Whitening and freckle-removing compositions, conditioners, medicaments, cosmetics

Abstract

The application discloses a whitening and freckle-removing composition, conditioner, medicine and cosmetic. The whitening and freckle-removing composition comprises a composition of Phyllanthus emblica, Passiflora edulis and discodermolide A; the Phyllanthus emblica comprises Phyllanthus emblica raw materials and / or Phyllanthus emblica extract, and the Passiflora edulis comprises Passiflora edulis raw materials and / or Passiflora edulis extract. The application has a significant inhibitory effect on tyrosinase activity by scientifically compounding discodermolide A and the second active component comprising one or more of Phyllanthus emblica, Passiflora edulis and Salvia, can inhibit the expression and function of the rate-limiting enzyme TYR in the melanin synthesis process, has a significant whitening and freckle-removing activity, is safe and efficient, and can be applied to various conditioners, medicines and cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic raw materials, in particular to a whitening and freckle-removing composition, a conditioner, a medicine and a cosmetic. BACKGROUND

[0002] With the acceleration of people's life pace and the growth of age, skin problems such as dull skin, pigment deposition, chloasma and sunburn are increasingly common, and the demand for products with multiple functions such as whitening and freckle-removing is increasing. Traditional chemical whitening agents are gradually eliminated due to safety controversies, and natural active ingredients become the preferred alternative due to their mildness and multiple effects. At present, a variety of natural active ingredients have been found to have whitening effects, but the absorption effect of natural active ingredients is not good, and it is difficult to fully exert their whitening effects. At the same time, a single natural active ingredient cannot meet the demand for functional diversification, and the mechanism of interaction and compatibility between multiple natural active ingredients is not clear. Therefore, it is very important to explore safe and effective natural active ingredients and their compatibility to achieve better whitening and freckle-removing effects. SUMMARY

[0003] Therefore, it is necessary to provide a safe and effective whitening and freckle-removing composition.

[0004] In a first aspect, the present application provides a whitening and freckle-removing composition, which comprises Phyllanthus emblica, Passiflora edulis and Ginkgo biloba extract A in its composition.

[0005] The Phyllanthus emblica comprises Phyllanthus emblica raw material and / or Phyllanthus emblica extract, and the Passiflora edulis comprises Passiflora edulis raw material and / or Passiflora edulis extract.

[0006] In some embodiments, the Phyllanthus emblica extract comprises water extract of Phyllanthus emblica raw material and / or alcohol extract of Phyllanthus emblica raw material; and / or, the Passiflora edulis extract comprises water extract of Passiflora edulis raw material and / or alcohol extract of Passiflora edulis raw material; and / or, the ratio of Phyllanthus emblica, Passiflora edulis and Ginkgo biloba extract A in the whitening and freckle-removing composition is (10-30) μg / mL:(50-150) μg / mL:(0.05-1) μM.

[0007] In some embodiments, the whitening and freckle-removing composition further comprises Salvia officinalis in its composition.

[0008] Optionally, the Salvia officinalis comprises Salvia officinalis raw material and / or Salvia officinalis extract.

[0009] Further optionally, the Salvia officinalis extract comprises water extract of Salvia officinalis raw material and / or alcohol extract of Salvia officinalis raw material.

[0010] Further optionally, the Salvia officinalis extract is Salvianan A.

[0011] In some embodiments, the whitening and freckle-removing composition comprises Phyllanthus emblica, Passiflora edulis, Ginkgo biloba, and Salvia officinalis;

[0012] Further optionally, the ratio of Phyllanthus emblica, Passiflora edulis, Ginkgo biloba, and Salvia officinalis is (0.1-200) μg / mL:(20-150) μg / mL:(0.05-3) μM:(0.05-3) μM.

[0013] In some embodiments, the whitening and freckle-removing composition inhibits tyrosinase activity and / or expression;

[0014] Optionally, the whitening and freckle-removing composition blocks the α-MSH-induced signal pathway to inhibit tyrosinase activity;

[0015] Further optionally, the whitening and freckle-removing composition inhibits the binding of melanocortin receptor 1 and α-MSH;

[0016] Further optionally, the whitening and freckle-removing composition inhibits the generation of cyclic adenosine monophosphate;

[0017] Further optionally, the whitening and freckle-removing composition inhibits the activation of protein kinase A.

[0018] In the second aspect, the present application further provides a conditioner, wherein the conditioner comprises the whitening and freckle-removing composition provided in the first aspect.

[0019] In some embodiments, the conditioner further satisfies at least one of the following features (1)-(4):

[0020] (1) the whitening and freckle-removing composition is added in an amount of 0.1 μg / mL-400 μg / mL;

[0021] (2) the conditioner further comprises an excipient;

[0022] (3) the conditioner has a form selected from the group consisting of a tablet, a granule, a capsule, a solution, a powder, a suspension, an emulsion, a paste, and an aerosol;

[0023] (4) the conditioner has a dosage form selected from the group consisting of an oral dosage, an injection, a spray, an inhalation, a drop, and a suppository.

[0024] In the third aspect, the present application further provides a medicine, wherein the medicine comprises the whitening and freckle-removing composition provided in the first aspect and a pharmaceutically acceptable excipient.

[0025] In some embodiments, the medicine satisfies at least one of the following features (1)-(3):

[0026] (1) the adding amount of the whitening and freckle-removing composition is 0.1 μg / mL-400 μg / mL;

[0027] (2) the form of the medicine includes tablet, granule, capsule, solution, powder, suspension, emulsion, paste or aerosol;

[0028] (3) the dosage form of the medicine includes oral agent, injection, spray, inhalation, drop or suppository.

[0029] In the fourth aspect, the present application further provides a cosmetic, which comprises the whitening and freckle-removing composition provided in the first aspect.

[0030] Optionally, the cosmetic further comprises a base and / or an auxiliary material.

[0031] Further optionally, the base comprises one or more of a solvent base, an oil base, a gum base and a powder base.

[0032] Further optionally, the auxiliary material comprises one or more of a humectant, a skin conditioner, a thickening agent, a moisturizer, a lubricant, an antioxidant, a surfactant, a pH regulator, a sunscreen, a fragrance, a colorant and a preservative.

[0033] Compared with the prior art, the technical scheme of the present application has the beneficial effects that:

[0034] The composition obtained by scientific compounding of the three components of Phyllanthus emblica, Passiflora edulis and Ginkgo biloba extract has a significant inhibitory effect on tyrosinase activity, can inhibit the expression and function of the rate-limiting enzyme TYR in the process of melanin synthesis, and has a significant whitening and freckle-removing activity. At the same time, the components in the composition of the present application are all natural plant raw materials or extracts, which are safe in source and suitable for being added in various conditioners, medicines and cosmetics. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present application, the following describes the technical scheme of the present application in a preferred embodiment of the present application. However, the present application can be realized in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0036] It should be noted that the experimental methods not specified in the following embodiments of the present application are usually carried out under conventional conditions or under the conditions recommended by the manufacturer. The various common chemical reagents used in the embodiments are all commercially available products.

[0037] Unless otherwise defined, 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 application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0038] The ranges disclosed herein are defined by their lower and upper limits. Ranges can be expressed as from one lower limit to one upper limit. When separated by a conjunction or phrase such as "and / or", ranges can comprise any combination of the upper and lower limits, i.e., ranges of "from x to y", "x-y", "x / y" and "x or y" are all specifically allowed ranges. It is specifically intended that the descriptions of ranges include and contemplate all possible sub-ranges.

[0039] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.

[0040] Melanin is a kind of biological pigment contained in animals and plants, which is generated and stored by melanocytes. The content and distribution of melanin affect the skin color. When the skin is exposed to strong ultraviolet radiation, ultraviolet radiation induces the generation of oxygen free radicals, promotes the expression of tyrosinase, stimulates the local skin to generate melanin, and thus causes skin pigmentation.

[0041] Tyrosinase (tyr) is an oxidoreductase widely existing in plants and animals, which is a key enzyme for the synthesis of melanin in organisms. The biosynthesis of melanin is a process in which l-tyrosine (l-tyr) in the body is catalyzed by tyrosinase to generate dopa, dopa is further oxidized to generate dopaquinone, and dopaquinone undergoes a series of chemical reactions to finally generate melanin. Because tyrosinase is involved in skin pigmentation, the inhibition of tyrosinase has been a long-term goal in the fields of skin care research, cosmetics, etc.

[0042] In order to reduce the generation of skin melanin, it is more important to explore safe and effective bioactive substances with whitening effect. The present application aims to provide one or more new bioactive substances to inhibit the activity of tyrosinase and thus control the amount of melanin generation, so as to achieve the effect of skin whitening and freckle removal.

[0043] In a first aspect, the present application provides a whitening and freckle-removing composition, which comprises a phyllanthus emblica, a passion fruit and a discodermolide A in the composition of the whitening and freckle-removing composition.

[0044] The phyllanthus emblica includes phyllanthus emblica raw materials and / or phyllanthus emblica extracts, and the passion fruit includes passion fruit raw materials and / or passion fruit extracts.

[0045] The three components of emblica, passion fruit and panlongqen A are combined scientifically in the present application to produce a significant synergistic tyrosinase activity inhibition effect, thereby achieving the effect of skin whitening and freckle removal.

[0046] In the present application, panlongqen A is extracted from artificially planted emblica or obtained by chemical synthesis.

[0047] In some embodiments, the emblica raw material includes the pulp, peel, flower, branch, stem, and the like of emblica. As a non-limiting example, the emblica includes emblica powder obtained by crushing the emblica raw material.

[0048] In some embodiments, the emblica extract includes substances extracted from the pulp, peel, flower, branch, stem, and the like of emblica.

[0049] In some embodiments, the emblica extract includes water extract of emblica raw material and / or alcohol extract of emblica raw material. As a non-limiting example, the emblica extract includes tannins, phenolic acids, flavonoids, terpenes, sterols, vitamins, and / or volatile oils.

[0050] In some embodiments, the passion fruit raw material includes the fruit, flower, branch, stem, and the like of passion fruit. As a non-limiting example, the passion fruit includes passion fruit powder obtained by crushing the passion fruit raw material.

[0051] In some embodiments, the passion fruit extract includes substances extracted from the fruit, flower, branch, stem, and the like of passion fruit. Further, the substances extracted from the fruit of passion fruit can better achieve the technical effects of the present application.

[0052] In some embodiments, the passion fruit extract includes water extract of passion fruit raw material and / or alcohol extract of passion fruit raw material. As a non-limiting example, the passion fruit extract includes flavonoids, triterpenes, and / or alkaloids, etc.

[0053] In some embodiments, the ratio of Phyllanthus emblica, Passiflora incarnata, and Ginkgo biloba in the whitening and freckle-removing composition is (0.1-200) pg / mL: (20-150) pg / mL: (0.05-3) pM. This includes, but is not limited to, 0.1 pg / mL:20 pg / mL:0.05 pM, 0.1 pg / mL:150 pg / mL:3 pM, 20 pg / mL:20 pg / mL:0.05 pM, 20 pg / mL:100 pg / mL:1 pM, 80 pg / mL:30 pg / mL:0.05 pM, 80 pg / mL:30 pg / mL:3 pM, 100 pg / mL:20 pg / mL:0.05 pM, 100 pg / mL:20 pg / mL:3 pM, or a range formed by any two of the foregoing and numerical values within the range. Further, the ratio of Phyllanthus emblica, Passiflora incarnata, and Ginkgo biloba in the whitening and freckle-removing composition is (20-100) pg / mL: (20-100) pg / mL: (1-3) pM.

[0054] In some embodiments, the whitening and freckle-removing composition further includes Salvia miltiorrhiza in the composition.

[0055] In some embodiments, the Salvia miltiorrhiza includes Salvia miltiorrhiza raw material and / or Salvia miltiorrhiza extract.

[0056] In some embodiments, the Salvia miltiorrhiza raw material includes flowers, branches, stems, roots, and the like of Salvia miltiorrhiza. As a non-limiting example, the Salvia miltiorrhiza includes Salvia miltiorrhiza powder obtained by crushing the Salvia miltiorrhiza raw material.

[0057] In some embodiments, the Salvia miltiorrhiza extract includes substances extracted from flowers, branches, stems, roots, and the like of Salvia miltiorrhiza.

[0058] In some embodiments, the Salvia miltiorrhiza extract includes water extract of Salvia miltiorrhiza raw material and / or alcohol extract of Salvia miltiorrhiza raw material. As a non-limiting example, the Salvia miltiorrhiza extract includes polysaccharides, alkaloids, and / or volatile oils, and the like. Further, the Salvia miltiorrhiza extract described herein is Salvianan A.

[0059] In some embodiments, the whitening and freckle-removing composition includes Phyllanthus emblica, Passiflora incarnata, Ginkgo biloba, and Salvia miltiorrhiza.

[0060] In some embodiments, the ratio of Phyllanthus emblica, Passiflora edulis, Ginkgolide B, and Salvia miltiorrhiza is (0.1-200) μg / mL:(20-150) μg / mL:(0.05-3) μM:(0.05-3) μM. This includes, but is not limited to, 0.1 μg / mL:20 μg / mL:0.05 μM:0.05 μM, 0.1 μg / mL:150 μg / mL:3 μM:3 μM, 20 μg / mL:20 μg / mL:0.05 μM:0.05 μM, 20 μg / mL:100 μg / mL:1 μM:1 μM, 80 μg / mL:30 μg / mL:0.05 μM:0.05 μM, 80 μg / mL:30 μg / mL:3 μM:3 μM, 100 μg / mL:20 μg / mL:0.05 μM:0.05 μM, 100 μg / mL:20 μg / mL:3 μM:3 μM, or a range formed by any two of the foregoing and values within the range. Further, the ratio of Phyllanthus emblica, Passiflora edulis, Ginkgolide B, and Salvia miltiorrhiza is (20-100) μg / mL:(20-100) μg / mL:(1-3) μM:(1-3) μM. Further still, the ratio of Phyllanthus emblica, Passiflora edulis, Ginkgolide B, and Salvia miltiorrhiza is (20-30) μg / mL:(80-100) μg / mL:(1-3) μM:(1-3) μM.

[0061] In some embodiments, the whitening and freckle-removing composition includes Phyllanthus emblica powder, Passiflora edulis extract, Ginkgolide B, and Salvianan A.

[0062] In some embodiments, the whitening and freckle-removing composition inhibits tyrosinase activity and / or expression, thereby controlling the amount of melanin production and achieving the whitening and freckle-removing effect.

[0063] In some embodiments, the whitening and freckle-removing composition inhibits tyrosinase activity and / or expression by blocking the α-MSH-induced signal pathway.

[0064] In some embodiments, the whitening and freckle-removing composition inhibits the binding of melanocortin receptor 1 and α-MSH, thereby inhibiting the production of cyclic adenosine monophosphate and the activation of protein kinase A, inhibiting the binding of melanocortin receptor 1 and α-MSH to block the α-MSH-induced signal pathway, thereby inhibiting tyrosinase activity and / or expression, reducing melanin production and accumulation, and achieving the whitening and freckle-removing effect.

[0065] The present application explicitly provides the functional activity mechanism of the whitening and freckle-removing composition, which can achieve the active effect and produce a significant effect.

[0066] In a second aspect, the present application also provides a conditioner, which comprises the whitening and freckle-removing composition according to the first aspect.

[0067] In some embodiments, the whitening and freckle-removing composition is added in the conditioner in an amount of 0.1 μg / mL to 400 μg / mL. This includes, but is not limited to, 0.1 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 120.6 μg / mL, 130 μg / mL, 150 μg / mL, 180 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, or a range formed by any two of the foregoing and a numerical value within the range.

[0068] In some embodiments, the conditioner further comprises an excipient. The conditioner can be in the form of a tablet, a granule, a capsule, a solution, a powder, a suspension, an emulsion, a paste, or an aerosol.

[0069] In some embodiments, the conditioner is in the form of an oral agent, an injection agent, a spray agent, an inhalation agent, a drop agent, or a suppository.

[0070] In a third aspect, the present application also provides a medicine, which comprises the whitening and freckle-removing composition according to the first aspect and a pharmaceutically acceptable excipient.

[0071] In some embodiments, the whitening and freckle-removing composition is added in the medicine in an amount of 0.1 μg / mL to 400 μg / mL. This includes, but is not limited to, 0.1 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 120.6 μg / mL, 130 μg / mL, 150 μg / mL, 180 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, or a range formed by any two of the foregoing and a numerical value within the range.

[0072] In some embodiments, the medicine is in the form of a tablet, a granule, a capsule, a solution, a powder, a suspension, an emulsion, a paste, or an aerosol.

[0073] In some embodiments, the medicine is in the form of an oral agent, an injection agent, a spray agent, an inhalation agent, a drop agent, or a suppository.

[0074] In a fourth aspect, the present application also provides a cosmetic, which comprises the whitening and freckle-removing composition according to the first aspect.

[0075] In some embodiments, the cosmetic further comprises a base and / or an adjuvant in the ingredients. The base comprises one or more of a solvent base, an oily base, a gum base, and a powder base. As non-limiting examples, the solvent base comprises one or more of water, alcohols, ketones, ethers, and aromatics. The oily base comprises one or more of greases, waxes, higher fatty acids, fatty alcohols, and esters. The gum base comprises one or more of starch, vegetable gum, gelatin, cellulose, polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP). The powder base comprises one or more of talc, kaolin, bentonite, mica, titanium white, zinc white, zinc stearate, magnesium stearate, calcium carbonate, and magnesium carbonate. The adjuvant comprises one or more of humectants, skin conditioning agents, thickening agents, moisturizers, lubricants, antioxidants, surfactants, pH adjusters, sunscreen agents, and fragrance, colorants, preservatives.

[0076] In some embodiments, the cosmetic has a form of a solution, a suspension, an emulsion, a gel, a paste, a cream, a powder, a spray, a foam, or a solid.

[0077] In some embodiments, the cosmetic comprises a cosmetic water, an essence cream, an essence liquid, a foundation, a cream, a mask, or a shampoo.

[0078] For experimental parameters not specified in the following specific examples, refer to the guidelines provided in the present application document, and also refer to experimental manuals in the art or other experimental methods known in the art, or refer to experimental conditions recommended by the manufacturer.

[0079] The raw materials and reagents involved in the following specific examples can be obtained commercially or prepared by those skilled in the art according to known means.

[0080] The sources and models of the related raw materials and cells involved in the specific embodiments of the present application are as follows:

[0081] The mouse melanoma cell B16-F10 was purchased from China Primoas Life Science Co., Ltd.

[0082] Roswell Park Memorial Institute (RPMI) 1640 medium and FBS fetal bovine serum were purchased from Gibco, USA.

[0083] The Cell Counting Kit (CCK-8) cell proliferation-toxicity detection kit was purchased from Japan Tongren Chemical Research Institute.

[0084] The tyrosinase activity detection kit was purchased from the official website of Merck Group, China.

[0085] Phyllanthus emblica powder was purchased from Hunan Langlin Biological Resources Co., Ltd.

[0086] Passion fruit extract was purchased from Xi'an Snot Biological Technology Co., Ltd.

[0087] Spiranthol A (CAS: 126192-35-6), Salvianan A (CAS: 2162956-63-8) were purchased from Tianjin Lummy Kangde New Drug Development Co., Ltd.

[0088] The rest of the various chemical reagents were conventional reagents.

[0089] Example 1, composition and preparation method thereof

[0090] The whitening and freckle-removing composition formula provided in the example is as follows:

[0091] Formula one: 20 μg / mL Phyllanthus emblica powder and 100 μg / mL passion fruit extract and 1 μM Spiranthol A.

[0092] Formula two: 200 μg / mL Phyllanthus emblica powder and 30 μg / mL passion fruit extract and 1 μM Spiranthol A.

[0093] Formula three: 0.1 μg / mL Phyllanthus emblica powder and 150 μg / mL passion fruit extract and 3 μM Spiranthol A.

[0094] Formula four: 80 μg / mL Phyllanthus emblica powder and 30 μg / mL passion fruit extract and 3 μM Spiranthol A.

[0095] Formula five: 100 μg / mL Phyllanthus emblica powder and 20 μg / mL passion fruit extract and 1 μM Spiranthol A.

[0096] Formula six: 20 μg / mL Phyllanthus emblica powder and 100 μg / mL passion fruit extract, 1 μM Spiranthol A and 1 μM Salvianan A.

[0097] The compositions of the above six groups all have the effects of whitening and freckle-removing.

[0098] Comparative example 1, composition and preparation method thereof

[0099] Formula seven: 20 μg / mL Phyllanthus emblica powder and 100 μg / mL passion fruit extract.

[0100] The formula of the example and the comparative example in the application was prepared by mixing the formula amount with sterile water.

[0101] B16-F10 cell is a commonly used melanoma cell line. The present application uses B16-F10 cell to study the effect of the composition on melanin formation and related biological processes. The specific test is as follows.

[0102] Test Example 1, cytotoxicity test of the composition

[0103] The CCK-8 method was used to screen the composition concentration that had no obvious inhibitory effect on the viability of B16-F10 cells. The specific method is as follows:

[0104] Logarithmic growth phase B16-F10 cells were taken, digested, and then cell suspension was prepared with RPMI 1640 complete medium containing 10% fetal bovine serum and 1% double antibody. 100 μL of cell suspension was inoculated into each well of a 96-well plate, and the cell number was 1×10 4 / well. After overnight culture in a 37°C, 5% CO2 incubator, when the cell confluence was about 80%, the cells were treated with 20 μg / mL of Phyllanthus emblica monomer, 100 μg / mL of Passiflora extract, 1 μM of Spiranthol A, 1 μM of Salvianan A, and seven groups of monomer combination formulations (Example 1 and Comparative Example 1).

[0105] A blank group containing only medium, a control group containing medium and cells without adding test substances, and an experimental group containing medium and cells with test substances (Example 1 and Comparative Example 1) were set up. The drug-treated cells were placed in a 37°C, 5% CO2 incubator for 24h. The original culture medium was replaced with culture medium containing 10% CCK-8, and the 96-well plate was placed in a constant temperature incubator for 4h. The OD value of each well at 450nm wavelength was measured, and the cell proliferation survival rate of different drugs was calculated according to formula (1). Five replicate wells were used for each concentration.

[0106] (1)

[0107] The results of the effect of each group on the survival rate of B16-F10 cells obtained by the above test method are shown in Table 1.

[0108] Table 1: Cell survival rate ( ± s, n = 5)

[0109]

[0110] As shown in Table 1, each monomer and composition at the corresponding concentration had no obvious inhibitory effect on the survival rate of B16-F10 cells. The maximum concentration of monomer and composition was used for subsequent tyrosinase activity inhibition rate experiment and Western blot (WB) experiment.

[0111] Test Example 2, Inhibition of Tyrosinase Activity by the Composition

[0112] Tyrosinase activity is related to the amount of melanin synthesis, and controlling the activity can control the amount of melanin production, and inhibiting tyrosinase activity can reduce the production of skin melanin. This test evaluates the whitening and freckle-removing efficacy of the composition by affecting the activity of tyrosinase.

[0113] Four monomers and seven groups of different formulations of compositions and a positive control inhibitor (kojic acid) were selected to evaluate the inhibitory effect of the test substance on tyrosinase activity.

[0114] The experimental method is as follows: according to the Sigma MAK257 kit instructions, centrifuge the kojic acid / tyrosinase / tyrosinase substrate 12000 rpm for 1 min. Dissolve the kojic acid in sterile water to prepare a 0.75 mM working solution, and place it on ice for standby. Dissolve the tyrosinase and tyrosinase substrate with 220 μL of tyrosinase detection buffer and 220 μL of sterile water, respectively, and place them on ice for standby.

[0115] (1) Add 20 μL of test drug to each well of a 96-well plate, 20 μL of 0.75 mM kojic acid as a positive control well, and 20 μL of tyrosinase detection buffer as a negative control well.

[0116] (2) Add 50 μL of enzyme-containing system (48 μL of tyrosinase detection buffer + 2 μL of tyrosinase) to each well, mix well by blowing, and stand for 10 min.

[0117] (3) Add 30 μL of substrate system (23 μL of tyrosinase detection buffer + 2 μL of tyrosinase substrate + 5 μL of tyrosinase enhancer) to each well, mix well by blowing, and stand for 30 min.

[0118] (4) Use a microplate reader to measure the absorbance values at 510 nm in kinetic mode for 30-60 min. Select two time points (T1 and T2) within the linear range and obtain the corresponding absorbance values (Abs1 and Abs2), and calculate the slope by formula (2).

[0119] (2)

[0120] Further, in order to detect the inhibitory effect of each monomer and composition on tyrosinase activity, the relative inhibition rate (%) is calculated by formula (3):

[0121] (3)

[0122] In the formula, Slope1 is the slope of the control group; Slope2 is the slope of the drug to be tested, including each single drug, combination or kojic acid.

[0123] Three groups were tested in parallel, and the results obtained according to the above test method and calculation method are shown in Table 2.

[0124] Table 2: Inhibition effect of tyrosinase activity

[0125]

[0126] The results show that the inhibition effect of the combination on tyrosinase activity is more obvious than that of the single drug after adding the single drug and the combination drug respectively. It is shown that the effect of the combination drug on inhibiting skin melanin synthesis is better than that of the single drug, and has a significant statistical significance. Compared with formula seven, the inhibition effect of formula one to formula six on tyrosinase activity is more significant, and it can be seen that the combination formula needs to be scientifically compounded with the single drug to better play the synergistic effect.

[0127] Test Example Three, Effect of Combination on TYR Protein Expression in B16-F10 Cells

[0128] TYR is a rate-limiting enzyme involved in the synthesis process of melanin. Overexpression of TYR in B16-F10 cells means that these cells have stronger ability to synthesize melanin. Therefore, the cell is an ideal model for studying the mechanism of melanin production and regulation, and the expression and function of TYR are important research objects. The mechanism of the whitening and freckle-removing efficacy of the combination is studied.

[0129] Four single molecules and seven groups of combination were used to treat B16-F10 cells, and Western blot experiment was performed after protein extraction to evaluate the expression level of TYR protein in B16-F10 cells.

[0130] The test method is as follows: logarithmic growth period B16-F10 cells were taken, digested and made into cell suspension with RPMI 1640 complete culture medium containing 10% fetal bovine serum and 1% double antibody, inoculated at 7x10 6The cells were cultured in 6 cm cell culture dishes, 5 mL per dish, in a 37℃, 5% CO2 incubator. After 24 hours of adaptive culture, the control group was replaced with fresh complete medium, and the drug intervention group was replaced with fresh complete medium containing the above monomers or compositions. After 24 hours of culture in a 37℃, 5% CO2 incubator, the cells were scraped off with a cell spatula, centrifuged at 1000 rpm for 5 min, and the total protein was extracted on ice. The protein concentration was determined by BCA kit; 30 μg of protein from each group was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% BSA, and incubated with TYR and GAPDH primary antibodies overnight at 4℃ on a shaker. The membrane was washed with TBS-T, and HRP-labeled secondary antibodies were added and incubated at room temperature for 1 h. The membrane was washed with TBS-T, and the chemiluminescence imaging system was used for imaging. The gray value of the obtained band was analyzed by Fiji software, and the protein expression level was obtained. The results are shown in Table 3.

[0131] Table 3: TYR protein expression level in B16-F10 cells

[0132]

[0133] As shown by the results in Table 3, after the B16-F10 cells were intervened by each monomer and each composition, the expression level of TYR protein was significantly reduced, and the effect of the composition was significantly better than that of the single drug group, with statistical significance. Compared with Formula Seven, the compositions of Formula One to Formula Six had a more significant effect on inhibiting the expression level of TYR protein in B16-F10 cells, indicating that the composition formula needs to be scientifically compounded with single drugs to better play a synergistic effect.

[0134] Data processing: The data were processed using GraphPad Prism 9.3.0 statistical software, and some experimental results were expressed as Mean ± SD.

[0135] This experiment studied the mechanism of the whitening and freckle-removing efficacy of the composition, and showed that the composition could significantly inhibit the expression and function of TYR, and regulate the activity of TYR by blocking the signal pathway induced by α-MSH. This process involves the binding of MC1R (melanocortin receptor 1) and α-MSH, which affects the generation of cAMP (cyclic adenosine monophosphate) and the activation of protein kinase A (PKA), and ultimately affects the expression and function of TYR. This application provides a theoretical basis for the study of the generation and regulation mechanism of melanin.

[0136] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not exist, they should be considered as within the scope of the present disclosure.

[0137] The above-described embodiments are merely illustrative of several embodiments of the present application, which are described in more detail and in a specific manner, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these are all within the scope of the present application. Therefore, the scope of protection of the patent of the present application should be subject to the appended claims.

Claims

1. A whitening and freckle-removing composition, characterized in that, The whitening and freckle-removing composition contains amla powder, passion fruit extract, and Panlongshen A; the amla powder was purchased from Hunan Langlin Biological Resources Co., Ltd., and the passion fruit extract was purchased from Xi'an Sinote Biotechnology Co., Ltd. The ratio of the amla powder, the passion fruit extract and the pangolin A is (0.1-200) μg / mL: (20-150) μg / mL: (0.05-3) μM.

2. The whitening and freckle-removing composition according to claim 1, characterized in that, The whitening and freckle-removing composition also includes Salvianan A.

3. The whitening and freckle-removing composition according to claim 2, characterized in that, The whitening and freckle-removing composition includes amla powder, passion fruit extract, pangolin A and Salvianan A; The ratio of Phyllanthus emblica powder, passion fruit extract, Panlongshen A and Salvianan A is (0.1-200) μg / mL : (20-150) μg / mL : (0.05-3) μM : (0.05-3) μM.

4. A conditioning agent, characterized in that, The conditioning agent comprises the whitening and freckle-removing composition according to any one of claims 1 to 3.

5. The conditioner according to claim 4, characterized in that, The amount of the whitening and freckle-removing composition added to the conditioning agent is 0.1 μg / mL-400 μg / mL.

6. The conditioner according to claim 4, characterized in that, The conditioner also includes excipients.

7. The conditioner according to claim 4, characterized in that, The conditioner may be in the form of tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes, or aerosols.

8. The conditioner according to claim 4, characterized in that, The dosage forms of the conditioning agents include oral preparations, injections, sprays, inhalers, drops, or suppositories.

9. A cosmetic product, characterized in that, The cosmetic product contains the whitening and freckle-removing composition according to any one of claims 1 to 3; The cosmetic ingredients also include a base; The matrix includes one or more of solvent-based matrix, oil-based matrix, colloidal matrix and powder matrix.

10. A cosmetic product, characterized in that, The cosmetic product contains the whitening and freckle-removing composition according to any one of claims 1 to 3; The cosmetic ingredients also include excipients; The excipients include one or more of the following: skin conditioning agents, thickeners, moisturizers, lubricants, antioxidants, surfactants, pH adjusters, sunscreens, and fragrances, colorants, and preservatives.

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