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

Abstract

The application discloses a whitening and freckle-removing composition, conditioner, medicine and cosmetic. The whitening and freckle-removing composition comprises broccoli, ganoderma lucidum and inula japonica in the composition; the broccoli comprises broccoli raw materials and / or broccoli extract, the ganoderma lucidum comprises ganoderma lucidum raw materials and / or ganoderma lucidum extract, and the inula japonica comprises inula japonica raw materials and / or inula japonica extract. The application has significant synergistic activity by scientific compounding of the broccoli and the inula japonica, has significant inhibitory effect on tyrosinase activity, can inhibit the expression and function of the rate-limiting enzyme TYR in the melanin synthesis process, has 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 there is an increasing demand for products with multiple functions such as whitening and freckle-removing. Traditional chemical whitening agents have been gradually eliminated due to safety controversies, and natural active ingredients have become the preferred alternative due to their mild and multi-effect characteristics. 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 broccoli, ganoderma lucidum and inula japonica in its composition.

[0005] The broccoli comprises broccoli raw materials and / or broccoli extract, the ganoderma lucidum comprises ganoderma lucidum raw materials and / or ganoderma lucidum extract, and the inula japonica comprises inula japonica raw materials and / or inula japonica extract.

[0006] In some embodiments, the broccoli, the inula japonica extract in the whitening and freckle-removing composition comprise Ivangustin; and / or,

[0007] The ratio of the broccoli, the ganoderma lucidum and the inula japonica in the whitening and freckle-removing composition is (200-1500) μg / mL:(20-1000) μg / mL:(1-10) μM.

[0008] In some embodiments, the whitening and freckle-removing composition further comprises sophora flavescens in its composition.

[0009] Optionally, the sophora flavescens comprises sophora flavescens raw materials and / or sophora flavescens extract.

[0010] Further optionally, the sophora flavescens extract comprises 2'-methoxy sophora flavescens flavone.

[0011] In some embodiments, the ratio of the Brassica napus, the Ganoderma lucidum, the Inula japonica and the Sophora flavescens is (200-1500) μg / mL:(20-1000) μg / mL:(1-10) μM:(1-10) μM.

[0012] In some embodiments, the whitening and freckle-removing composition inhibits tyrosinase activity and / or expression;

[0013] Optionally, the whitening and freckle-removing composition blocks the α-MSH-induced signal pathway to inhibit tyrosinase activity;

[0014] Further optionally, the whitening and freckle-removing composition inhibits the binding of melanocortin receptor 1 and α-MSH;

[0015] Further optionally, the whitening and freckle-removing composition inhibits the generation of cyclic adenosine monophosphate;

[0016] Further optionally, the whitening and freckle-removing composition inhibits the activation of protein kinase A.

[0017] In the second aspect, the present application further provides a conditioning agent, which comprises the whitening and freckle-removing composition provided in the first aspect.

[0018] In some embodiments, the conditioning agent further satisfies at least one of the following features (1)-(4):

[0019] (1) the addition amount of the whitening and freckle-removing composition is 20 μg / mL-2600 μg / mL;

[0020] (2) the conditioning agent further comprises an auxiliary material;

[0021] (3) the form of the conditioning agent comprises a tablet, a granule, a capsule, a solution, a powder, a suspension, an emulsion, a paste or an aerosol;

[0022] (4) the dosage form of the conditioning agent comprises an oral agent, an injection agent, a spray agent, an inhalation agent, a drop agent or a suppository.

[0023] In the third aspect, the present application further provides a medicine, which comprises the whitening and freckle-removing composition provided in the first aspect and a pharmaceutically acceptable auxiliary material.

[0024] In some embodiments, the medicine satisfies at least one of the following features (1)-(3):

[0025] (1) the addition amount of the whitening and freckle-removing composition is 20 μg / mL-2600 μg / mL;

[0026] (2) the form of the medicine includes tablet, granule, capsule, solution, powder, suspension, emulsion, paste or aerosol;

[0027] (3) the dosage form of the medicine includes oral agent, injection, spray, inhalation, drop or suppository.

[0028] In a fourth aspect, the present application further provides a cosmetic product, which comprises the whitening and freckle-removing composition according to the first aspect.

[0029] Optionally, the cosmetic product further comprises a base and / or an auxiliary material.

[0030] Further optionally, the base comprises one or more of a solvent base, an oil base, a gum base and a powder base.

[0031] Further optionally, the auxiliary material comprises one or more of a moisturizer, a skin conditioner, a thickening agent, a humectant, a lubricant, an antioxidant, a surfactant, a pH adjuster, a sunscreen, a fragrance, a colorant and a preservative.

[0032] Compared with the prior art, the technical scheme of the present application has the beneficial effects that:

[0033] The composition obtained by scientific compounding of broccoli, ganoderma lucidum and inula japonica 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 to various conditioners, medicines and cosmetics. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present application, the following describes the technical scheme of the present application in a more comprehensive manner. 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.

[0039] 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.

[0040] 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 is further subjected to 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.

[0041] 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.

[0042] In a first aspect, the present application provides a whitening and freckle-removing composition, which comprises broccoli, ganoderma lucidum and inula japonica in its composition, wherein the broccoli comprises broccoli raw material and / or broccoli extract, the ganoderma lucidum comprises ganoderma lucidum raw material and / or ganoderma lucidum extract, and the inula japonica comprises inula japonica raw material and / or inula japonica extract.

[0043] The application uses Brassica napus, Ganoderma lucidum and Inula japonica in combination, and scientific compatibility produces a significant synergistic tyrosinase activity inhibition effect, thereby achieving the effect of skin whitening and freckle removal.

[0044] In some embodiments, the Brassica napus raw material includes roots, branches, stems, leaves, seeds and other parts of Brassica napus. As a non-limiting example, the Brassica napus includes crushing the Brassica napus raw material.

[0045] In some embodiments, the Brassica napus extract includes substances extracted from roots, branches, stems, leaves, seeds and other parts of Brassica napus.

[0046] In some embodiments, the Brassica napus extract includes water extract of Brassica napus raw material and / or alcohol extract of Brassica napus raw material. As a non-limiting example, the Brassica napus extract includes active ingredients such as sulforaphane.

[0047] In some embodiments, the Ganoderma lucidum includes Ganoderma lucidum raw material and / or Ganoderma lucidum extract.

[0048] In some embodiments, the Ganoderma lucidum raw material includes mycelium and / or fruiting body of Ganoderma lucidum. As a non-limiting example, the Ganoderma lucidum includes crushing the Ganoderma lucidum raw material.

[0049] In some embodiments, the Ganoderma lucidum extract includes substances extracted from mycelium and / or fruiting body of Ganoderma lucidum.

[0050] In some embodiments, the Ganoderma lucidum extract includes water extract of Ganoderma lucidum raw material and / or alcohol extract of Ganoderma lucidum raw material. As a non-limiting example, the Ganoderma lucidum extract includes active ingredients such as polysaccharides, triterpenes and alkaloids.

[0051] In some embodiments, the Inula japonica raw material includes flowers, branches, stems, roots, seeds and other parts of Inula japonica. As a non-limiting example, the Inula japonica includes crushing the Inula japonica raw material.

[0052] In some embodiments, the Inula japonica extract includes substances extracted from flowers, branches, stems, roots, seeds and other parts of Inula japonica.

[0053] In some embodiments, the Inula japonica extract includes water extract of Inula japonica raw material and / or alcohol extract of Inula japonica raw material. As a non-limiting example, the Inula japonica extract includes sesquiterpenes, diterpenes, flavonoids, steroids, ketones, alcohols, phenolic acids and polysaccharides. Further, the Inula japonica extract described in the application includes Ivangustin (CAS: 14164-59-1).

[0054] In some embodiments, the ratio of the Brassica napus, the Ganoderma lucidum, and the Inula japonica in the whitening and freckle-removing composition is (200-1500) pg / mL: (20-1000) pg / mL: (1-10) pM. This includes, but is not limited to, 200 pg / mL:20 pg / mL:1 pM, 200 pg / mL:1000 pg / mL:1 pM, 200 pg / mL:1000 pg / mL:5 pM, 200 pg / mL:1000 pg / mL:10 pM, 300 pg / mL:20 pg / mL:1 pM, 500 pg / mL:100 pg / mL:1 pM, 500 pg / mL:100 pg / mL:5 pM, 1000 pg / mL:200 pg / mL:5 pM, 1500 pg / mL:20 pg / mL:1 pM, 1500 pg / mL:50 pg / mL:5 pM, 1500 pg / mL:1000 pg / mL:1 pM, 1500 pg / mL:1000 pg / mL:10 pM, or a range formed by any two of the foregoing and numerical values within the range. Further, the ratio of the Brassica napus, the Ganoderma lucidum, and the Inula japonica in the whitening and freckle-removing composition is (200-1000) pg / mL: (100-1000) pg / mL: (1-10). Further, the ratio of the Brassica napus, the Ganoderma lucidum, and the Inula japonica in the whitening and freckle-removing composition is (200-1000) pg / mL: (200-1000) pg / mL: (5-10) pM.

[0055] In some embodiments, the whitening and freckle-removing composition further includes Sophora flavescens in the composition.

[0056] In some embodiments, the Sophora flavescens raw material includes flowers, leaves, branches, stems, roots, seeds, and the like of Sophora flavescens. As a non-limiting example, the Sophora flavescens includes Sophora flavescens raw material that is crushed.

[0057] In some embodiments, the Sophora flavescens extract includes substances extracted from flowers, leaves, branches, stems, roots, seeds, and the like of Sophora flavescens.

[0058] In some embodiments, the Sophora flavescens extract includes water extract of Sophora flavescens raw material and / or alcohol extract of Sophora flavescens raw material. As a non-limiting example, Sophora flavescens extract includes flavonoids, phenols, alkaloids, and the like. Further, the Sophora flavescens extract described in the present application includes 2'-methoxySophora flavescens flavone (CAS: 270249-38-2).

[0059] In some embodiments, the ratio of the Brassica napus, the Ganoderma lucidum, the Inula japonica, and the Sophora flavescens in the whitening and freckle-removing composition is (200-1500) pg / mL:(20-1000) pg / mL:(1-10) pM:(1-10) pM. This includes, but is not limited to, 200 pg / mL:20 pg / mL:1 pM:1 pM, 200 pg / mL:1000 pg / mL:1 pM:1 pM, 200 pg / mL:1000 pg / mL:5 pM:5 pM, 200 pg / mL:1000 pg / mL:10 pM:10 pM, 300 pg / mL:20 pg / mL:1 pM:1 pM, 500 pg / mL:100 pg / mL:1 pM:1 pM, 500 pg / mL:100 pg / mL:1 pM:5 pM, 500 pg / mL:100 pg / mL:5 pM:5 pM, 500 pg / mL:100 pg / mL:1 pM:10 pM, 1000 pg / mL:200 pg / mL:5 pM:5 pM, 1500 pg / mL:20 pg / mL:1 pM:1 pM, 1500 pg / mL:50 pg / mL:5 pM:5 pM, 1500 pg / mL:1000 pg / mL:1 pM:1 pM, 1500 pg / mL:1000 pg / mL:10 pM:10 pM, or a range formed by any two of the foregoing and numerical values within the range.

[0060] Further, the ratio of the Brassica napus, the Inula japonica, the Ganoderma lucidum, and the Sophora flavescens in the whitening and freckle-removing composition is (200-1000) pg / mL:(100-1000) pg / mL:(1-10) pM:(1-10) pM. Further, the ratio of the Brassica napus, the Inula japonica, the Ganoderma lucidum, and the Sophora flavescens in the whitening and freckle-removing composition is (200-1000) pg / mL:(100-1000) pg / mL:(1-5) pM:(1-10) pM. Further, the ratio of the Brassica napus, the Inula japonica, the Ganoderma lucidum, and the Sophora flavescens in the whitening and freckle-removing composition is (400-600) pg / mL:(100-200) pg / mL:(1-5) pM:(1-10) pM.

[0061] In some embodiments, the composition of the whitening and freckle-removing composition includes Brassica napus seed extract, Ganoderma lucidum extract, Ivangustin, and 2'-methoxySophora flavescens flavone.

[0062] In some embodiments, the whitening and freckle-removing composition inhibits tyrosinase activity and / or expression, thereby controlling the amount of melanin production, and thus 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 tyrosinase activity and / or expression by blocking the α-MSH-induced signal pathway.

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

[0066] In the second aspect, the present application further provides a conditioner, which comprises the whitening and freckle-removing composition provided in the first aspect.

[0067] In some embodiments, the whitening and freckle-removing composition is added in the conditioner in an amount of 20 μg / mL-2600 μg / mL, including but not limited to 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL, 1100 μg / mL, 1200 μg / mL, 1300 μg / mL, 1400 μg / mL, 1500 μg / mL, 2000 μg / mL, 2500 μg / mL, 2600 μg / mL, or a range formed by any two of the foregoing and numerical values within the range. Further, the whitening and freckle-removing composition is added in the conditioner in an amount of 500 μg / mL-800 μg / mL.

[0068] In some embodiments, the conditioner further comprises an auxiliary material. 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, a spray, an inhalant, a drop, or a suppository.

[0070] In the third aspect, the present application further provides a medicine, which comprises the whitening and freckle-removing composition provided in the first aspect and a pharmaceutically acceptable auxiliary material.

[0071] In some embodiments, the amount of the whitening and spot-removing composition added to the medicament is 20 μg / mL to 2600 μg / mL. This includes, but is not limited to, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL, 1100 μg / mL, 1200 μg / mL, 1300 μg / mL, 1400 μg / mL, 1500 μg / mL, 2000 μg / mL, 2500 μg / mL, 2600 μg / mL, or any range formed by both of the foregoing, and values ​​within that range. Further, the amount of the whitening and spot-removing composition added to the medicament is 500 μg / mL to 800 μg / mL.

[0072] In some embodiments, the form of the drug includes tablets, granules, capsules, solutions, powders, suspensions, emulsions, pastes, or aerosols.

[0073] In some embodiments, the dosage form of the drug includes oral, injectable, spray, inhaler, drop, or suppository.

[0074] Fourthly, this application also provides a cosmetic product, the ingredients of which include the whitening and freckle-removing composition provided in the first aspect.

[0075] In some embodiments, the cosmetic ingredients further include a matrix and / or excipients. The matrix includes one or more of solvent-based matrices, oil-based matrices, gel-based matrices, and powder-based matrices. As a non-limiting example, the solvent-based matrix includes one or more of water, alcohols, ketones, ethers, and fragrances. The oil-based matrix includes one or more of oils, waxes, higher fatty acids, fatty alcohols, and esters. The gel-based matrix includes one or more of starch, plant gums, gelatin, cellulose, polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP). The powder-based matrix includes one or more of talc, kaolin, bentonite, mica, titanium dioxide, zinc dioxide, zinc stearate, magnesium stearate, calcium carbonate, and magnesium carbonate. The excipients include one or more of moisturizers, skin conditioning agents, thickeners, lubricants, antioxidants, surfactants, pH adjusters, sunscreens, and fragrances, colorants, and preservatives.

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

[0077] In some embodiments, the cosmetics include toners, serums, essences, foundations, creams, masks, or shampoos.

[0078] The experimental parameters not written in the following specific examples are preferably referred to the guidance given in the present application document, and can also be referred to the experimental manual in the art or other experimental methods known in the art, or the experimental conditions recommended by the manufacturer.

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

[0080] The sources and models of the related raw materials and cells involved in the specific examples 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] The ganoderma extract was purchased from Shaanxi Jiahuo Biological Technology Co., Ltd.

[0086] The broccoli seed extract was purchased from Xi'an Yiao Biological Technology Co., Ltd.

[0087] 2'-Methoxykurarinone is a compound isolated from the roots of Sophora flavescens. The 2'-Methoxykurarinone (CAS: 270249-38-2) used in the present application was purchased from Tianjin Yikangkang New Drug Development Co., Ltd.

[0088] Ivangustin is a sesquiterpene isolated from the medicinal plant Inula britannica. The Ivangustin (CAS: 14164-59-1) used in the present application was purchased from Tianjin Yikangkang New Drug Development Co., Ltd.

[0089] The remaining various chemical reagents are conventional reagents.

[0090] Example 1, composition and preparation method thereof

[0091] The whitening and freckle-removing composition provided in the present example has the following formula:

[0092] Formula One: 500 μg / mL broccoli seed extract, 100 μg / mL Ganoderma extract, and 1 μM Ivangustin.

[0093] Formula Two: 300 μg / mL broccoli seed extract, 20 μg / mL Ganoderma extract, and 1 μM Ivangustin.

[0094] Formula Three: 1500 μg / mL broccoli seed extract, 50 μg / mL Ganoderma extract, and 5 μM Ivangustin.

[0095] Formula Four: 1000 μg / mL broccoli seed extract, 200 μg / mL Ganoderma extract, and 5 μM Ivangustin.

[0096] Formula Five: 200 μg / mL broccoli seed extract, 1000 μg / mL Ganoderma extract, and 10 μM Ivangustin.

[0097] Formula Six: 500 μg / mL broccoli seed extract, 100 μg / mL Ganoderma extract, 1 μM Ivangustin, and 1 μM 2'-methoxykopsan.

[0098] Formula Seven: 500 μg / mL broccoli seed extract, 100 μg / mL Ganoderma extract, 5 μM Ivangustin, and 10 μM 2'-methoxykopsan.

[0099] The compositions of the above seven groups all have the effects of whitening and removing freckles.

[0100] Comparative Example 1: Composition and method for preparing the same

[0101] Formula Eight: 500 μg / mL broccoli seed extract and 100 μg / mL Ganoderma extract.

[0102] The formulas of the examples and comparative examples in the present application are prepared by mixing the formula amount with sterile water.

[0103] B16-F10 cell is a commonly used melanoma cell line. The present application uses B16-F10 cell to study the effects of the composition on the formation of melanin and related biological processes. The specific test is as follows.

[0104] Test Example One: Cytotoxicity test of the composition

[0105] The CCK-8 method is used to screen the composition concentration that has no obvious inhibitory effect on the viability of B16-F10 cells. The specific method is as follows:

[0106] B16-F10 cells in logarithmic growth phase were digested and then suspended in RPMI 1640 complete medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics. The suspension was then seeded into 96-well plates at a density of 100 μL per well (1 × 10⁶ cells). 4 / well, cultured overnight in a 37°C, 5% CO2 incubator. When the cell confluence was about 80%, the cells were treated with eight formulations of single drugs (Example 1 and Comparative Example 1) consisting of 500 μg / mL broccoli seed extract, 100 μg / mL Ganoderma lucidum extract, 1 μM Ivangustin and 1 μM 2'-methoxymatrine.

[0107] The following groups were set up: a blank group containing only culture medium, a control group containing culture medium and cells without the test substance, and an experimental group containing culture medium and cells containing the test substance (Example 1 and Comparative Example 1). After drug treatment, the cells were placed in a 37°C, 5% CO2 incubator for 24 hours. The original culture medium was then replaced with a medium containing 10% CCK-8, and the 96-well plate was placed in an incubator for 4 hours. The OD value of each well was measured at a wavelength of 450 nm. The cell proliferation and survival rate of different drugs were calculated according to formula (1). Five replicates were performed for each concentration.

[0108] (1)

[0109] The results of the effects of each group on the survival rate of B16-F10 cells obtained by the above experimental method are shown in Table 1.

[0110] Table 1: Cell viability ( ±s, n=5)

[0111]

[0112] The results in Table 1 show that none of the single drugs or combinations significantly inhibited the survival rate of B16-F10 cells at their respective concentrations. Subsequent tyrosinase activity inhibition rate experiments and Western blot (WB) experiments were conducted using the maximum concentration of the single drug and the combination.

[0113] Experimental Example 2: Inhibitory effect of the composition on tyrosinase activity

[0114] Tyrosinase activity is related to melanin synthesis; controlling its activity can control melanin production, and inhibiting tyrosinase activity can reduce melanin production in the skin. This experiment evaluates the skin-whitening and spot-removing efficacy of the composition by its effect on tyrosinase activity.

[0115] Four single drugs and eight different formulations, along with a positive control inhibitor (kojic acid), were selected to evaluate the inhibitory effect of the test substances on tyrosinase activity.

[0116] The experimental method is as follows: according to the Sigma MAK257 kit instructions, centrifuge kojic acid / tyrosinase / tyrosinase substrate 12000 rpm for 1 min. Prepare a 0.75 mM working solution of kojic acid with sterile water and place on ice for later use. Dissolve tyrosinase and tyrosinase substrate with 220 μL of tyrosinase detection buffer and 220 μL of sterile water, respectively, and place on ice for later use.

[0117] (1) Add 20 μL of test drug wells, 20 μL of 0.75 mM kojic acid as positive control wells, and 20 μL of tyrosinase assay buffer as negative control wells in sequence in a 96-well plate.

[0118] (2) Add 50 μL of enzyme-containing system (48 μL of tyrosinase assay buffer + 2 μL of tyrosinase) to each well, mix by blowing, and stand for 10 min.

[0119] (3) Add 30 μL of substrate system (23 μL of tyrosinase assay buffer + 2 μL of tyrosinase substrate + 5 μL of tyrosinase enhancer) to each well, mix by blowing, and stand for 30 min.

[0120] (4) Measure the absorbance values at 510 nm in kinetic mode for 30-60 min using a microplate reader. 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).

[0121] (2)

[0122] Further, in order to detect the inhibitory effect of each single drug and combination on tyrosinase activity, the relative inhibition rate (%) is calculated by formula (3):

[0123] (3)

[0124] Wherein Slope1 is the slope of the control group; Slope2 is the slope of the test drug, including each single drug, combination or kojic acid.

[0125] Three groups were tested in parallel, and the results obtained according to the above test method and calculation method are shown in Table 2.

[0126] Table 2: Inhibition effect of tyrosinase activity

[0127]

[0128] The results show that the combination of drugs has a more obvious inhibitory effect on tyrosinase activity than single drugs. This indicates that the combination of drugs has a better effect on inhibiting the synthesis of skin melanin than single drugs. Compared with formula eight, the tyrosinase activity inhibition effect of formula one to formula seven is more significant. It can be seen that the combination formula needs to be scientifically compounded with single drugs to better play a synergistic effect.

[0129] Test Example Three, Effect of the Combination on TYR Protein Expression in B16-F10 Cells

[0130] TYR is a rate-limiting enzyme involved in the synthesis of melanin. B16-F10 cells overexpress TYR, which means that these cells have a stronger ability to synthesize melanin. Therefore, this application uses this cell as an ideal model for studying the mechanism of melanin production and regulation, and takes the expression and function of TYR as an important research object to study the mechanism of the whitening and freckle-removing efficacy of the combination.

[0131] Four single drugs and eight groups of combination compositions were used to treat B16-F10 cells. After protein extraction, Western blot experiment was performed to evaluate the expression level of TYR protein in B16-F10 cells.

[0132] The test method is as follows: take B16-F10 cells in logarithmic growth phase, digest them with RPMI 1640 complete culture medium containing 10% fetal bovine serum and 1% double antibody to prepare a cell suspension, inoculate 7x10 6 in 6 cm cell culture dishes, 5 mL per dish, and culture in a 37°C, 5% CO2 incubator. After 24 hours of adaptive culture, the control group was replaced with fresh complete culture medium, and the drug intervention group was replaced with fresh complete culture medium containing the above single drug or combination. After 24 hours of culture in a 37°C, 5% CO2 incubator, the cells were scraped off with a cell spatula, centrifuged at 1000 rpm for 5 min, lysed on ice to extract total cell protein, and 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°C on a shaker. The membrane was washed with TBS-T, HRP-labeled secondary antibody was added and incubated at room temperature for 1 h, the membrane was washed with TBS-T, and the chemiluminescence imaging system was used to take pictures. The gray value of the band was analyzed by Fiji software to obtain the protein expression level. The results are shown in Table 3.

[0133] Table 3: TYR Protein Expression Level in B16-F10 Cells

[0134]

[0135] As shown by the results in Table 3, after the B16-F10 cells were intervened by each single drug and each composition, the expression level of TYR protein was significantly reduced, and the effect of the composition was obviously better than that of the single drug group. Compared with formula eight, the compositions of formula one to formula seven had a more significant effect on inhibiting the expression level of TYR protein in B16-F10 cells, which indicated that the composition formula needed to be scientifically compounded with the single drug to better play a synergistic effect.

[0136] Data processing, the data were processed by GraphPad Prism 9.3.0 statistical software, and part of the experimental results were expressed as Mean ± SD.

[0137] The experiment showed that the composition could significantly inhibit the expression and function of TYR by studying the mechanism of the whitening and freckle-removing efficacy of the composition, and by blocking the signal pathway induced by α-MSH to regulate the activity of TYR. This process involves the binding of MC1R (melanocortin receptor 1) and α-MSH, which in turn 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. The present application provides a theoretical basis for studying the generation and regulation mechanism of melanin.

[0138] Each technical feature of the above-described embodiments can be combined arbitrarily, and to make the description concise, each technical feature in the above-described embodiments is not described in all possible combinations, however, as long as the combination of these technical features does not exist contradictory, it should be considered as the scope of the present application.

[0139] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A skin whitening and freckle-removing composition, characterized by comprising, The composition of the whitening and freckle-removing composition comprises Brassica napus L. seed extract, Ganoderma lucidum extract and Inula japonica extract; The Brassica napus L. seed extract is purchased from Xi'an Youao Biological Technology Co., Ltd.; the Ganoderma lucidum extract is purchased from Shaanxi Jiahe Biological Technology Co., Ltd.; and the Inula japonica extract is Ivangustin.

2. The whitening and freckle-removing composition according to claim 1, characterized by, The ratio of the Brassica napus L. seed extract, the Ganoderma lucidum extract and the Inula japonica extract in the whitening and freckle-removing composition is (200-1500) μg / mL:(20-1000) μg / mL:(1-10) μM.

3. The whitening and freckle-removing composition according to claim 1 or 2, characterized by, The composition of the whitening and freckle-removing composition further comprises Sophora flavescens extract; The Sophora flavescens extract is 2'-methoxySophora flavescens flavone.

4. The whitening and freckle-removing composition according to claim 3, characterized by, The ratio of the Brassica napus L. seed extract, the Ganoderma lucidum extract, the Inula japonica extract and the Sophora flavescens extract is (200-1500) μg / mL:(20-1000) μg / mL:(1-10) μM:(1-10) μM.

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

6. The conditioning agent of claim 5, wherein, The conditioning agent further satisfies at least one of the following characteristics (1)-(4): (1) the addition amount of the whitening and freckle-removing composition is 20 μg / mL-2600 μg / mL; (2) the composition of the conditioning agent further comprises an auxiliary material; (3) the form of the conditioning agent comprises sheet, granule, capsule, solution, powder, suspension, emulsion, paste or aerosol; (4) the dosage form of the conditioning agent comprises oral agent, injection, spray, inhalation, drop or suppository.

7. A cosmetic product, characterized by, The composition of the cosmetic comprises the whitening and freckle-removing composition according to any one of claims 1-4; The composition of the cosmetic further comprises an auxiliary material; The auxiliary material comprises one or more of moisturizer, skin conditioner, thickening agent, humectant, lubricant, antioxidant, surfactant, pH regulator, sunscreen, essence and flavor, colorant and preservative.

8. A cosmetic product, characterized by, The composition of the cosmetic comprises the whitening and freckle-removing composition according to any one of claims 1-4; The composition of the cosmetic further comprises a base; The base comprises one or more of solvent base, oily base, gum base and powder base.

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