except that distilled water is used instead of the sample solution in 1).

4) Blank in the blank test was measured in the same manner as in 3) except that methanol was used instead of the 0.1 mM DPPH solution in 3) to measure the absorbance Bo.

The free radical scavenging rate was calculated by the following Equation 2, and the results are shown in Table 2 below.

In Equation 2, St is the absorbance at 560 nm after free radical scavenging of the sample solution, Bt is the absorbance at 560 nm after free radical scavenging of the blank test solution, and So is at 560 nm before the reaction without free radical scavenging of the sample solution. Absorbance, Bo is the absorbance at 560 nm before the reaction in the absence of free radicals of the blank test solution.

Free radical scavenging rate (%) Treatment concentration (ppm) 6-methoxynalingenin (%)  6-methoxynaringenin-7-O-glucoside (%) One 23.1 19.1 5 45.3 40.4 10 68.5 61.9 25 83.1 78.5 50 90.5 88.2

As can be seen from Table 2, in the radical scavenging test by DPPH it can be seen that the antioxidant capacity of 6-methoxynalingenin and 6-methoxynaringenin-7-O-glucoside increased concentration-dependently.

Experimental Example  3. MMP -1 production inhibitory effect

The inhibitory effect of MMP-1 production, a collagen degrading enzyme, was measured on 6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside prepared according to Preparation Example 1.

Fibroblasts (Korean Cell Line Bank, South Korea), which are human normal skin cells, were seeded in 48-well microplates (Nunc, Denmark) at 1 × 10 ⁶ cells per well, and treated with DMEM medium (Sigma, United States) and 37 ° C. After 24 hours of incubation, 6-methoxynalingenin as a control and serum-free DMEM medium and 6-methoxynaringenin-7-O-glucoside prepared in Preparation Example 1 were added And 6-methoxynalingenin-7-O-glucoside-free serum incubated for 48 hours in DMEM medium.

After the additional incubation, the supernatant of each well was collected and the amount (ng / ml) of newly synthesized MMP-1 was measured using an MMP-1 assay kit (Amersham, USA), and MMP- according to Equation 3 below. 1 production inhibition rate was calculated, the results are shown in Table 3.

Inhibition rate of MMP-1 production (%) Treatment concentration (ppm) 6-methoxynalingenin (%)  6-methoxynaringenin-7-O-glucoside (%) One 15.14 11.45 2.5 29.12 22.12 5 48.23 45.88 10 64.26 60.54 25 86.25 80.31

As can be seen from Table 3, it can be seen that 6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside reduce the activity of MMP-1 in a concentration-dependent manner.

Experimental Example  4. Collagen Synthesis Enhancement Effect

Fibroblasts, human normal epithelial cells, were seeded in 48-well microplates at 1 × 10 ⁶ cells per well and incubated in DMEM medium for 24 hours. Subsequently, 6-methoxynaringenin and 6-methoxy were prepared in serum-free DMEM medium and control group to which 6-methoxynaringenin or 6-methoxynaringenin-7-O-glucoside prepared in Preparation Example 1 was added. Incubated further for 48 hours in serum-free DMEM medium without naringenin-7-O-glucoside. After further incubation, the supernatant of each well was collected and measured in ng / ml by measuring the amount of procollagen (procollagen) type I C-peptide (PICP) using a collagen kit (Takara, Japan). Was measured. Collagen production increase rate was calculated according to the following equation 4, the results are shown in Table 4.

Collagen production increase rate (%) Treatment concentration (ppm) 6-methoxynalingenin (%)  6-methoxynaringenin-7-O-glucoside (%) One 20.1 18.24 2.5 38.4 41.12 5 41.5 41.87 10 72.2 78.50 25 113.5 121.35

As can be seen from Table 4, it can be seen that the collagen production increase rate of 6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside increases in a concentration-dependent manner.

Experimental Example  5. Inhibitory Effects of Melanogenesis Using B16F1 Melanin-forming Cells

To determine the whitening effect of 6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside prepared according to Preparation Example 1, the degree of inhibition of melanin production on B16F1 melanin forming cells was measured to determine the whitening effect. It was.

The B16F1 melanin forming cells used in Experimental Example 5 are cell strains derived from mice, and secrete a black pigment called melanin, which were obtained from ATCC (American Type Culture Collection). During the artificial culture of these cells, samples were treated to compare the degree of reduction of melanin pigment.

The melanin biosynthesis inhibitory effect of B16F1 melanin forming cells was measured as follows.

Dispense B16F1 melanin forming cells into 6-well plates at a concentration of 2 X 10 ⁵ per well and attach cells to 6-methoxynaringenin or 6-methoxynaringenin-7-O-glucoside at concentrations that do not cause toxicity. Treated for 72 hours. After incubation for 72 hours, the cells were detached with trypsin-EDTA, the number of cells was measured, and the cells were recovered by centrifugation. Quantification of intracellular melanin was carried out with a slight modification of the method of Lotan (Cancer Res., 40: 3345-3350, 1980). Specifically, the cell pellet was washed once with PBS, and then 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells. After centrifugation (3,000 rpm, 10 min), 1N NaOH (10% DMSO) was added to the cell filtrate to dissolve the extracted melanin and the absorbance of melanin was measured at 405 nm with a microplate reader. The inhibition rate (%) of melanin formation was measured.

Melanin production inhibition rate (%) of the B16F1 melanin forming cells was calculated by the following Equation 5, the results are shown in Table 5. IC ₅₀ values in Table 5 refer to concentrations of substances that inhibit melanin production by 50%. At this time, hydroquinone and arbutin, which are known to have melanin production inhibitory effects, were used as a comparison group.

In Equation 5, A is the amount of melanin in the well to which the sample is not added, and B is the amount of melanin in the well to which the sample is added.

Melanin inhibition rate (%) Name of sample IC ₅₀ 6-methoxynalingenin 0.0004% 6-methoxynaringenin-7-O-glucoside 0.0004% Hydroquinone 0.001% Arbutin 0.02%

As can be seen from Table 5, 6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside significantly inhibit melanin production in B16F1 melanin forming cells, especially than arbutin or hydroquinone used as a positive control. It can be seen that the effect is much greater.

Experimental Example  6. Inhibitory Effects of Ultraviolet Irradiation on Inflammatory Cytokine Expression

The inhibitory effect of inflammatory cytokine expression expressed by ultraviolet irradiation was evaluated for 6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside prepared according to Preparation Example 1.

Fibroblasts isolated from human epidermal tissue (Fibroblast) were placed in a 24-well test plate each 5 x 10 ⁴ and attached for 24 hours. Each well was washed once with PBS and 500 占 퐇 of PBS was added to each well. The fibroblasts were irradiated with 10 mJ / cm 2 of UV light using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), after which PBS was removed and FBS was not added to the cell culture medium (DMEM). Medium) 350 μl was added.

The samples were treated with 6-methoxynaringenin or 6-methoxynaringenin-7-O-glucoside and then incubated for 5 hours. By taking 150 µl of the culture supernatant and quantifying IL-1α, the effect of inhibiting the inflammatory cytokine expression of 6-methoxynaringenin or 6-methoxynaringenin-7-O-glucoside was determined. The amount of IL-1α was quantified using an enzyme immunoassay (Enzyme-linked Immunosorbent Assay), and the expression inhibition rate of inflammatory cytokines (IL-1α) was calculated by Equation 6 below, and the results are shown in Table 6. .

In Equation 6, Bo is the amount of IL-1α produced in the wells which have not been irradiated with UV light, and Bt is the amount of IL-1α produced in the wells which have not been irradiated with UV light, and St is the amount of IL-1α IL-1α production of wells.

Inhibitory rate of inflammatory cytokine expression (%) Treatment concentration (ppm) 6-methoxynalingenin (%)  6-methoxynaringenin-7-O-glucoside (%) One 15.1 23.99 5 23.4 35.78 10 54.5 61.33 50 61.2 67.65 100 73.5 78.99

As can be seen from Table 6, it can be seen that 6-methoxynaringenin and 6-methoxynaringenin-7-O-glucoside inhibit the production of IL-1α, an inflammatory cytokine caused by ultraviolet light.

Manufacturing example  2. Example  1 to 3 and Comparative Example  1 ~ 2 Composition Preparation

To measure the effect on cosmetics comprising 6-methoxynaringenin or 6-methoxynaringenin-7-O-glucoside, 6-methoxynaringenin and / or 6-methoxynaringenin-7-O A composition comprising glycerin, 1,3 butylene glycol and sorbitol as a moisturizing agent instead of a cosmetic comprising a glucoside (Examples 1 to 3), 6-methoxynaringenin or 6-methoxynaringenin-7-O-glucoside (Comparative Example 1) and a composition (Comparative Example 2) not containing all of them were prepared as shown in Table 7 below. The content units in Table 7 are by weight.

ingredient Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 6-methoxynalingenin 5.0 - 2.5 - - 6-methoxynaringenin-7-O-glucoside - 5.0 2.5 - - glycerin - - 5.0 1,3-butylene glycol - - - 2.5 - Sorbitol - - - 2.5 - EDTA-2Na 0.02 0.02 0.02 0.02 0.02 Purified water to 100 to 100 to 100 to 100 to 100 Cetostearyl alcohol 2.0 2.0 2.0 2.0 2.0 Glyceryl stearate 1.5 1.5 1.5 1.5 1.5 Microcrystalline 0.7 0.7 0.7 0.7 0.7 Squalane 5.0 5.0 5.0 5.0 5.0 Liquid paraffin 3.0 3.0 3.0 3.0 3.0 Trioctanoin 5.0 5.0 5.0 5.0 5.0 Polysorbate 1.2 1.2 1.2 1.2 1.2 Sorbitan stearate 0.5 0.5 0.5 0.5 0.5 Tocopheryl acetate 0.2 0.2 0.2 0.2 0.2 Cyclomethicone 3.0 3.0 3.0 3.0 3.0 BHT 0.05 0.05 0.05 0.05 0.05 Incense, preservative Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount

Experimental Example  7. Cosmetics  Anti-inflammatory effect of the composition (ear edema experiment)

The anti-inflammatory effect was measured using the cosmetic composition prepared in Preparation Example 2 (Example 1, Example 2, Example 3 and Comparative Example 1). Sixteen healthy rats were divided into four groups of four per sample. First, both ears of rats were wiped well with alcohol (ethanol), and 4 μl of each sample was applied. After 1 hour, 20 μl of acetone was applied to the left ear, and arachidonic acid, an inflammation-positive control agent, was applied to the right ear. After 1 hour, the thickness of both ears was repeatedly measured using a micrometer, and the average value was obtained. The anti-inflammatory effect was calculated according to Equation 7 below, and the results are shown in Table 8.

In Equation 7, A is swelled thickness in the experimental group, B is swelled thickness in the control group.

Swelling Anti-inflammatory effect (%)
Swelling thickness Difference from control
(BA) Control (purified water) 2.87 (B) - Comparative Example 1 2.84 (A) 0.03 1.04 Example 1 1.88 (A) 0.99 34.50 Example 2 1.77 (A) 1.10 38.32 Example 3 1.82 (A) 1.05 36.58

As can be seen in Table 8, the cosmetic composition of Example 1, Example 2 or Example 3 containing 6-methoxy naringenin or 6-methoxynaringenin-7-O-glucoside of the present invention is Comparative Example 1 It can be seen that the anti-inflammatory effect is significantly superior to the cosmetic composition of.

Experimental Example  8. Skin irritation test

Samples were prepared by adding lactic acid 0.3% as an irritant to the cosmetic compositions according to Examples 1, 2 and Comparative Example 1 so that the pH was 5.5. 0.3% lactic acid solution alone and 5 g of 20 cm of each of the prepared samples were patched for 24 hours, and then removed for 1 hour and 24 hours. State changes were read.

The stimulation rate was calculated according to Equation 8 below, and the results are shown in Table 9 below.

Criteria

-: No erythema or unusual phenomenon

+ -: slightly reddish from the surroundings

+: Significantly reddened than surrounding

++: reddens and swells more heavily than the surroundings

sample
Judgment result Stimulation rate
(%) ++ + + - - Example 1 + lactic acid 0.3% - 4 6 20 15.5 Example 2 + lactic acid 0.3% - 2 4 24 8.89 Example 3 + lactic acid 0.3% - 3 5 22 12.2 Comparative Example 1 + lactic acid 0.3% 3 15 11 One 55.55 Lactic acid 0.3% 7 15 8 - 65.55

As can be seen in Table 9, the cosmetic compositions of Examples 1, 2 and 3 according to the present invention can be seen that the stimulation rate is reduced in comparison with the comparison in the stimulation alleviation effect by lactic acid.

Experimental Example  9. Moisturizing effect

The clinical moisturizing effect on the cosmetic composition prepared in Preparation Example 2 (Examples 1,2,3 and Comparative Examples 1,2) was measured as follows.

The survey divided 100 healthy adult men and women who feel dry skin into 5 groups (A, B, C, D, E) by 20 randomly, and then, in Examples 1 and 2, respectively. Formulations 3, and 3 were applied to groups D and E, respectively, for 8 weeks twice a day for the formulations of Comparative Examples 1 and 2. Moisturizing effect was evaluated using Corneometer CM 820 (Corage + Khazaka, Germany) every two weeks from the start of the practical test and after the end, and the results are shown in Table 10 below.

Before use After 2 weeks of use After 4 weeks of use Example 1 23.5 38.6 45.7 Example 2 23.2 36.4 39.6 Example 3 23.6 37.2 42.0 Comparative Example 1 24.5 29.9 31.8 Comparative Example 2 22.8 30.5 31.9

As can be seen in Table 10, the cosmetics containing 6-methoxynalingenin or 6-methoxynaringenin-7-O-glucoside (Examples 1 and 2) compared to the general cosmetics containing other moisturizing agents (Comparative Examples 1, 2) It can be seen that the moisturizing effect of (3) is excellent.

Manufacturing example  3. Example  4-7 and Comparative Example  3-4 composition preparation

As shown in Table 11, cosmetics containing 6-methoxynalingenin (Examples 4-7), cosmetics not containing 6-methoxynaringenin (Comparative Example 3), and 6-methoxynaringenin Instead, a cosmetic composition (comparative example 4) containing methylparaben and imidazolidinylurethane was prepared as a preservative. The content units in Table 11 are by weight.

ingredient Example Comparative Example 4 5 6 7 3 4 Purified water To 100 To 100 To 100 To 100 To 100 To 100 glycerin 4.0 4.0 4.0 4.0 4.0 4.0 Butylene glycol 2.0 2.0 2.0 2.0 2.0 2.0 Propylene glycol 2.0 2.0 2.0 2.0 2.0 2.0 EDTA-2Na 0.02 0.02 0.02 0.02 0.02 0.02 Polysorbate 60 1.0 1.0 1.0 1.0 1.0 1.0 Glyceryl stearate 1.5 1.5 1.5 1.5 1.5 1.5 Wax 4.0 4.0 4.0 4.0 4.0 4.0 Macadamia Nut Oil 5.0 5.0 5.0 5.0 5.0 5.0 Squalane 3.0 3.0 3.0 3.0 3.0 3.0 Spices a very small amount a very small amount a very small amount a very small amount a very small amount a very small amount 6-methoxynalingenin 0.1 0.5 1.0 2.0 - - Methylparaben - - - - - 0.2 Imidazolidinyl urea - - - - - 0.2

Experimental Example  10: antimicrobial activity test

The antibacterial effect on bacteria and fungi was measured using the cosmetics prepared according to Preparation Example 3.

First, in the case of bacteria, E. coli ( Esherichia ) in the cosmetic of Example 4-7, 20-30 g of the cosmetic of Comparative Example 3-4 coli ; ATCC 8739), Pseudomonas aeruginosa (ATCC9027) and Staphylococcus aureus (ATCC6538) were mixed and added so that the initial concentration per sample was 10 ⁷ cfu / g. 1 g of each cosmetic was taken at 1, 7, 14, 21 and 28 days intervals while culturing them in a thermostat at 30-32 ° C for 4 weeks, and the number of viable cells was measured.

Next, in the case of the fungus, Candida albicans (Candida albicans ; ATCC10231), Penicillium cytrinum citrinum ; KCTC2123) and Aspergillus niger ; ATCC16404) was mixed to add 10 ⁷ cfu / g per sample, and then cultured in a 25 ° C. incubator at 7 days intervals to observe the presence of odor and mycelia and spores on the sample surface.

The results are shown in Table 12 below.

Cosmetics Bacteria (cfu / g) mold Initial number of bacteria Day 1 Day 7 Day 14 Day 21 Day 28 Example 4 1 x 10 ⁷ 41000 500 250 <250 <250 - Example 5 1 x 10 ⁷ 45000 300 200 <100 <100 - Example 6 1 x 10 ⁷ 44000 150 <100 <100 <100 - Example 7 1 x 10 ⁷ 45000 <100 <100 <100 <100 - Comparative Example 3 1 x 10 ⁷ 54000000 30000000 > 1x10 ⁸ > 1x10 ⁸ > 1x10 ⁸ +++ Comparative Example 4 1 x 10 ⁷ 40000 300 <100 <100 <100 -

-: No spawning and mycelium spawning for 8 weeks and good

+: Molding on the wall or lid within 4 weeks

++: mending within 4 weeks and mold on part of the surface

+++: Mildew and mold on the entire surface within 4 weeks

As can be seen in Table 12, the cosmetic composition of Comparative Example 3, which does not use a preservative, it can be seen that within four weeks of deodorization and mold on the entire surface, the bacterial bacteria significantly increased even after one day. However, the cosmetic composition of Examples 4 to 7 containing 6-methoxynalingenin showed a concentration-dependent antimicrobial effect against bacteria and fungi, and when used in a content of 2.0% after about 28 days Preservatives similar to or better than conventional preservatives (Comparative Example 4) were obtained, and even when used in an amount of 0.5% or more when judged as the whole culture day, it was found that much better antiseptics were obtained than conventional preservatives.

Experimental Example  11. Skin wrinkle improvement

A clinical experiment was conducted on the skin wrinkle improvement effect of the cosmetic comprising 6-methoxynalingenin-7-O-glucoside of the present invention. Clinical trials were evaluated through the actual use test of each cosmetic.

Thirty test subjects (women aged 20 to 35 years) were coated with Example 2 on the right side of the face and Comparative Example 1 on the left side of the face for two consecutive days, two times a day.

After completion of the experiment, the skin wrinkle improvement effect was evaluated by visual observation by an experienced doctor before and after using the product for two months. The experimental results are shown in Table 13 below.

Effectiveness was calculated according to Equation 9 below.

Wrinkle improvement effect Effective rate (%) Great slightly none Example 2 24 4 2 93.3 Comparative Example 1 3 4 23 23.3

As can be seen from Table 13, the cosmetic composition according to Example 2 of the present invention showed a higher wrinkle improvement effect than the cosmetic composition according to Comparative Example 1. In addition, the skin irritation could not be observed in the skin of the subjects applying the cosmetic composition according to Example 2 of the present invention to the skin.

Claims (9)

delete 6-methoxynalingenin or 6-methoxynalingenin-7-O-glucoside as an active ingredient, and in addition to the active ingredient comprising any one of antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings A cosmetic composition for preventing skin aging or skin wrinkles, comprising an adjuvant and a carrier. 6-methoxynaringenin or 6-methoxynaringenin-7-O-glucoside as an active ingredient, and in addition to the active ingredient comprising any one of antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors A cosmetic composition for skin whitening, comprising an adjuvant and a carrier. 6-methoxynaringenin or 6-methoxynaringenin-7-O-glucoside as an active ingredient, and in addition to the active ingredient comprising any one of antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors An anti-inflammatory cosmetic composition comprising an adjuvant and a carrier. 6-methoxynaringenin or 6-methoxynaringenin-7-O-glucoside as an active ingredient, and in addition to the active ingredient comprising any one of antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors An antimicrobial cosmetic composition comprising an adjuvant and a carrier. delete 6. The method according to any one of claims 2 to 5,
The 6-methoxy naringenin or 6-methoxy naringenin-7-O-glucoside is a cosmetic composition, characterized in that contained in 0.000001 to 30.0% by weight based on the total weight of the cosmetic composition. 6. The method according to any one of claims 2 to 5,
The 6-methoxy naringenin or 6-methoxy naringenin-7-O-glucoside is 0.0001 to 10.0% by weight relative to the total weight of the cosmetic composition cosmetic composition. 6. The method according to any one of claims 2 to 5,
The cosmetic composition is a formulation selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, cleansing, oils, powder foundations, emulsion emulsions, wax foundations, packs, massage creams and sprays. Cosmetic composition, characterized in that consisting of.