Whitening and freckle-removing composition, gel, preparation method therefor, and use thereof

Abstract

Provided are a whitening and freckle-removing composition, a gel, a preparation method therefor, and a use thereof, relating to the technical field of skin-care products. The whitening and freckle-removing composition comprises tranexamic acid, nicotinamide, 4-butylresorcinol, licorice extract, 3-o-ethyl ascorbic acid, and glutathione. For the whitening composition, by inhibiting the activity of tyrosinase, blocking the transfer of melanin from melanocytes to keratinocytes, and inhibiting the formation and accumulation of melanin by means of multiple mechanisms including oxidation resistance, and by means of the synergistic action of all components, cosmetic benefits of whitening and freckle-removing are achieved. The whitening and freckle-removing gel has a simple preparation method, has a mild and non-sensitizing formula, is safe and non-irritating, and is suitable for large-scale production and application.

Description

A whitening and freckle-removing composition, a gel and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of skin care, in particular to a whitening and freckle-removing composition, a gel and a preparation method and application thereof. BACKGROUND

[0002] Melanin is mainly produced by melanocytes in the basal layer of the epidermis of human skin, and it plays a crucial role in protecting the skin from ultraviolet radiation and oxidative stress caused by external pollutants. When the skin is excessively exposed to sunlight, tyrosinase in the melanocytes in the basal layer of the skin is activated, thereby converting intracellular tyrosine into melanin. Excessive secretion of melanin will gradually transfer to keratinocytes, causing the skin to darken. Abnormal accumulation of melanin in the basal layer will cause hyperpigmentation, leading to melasma, freckles, age spots and other conditions, affecting people's quality of life.

[0003] The production of melanin includes several different links such as initiation, production, transport and metabolism. With the development of consumer product development technology, in order to prevent skin darkening, spots, freckles and other conditions, and achieve whitening effects, it has been found that melanin can be reduced in the following ways to achieve skin whitening and reduce pigmented spots: (1) preventing the proliferation of melanocytes; (2) inhibiting tyrosinase activity, preventing tyrosine oxidation or blocking melanin transfer; (3) accelerating the metabolism and exfoliation of keratin melanin, etc. At present, there are numerous freckle-removing products on the market, but most of the freckle-removing and whitening products on the market are mainly composed of chemical ingredients, and the whitening strategies are too single. Short-term use may have some effect, but long-term use will cause the skin to develop resistance and rebound, and the effect will deteriorate. For example, single whitening products that inhibit tyrosinase are prone to rebound after use. Therefore, it is necessary to develop a multi-strategy synergistic whitening product that not only inhibits the synthesis of pigments but also blocks the transport of pigments and enhances the antioxidant capacity of the skin, to stably and persistently inhibit the formation and accumulation of pigmented spots. SUMMARY

[0004] The present application aims to overcome the defects in the prior art and provide a whitening and freckle-removing composition, a gel and a preparation method and application thereof. The whitening and freckle-removing composition of the present application not only inhibits the synthesis of pigments but also blocks the transport of pigments and enhances the antioxidant capacity of the skin, and is a multi-strategy synergistic whitening product.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a whitening and freckle-removing composition, which comprises the following components in parts by weight: 1-10 parts of tranexamic acid, 0.1-5 parts of nicotinamide, 0.01-0.5 parts of 4-butylresorcinol, 0.001-0.1 parts of glycyrrhiza extract, 1-10 parts of 3-o-ethyl ascorbic acid and 0.5-5 parts of glutathione.

[0007] Preferably, the whitening and freckle-removing composition further comprises a humectant, a pH regulator, a soothing agent, a skin conditioner;

[0008] The humectant comprises butanediol and polyethylene glycol-400, and the humectant is in a weight fraction of 1-10 parts.

[0009] The pH regulator comprises lactic acid or arginine, and the pH regulator is in a weight fraction of 0.5-5 parts.

[0010] The soothing agent comprises dipotassium glycyrrhizinate, and the soothing agent is in a weight fraction of 0.5-5 parts.

[0011] The skin conditioner comprises one or more of a whitening and freckle-removing component, an anti-aging component and an antioxidant component.

[0012] Preferably, the whitening and freckle-removing component comprises one or more of alpha arbutin, phloretin, resveratrol, nonapeptide-1 and resorcinol derivatives, and the whitening and freckle-removing component is in a weight fraction of 0.5-5 parts.

[0013] Preferably, the anti-aging component comprises one or more of ceramide and derivatives thereof, polypeptide, carnosine, ekdine, boswella, polydeoxyribonucleotide, nicotinamide adenine dinucleotide and glyceroglucoside, and the anti-aging component is in a weight fraction of 1-10 parts.

[0014] Preferably, the antioxidant component comprises one or more of butylated hydroxyanisole, coenzyme Q10, astaxanthin, vitamin E, ergothioneine, superoxide dismutase and ascorbic acid and derivatives thereof, and the antioxidant component is in a weight fraction of 1-10 parts.

[0015] Preferably, the skin conditioner further comprises a plant extract, and the plant extract comprises one or more of aloe extract, gentian extract, sophora flavescens extract, ginseng extract, tea extract, wild soybean seed extract and centella asiatica extract, and the plant extract is in a weight fraction of 0.01-5 parts.

[0016] The present application further provides use of the whitening and freckle-removing composition in preparation of a skin care product, and the skin care product comprises a gel, a water agent, an essence, a lotion, a cream, an ointment or a mask.

[0017] The application further provides a whitening and freckle-removing gel made of the following components with mass percentage:

[0018] A: sodium hyaluronate 2.5-3.5%, polyvinylpyrrolidone 0.8-1.2%, and polyvinyl alcohol 2.5-3.5%;

[0019] B: tranexamic acid 2.5-3.5%, arginine 0.8-1.0%, glutathione 0.8-1.2%, 3-o-ethyl ascorbic acid 1.8-2.3%, glycyrrhizic acid dipotassium 0.4-0.5%, nicotinamide 0.5-0.7%, carnosine 0.09-0.11%, wild soybean seed extract 0.11-0.13%, and lactic acid 0.09-0.12%;

[0020] C: polyethylene glycol-4000 0.6-0.8%, butanediol 2.5-3.5%, glycyrrhiza extract 0.008-0.01%, and 4-butyl resorcinol 0.04-0.06%;

[0021] the rest of water.

[0022] The application further provides a preparation method of the whitening and freckle-removing gel, comprising the following steps:

[0023] (1) mixing A with water accounting for 45-50% of the total mass of the gel, stirring, dissolving at 87-92°C for 50-70 min to obtain a solution;

[0024] (2) mixing B with water accounting for 30-36% of the total mass of the gel, filtering after dissolving to obtain a filtrate;

[0025] (3) mixing the components in C to obtain a mixture;

[0026] (4) after the solution is cooled to 20-25°C, mixing the solution with the filtrate and the mixture, stirring for 50-70 min to obtain the whitening and freckle-removing gel.

[0027] By adopting the technical scheme, the whitening and freckle-removing composition has the following beneficial effects: the whitening and freckle-removing composition comprises tranexamic acid, nicotinamide, 4-butylresorcinol, licorice extract, 3-o-ethyl ascorbic acid and glutathione. The tranexamic acid and the 4-butylresorcinol in the whitening and freckle-removing composition provided by the application are a kind of protease inhibitor, which can inhibit the activity of tyrosinase, a key enzyme in the process of melanin production. The licorice extract is extracted from the root of Glycyrrhiza glabra, can penetrate into the inside of the skin and maintain high activity, and can effectively inhibit the activity of various enzymes (such as tyrosinase) in the process of melanin production. At the same time, the licorice extract also has the effects of preventing rough skin and anti-inflammatory and antibacterial. The nicotinamide can inhibit the transfer of melanin to keratinocytes. The 3-o-ethyl ascorbic acid is a derivative of vitamin C, which has good stability and strong antioxidant property. In addition, the 3-o-ethyl ascorbic acid also has the effects of promoting wound healing and anti-inflammatory. The glutathione is a kind of tripeptide containing thiol groups, which is combined by glutamic acid, cysteine and glycine, and has the effects of antioxidant and integrated detoxification. The whitening and freckle-removing composition provided by the application mainly plays a role through the key nodes related to melanin deposition (inhibition of tyrosinase activity, inhibition of melanin transfer and antioxidant), and the components synergistically improve the whitening effect. The preparation method of the whitening and freckle-removing gel of the application is simple, the formula is mild and non-allergic, safe and non-irritating, and is suitable for large-scale production and application. BRIEF DESCRIPTION OF DRAWINGS

[0028] Fig. 1 is a graph of the whitening effect of the whitening and freckle-removing composition on a zebrafish model.

[0029] Fig. 2 is a cytotoxicity result graph of the whitening and freckle-removing composition.

[0030] Fig. 3 is a graph of the inhibitory effect of tranexamic acid on melanin. DETAILED DESCRIPTION

[0031] The application provides a whitening and freckle-removing composition, which comprises the following components in parts by weight: 1-10 parts of tranexamic acid, 0.1-5 parts of nicotinamide, 0.01-0.5 parts of 4-butylresorcinol, 0.001-0.1 parts of licorice extract, 1-10 parts of 3-o-ethyl ascorbic acid and 0.5-5 parts of glutathione.

[0032] In the whitening and freckle-removing composition provided by the application, the part by weight of the tranexamic acid is 1-10 parts, and is further preferably 3-7 parts, and is more preferably 5 parts;

[0033] The part by weight of the nicotinamide is 0.1-5 parts, and is further preferably 1-4 parts, and is more preferably 3 parts;

[0034] The part by weight of the 4-butylresorcinol is 0.01-0.5 parts, and is further preferably 0.1-0.4 parts, and is more preferably 0.2 parts;

[0035] The licorice extract is 0.001-0.1 parts by weight, further preferably 0.01-0.08 parts by weight, and more preferably 0.05 parts by weight;

[0036] The 3-o-ethyl ascorbic acid is 1-10 parts by weight, further preferably 3-8 parts by weight, and more preferably 5 parts by weight;

[0037] The glutathione is 0.5-5 parts by weight, further preferably 1-3 parts by weight, and more preferably 2 parts by weight.

[0038] The coagulation acid is a protease inhibitor, which can reduce the activity of tyrosinase, thereby inhibiting the formation of melanin; at the same time, the coagulation acid can also inhibit plasmin, eliminate skin inflammation and blood vessel expansion, and play an antioxidant role in the use process.

[0039] The licorice extract is extracted from the roots of Glycyrrhiza glabra, can penetrate into the skin and maintain high activity, and can effectively inhibit the activity of various enzymes (such as tyrosinase) in the melanin production process; at the same time, the licorice extract also has the effects of preventing rough skin and anti-inflammatory and antibacterial.

[0040] The nicotinamide has the following effects: interfering with the transport process of melanin, nicotinamide can interfere with the transport of melanin from melanocytes to keratinocytes, reducing the deposition of melanin in the epidermis; accelerating metabolism: nicotinamide can accelerate the shedding of keratin containing melanocytes, promote the synthesis of collagen, and prevent excessive deposition of melanin; maintaining skin water content: nicotinamide helps maintain skin moisture balance, enhances skin barrier, and thus moisturizes the skin.

[0041] The 4-butyl resorcinol not only shows strong inhibitory effect on tyrosinase, but also further regulates the activity of dihydroxyindole carboxylic acid oxidase (TRP-1), which is also involved in the melanin metabolism process.

[0042] The 3-o-ethyl ascorbic acid is a derivative of vitamin C, which has good stability and strong antioxidant property; in addition, the 3-o-ethyl ascorbic acid also has the effects of promoting wound healing and anti-inflammatory.

[0043] The glutathione is a tripeptide containing thiol groups, which is combined by glutamic acid, cysteine and glycine, and has antioxidant and detoxification effects.

[0044] The components in the whitening and freckle-removing composition of the present application have a synergistic effect, which can effectively inhibit the generation and deposition of skin melanin, and achieve the effect of whitening.

[0045] The whitening and freckle-removing composition also comprises a moisturizing agent, a pH adjusting agent, a soothing agent, a skin conditioning agent;

[0046] The moisturizing agent comprises butylene glycol and polyethylene glycol-400, and the weight percentage of the moisturizing agent is 1-10 parts, further preferably 2-8 parts, and more preferably 5 parts;

[0047] The pH adjusting agent comprises lactic acid or arginine, and the weight percentage of the pH adjusting agent is 0.5-5 parts, further preferably 1-4 parts, and more preferably 3 parts;

[0048] The soothing agent comprises dipotassium glycyrrhizinate, and the weight percentage of the soothing agent is 0.5-5 parts, further preferably 2-4 parts, and more preferably 3 parts;

[0049] The skin conditioning agent comprises one or more of a whitening and freckle-removing component, an anti-aging and protective component, an antioxidant component, and a plant extract. The whitening and freckle-removing component comprises one or more of alpha arbutin, phloretin, resveratrol, nonapeptide-1, and a resorcinol derivative; the resorcinol derivative comprises one or more of phenylethyl resorcinol (377), peptan, hexyl resorcinol, and dimethoxytoluyl resorcinol; and the weight percentage of the whitening and freckle-removing component is 0.5-5 parts, further preferably 2-4 parts, and more preferably 3 parts;

[0050] The anti-aging and protective component comprises one or more of a ceramide and a derivative thereof, a polypeptide, a carnosine, an ekdysone, a boswellin, a polydeoxyribonucleotide (PDRN), a nicotinamide adenine dinucleotide (NMN), and a glycerol glucoside; the ceramide and the derivative thereof comprise glucosylceramide; the polypeptide comprises one or more of oligopeptide-1, acetyl hexapeptide-3, acetyl octapeptide-1, pentapeptide-3, and blue copper peptide; and the weight percentage of the anti-aging and protective component is 1-10 parts, further preferably 3-7 parts, and more preferably 5 parts;

[0051] The antioxidant component comprises one or more of butylated hydroxyanisole, coenzyme Q10, astaxanthin, vitamin E, ergothioneine, superoxide dismutase (SOD), and ascorbic acid and a derivative thereof; the ascorbic acid and the derivative thereof comprise one or more of ascorbic acid glucoside, ascorbic acid magnesium phosphate, ascorbic acid sodium phosphate, ascorbic acid palmitate, and ascorbic acid tetraisopalmitate; and the weight percentage of the antioxidant component is 1-10 parts, further preferably 2-8 parts, and more preferably 5 parts.

[0052] The present application also provides the use of the whitening and freckle-removing composition in the preparation of skin care products, including gels, lotions, serums, emulsions, creams, ointments or masks. The present application does not have special limitations on the preparation method of the gels, lotions, serums, emulsions, creams, ointments or masks, and they can be prepared by known methods with the addition of conventional emulsifiers, thickeners or other substances.

[0053] Gel-type skin care products can be prepared by adding water-soluble polymers such as polyvinyl alcohol, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose and guar gum, etc.

[0054] Lotion-type and cream-type skin care products can be prepared by adding fatty alcohols such as 1,2-pentanediol, glycerol, butanediol, xylitol, panthenol, etc., emulsifiers including hexyldecanol, tridecyl alcohol polyether-9, polyethylene glycol-40 hydrogenated castor oil, polysorbate-20, etc., preservative systems such as phenoxyethanol, etc., vegetable oils such as almond oil, camellia oil, jojoba oil, etc.

[0055] Ointment-type compositions can be prepared by adding emulsifiers and waxes (e.g., beeswax, paraffin wax and petrolatum, etc.) and the like.

[0056] The present application also provides a whitening and freckle-removing gel made of the following components in mass percentage:

[0057] A: sodium hyaluronate 2.5-3.5%, polyvinylpyrrolidone 0.8-1.2% and polyvinyl alcohol 2.5-3.5%;

[0058] B: tranexamic acid 2.5-3.5%, arginine 0.8-1.0%, glutathione 0.8-1.2%, 3-o-ethyl ascorbic acid 1.8-2.3%, dipotassium glycyrrhizinate 0.4-0.5%, nicotinamide 0.5-0.7%, carnosine 0.09-0.11%, wild soybean seed extract 0.11-0.13% and lactic acid 0.95-1.5%;

[0059] C: polyethylene glycol-400 0.6-0.8%, butanediol 2.5-3.5%, glycyrrhiza extract 0.008-0.01% and 4-butylresorcinol 0.04-0.06%;

[0060] The balance is water.

[0061] The mass percentage of the sodium hyaluronate is 2.5-3.5%, further preferably 2.8-3.2%, and more preferably 3%; the mass percentage of the polyvinylpyrrolidone is 0.8-1.2%, further preferably 0.9-1.1%, and more preferably 1.0%; the mass percentage of the polyvinyl alcohol is 2.5-3.5%, further preferably 2.7-3.3%, and more preferably 3%; the mass percentage of the tranexamic acid is 2.5-3.5%, further preferably 2.7-3.2%, and more preferably 3%; the mass percentage of the arginine is 0.8-1.0%, further preferably 0.85-0.95%, and more preferably 0.9%; the mass percentage of the glutathione is 0.8-1.2%, further preferably 0.9-1.1%, and more preferably 1%; the mass percentage of the 3-o-ethyl ascorbic acid is 1.8-2.3%, further preferably 1.9-2.2%, and more preferably 2%; the mass percentage of the dipotassium glycyrrhizinate is 0.4-0.5%, further preferably 0.42-0.47%, and more preferably 0.44%; the mass percentage of the nicotinamide is 0.5-0.7%, further preferably 0.55-0.65%, and more preferably 0.6%; the mass percentage of the carnosine is 0.09-0.11%, further preferably 0.095-0.108%, and more preferably 0.105%; the mass percentage of the wild soybean seed extract is 0.11-0.13%, further preferably 0.115-0.125%, and more preferably 0.12%; the mass percentage of the lactic acid is 0.09-0.12%, further preferably 0.093-0.1%, and more preferably 0.098%. The mass percentage of the polyethylene glycol is 0.6-0.8%, further preferably 0.65-0.75%, and more preferably 0.7%; the mass percentage of the butanediol is 2.5-3.5%, further preferably 2.8-3.2%, and more preferably 3%; the mass percentage of the licorice extract is 0.008-0.01%, further preferably 0.0085-0.0095%, and more preferably 0.0089%; and the mass percentage of the 4-butyl resorcinol is 0.04-0.06%, further preferably 0.045-0.055%, and more preferably 0.05%.

[0062] The application further provides a preparation method of the whitening and freckle-removing gel, comprising the following steps:

[0063] (1) mixing and stirring A with 45-50% mass percentage of water, dissolving at 87-92°C for 50-70 min to obtain a solution;

[0064] (2) mixing B with 30-36% mass percentage of water, dissolving and filtering to obtain a filtrate;

[0065] (3) mixing the components in C to obtain a mixture;

[0066] (4) after the solution is cooled to 20-25℃, mixing the solution with the filtrate and the mixture, and stirring for 50-70 min to obtain the whitening and freckle-removing gel.

[0067] In the present application, all the components in A are dissolved in water and stirred and dissolved. The mass percentage of the water is 45-50%, further preferably 46-48%, and more preferably 46.92%. The water is preferably water for injection, and is fully stirred and dissolved. The temperature of the dissolution is 87-92℃, further preferably 89-91℃, and more preferably 90℃. The time of the dissolution is 50-70 min, further preferably 55-65 min, and more preferably 60 min, to obtain a solution.

[0068] In the present application, all the components in B are dissolved in water, and then filtered to obtain a filtrate. The oscillation is preferably performed in a vortex shaker, and the frequency of the oscillation is not particularly limited in the present application, as long as the powder is fully dissolved. The water is preferably water for injection, and the filtration is preferably performed using a water filter.

[0069] In the present application, all the components in C are stirred and dissolved to obtain a mixture. After the solution is cooled to 20-25℃, the solution, the filtrate and the mixture are mixed, fully stirred and uniformly mixed to obtain the whitening and freckle-removing gel.

[0070] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0071] The mouse melanoma B16-F10 cells in the examples of the present application are purchased from Shanghai Zeptometrix Bio-Technology Co., Ltd. The L929 cells are purchased from the National Standard Institute of Biological and Medical Cell Resource (NSTI-BMCR). The wild soybean seed extract is purchased from Hunan Yaxiu Biological Technology Co., Ltd. The licorice extract is purchased from Hangzhou Xiwang Biological Technology Co., Ltd. If no special instructions are given, other reagents can be purchased through commercial channels.

[0072] Example 1

[0073] The whitening and freckle-removing composition comprises the following components: trans-4-hydroxyproline 1 g, nicotinamide 0.1 g, 4-butylresorcinol 0.015 g, licorice extract 0.001 g, 3-o-ethyl ascorbic acid 1 g and glutathione 0.5 g.

[0074] Example 2

[0075] A whitening and freckle-removing composition comprises the following components: tranexamic acid 5 g, nicotinamide 3 g, 4-butylresorcinol 0.3 g, licorice extract 0.05 g, 3-o-ethyl ascorbic acid 5 g, and glutathione 3 g.

[0076] Example 3

[0077] A whitening and freckle-removing composition comprises the following components: tranexamic acid 10 g, nicotinamide 5 g, 4-butylresorcinol 0.5 g, licorice extract 0.1 g, 3-o-ethyl ascorbic acid 10 g, and glutathione 5 g.

[0078] Example 4

[0079] Accurately weighed 9.38 g (46.92 wt%) of water for injection in a 50 mL centrifuge tube, then accurately weighed 0.60 g of sodium hyaluronate (3 wt%), 0.20 g of polyvinylpyrrolidone (1 wt%), and 0.60 g of polyvinyl alcohol (3 wt%), then stirred well and dissolved at 90°C for 1 h to obtain solution A. Then, another 10 mL centrifuge tube was weighed and 3.59 g of water for injection (35.88 wt%), 0.30 g of tranexamic acid (3 wt%), 0.09 g of arginine (0.9 wt%), 0.10 g of glutathione (1 wt%), 0.20 g of 3-o-ethyl ascorbic acid (2 wt%), 0.044 g of dipotassium glycyrrhizinate (0.44 wt%), 0.06 g of nicotinamide (0.6 wt%), 0.0105 g of carnosine (0.105 wt%), 0.012 g of wild soybean seed extract (0.12 wt%), and 0.098 g of lactic acid (0.98 wt%) were added. The powder was completely dissolved by shaking well, then filtered using a water-based filter to obtain filtrate B. Another 2 mL centrifuge tube was accurately weighed with 0.07 g of polyethylene glycol-400 (0.7 wt%), 0.03 g of butanediol (0.3 wt%), 0.00089 g of licorice extract (0.0089 wt%), and 0.005 g of 4-butylresorcinol (0.05 wt%), and stirred to facilitate dissolution. After dissolution, a mixture was obtained. When the solution was cooled to 25°C, the solution was mixed with the filtrate and the mixture, and stirred well to obtain a whitening and freckle-removing gel.

[0080] Experimental Example 1:

[0081] Take a 10 mL centrifuge tube, weigh and add 4.57 g of water for injection (91.44 wt%), 0.15 g of tranexamic acid (3 wt%), 0.045 g of arginine (0.9 wt%), 0.05 g of glutathione (1 wt%), 0.01 g of 3-o-ethyl ascorbic acid (2 wt%), 0.03 g of nicotinamide (0.6 wt%), mix well, and filter using a water filter to obtain filtrate A. Take another 2 mL centrifuge tube, accurately weigh 0.035 g of polyethylene glycol-400 (0.7 wt%), 0.015 g of butanediol (0.3 wt%), 0.00045 g of licorice extract (0.0089 wt%), and 0.0025 g of 4-butylresorcinol (0.05 wt%), and stir to promote dissolution to obtain solution B. Then, mix filtrate A and solution B uniformly to obtain a whitening composition. Further dilute the above whitening composition into a DMEM medium solution at a mass concentration of 1%, sterilize through a 0.22 μm filter, and reserve for use.

[0082] Mouse melanoma cells B16-F10 cells are inoculated into a 6-well plate at a density of 2 x 10 5 cells / well, and after 48 h, the plate is taken out, the medium in the well is aspirated, washed once with PBS, and the above DMEM medium containing whitening and freckle-removing ingredients is added. DMEM medium without any active ingredients is used as a blank control, 3 repeats per well, and the culture is continued for 48 h. After the culture is completed, the cells are lysed, the intracellular melanin is extracted by centrifugation, 200 μL of 10% DMSO in 1 mol / L sodium hydroxide solution is added, and then the mixture is heated in a water bath at 80°C for 2-3 h. After the melanin is completely dissolved, 180 μL is taken into a 96-well plate, the absorbance value at 492 nm is detected using an enzyme marker, and the melanin inhibition rate is calculated according to the following formula. Melanin inhibition rate = [(OD 空白组 - OD 实验组 ) / OD 空白组 ] x 100%

[0083] Table 1: Results of melanin inhibition experiment

[0084] From the melanin inhibition rate, it can be seen that the melanin inhibition rate of the whitening and freckle-removing composition at a mass concentration of 1% is about 82.27%. This shows that the composition has excellent whitening efficacy.

[0085] Experimental Example 2

[0086] The whitening and freckle-removing gel prepared in Example 4 is dissolved in a DMEM medium solution at a mass concentration of 1%, sterilized through a 0.22 μm filter, and reserved for use.

[0087] Mouse melanoma cells B16-F10 cells are inoculated into a 6-well plate at a density of 2 x 105 The B16-F10 cells were inoculated into the 6-well plates at a density of 1 cell / well, and after 48 h, the plates were taken out, the culture medium in the wells was discarded, and the cells were washed once with PBS. The DMEM medium containing the whitening and freckle-removing gel was added, and the DMEM medium without any whitening active ingredient was used as a blank control. Each well was repeated 3 times, and the culture was continued for 48 h. After the culture, the cells were lysed, the intracellular melanin was extracted by centrifugation, 200 μL of 10% DMSO-containing 1 mol / L sodium hydroxide solution was added, and then the mixture was heated in a water bath at 80°C for 2-3 h. After the melanin was completely dissolved, 180 μL of the solution was taken into a 96-well plate, the absorbance value at 492 nm was detected by using an enzyme marker, and the melanin inhibition rate was calculated according to the following formula. Melanin inhibition rate = [(OD 空白组 -OD 实验组 ) / OD 空白组 ] x 100%

[0088] Table 2: Results of the melanin inhibition experiment

[0089] According to the melanin inhibition rate, the melanin inhibition rate of the whitening and freckle-removing gel with a mass concentration of 1% was about 80.09%. This indicates that the gel has excellent whitening effect.

[0090] Experimental Example 3

[0091] The whitening and freckle-removing gel prepared in Example 4 was dissolved in the DMEM medium solution at a mass concentration of 1%, filtered through a 0.22 μm filter membrane, and prepared for use.

[0092] The B16-F10 cells were inoculated into the 96-well plates at a density of 8000 cells / well, and then incubated in a culture box at 37°C, 5% CO2, and saturated humidity for 48 h. After 48 h, the 96-well plates were taken out, the test solution was added, 4 groups of parallel samples were set for each concentration, a blank control and a positive control of 0.05% kojic acid were set, and each well was repeated 3 times. After the administration, the incubation was continued for 24 h. After 24 h, the old culture medium was discarded, each well was washed once with 100 μL of PBS, and then 40 μL of a cell lysis solution containing 1 mM PMSF was added to each well. The plates were placed in a 4°C incubator for lysis for 30 min. After the lysis, the plates were placed in a 37°C incubator for 5 min, 100 μL of 1 mg / mL L-DOPA was added to each well, and the incubation was continued in the 37°C incubator for 2 h. After the incubation, the absorbance value at 492 nm was detected by using an enzyme marker, and the tyrosinase inhibition rate was calculated according to the following formula. Tyrosinase inhibition rate = [(OD 空白组 -OD 实验组 ) / OD 空白组 ] x 100%

[0093] Table 3 Tyrosinase inhibition rate experimental results

[0094] Experimental Example 4 Zebrafish experiment

[0095] The whitening and freckle-removing composition prepared in Experimental Example 1 was diluted with standard dilution water to a mass concentration of 1% for use. Wild-type AB strain zebrafish that had developed normally for 6 hours after fertilization were selected and placed in a 6-well plate, with 15 zebrafish embryos per well. Then, the ultrapure water in the 6-well plate was removed without damaging the embryos. Then, 3 mL of the 1% composition dilution was quickly added to the wells, and a group of blank controls was set up (the blank control group was directly added with 3 mL of ultrapure water). Then, the 6-well plate was wrapped with tin paper and placed in a biochemical incubator at a temperature of 28.5±0.2°C for continued incubation for 45 hours. After the incubation was completed, 10 zebrafish were randomly selected from each group and placed under a dissecting microscope for observation and photographing. Then, the ImageJ image processing software was used to analyze the melanin signal intensity of the head of the zebrafish, and FIG. 1 is an example graph of the results of the two groups. The results showed that the whitening efficacy of the whitening and freckle-removing composition at a concentration of 1% reached 41%.

[0096] Experimental Example 5 Cytotoxicity experiment

[0097] In order to confirm whether the whitening and freckle-removing composition described in the present application has cytotoxicity, it was evaluated using the L929 cell model and the CCK8 evaluation method. L929 cells were inoculated in a 96-well plate at a number of 8000 per well, and then the plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for incubation for 24 hours. After 24 hours, the plate was taken out, the old culture medium in the wells was aspirated, and 100 μL of DMEM culture medium containing gradient mass concentrations (0.1%, 1%, and 5%) of the whitening and freckle-removing composition prepared in Experimental Example 1 was added to the corresponding wells, with 6 groups in parallel for each concentration, while a blank control (DMEM culture medium without any active ingredients was added) and 1% zinc diethyldithiocarbamate (ZDEC) were set up as positive controls. After administration, incubation was continued for 24 hours. After 24 hours, the plate was taken out, each well was washed once with 100 μL of PBS, and then 100 μL of a solution containing 10% CCK8 prepared with serum-free DMEM culture medium was added to each well. Then, the plate was placed back in the incubator for continued incubation for 50 minutes. After the incubation was completed, the absorbance was measured using a microplate reader, and the results are shown in FIG. 2. The results showed that the cell viability at all concentrations was close to or even exceeded 100%, and it can be considered that the whitening and freckle-removing composition has no cytotoxicity.

[0098] Experimental Example 6 Clinical experiment

[0099] The whitening and freckle-removing gel prepared in Example 4 was used to conduct an experiment to test its irritation to human skin. A total of 30 subjects were recruited, including 9 males and 21 females, aged between 22 and 57 years, with an average age of 38.8±10.5 years. The specific test method was as follows: about 1 g of the gel was attached to a hydrocolloid dressing, and then qualified spot tester materials were selected. In a closed patch test method, the test object was fixed to the back of the subject with a low-sensitization adhesive tape. After 24 h, the test object was removed, and the skin reaction was observed at 0.5, 24, and 48 h after removal, respectively. The results were recorded according to the skin reaction grading standard in the “Cosmetic Safety Technology Specification” (2015 edition). The results showed that 0 out of 30 people had skin adverse reactions.

[0100] Further, the whitening efficacy of the composition was tested. A total of 30 subjects were recruited, including 3 males and 27 females. In addition, ascorbic acid with a concentration of 7% was selected as a positive control in this clinical trial, and no negative control was applied. The specific test method was as follows: the back skin of the subject was irradiated with a sunlight simulator, once a day, for 4 consecutive days. After 4 days of darkening, the areas with an ITA0 value difference greater than 5 from the mean value of all test areas were removed. Then, normal skin areas with an application area not less than 6 cm 2 were selected, and the test object was applied continuously for 4 weeks, once a day. Then, the melanin value, skin ITA0 value, and visual skin color grade evaluation by the test observation doctor were tested before application (0 weeks) and at 1, 2, 3, and 4 weeks, respectively. After 4 weeks, the changes in skin brightness and skin melanin values of different groups were shown in Table 4.

[0101] Table 4 Clinical test results of whitening and freckle-removing gel

[0102] The experimental results show that the whitening and freckle-removing composition has better whitening and lightening effect compared with the negative control and the positive control. After 4 weeks of use, the skin brightness value of the test group increases from 25.41±4.03 to 32.08±3.64, increasing by 26.24%; the skin melanin value decreases from 172.74±40.95 in the 0th week to 126.45±21.98, decreasing by about 26.79%. However, the skin brightness value of the negative control group changes from 25.68±4.47 to 27.76±4.65, almost no change; the skin melanin value changes from 163.32±38.68 to 145.13±36.01, decreasing by 11%. The skin brightness value of the positive control group increases from 25.88±4.23 to 31.17±4.05, increasing by about 20.44%; the skin melanin value decreases from 161.06±38.20 to 130.00±31.39, decreasing by about 19.28%. In summary, the whitening and freckle-removing composition has better whitening and lightening effect compared with the negative control and the positive control.

[0103] Experimental Example 7

[0104] The melanin inhibition rate of tranexamic acid alone was tested using a B16-F10 mouse skin melanoma cell model. Commercially available tranexamic acid injection with a concentration of 5% was diluted to concentrations of 3%, 2.5%, 1.5%, 1%, and 0.5% using DMEM low-glucose medium. The control group was DMEM medium without any whitening ingredients. Then, all the test solutions were added to B16-F10 cells for co-culture for 48 h, with three replicates for each group. After the culture ended, the cells were lysed, and the intracellular melanin was extracted by centrifugation, 200 μL of 10% DMSO-containing 1 mol / L sodium hydroxide solution was added, and then the mixture was heated in a water bath at 80°C for 2-3 h. After the melanin was completely dissolved, 180 μL was taken and added to a 96-well plate, and the absorbance was detected using an enzyme marker, and the results are shown in FIG. 3.

[0105] The results show that the melanin inhibition effect of tranexamic acid is positively correlated with the concentration. When the concentration of tranexamic acid is 0.5%, it almost shows no melanin inhibition effect; when the concentration is 1%, the melanin inhibition rate is 7.04%; when the concentration is 1.5%, the melanin inhibition rate is 7.76%; when the concentration is 2.5%, the melanin inhibition rate is 79.14%; and when the concentration is 3%, the melanin inhibition rate is 78.66%. In Experimental Example 1, the melanin inhibition rate of the composition with a mass concentration of 1% was tested, in which the actual concentration of tranexamic acid is 0.03%, while in this experimental example, the concentration of tranexamic acid is 2.5-3% only when the melanin inhibition rate is high, but high-concentration tranexamic acid not only has cytotoxicity but also has skin irritation, and cannot be directly used on the skin.

[0106] Experimental Example 8

[0107] Comparison of the melanin production inhibitory effect of the whitening and freckle-removing gel of the present invention and RESTRUCTURER of Ino Skin Family.

[0108] The melanin production inhibitory effect of the whitening and freckle-removing gel of the present invention and RESTRUCTURER of Ino Skin Family were compared using B16-F10 cells. The whitening and freckle-removing composition of the present invention was prepared in the same manner as in Example 4. The whitening and freckle-removing gel of the present invention and RESTRUCTURER were added to DMEM medium at an addition amount of 1%. Meanwhile, DMEM medium containing no active ingredient was set as a blank control group. Subsequently, all of the test solutions were added to B16-F10 cells to be co-cultured for 48 h, and three parallels were set for each group. After the culture, the cells were lysed, and the intracellular melanin was extracted by centrifugation, 200 μL of 1 mol / L sodium hydroxide solution containing 10% DMSO was added, and then the mixture was heated in a water bath at 80°C for 2-3 h. After the melanin was completely dissolved, 180 μL of the solution was taken in a 96-well plate, and the absorbance was measured using a microplate reader. The results showed that the whitening and freckle-removing gel of the present invention had a better whitening effect, and the melanin inhibition rate was about 80.09%, while the melanin inhibition rate of RESTRUCTURER was only 52.00%.

[0109] Experimental Example 9

[0110] 1. A solution containing ascorbic acid, glutathione and tranexamic acid was prepared according to the mass percentage in Table 5. For example, as shown in Comparative Example 1, 10 mL of DMEM low-sugar culture medium was taken in a 15 mL centrifuge tube, 0.0005 g of 3-o-ethyl ascorbic acid (0.0050 wt%), 0.005 g of glutathione (0.05 wt%) and 0.10 g of tranexamic acid (1 wt%) were accurately weighed and added, and the powder was fully dissolved by shaking.

[0111] B16-F10 cells were seeded in a 6-well plate at a density of 2 x 10 5 cells / well, and after 48 h, the plate was taken out, the culture medium in the wells was aspirated, and 2 mL of each of the prepared test solutions was added to the corresponding wells, and three parallels were set for each group, and a blank control was set at the same time, and then the incubation was continued for 48 h. After the culture, the cells were lysed, and the intracellular melanin was extracted by centrifugation, 200 μL of 1 mol / L sodium hydroxide solution containing 10% DMSO was added, and then the mixture was heated in a water bath at 80°C for 2-3 h. After the melanin was completely dissolved, 180 μL of the solution was taken in a 96-well plate, and the test absorbance value at 492 nm was measured, and the melanin inhibition rate was calculated, and the results are shown in Table 5.

[0112] Table 5 Melanin inhibition rate of the whitening and freckle-removing compositions of Comparative Examples 1-9

[0113] The results show that the melanin inhibition rate of the composition with the best melanin inhibition effect in Table 5 is about 65.56%, which is weaker than the whitening and freckle-removing composition of the present application. In addition, the experiment also tests 3-o-ethyl ascorbic acid with a concentration of 0.02%, glutathione with a concentration of 0.01%, and tranexamic acid with a concentration of 0.03% (equivalent to the ingredient concentration in the whitening and freckle-removing composition diluted to a concentration of 1%). However, the melanin inhibition rate of the three ingredients is only 2.33%, which is much smaller than 1% of the whitening and freckle-removing composition. This shows that the addition of other ingredients (nicotinamide, 4-butylresorcinol, licorice extract) of the whitening and freckle-removing composition of the present application is necessary, which can synergistically act with 3-o-ethyl ascorbic acid, glutathione, and tranexamic acid to improve the whitening effect.

[0114] 2. Prepare a solution containing glutathione, tranexamic acid, and nicotinamide according to the mass percentage in Table 6 and Table 7. For example, as shown in Comparative Example 10, 10 mL of DMEM low-glucose culture medium is taken into a 15 mL centrifuge tube, and 0.01 g of ascorbic acid (0.10 wt%), 0.01 g of tranexamic acid (0.10 wt%), and 0.05 g of nicotinamide (0.50 wt%) are accurately weighed and added, and the powder is fully dissolved by shaking.

[0115] B16-F10 cells are inoculated into a 6-well plate at a density of 2×10 5 cells / well, and after 48 h, the plate is taken out, the culture medium in the well is aspirated, and 2 mL of each gradient concentration of the test solution is added to the corresponding well, with 3 groups in parallel for each concentration, and a blank control is set. After administration, incubation is continued for 48 h. After the end of the culture, the cells are lysed, and the intracellular melanin is extracted by centrifugation, 200 μL of 1 mol / L sodium hydroxide solution containing 10% DMSO is added, and then it is placed in a water bath at 80°C for heating for 2-3 h. After the melanin is completely dissolved, 180 μL is taken to a 96-well plate, the test absorbance value at 492 nm is detected, and the melanin inhibition rate is calculated, and the results are shown in Table 6.

[0116] Table 6 Melanin inhibition rate of the whitening and freckle-removing composition of Comparative Examples 10-13

[0117] The results of Table 6 show that the whitening effect of the whitening and freckle-removing composition with different proportions of glutathione, tranexamic acid, and nicotinamide is weaker than the whitening and freckle-removing composition of the present application.

[0118] Table 7 Melanin inhibition rate of the whitening and freckle-removing composition of Comparative Examples 14-17

[0119] Table 7 shows that the melanin inhibition rate of the combination of nicotinamide with a concentration of 0.006%, 4-butylresorcinol with a concentration of 0.0005% and licorice extract with a concentration of 0.000089% (equivalent to the combination of the three ingredients in the 1% whitening and freckle-removing composition) is only 22.89%, which is much less than the whitening and freckle-removing composition with a concentration of 1%. After increasing the concentration of the three ingredients by 10 times, the melanin inhibition rate is increased, but is still less than the 1% whitening and freckle-removing composition. This indicates that the addition of 3-o-ethyl ascorbic acid, glutathione and tranexamic acid in addition to the three ingredients in the combination is also necessary. There is a synergistic whitening effect among the tranexamic acid, nicotinamide, 4-butylresorcinol, licorice extract, 3-o-ethyl ascorbic acid of the present application.

[0120] From the above examples, it can be seen that the present application provides a whitening and freckle-removing composition, a gel and a preparation method and application thereof. The whitening and freckle-removing composition of the present application inhibits the formation and accumulation of melanin from multiple strategies, and each component synergistically plays a role, thereby achieving the cosmetic effects of whitening and freckle-removing.

[0121] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A skin whitening and freckle-removing composition, characterized by comprising: The composition comprises the following components in parts by weight: 3-7 parts of tranexamic acid, 1-4 parts of nicotinamide, 0.1-0.4 parts of 4-butylresorcinol, 0.01-0.08 parts of liquorice extract, 3-8 parts of 3-o-ethyl ascorbic acid and 1-3 parts of glutathione.

2. The whitening and freckle-removing composition according to claim 1, characterized by, The whitening and freckle-removing composition further comprises a moisturizing agent, a pH adjusting agent, a soothing agent and a skin conditioning agent; The moisturizing agent comprises butylene glycol and polyethylene glycol-400, and the moisturizing agent is in a weight fraction of 1-10 parts; The pH adjusting agent comprises lactic acid or arginine, and the pH adjusting agent is in a weight fraction of 0.5-5 parts; The soothing agent comprises dipotassium glycyrrhizinate, and the soothing agent is in a weight fraction of 0.5-5 parts.

3. The whitening and freckle-removing composition according to claim 2, characterized by, The skin conditioning agent comprises one or more of a whitening and freckle-removing component, an anti-aging component and an antioxidant component.

4. The whitening and freckle-removing composition according to claim 3, characterized by, The whitening and freckle-removing component comprises one or more of phloretin, resveratrol, nonapeptide-1 and resorcinol derivatives, and the whitening and freckle-removing component is in a weight fraction of 0.5-5 parts.

5. The whitening and freckle-removing composition according to claim 3, characterized by, The anti-aging component comprises one or more of ceramide and its derivatives, carnosine, ektoine, boswella, polydeoxyribonucleotide and nicotinamide adenine dinucleotide, and the anti-aging component is in a weight fraction of 1-10 parts.

6. The whitening and freckle-removing composition according to claim 3, wherein The antioxidant component comprises one or more of butylated hydroxyanisole, coenzyme Q10, astaxanthin, vitamin E, ergothioneine, superoxide dismutase and ascorbic acid and its derivatives, and the antioxidant component is in a weight fraction of 1-10 parts.

7. The whitening and freckle-removing composition according to claim 3, characterized by, The skin conditioning agent further comprises a plant extract comprising one or more of aloe extract, gentian extract, sophora flavescens extract, ginseng extract, tea leaf extract, wild soybean seed extract and gynura extract, and the plant extract is in a weight fraction of 0.01-5 parts.

8. Use of the whitening and freckle-removing composition according to any one of claims 1-7 in the preparation of a skin care product, wherein the skin care product comprises a gel, an essence, a lotion, a cream, an ointment or a mask.

9. A skin whitening and freckle removing gel, characterized by, The composition is prepared from the following components in mass percentage: A: 2.5-3.5% of sodium hyaluronate, 0.8-1.2% of polyvinylpyrrolidone and 2.5-3.5% of polyvinyl alcohol; B: 2.5-3.5% of tranexamic acid, 0.8-1.0% of arginine, 0.8-1.2% of glutathione, 1.8-2.3% of 3-o-ethyl ascorbic acid, 0.4-0.5% of dipotassium glycyrrhizinate, 0.5-0.7% of nicotinamide, 0.09-0.11% of carnosine, 0.11-0.13% of wild soybean seed extract and 0.09-0.12% of lactic acid; C: 0.6-0.8% of polyethylene glycol-4000, 2.5-3.5% of butylene glycol, 0.008-0.01% of liquorice extract and 0.04-0.06% of 4-butylresorcinol; the balance being water.

10. The process for the preparation of the whitening and freckle-removing gel of claim 9, characterized by, The method comprises the following steps: (1) mixing A with water in a total mass of 45-50% of the gel, stirring and dissolving the mixture at 87-92°C for 50-70 min to obtain a solution; (2) mixing B with water in a total mass of 30-36% of the gel, filtering the solution after dissolving to obtain a filtrate; (3) mixing the components in C to obtain a mixture; (4) After the solution is reduced to 20-25 °C, it is mixed with the filtrate and the mixture, and stirred for 50-70 min to obtain the whitening and freckle-removing gel.

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