A composition containing phenylethyl resorcinol with whitening effect and a preparation method and application thereof

Abstract

The present application belongs to the technical field of cosmetics, and discloses a composition containing phenylethyl resorcinol and having a whitening effect, and a preparation method and application thereof.The composition comprises components: glabridin, phenylethyl resorcinol and white wine resveratrol.The composition of the present application shows a certain synergy in the four whitening effect paths of resisting oxidative stress, inhibiting melanin production, inhibiting tyrosinase activity and inhibiting MITF-related gene protein expression, has excellent whitening effect, and can achieve excellent whitening effect at a low concentration, can obviously reduce the addition amount of each component in the product, and can also avoid the problem of irritation caused by the excessively high addition amount of a single component.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a composition containing phenylethyl resorcinol with whitening effect and a preparation method and application thereof. BACKGROUND

[0002] Melanin has the effect of protecting the skin from ultraviolet radiation and avoiding cell DNA damage, plays a crucial role in protecting the skin from ultraviolet irradiation and oxidative stress, and is the main physiological defense against solar radiation. However, its rapid growth or uneven distribution will lead to excessive deposition of local melanin in the skin, which will lead to age spots, freckles and chloasma and even skin cancer, causing great distress to patients.

[0003] At present, the whitening drugs commonly used in clinical practice, such as benzenediol, hydroquinone, and kojic acid, have good ability to inhibit melanin production, but these substances have irritant and cytotoxic properties, which can cause pigment loss, allergies, and even skin cancer and other adverse reactions. Therefore, the pursuit of high-efficiency and safe melanin inhibitors is the research direction of whitening. One of the main research directions is to extract active substances from plant raw materials and apply them to whitening raw materials. Although the whitening raw materials prepared from plant extracts have good whitening effect, the extracts have high irritancy and cytotoxicity due to the use of a large amount of organic solvents in the extraction process, and the safety of the extracts to human skin cannot be guaranteed. At the same time, the purity of the active ingredients in the plant extracts is low, and a series of purification is needed to obtain high-efficiency whitening raw materials. The purification process is complex, time-consuming, low in efficiency, cumbersome in operation steps, and high in cost. In addition, due to the different strengths of the activities of various components, a large amount of components need to be added to achieve a certain whitening effect, which not only increases the price cost, but also is not conducive to the health of human skin and is easy to irritate and damage the skin.

[0004] Therefore, it is a technical problem that needs to be solved by those skilled in the art to develop whitening substances with good whitening effect and more mildness and safety. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a composition in which glabridin, phenylethyl resorcinol and resveratrol play a synergistic effect, and by regulating four whitening mechanism paths of inhibiting tyrosinase activity, inhibiting melanin production, resisting oxidative stress and inhibiting small eye malformation related transcription factor (MITF) related gene protein expression, excellent whitening effect is achieved. Further, the composition exhibits excellent whitening effect at a lower concentration, reducing the side effects such as irritation caused by the use of a single high-concentration chemical component, and is safer.

[0006] The first aspect of the present application provides a composition comprising components: glabridin, phenylethyl resorcinol and resveratrol.

[0007] According to some embodiments of the present application, the glabridin is added in the composition in an amount of 10wt%-50wt%.

[0008] According to some embodiments of the present application, the phenylethyl resorcinol is added in the composition in an amount of 10wt%-70wt%.

[0009] According to some embodiments of the present application, the resveratrol is added in the composition in an amount of 15wt%-80wt%.

[0010] The second aspect of the present application provides a method for preparing the composition, comprising the following steps:

[0011] mixing the glabridin, phenylethyl resorcinol and resveratrol to obtain the composition.

[0012] The third aspect of the present application provides a cosmetic comprising the composition of the present application.

[0013] According to some embodiments of the present application, the dosage form of the cosmetic is selected from the group consisting of cream, emulsion, serum, water agent, oil agent, gel or mask.

[0014] According to some embodiments of the present application, the cosmetic further comprises an excipient acceptable in cosmetics.

[0015] According to some embodiments of the present application, the excipient comprises at least one of emollients, emulsifiers, thickening agents, humectants, pH adjusting agents and preservatives.

[0016] According to some embodiments of the present application, the composition is added in the cosmetic in an amount of ≤20wt%. Further, the composition is added in the cosmetic in an amount of 0.01wt%-20wt%. Still further, the composition is added in the cosmetic in an amount of 0.1wt%-20wt%. Yet further, the composition is added in the cosmetic in an amount of 0.3wt%-10wt%.

[0017] The fourth aspect of the present application provides an emulsion comprising the composition of the present application.

[0018] According to some embodiments of the present application, the emulsion comprises components: betaine, xanthan gum, polyacryloyldimethyl taurate, C14-22 alcohol, C12-20 alkyl glucoside, dimethicone, isononyl isononanoate, cetearyl alcohol, cyclopentasiloxane, p-hydroxyacetophenone, 1,2-hexanediol, propylene glycol, butylene glycol, glabriden, benzene ethyl resorcinol, resveratrol and water.

[0019] The fifth aspect of the present application provides a cream, comprising the composition of the present application.

[0020] According to some embodiments of the present application, the cream comprises components: butylene glycol, disodium EDTA, xanthan gum, betaine, allantoin, glycerin, glyceryl polyacrylate, cetearyl alcohol, cetearyl glucoside, glyceryl stearate, PEG-100 stearate, hydrogenated poly (C6-14 alkene), tocopherol (vitamin E), p-hydroxyacetophenone, 1,2-hexanediol, polyacrylate-13, polyisobutylene, polysorbate-20, sorbitan isostearate, glabriden, benzene ethyl resorcinol, resveratrol and water.

[0021] The beneficial effects of the present application relative to the prior art are as follows:

[0022] 1) The composition of the present application exhibits a certain synergy in the four whitening efficacy paths of resisting oxidative stress, inhibiting melanin production, inhibiting tyrosinase activity and inhibiting MITF-related gene protein expression through the combination of glabriden, benzene ethyl resorcinol and resveratrol, has excellent whitening effect, and the composition of the present application can also achieve excellent whitening effect at low concentration, can significantly reduce the addition amount of each component in the product, and can also avoid the problem of irritation caused by the excessive addition amount of a single component.

[0023] 2) The preparation method of the composition of the present application is simple and easy to operate, the raw materials are easy to obtain, and the composition can be produced on a large scale. BRIEF DESCRIPTION OF DRAWINGS

[0024] The present application will be further described below in conjunction with the drawings and examples.

[0025] Figure 1 The statistical graph of MITF-related gene expression results in test example 3. DETAILED DESCRIPTION

[0026] In order to make those skilled in the art more clearly understand the technical solutions of the present application, the following examples are listed for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0027] The raw materials, reagents or devices used in the following examples, if not specifically stated, can be obtained from conventional commercial channels or can be obtained by known existing methods.

[0028] Example 1

[0029] The present example provides a composition composed of a mixture of glabridin, phenethyl resorcinol and resveratrol powders in a molar ratio of 1:1:1 (mass ratio glabridin 42.30wt%, phenethyl resorcinol 27.94wt%, resveratrol 29.76wt%).

[0030] Example 2

[0031] The present example provides a composition composed of a mixture of glabridin, phenethyl resorcinol and resveratrol powders in a molar ratio of 2.33:2.47:1 (mass ratio glabridin 50wt%, phenethyl resorcinol 35wt%, resveratrol 15wt%).

[0032] Example 3

[0033] The present example provides a composition composed of a mixture of glabridin, phenethyl resorcinol and resveratrol powders in a molar ratio of 1:7.11:1.44 (mass ratio glabridin 15wt%, phenethyl resorcinol 70wt%, resveratrol 15wt%).

[0034] Example 4

[0035] The present example provides a composition composed of a mixture of glabridin, phenethyl resorcinol and resveratrol powders in a molar ratio of 1:1.52:11.32 (mass ratio glabridin 10wt%, phenethyl resorcinol 10wt%, resveratrol 80wt%).

[0036] Comparative Example 1

[0037] The present comparative example provides a single component containing only phenethyl resorcinol powder, which is used in the same amount as the composition of Example 1.

[0038] Comparative Example 2

[0039] The present comparative example provides a single component containing only resveratrol powder, which is used in the same amount as the composition of Example 1.

[0040] Comparative Example 3

[0041] The present comparative example provides a single component containing only glabridin powder, which is used in the same amount as the composition of Example 1.

[0042] Comparative Example 4

[0043] This comparative example provides a single component containing only the powder of nicotinamide in the same amount as the composition of Example 1.

[0044] Comparative Example 5

[0045] This comparative example provides a single component containing only the powder of phloretin in the same amount as the composition of Example 1.

[0046] Comparative Example 6

[0047] This comparative example provides a single component containing only the powder of phloretin in the same amount as the composition of Example 1.

[0048] Comparative Example 7

[0049] This comparative example provides a composition composed of the powder of glabridin and the powder of benzene-ethyl resorcinol mixed at a molar ratio of 1:1 (mass ratio of glabridin 60.22 wt%, benzene-ethyl resorcinol 39.78 wt%), in the same amount as the composition of Example 1.

[0050] Comparative Example 8

[0051] This comparative example provides a composition composed of the powder of glabridin and the powder of resveratrol mixed at a molar ratio of 1:1 (mass ratio of glabridin 58.70 wt%, resveratrol 41.30 wt%), in the same amount as the composition of Example 1.

[0052] Comparative Example 9

[0053] This comparative example provides a composition composed of the powder of benzene-ethyl resorcinol and the powder of resveratrol mixed at a molar ratio of 1:1 (mass ratio of benzene-ethyl resorcinol 48.42 wt%, resveratrol 51.58 wt%), in the same amount as the composition of Example 1.

[0054] Comparative Example 10

[0055] This comparative example provides a composition composed of the powder of glabridin, the powder of nicotinamide, and the powder of resveratrol mixed at a molar ratio of 1:1:1 (mass ratio of glabridin 48.07 wt%, nicotinamide 18.10 wt%, resveratrol 33.83 wt%), in the same amount as the composition of Example 1. The difference compared to Example 1 is that benzene-ethyl resorcinol is replaced by nicotinamide.

[0056] Comparative Example 11

[0057] The comparative example provides a composition composed of three powders of glabridin, denatonium acid and phenethyl resorcinol in a molar ratio of 1:1:1 (mass ratio glabridin 46.62wt%, denatonium acid 22.59wt%, phenethyl resorcinol 30.79wt%), which is used in the same amount as the composition of Example 1. Compared with Example 1, the only difference is that resveratrol is replaced by denatonium acid.

[0058] Comparative Example 12

[0059] The comparative example provides a composition composed of three powders of phloretin, phenethyl resorcinol and resveratrol in a molar ratio of 1:1:1 (mass ratio phloretin 38.27wt%, phenethyl resorcinol 29.89wt%, resveratrol 31.84wt%), which is used in the same amount as the composition of Example 1. Compared with Example 1, the only difference is that glabridin is replaced by phloretin.

[0060] Comparative Example 13

[0061] The comparative example provides a single component containing only polygonum cuspidatum glycoside powder, which is used in the same amount as the composition of Example 1.

[0062] Comparative Example 14

[0063] The comparative example provides a composition composed of three powders of glabridin, resveratrol and polygonum cuspidatum glycoside in a molar ratio of 1:1:1 (mass ratio glabridin 34.40wt%, resveratrol 24.20wt%, polygonum cuspidatum glycoside 41.40wt%), which is used in the same amount as the composition of Example 1. Compared with Example 1, the only difference is that phenethyl resorcinol is replaced by polygonum cuspidatum glycoside.

[0064] Test Example 1 In vitro antioxidant experiment

[0065] 1.1 Preparation of sample solution

[0066] Dimethyl sulfoxide (DMSO) was used as the solvent, and comparative example 1 (phenethyl resorcinol powder) was prepared into sample solutions with molar concentrations of 10mM, 5mM, 1mM, 0.5mM and 0.1mM, respectively, for testing.

[0067] DMSO was used as the solvent, and comparative example 2 (resveratrol powder) was prepared into sample solutions with molar concentrations of 10mM, 5mM, 1mM, 0.5mM and 0.1mM, respectively, for testing.

[0068] DMSO was used as the solvent, and comparative example 3 (glabridin powder) was prepared into sample solutions with molar concentrations of 10mM, 5mM, 1mM, 0.5mM and 0.1mM, respectively, for testing.

[0069] Comparative Example 7 (a mixture of glabriden and phenylethyl resorcinol in equimolar ratio) was prepared into sample solution 1 (containing 10 mM of glabriden and 10 mM of phenylethyl resorcinol), sample solution 2 (containing 5 mM of glabriden and 5 mM of phenylethyl resorcinol), sample solution 3 (containing 1 mM of glabriden and 1 mM of phenylethyl resorcinol), sample solution 4 (containing 0.5 mM of glabriden and 0.5 mM of phenylethyl resorcinol), and sample solution 5 (containing 0.1 mM of glabriden and 0.1 mM of phenylethyl resorcinol) using DMSO as a solvent.

[0070] Comparative Example 8 (a mixture of glabriden and resveratrol in equimolar ratio) was prepared into sample solution 1 (containing 10 mM of glabriden and 10 mM of resveratrol), sample solution 2 (containing 5 mM of glabriden and 5 mM of resveratrol), sample solution 3 (containing 1 mM of glabriden and 1 mM of resveratrol), sample solution 4 (containing 0.5 mM of glabriden and 0.5 mM of resveratrol), and sample solution 5 (containing 0.1 mM of glabriden and 0.1 mM of resveratrol) using DMSO as a solvent.

[0071] Comparative Example 9 (a mixture of phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared into sample solution 1 (containing 10 mM of resveratrol and 10 mM of phenylethyl resorcinol), sample solution 2 (containing 5 mM of resveratrol and 5 mM of phenylethyl resorcinol), sample solution 3 (containing 1 mM of resveratrol and 1 mM of phenylethyl resorcinol), sample solution 4 (containing 0.5 mM of resveratrol and 0.5 mM of phenylethyl resorcinol), and sample solution 5 (containing 0.1 mM of resveratrol and 0.1 mM of phenylethyl resorcinol) using DMSO as a solvent.

[0072] Comparative Example 10 (a mixture of glabriden, nicotinamide, and resveratrol in equimolar ratio) was prepared into sample solution 1 (containing 10 mM of glabriden, 10 mM of nicotinamide, and 10 mM of resveratrol), sample solution 2 (containing 5 mM of glabriden, 5 mM of nicotinamide, and 5 mM of resveratrol), sample solution 3 (containing 1 mM of glabriden, 1 mM of nicotinamide, and 1 mM of resveratrol), sample solution 4 (containing 0.5 mM of glabriden, 0.5 mM of nicotinamide, and 0.5 mM of resveratrol), and sample solution 5 (containing 0.1 mM of glabriden, 0.1 mM of nicotinamide, and 0.1 mM of resveratrol) using DMSO and deionized water as solvents.

[0073] Comparative Example 11 (a mixture of glabriden, phenylethyl resorcinol and tannic acid in equimolar ratio) was prepared into sample solution 1 (containing 10 mM glabriden, 10 mM phenylethyl resorcinol and 10 mM tannic acid), sample solution 2 (containing 5 mM glabriden, 5 mM phenylethyl resorcinol and 5 mM tannic acid), sample solution 3 (containing 1 mM glabriden, 1 mM phenylethyl resorcinol and 1 mM tannic acid), sample solution 4 (containing 0.5 mM glabriden, 0.5 mM phenylethyl resorcinol and 0.5 mM tannic acid) and sample solution 5 (containing 0.1 mM glabriden, 0.1 mM phenylethyl resorcinol and 0.1 mM tannic acid) using DMSO and deionized water as solvents, and the tests were carried out.

[0074] Example 1 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared into sample solution 1 (containing 10 mM glabriden, 10 mM phenylethyl resorcinol and 10 mM resveratrol), sample solution 2 (containing 5 mM glabriden, 5 mM phenylethyl resorcinol and 5 mM resveratrol), sample solution 3 (containing 1 mM glabriden, 1 mM phenylethyl resorcinol and 1 mM resveratrol), sample solution 4 (containing 0.5 mM glabriden, 0.5 mM phenylethyl resorcinol and 0.5 mM resveratrol) and sample solution 5 (containing 0.1 mM glabriden, 0.1 mM phenylethyl resorcinol and 0.1 mM resveratrol) using DMSO as solvent, and the tests were carried out.

[0075] Example 2 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in a molar ratio of 2.33:2.47:1) was prepared into sample solutions with the same concentrations as those of Example 1 using DMSO as solvent, and the tests were carried out.

[0076] Example 3 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in a molar ratio of 1:7.11:1.44) was prepared into sample solutions with the same concentrations as those of Example 1 using DMSO as solvent, and the tests were carried out.

[0077] Example 4 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in a molar ratio of 1:1.52:11.32) was prepared into sample solutions with the same concentrations as those of Example 1 using DMSO as solvent, and the tests were carried out.

[0078] 1.2 Experimental principle

[0079] DPPH, 1, 1-diphenyl-2-picrylhydrazyl radical, is a stable nitrogen-centered free radical with an absorption maximum at 519 nm. The ethanolic solution of DPPH is dark purple. When a free radical scavenger is added to the DPPH solution, the single electron of DPPH is paired and the color becomes lighter, and the absorbance at the maximum wavelength decreases. Therefore, the ability of a substance to scavenge DPPH free radicals can be determined by the degree of decrease in absorbance, and the antioxidant capacity of the substance can be evaluated.

[0080] 1.3 Experimental method

[0081] Measurement of A0: 50 μL of anhydrous ethanol was added to the enzyme plate, and then 100 μL of DPPH solution was added to each well, which was then incubated at 37°C for 30 min, and then the absorbance A0was measured at 519 nm in the enzyme marker.

[0082] Measurement of A: 50 μL of sample solution of different concentrations was added to each well of the enzyme plate, and then 100 μL of DPPH solution was added to each well, which was then incubated at 37°C for 30 min, and then the absorbance A was measured at 519 nm in the enzyme marker.

[0083] (1) DPPH scavenging rate:

[0084] DPPH scavenging rate (%) = (A0- A) / A0x 100;

[0085] (2) Half scavenging concentration:

[0086] The sample concentration was taken as the abscissa and the DPPH scavenging rate as the ordinate, and a curve was drawn and the sample concentration when the DPPH scavenging rate was 50% was calculated, i.e. the half scavenging concentration (IC50). The smaller the IC50, the stronger the ability of the sample to scavenge DPPH free radicals.

[0087] 1.4 Experimental results

[0088] The results are shown in Table 1.

[0089] Table 1 DPPH free radical scavenging rate IC50 value

[0090]

[0091] Glabrae has excellent anti-inflammatory, antioxidant and anti-melanin formation effect, and is a recognized whitening ingredient; and resveratrol and phenethyl resorcinol are excellent antioxidants. From the test results in Table 1, it can be seen that phenethyl resorcinol, resveratrol and glabridin at various concentrations all exhibit in vitro antioxidant activity, among which phenethyl resorcinol has the strongest DPPH radical scavenging activity, better than resveratrol, and glabridin has relatively weak effect; the antioxidant activity of the combination of phenethyl resorcinol + resveratrol + glabridin (Examples 1-4) is significantly higher than that of the three individual components (Comparative Examples 1-3), and in each example, phenethyl resorcinol, resveratrol and glabridin are mixed in different proportions, all of which have excellent in vitro antioxidant activity, and the antioxidant activity is comparable. These results all show that the composition exhibits excellent synergistic effect, and the in vitro antioxidant activity at low concentration is much stronger than that of each monomer compound. From the results of Comparative Example 7, Comparative Example 8 and Comparative Example 9, it can be seen that if any component in the combination of glabridin, resveratrol and phenethyl resorcinol is removed to form a combination of two, no obvious synergistic effect is exhibited; and from the data of Comparative Example 10 and Comparative Example 11, it can be found that if any one of the components in the combination of glabridin, resveratrol and phenethyl resorcinol is replaced by other whitening active substances, no obvious synergistic effect is exhibited.

[0092] The above results show that the combination of phenethyl resorcinol + resveratrol + glabridin has excellent in vitro antioxidant activity and has a unique property, and the combination significantly enhances the antioxidant activity.

[0093] Test Example 2 Cell Melanin and Tyrosinase Inhibition Test

[0094] 2.1 Preparation of sample solution

[0095] Comparative Example 1 (phenethyl resorcinol powder) was prepared into a sample solution with a molar concentration of 1 μM using DMSO as a solvent for the test.

[0096] Comparative Example 2 (resveratrol powder) was prepared into a sample solution with a molar concentration of 1 μM using DMSO as a solvent for the test.

[0097] Comparative Example 3 (glabridin powder) was prepared into a sample solution with a molar concentration of 1 μM using DMSO as a solvent for the test.

[0098] Comparative Example 4 (trans-amic acid powder) was prepared into a sample solution with a molar concentration of 1 μM using deionized water as a solvent for the test.

[0099] Comparative Example 5 (nicotinamide powder) was prepared into a sample solution with a molar concentration of 1 μM using deionized water as a solvent for the test.

[0100] Example 1 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared as a sample solution (containing 1 μM glabriden, 1 μM phenylethyl resorcinol and 1 μM resveratrol) in DMSO and tested.

[0101] Example 2 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared as a sample solution (containing 1 μM glabriden, 1 μM phenylethyl resorcinol and 1 μM resveratrol) in DMSO and tested.

[0102] Example 3 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared as a sample solution (containing 1 μM glabriden, 1 μM phenylethyl resorcinol and 1 μM resveratrol) in DMSO and tested.

[0103] Example 4 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared as a sample solution (containing 1 μM glabriden, 1 μM phenylethyl resorcinol and 1 μM resveratrol) in DMSO and tested.

[0104] Example 5 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared as a sample solution (containing 1 μM glabriden, 1 μM phenylethyl resorcinol and 1 μM resveratrol) in DMSO and tested.

[0105] Example 6 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared as a sample solution (containing 1 μM glabriden, 1 μM phenylethyl resorcinol and 1 μM resveratrol) in DMSO and tested.

[0106] Example 7 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared as a sample solution (containing 1 μM glabriden, 1 μM phenylethyl resorcinol and 1 μM resveratrol) in DMSO and tested.

[0107] Example 8 (a mixture of glabriden, phenylethyl resorcinol and resveratrol in equimolar ratio) was prepared as a sample solution (containing 1 μM glabriden, 1 μM phenylethyl resorcinol and 1 μM resveratrol) in DMSO and tested.

[0108] 2.2 Experimental principle

[0109] Melanin is a high-molecular-weight biological pigment synthesized within melanocytes. Its synthesis pathway proceeds as follows: within melanocytes, tyrosine is catalyzed by tyrosinase to form dopa, which is then dehydrogenated to form dopaquinone. Dopaquinone then rearranges into 5,6-quinone indole, which then polymerizes and binds to structural proteins within the melanosome to form melanopsin, or melanin. After synthesis, melanin is transported from melanocytes to keratinocytes. As keratinocytes mature, it diffuses throughout the epidermal layers and eventually disappears with epidermal shedding.

[0110] B16 mouse skin melanoma cells, whose melanin synthesis function is largely similar to that of normal human skin melanocytes, are widely used as test cells for evaluating the efficacy of whitening chemicals. α-MSH, an α-melanogenic hormone, can stimulate melanin secretion in B16 cells. Using this α-MSH-induced B16 cell model, the inhibitory effect of test substances on melanin synthesis was determined.

[0111] 2.3 Experimental methods

[0112] 2.3.1 Cell culture

[0113] B16 cells were cultured with 1640 complete medium (1640 medium + 10% FBS + double antibody) in a carbon dioxide incubator at 37°C and 5% CO2, and passaged every 2 to 3 days.

[0114] 2.3.2 Determination of intracellular melanin content

[0115] B16 cells in the logarithmic growth phase were selected and digested with 0.25% trypsin, and the cells were diluted with 1640 complete medium to a density of 5 × 10 4 A cell suspension of 100 cells / mL was seeded into a 6-well culture plate, 2 mL per well, and cultured in a 37°C, 5% CO2 incubator. After cell attachment was observed under a microscope, culture medium containing the sample and 1 μM α-MSH was added, along with a control (no sample, 1 μM α-MSH). After incubation at 37°C, 5% CO2 for 48 hours, the supernatant was discarded, and 0.5 mL of 0.25% trypsin solution was added to each well and digested at room temperature for 1 minute. Digestion was terminated by adding 1 mL of culture medium, and the cells were dissociated into a single-cell suspension by pipetting. Cell counts were performed, and the cell suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and 1 mL of 1 mol / L NaOH solution (containing 10% DMSO) was added. The cells were incubated in an 80°C water bath for 30 minutes, and the absorbance was measured at 490 nm using a microplate reader.

[0116] 2.3.3 Determination of intracellular tyrosinase activity

[0117] B16 cells in the logarithmic growth phase were selected and digested with 0.25% trypsin, and the cells were diluted with 1640 complete medium to a density of 5 × 104 The cells were inoculated in 6-well plates at a density of 1 x 105 cells / mL, 2 mL per well, and cultured in a 37°C, 5% CO2 incubator. After the cells adhered to the wall of the well, the sample and α-MSH (1 μM) were added, and a control group (without sample and with 1 μM α-MSH) was set up. After incubation at 37°C, 5% CO2 for 48 h, the supernatant was discarded, 0.5 mL of 0.25% trypsin was added to each well, and the cells were digested at room temperature for 1 min. The digestion was stopped by adding 1 mL of medium, and the cells were blown into a single cell suspension. The cells were counted, and the cell suspension was centrifuged at 1000 r / min for 5 min. The supernatant was discarded, 1 mL of 0.5% deoxycholate sodium solution was added, and the cells were lysed on ice for 15 min to prepare a tyrosinase-containing extract. After preheating at 37°C, 0.5 mL of 0.3% dopa solution was added, and the absorbance was measured at 475 nm after reaction at 37°C for 10 min.

[0118] 2.3.4 Result processing

[0119] (1) Melanin synthesis inhibition rate:

[0120] Melanin synthesis inhibition rate (%) = [1 - (As / Ds) / (Ac / Dc)] x 100

[0121] wherein As is the absorbance of the sample group, Ac is the absorbance of the control group, Ds is the cell concentration of the sample group, and Dc is the cell concentration of the control group.

[0122] (2) Tyrosinase activity inhibition rate:

[0123] Tyrosinase activity inhibition rate (%) = [1 - (As / Ds) / (Ac / Dc)] x 100

[0124] wherein As is the absorbance of the sample group, Ac is the absorbance of the control group, Ds is the cell concentration of the sample group, and Dc is the cell concentration of the control group.

[0125] 2.4 Experimental results

[0126] The results are shown in Table 2.

[0127] Table 2 Cell melanin and tyrosinase inhibition rate

[0128]

[0129] Melanin is an important factor affecting the color of human skin, and excessive melanin can cause skin color to darken, and in severe cases, can cause skin diseases such as chloasma and melanoma, which seriously affect people's physical and mental health. As can be seen from the data in Table 2, the inhibition rate of phenylethyl resorcinol (Comparative Example 1) on cell melanin synthesis is stronger than that of glabridin (Comparative Example 3) and resveratrol (Comparative Example 2). When glabridin, resveratrol and phenylethyl resorcinol are compounded to form a composition (Example 1), the inhibition rate on cell melanin production can reach 27.8%, which is significantly higher than all the comparative examples.

[0130] Tyrosinase is an oxidase and a rate-limiting enzyme for regulating melanin production. As can be seen from the data in Table 2, the inhibition of each substance on cell tyrosinase activity has the same trend as the inhibition of melanin synthesis. The inhibition effect of resveratrol (Comparative Example 2) on cell tyrosinase activity is relatively poor, only 12.8%, which is slightly lower than that of phenylethyl resorcinol (Comparative Example 1); while glabridin (Comparative Example 3) has the strongest tyrosinase inhibition rate (28.3%) among the three compounds. When glabridin, resveratrol and phenylethyl resorcinol are compounded to form a composition (Example 1), the inhibition rate on cell tyrosinase can reach 39.8%, which is much higher than other comparative examples. Both of these results show that the compounded composition (Example 1) has excellent synergistic effect, showing stronger inhibition effect on cell melanin and tyrosinase than single compound at the same concentration. At the same time, any one component of the glabridin, resveratrol and phenylethyl resorcinol composition is removed to form a combination of the two, and neither has obvious synergistic effect, and replacing any one component of the glabridin, resveratrol and phenylethyl resorcinol composition with other whitening active substances also does not form obvious synergistic whitening effect.

[0131] The above results show that the combination of glabridin + resveratrol + phenylethyl resorcinol has obvious synergistic effect on inhibiting melanin synthesis and inhibiting tyrosinase activity, and has a unique property. The composition significantly enhances the whitening effect.

[0132] Test Example 3 Cell Melanin and Tyrosinase Related Gene and Protein Expression Inhibition Test

[0133] 3.1 Preparation of sample solution

[0134] Comparative Example 1 (phenylethyl resorcinol powder) was prepared into a sample solution with a molar concentration of 1 μM with DMSO as the solvent for the test.

[0135] Comparative Example 2 (resveratrol powder) was prepared into a sample solution with a molar concentration of 1 μM with DMSO as the solvent for the test.

[0136] The sample solution of Comparative Example 3 (glabridin powder) was prepared in DMSO as a solvent to have a molar concentration of 1 μM, and the test was performed.

[0137] The sample solution of Comparative Example 13 (polygonum cuspidatum glycoside powder) was prepared in DMSO as a solvent to have a molar concentration of 1 μM, and the test was performed.

[0138] The sample solution of Comparative Example 14 (a mixture of glabridin, resveratrol, and polygonum cuspidatum glycoside powders in an equimolar ratio) was prepared in DMSO as a solvent to have 1 μM of glabridin, 1 μM of resveratrol, and 1 μM of polygonum cuspidatum glycoside, and the test was performed.

[0139] The sample solution of Example 1 (a mixture of glabridin, phenylethyl resorcinol, and resveratrol powders in an equimolar ratio) was prepared in DMSO as a solvent to have 1 μM of glabridin, 1 μM of phenylethyl resorcinol, and 1 μM of resveratrol, and the test was performed.

[0140] 3.2 Experimental Principle

[0141] B16 mouse skin melanoma cells, whose melanin synthesis function is basically the same as that of human normal skin melanocytes, are widely used as test cells for determining the efficacy of whitening chemicals. α-MSH is an α-melanocyte-stimulating hormone that can promote B16 cells to secrete melanin. The effects of a test substance on the expression of MITF pathway-related genes of B16 cells were determined by inducing a B16 cell melanization model using α-MSH.

[0142] 3.3 Experimental Method

[0143] 3.3.1 Cell Culture

[0144] B16 cells were cultured in 1640 complete medium (1640 medium + 10% FBS by volume + double antibody) at 37°C in a carbon dioxide incubator with 5% CO2, and were subcultured every 2 to 3 days.

[0145] 3.3.2 Extraction of Cell RNA and Preparation of Protein

[0146] RNA: Add cell lysis solution to each well of the six-well plate to lyse the cells. Add chloroform (1:5 volume ratio to the lysis solution) to the lysis solution, shake well, and then let stand at room temperature for 5 min before placing it in a 4°C centrifuge at 12000 rpm for 15 min. Absorb the upper colorless liquid into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min in a 4°C centrifuge, and discard the supernatant. Add 500 μL of 75% ethanol. Centrifuge at 7500 rpm for 5 min in a 4°C centrifuge, discard the supernatant, and let stand at room temperature to dry for 1-2 min. Add an appropriate amount of DEPC water to fully dissolve the RNA precipitate.

[0147] Protein: Add cell lysis solution to each well of the six-well plate to lyse the cells. Lyse the cells on ice for 15 min, centrifuge at 12000 rpm for 15 min, and collect the supernatant, which is the extracted protein sample. After collecting the cell lysate, measure the protein concentration using a BCA kit, mix the cell lysate with loading buffer, and boil for 5 min to completely denature it.

[0148] 3.3.3 Synthesis of cDNA

[0149] The reverse transcription kit used in this experiment is (HiScript® II Q RT SuperMix for qPCR (+gDNA wiper)) purchased from Nanjing Novogene Biotech Co., Ltd. According to the kit instructions, follow the steps.

[0150] 3.3.4 qRT-PCR (real-time fluorescent quantitative PCR reverse transcription)

[0151] Prepare the mixture as follows in Table 3:

[0152] Table 3 qRT-PCR reaction system

[0153]

[0154] Then perform the qPCR reaction according to the kit instructions.

[0155] 3.3.5 Electrophoresis and membrane transfer

[0156] Prepare the electrophoresis gel according to the instructions of the PAGE gel quick preparation kit. Add the prepared protein sample and protein marker to the corresponding gel well, separate the gel (constant voltage) at 80-100 V for 30 min, concentrate the gel (constant voltage) at 110-130 V for 60-100 min, and turn off the voltage; after taking out the gel, cut the gel as needed, and cut a suitable PVDF membrane according to the actual size and activate it in methanol. According to the order, place the gel and PVDF membrane on the membrane transfer clamp, and transfer the membrane at 300 mA for 40-120 min.

[0157] 3.3.6 Blocking and incubation

[0158] After membrane transfer, appropriately cut the PVDF membrane, wash the membrane with TBST for 5 min, then soak the PVDF membrane with 5% skimmed milk powder, and incubate at 4°C for 60 min with slow shaking on a shaker; after washing off the skimmed milk powder with TBST, soak the diluted specific primary antibody in the PVDF membrane and incubate overnight at 4°C; then recover the primary antibody, wash the membrane with TBST, and soak the diluted secondary antibody in the PVDF membrane and incubate at room temperature for 60 min, then discard the secondary antibody and wash the membrane with TBST.

[0159] 3.3.7 Development

[0160] According to the instructions of the kit, prepare the developing solution, soak the PVDF in the developing solution for 1 min, develop the PVDF membrane with gel imaging, and collect the protein band picture; use image software to collect the protein band gray value and analyze and process.

[0161] 3.4 Experimental results

[0162] The MITF-related gene expression results are shown in Figure 1 .

[0163] MITF is the only factor in the MiT family that plays an important role in normal melanocyte development. MITF-M is a melanocyte-specific regulatory factor that plays a key role in melanoma and hyperpigmentation diseases. MITF-M directly regulates the transcription of related pigment genes, including tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), tyrosinase-related protein-2 (TRP2), glycoprotein nonmetastatic melanoma protein b (GPNMB), and premelanosome protein (PMEL).

[0164] Figure 1The qPCR results show the effect of each sample on the mRNA expression level of the related genes in the MITF pathway. TYR, TYRP1, and TRP2 are three enzymes in the tyrosinase gene family, in which TRY is a key enzyme in the melanin synthesis reaction, which is involved in two melanin production processes, while TYRP-1 and TRP-2 play an important role in the process of eumelanin synthesis. TYRP-1 has 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase activity, and TRP-2 can rapidly convert dopachrome to DHICA. The mRNA expression levels of TYR, TYRP1, and TRP2 were determined by qPCR. The results are shown in Table 1. Figure 1 It can be seen from the data that phenylethyl resorcinol (Comparative Example 1), resveratrol (Comparative Example 2), glabridin (Comparative Example 3), and polydatin (Comparative Example 13) can all reduce the mRNA expression levels of TYR, TYRP1, and TRP2. After phenylethyl resorcinol, resveratrol, and glabridin are combined, the composition (Example 1) exhibits excellent synergistic effects and can significantly inhibit the mRNA expression levels of TYR, TYRP1, and TRP2, and the inhibition rates are all significantly higher than those of Comparative Examples 1, 2, 3, and 13. After polydatin is used to replace phenylethyl resorcinol and combined with glabridin and resveratrol (Comparative Example 14), the inhibition effect on the mRNA expression of TYR, TYRP1, and TRP2 does not exhibit synergistic effects.

[0165] PMEL is the structural basis of fibrils in melanosome proteins, and GPNMB plays an important role in the formation of late-stage melanosome, which may be related to the transport or transfer of melanosome to keratinocytes. Figure 1 The results show that phenylethyl resorcinol (Comparative Example 1), resveratrol (Comparative Example 2), glabridin (Comparative Example 3), and polydatin (Comparative Example 13) can all reduce the mRNA expression levels of PMEL and GPNMB. Similarly, the composition (Example 1) exhibits synergistic effects and excellent inhibition effects on the mRNA expression of PMEL and GPNMB, reaching 55.19% and 46.09%, respectively, which are significantly stronger than those of Comparative Examples 1, 2, 3, and 13. After polydatin is used to replace phenylethyl resorcinol and combined with glabridin and resveratrol (Comparative Example 14), the inhibition effect on the mRNA expression of PMEL and GPNMB does not exhibit synergistic effects.

[0166] The above qPCR results all show that the composition combined with phenylethyl resorcinol, resveratrol, and glabridin has excellent synergistic effects and exhibits excellent whitening effects at a low concentration, which can significantly inhibit the expression of important genes in the MITF signaling pathway.

[0167] The expression level of TYR protein in the cells was determined by immunoblotting. As shown in Table 4.

[0168] Table 4 TYR protein expression inhibition rate result statistics

[0169]

[0170] As can be seen from the data in Table 4, resveratrol, glabridin and phenethyl resorcinol can all inhibit the expression of TYR protein in melanocytes, and the inhibition effect of phenethyl resorcinol is stronger than that of resveratrol and glabridin, which is similar to the result of qPCR. At the same time, resveratrol, glabridin and phenethyl resorcinol show a synergistic effect after compounding, and the TYR protein expression inhibition rate of the composition is 64.88%, which is 2.42 times that of phenethyl resorcinol, and is significantly higher than that of the monomer compound at the same concentration.

[0171] Therefore, both qPCR and Western blotting experiments show that the composition compounded by phenethyl resorcinol, resveratrol and glabridin has excellent synergistic effect, can significantly inhibit the expression of important genes and proteins in the MITF signaling pathway, and shows excellent whitening effect. The composition can achieve excellent whitening effect at low concentration, which can greatly reduce the addition amount of each component in cosmetics, and is conducive to avoiding a series of skin problems caused by excessive addition of single component.

[0172] Application Example 1

[0173] The present application provides an emulsion containing the composition of the present application, and the specific formula of the emulsion is shown in Table 5, and the total mass fraction is 100%.

[0174] Table 5 Emulsion Formula

[0175]

[0176] The above emulsion can be prepared by conventional preparation methods in the art.

[0177] Application Example 2

[0178] The present application provides a cream containing the composition of the present application, and the specific formula of the cream is shown in Table 6, and the total mass fraction is 100%.

[0179] Table 6 Cream Formula

[0180]

[0181] The above cream can be prepared by conventional preparation methods in the art.

[0182] It is not difficult to conceive that the emulsion of application example 1 and the cream of application example 2, since both use the composition of the present application, the glabridin, the hydroxytyrosol and the resveratrol in the composition play a synergistic effect, through the regulation of the four whitening mechanism paths of inhibiting tyrosinase activity, inhibiting melanin production, resisting oxidative stress and inhibiting MITF related gene protein expression, thereby having excellent whitening effect. Therefore, these cosmetics, since the composition of the present application is added therein, thereby also have excellent whitening effect.

[0183] The above describes the preferred embodiments of the present application, but the present application is not limited to the above-mentioned embodiments, and those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the present application. These equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. A composition, characterized in that The composition consists of the following components: 10 wt%-50 wt% of glabridin, 10 wt%-70 wt% of phenylethylresorcinol and 15 wt%-80 wt% of resveratrol.

2. The method for preparing the composition according to claim 1, wherein The following steps are involved: The glabridin, phenylethylresorcinol and resveratrol are mixed to obtain the composition.

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