Melanin precursor composition, its preparation method and application

Abstract

The present invention relates to a melanin precursor composition, a preparation method and an application thereof. The melanin precursor composition comprises the following raw materials for preparation by weight: 14-20 parts of soybeans, 12-18 parts of black truffles, 10-15 parts of grape seeds, 8-12 parts of cocoa beans, 6-10 parts of Chinese gallnuts, and 6-10 parts of holly leaves. The melanin precursor composition is prepared from the above raw materials through pretreatment, primary extraction, complex protease hydrolysis, secondary extraction, Saccharomyces cerevisiae fermentation, and enzymatic reaction. The melanin precursor composition can effectively solve the problems existing in the existing hair dyes, such as low dyeing efficiency, poor dyeing effect, and poor dyeing durability, while ensuring safety and good use experience, and has good market prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of hair cosmetics, and particularly relates to a melanin precursor composition, a preparation method thereof, and an application thereof. Background Art

[0002] Traditional hair dyes usually contain p-phenylenediamine and other potentially harmful chemicals. Although these ingredients can provide quick and long-lasting coloring effects, they may cause skin irritation, even allergic reactions, and long-term use may damage the hair structure. The application of biosynthesized melanin precursors in hair coloring products represents an innovative direction in hair dyeing technology. It aims to provide a safer, more natural, and healthier hair dyeing solution, bringing many potential benefits and meeting consumers' demands for high-quality, low-irritation hair dyeing products.

[0003] The invention patent application with the application number 201811092804.3 discloses a preparation method of an all-natural enzyme-fermented black hair dye, and the steps are as follows: Step 1, preparing a tyrosinase solution from black beans; Step 2, preparing a tyramine substrate solution; Step 3, preparing melanin precursors in a biological fermentation system; Step 4, adding auxiliaries and mordants to the melanin precursors prepared in Step 3 to make a hair dye. Using all-natural black beans and food-grade tyramine as raw materials and adopting a green biological fermentation method, an all-natural black hair dye with a blackening effect is successfully prepared. It effectively overcomes the use of components harmful to the human body, such as ammonia water, hydrogen peroxide, p-phenylenediamine and its derivatives, in chemically synthesized hair dyes; and overcomes the problem of difficult pigment extraction in natural hair dyes. However, in this invention, the raw material source for extracting melanin precursors is single, the dyeing efficiency is not ideal enough, and additional tyrosine needs to be added, increasing the cost, and the dyeing durability is not good.

[0004] The invention patent application with the application number CN201210021828.6 discloses a method for preparing melanin and its precursor substances by using a composite enzyme. It uses a composite enzyme containing tyrosinase extracted from the fermentation broth of melanin-producing Bacillus subtilis, and uses tyrosine and / or its derivatives as substrates to generate melanin and its precursor substances through an enzymatic reaction. The composite enzyme used in the present invention has a stable source, high enzyme activity, and high production efficiency; the production of a series of melanin precursor substances by the enzymatic reaction method is simple and easy to operate, and further through the method of carrier immobilization, the enzyme can be reused repeatedly, further reducing the cost and increasing the production efficiency. The prepared melanin precursor substances can be used as effective hair dyes and antioxidants. However, in this invention, tyrosine and / or its derivatives need to be used as substrates, natural plant raw materials are not used as the raw material source for extracting melanin precursors, the cost is relatively high, and the hair dyeing effect and dyeing durability are not verified, and the technical effects are unknown.

[0005] It can be seen from this that existing natural hair dyes containing melanin precursors often have problems such as low dyeing efficiency, poor dyeing effect, and poor dyeing durability. Summary of the Invention

[0006] The present invention aims to provide a melanin precursor composition, its preparation method and application, which can effectively solve the problems of existing hair dyes such as low dyeing efficiency, poor dyeing effect, and poor dyeing durability, while ensuring safety and good use experience, and having good market prospects.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A melanin precursor composition, characterized in that, by weight, the composition comprises the following preparation raw materials: 14-20 parts of soybeans, 12-18 parts of black truffles, 10-15 parts of grape seeds, 8-12 parts of cocoa beans, 6-10 parts of Chinese gallnuts, 6-10 parts of holly leaves, and the composition is prepared from the above raw materials through pretreatment, primary extraction, compound protease hydrolysis, secondary extraction, Saccharomyces cerevisiae fermentation, and enzymatic reaction.

[0009] Further, by weight, the composition comprises the following preparation raw materials: 16-18 parts of soybeans, 14-16 parts of black truffles, 12-14 parts of grape seeds, 10-12 parts of cocoa beans, 8-10 parts of Chinese gallnuts, 8-10 parts of holly leaves.

[0010] Further, by weight, the composition comprises the following preparation raw materials: 17 parts of soybeans, 15 parts of black truffles, 13 parts of grape seeds, 10 parts of cocoa beans, 8 parts of Chinese gallnuts, 8 parts of holly leaves.

[0011] Soybeans, also known as soybeans, are annual herbaceous plants of the genus Glycine in the legume family. Soybeans are rich in protein, fat, carbohydrates, carotene, vitamin B1, vitamin B2, niacin, etc., and also contain health care factors such as soybean oligosaccharides, isoflavones, saponins, nucleic acids, etc. that are extremely precious to human health. Soybeans are rich in vitamin E, which can not only destroy the chemical activity of free radicals, have antioxidant effects, inhibit skin aging, etc., but also prevent pigmentation on the skin. Soybeans contain a variety of physiologically active substances that are very beneficial to human health, such as soybean isoflavones, soybean lecithin, soybean polypeptides, soybean dietary fiber, etc. Soybean protein is rich in various amino acids, especially tyrosine, lysine, and tryptophan, and tyrosine can be used to synthesize melanin precursor substances.

[0012] Black truffle, also known as tuber, is an edible fungus that grows underground. Research data shows that black truffle contains rich proteins, various amino acids (such as tyrosine, lysine, tryptophan, leucine, etc.), unsaturated fatty acids, various vitamins, essential trace elements such as zinc, manganese, iron, calcium, phosphorus, selenium, and a large number of metabolites such as sphingolipids, cerebrosides, ceramides, triterpenes, androstenone, adenosine, truffle acid, sterols, truffle polysaccharides, and truffle polypeptides, which have extremely high nutritional and health care value; polysaccharides, polypeptides, and triterpenes have the effects of enhancing immunity, anti-aging, and anti-fatigue, and can be used for beauty and health care; tyrosine contained in black truffle is the key amino acid for melanin synthesis and can be used to synthesize melanin precursor substances.

[0013] Grape seeds are the seeds of grapes and contain polyphenols, oils, crude proteins, amino acids, vitamins, and various trace elements, etc. Grape seeds contain high concentrations of polyphenolic compounds such as proanthocyanidins, which can be used as precursors for melanin production. Grape seed extract has antioxidant and free radical scavenging effects, preventing free radicals from damaging human cells, and has the effects of improving skin elasticity and luster, whitening, moisturizing, removing spots, reducing wrinkles, keeping the skin soft, smooth, removing acne, and healing scars.

[0014] Cocoa beans are the fruits of the evergreen cocoa tree of the Sterculiaceae family and are the flat seeds of oblong nuts. Cocoa beans contain oleic acid, linoleic acid, stearic acid, palmitic acid, protein, vitamins A, B1, B3, B5, B6, D, E, and minerals calcium, magnesium, copper, potassium, sodium, iron, zinc; cellulose, polyphenols, including oligomeric flavonoid substances, among which the content of polyphenolic compounds is relatively high and contributes to melanin synthesis. The rich proanthocyanidins, catechins, and vitamin E in cocoa beans have strong antioxidant effects and can be used as precursor substances for melanin production. These antioxidants, together with vitamins A and zinc in cocoa, can beautify the skin, remove acne and scars.

[0015] Chinese gallnut is a traditional Chinese medicine. The young leaves of Rhus chinensis are invaded and parasitized by Chinese gallnut aphids, and galls are formed, which are the Chinese gallnuts and can be used for medicinal purposes. They contain rich components such as tannic acid, gallic acid, and tannin, and have biological activities such as antioxidant, anti-inflammatory, and antibacterial; tannic acid and gallic acid show certain potential in the regulation of melanin synthesis. Research shows that gallic acid can activate tyrosinase, thereby promoting the synthesis of melanin precursor substances, and tannic acid also has the effect of promoting tyrosinase activity at low concentrations; Chinese gallnut contains tannin, which belongs to polyphenolic compounds and can be used for melanin synthesis.

[0016] Holly leaves are the leaves of the evergreen tree Ilex in the genus Ilex of the Aquifoliaceae family. The main chemical components include protocatechuic acid, which is a phenolic acid, an intermediate product of tyrosine metabolism, and also a precursor in the melanin synthesis pathway, contributing to the synthesis of melanin precursors; it also contains volatile oils that can provide aroma for products; flavonoids, which have antioxidant and protective effects and help improve hair quality; Holly leaf extract can also be added as an auxiliary component to hair dyes to provide additional hair care effects, such as improving scalp health, increasing hair luster, or acting as a preservative, etc.

[0017] The present invention also provides a preparation method of the melanin precursor composition described in any one of the above, comprising the following steps:

[0018] S1 Pretreatment: Clean, remove impurities, dry, sterilize, and ultrafinely pulverize all raw materials by weight to obtain soybean powder, black truffle powder, grape seed powder, cocoa bean powder, Chinese gallnut powder, and holly leaf powder;

[0019] S2 Primary extraction: Mix the soybean powder and black truffle powder obtained in step S1, use phosphate buffer solution as the extraction solvent, under the conditions of pH value of 4 - 7 and temperature of 25 - 45 °C, continuously stir and extract for 2 - 4 hours, filter to remove insoluble substances, and collect the supernatant;

[0020] S3 Composite protease hydrolysis: Take the supernatant obtained in step S2, use composite protease, and carry out hydrolysis under suitable pH value and temperature conditions for 6 - 10 hours to obtain a hydrolysate. Inactivate the enzyme in the above hydrolysate, and then separate and purify it by high - performance liquid chromatography to obtain extract A;

[0021] S4 Secondary extraction: Mix the grape seed powder, cocoa bean powder, Chinese gallnut powder, and holly leaf powder obtained in step S1 as raw materials, select an ethanol aqueous solution with a mass concentration of 50 - 70% as the solvent, the mass ratio of the raw materials to the solvent is 1:10 to 2:10, extract at 60 - 75 °C for 6 - 8 hours, filter to remove solid residues, and concentrate the filtrate by vacuum distillation to remove most of the solvent to obtain an extract;

[0022] S5 Saccharomyces cerevisiae fermentation: Cool the extract obtained in step S4 to 25 - 30 °C, adjust the pH value to 4.5 - 5.5, add Saccharomyces cerevisiae for fermentation, and continue for 18 - 24 hours to obtain fermentation broth B;

[0023] S6 Enzymatic reaction: Mix extract A obtained in step S3 and fermentation broth B obtained in step S5, control the temperature at 30 - 40 °C and pH value at 6.5 - 7.5, add tyrosinase and polyphenol oxidase, continuously react for 6 - 8 hours, inactivate the enzyme, and obtain enzyme - hydrolyzed solution C;

[0024] S7 Post-treatment: The enzymolysis solution C from step S6 is purified by column chromatography, and finally a melanin precursor composition in powder form is obtained by freeze-drying.

[0025] Preferably, in step S3, the compound protease is composed of trypsin, papain, and flavor protease in a mass ratio of (1 - 3):(2 - 3):(1 - 2).

[0026] Preferably, in step S3, the pH value is 7 - 8 and the temperature is 25 - 37 °C.

[0027] Preferably, in step S6, the mass ratio of tyrosinase to polyphenol oxidase is (2 - 3):(1 - 2).

[0028] The present invention also provides the application of any of the above-mentioned melanin precursor compositions in the preparation of hair dyes.

[0029] The present invention also provides a hair dye, comprising 8 - 15% by weight of any of the above-mentioned melanin precursor compositions and cosmetically acceptable excipients.

[0030] Further, the excipients include surfactants, thickeners, antioxidants, pH regulators, fragrances, and water.

[0031] Further, the surfactant includes one or more of cocamidopropyl betaine, sodium cocoyl glutamate, and cetearyl glucoside.

[0032] Further, the thickener includes one or more of guar gum, xanthan gum, carrageenan, and gum arabic.

[0033] Further, the antioxidant includes one or more of vitamin E, vitamin C, and resveratrol.

[0034] Further, the pH regulator includes one or more of citric acid, sodium citrate, lactic acid, sodium lactate, malic acid, and sodium bicarbonate.

[0035] Further, the fragrance includes one or more of lavender essential oil, ylang-ylang essential oil, peppermint essential oil, and neroli essential oil.

[0036] Further, the hair dye includes the following raw materials in parts by weight: 8 - 15 parts of melanin precursor composition, 2 - 6 parts of surfactant, 8 - 10 parts of thickener, 1 - 3 parts of antioxidant, 0.3 - 0.8 parts of pH regulator, 0.4 - 0.9 parts of fragrance, and the balance is water; the total weight parts is 100 parts.

[0037] The present invention also provides a method for preparing the above hair dye, which comprises the following steps: Based on 100 parts by weight, an appropriate amount of water is added to an emulsifying pan and heated to 40-50 °C. A surfactant, a thickening agent, and an antioxidant are added to the emulsifying pan and stirred evenly. The temperature is lowered to 30-35 °C, and a melanin precursor composition is added and stirred evenly. Then, an appropriate amount of pH regulator, essence, and the remaining water are added, and stirring is continued until evenly mixed. It is cooled to room temperature to obtain the hair dye.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The melanin precursor composition of the present invention uses specific plant raw materials, namely soybeans, black truffles, grape seeds, cocoa beans, Chinese gallnuts, and holly leaves, as raw materials for extracting melanin synthesis starting substances. They coordinate and cooperate with each other, and the prepared hair dye has the effects of high dyeing efficiency, good dyeing effect, and good dyeing durability.

[0040] 2. The present invention uses natural raw materials and combines complex protease hydrolysis, Saccharomyces cerevisiae fermentation, and enzymatic reactions. Saccharomyces cerevisiae can help transform the components in the above plant extracts to produce intermediate products that can be used for melanin synthesis; tyrosinase and polyphenol oxidase are key enzymes for melanin synthesis, and they participate in the biosynthesis of melanin; tyrosinase catalyzes the conversion of tyrosine to dopa and further to melanin; while polyphenol oxidase can catalyze the oxidation polymerization of polyphenolic substances to form pigments. The melanin precursor composition prepared through the above steps can significantly improve the dyeing effect.

[0041] 3. The present invention does not add p-phenylenediamine and other potentially harmful chemical substances, is mild and non-irritating, has a good use experience, and has a good market prospect. Detailed implementation manners

[0042] Next, in combination with the embodiments of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0043] It should be understood that when used in this specification and the appended claims, the terms "comprises" and "comprising" indicate the presence of the described features, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0044] It should also be understood that the terms used in the specification of this application are merely for the purpose of describing specific embodiments and are not intended to limit this application. As used in the specification of this application and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include the plural forms.

[0045] It should be further understood that the term "and / or" used in the specification of this application and the appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0046] Some embodiments of this application will be described in detail below. Without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0047] Embodiment 1

[0048] This embodiment provides a melanin precursor composition. By weight, the composition includes the following preparation raw materials: 14 parts of soybeans, 12 parts of black truffles, 10 parts of grape seeds, 8 parts of cocoa beans, 6 parts of Chinese gallnuts, and 6 parts of holly leaves;

[0049] The preparation method of the above melanin precursor composition includes the following steps:

[0050] S1 Pretreatment: Wash and remove impurities, dry, sterilize, and ultrafinely pulverize all raw materials by weight to obtain soybean powder, black truffle powder, grape seed powder, cocoa bean powder, Chinese gallnut powder, and holly leaf powder;

[0051] S2 Primary extraction: Mix the soybean powder and black truffle powder obtained in step S1, use phosphate buffer solution as the extraction solvent, and under the conditions of pH value of 4 and temperature of 25°C, continuously stir and extract for 4 hours, filter to remove insoluble substances, and collect the supernatant;

[0052] S3 Composite protease hydrolysis: Take the supernatant obtained in step S2, use a composite protease composed of trypsin, papain, and flavor protease in a mass ratio of 1:2:1, and carry out hydrolysis under the conditions of pH value of 7 and temperature of 25°C for 10 hours to obtain a hydrolysis solution. Inactivate the enzyme in the above hydrolysis solution, and then separate and purify it by high performance liquid chromatography to obtain extract A;

[0053] S4 Secondary extraction: Mix the grape seed powder, cocoa bean powder, Chinese gallnut powder, and holly leaf powder obtained in step S1 as raw materials, select an ethanol aqueous solution with a mass concentration of 50% as the solvent, the mass ratio of the raw materials to the solvent is 1:10, extract at 60°C for 8 hours, filter to remove solid residues, and concentrate the filtrate by vacuum distillation to remove most of the solvent to obtain an extract;

[0054] S5 Saccharomyces cerevisiae fermentation: Cool the extract in step S4 to 25 °C, adjust the pH value to 4.5, add Saccharomyces cerevisiae for fermentation, and continue for 24 hours to obtain fermentation broth B;

[0055] S6 Enzymatic reaction: Mix the extract A in step S3 with the fermentation broth B in step S5, control the temperature at 30 °C and the pH value at 6.5, add tyrosinase and polyphenol oxidase with a mass ratio of 3:1, continue the reaction for 8 hours, inactivate the enzymes, and obtain enzymolysis solution C;

[0056] S7 Post-treatment: Purify the enzymolysis solution C in step S6 by column chromatography, and finally obtain the melanin precursor composition in powder form by freeze-drying.

[0057] Example 2

[0058] This example provides a melanin precursor composition. By weight, the composition includes the following preparation raw materials: 17 parts of soybeans, 15 parts of black truffles, 13 parts of grape seeds, 10 parts of cocoa beans, 8 parts of Chinese gallnuts, and 8 parts of holly leaves;

[0059] The preparation method of the above melanin precursor composition includes the following steps:

[0060] S1 Pretreatment: Wash and remove impurities, dry, sterilize, and ultrafinely crush all raw materials by weight to obtain soybean powder, black truffle powder, grape seed powder, cocoa bean powder, Chinese gallnut powder, and holly leaf powder;

[0061] S2 Primary extraction: Take the soybean powder and black truffle powder in step S1, use phosphate buffer solution as the extraction solvent, under the conditions of pH value of 6 and temperature of 40 °C, continuously stir and extract for 3 hours, filter to remove insoluble substances, and collect the supernatant;

[0062] S3 Composite protease hydrolysis: Take the supernatant in step S2, use a composite protease composed of trypsin, papain, and flavor protease with a mass ratio of 2:2:1, carry out hydrolysis under the conditions of pH value of 7.5 and temperature of 35 °C for 8 hours to obtain a hydrolysis solution, inactivate the above hydrolysis solution, and then separate and purify it by high performance liquid chromatography to obtain extract A;

[0063] S4 Secondary extraction: Take the grape seed powder, cocoa bean powder, Chinese gallnut powder, and holly leaf powder in step S1 and mix them as raw materials, select an ethanol aqueous solution with a mass concentration of 65% as the solvent, the mass ratio of the raw materials to the solvent is 1:15, extract at 70 °C for 7 hours, filter to remove solid residues, and concentrate the filtrate by vacuum distillation to remove most of the solvent to obtain an extract;

[0064] S5 Saccharomyces cerevisiae fermentation: Cool the extract in step S4 to 28°C, adjust the pH value to 5, add Saccharomyces cerevisiae for fermentation, and continue for 20 hours to obtain fermentation broth B;

[0065] S6 Enzymatic reaction: Mix the extract A in step S3 with the fermentation broth B in step S5, control the temperature at 32°C and the pH value at 7, add tyrosinase and polyphenol oxidase with a mass ratio of 2:1, continue the reaction for 7 hours, inactivate the enzymes, and obtain enzymolysis solution C;

[0066] S7 Post-treatment: Purify the enzymolysis solution C in step S6 by column chromatography, and finally obtain the melanin precursor composition in powder form by freeze-drying.

[0067] Example 3

[0068] This example provides a melanin precursor composition. By weight, the composition includes the following preparation raw materials: 20 parts of soybeans, 18 parts of black truffles, 15 parts of grape seeds, 12 parts of cocoa beans, 10 parts of Chinese gallnuts, and 10 parts of holly leaves;

[0069] The preparation method of the above melanin precursor composition includes the following steps:

[0070] S1 Pretreatment: Wash and remove impurities from all raw materials by weight, dry, sterilize, and ultrafinely pulverize to obtain soybean powder, black truffle powder, grape seed powder, cocoa bean powder, Chinese gallnut powder, and holly leaf powder;

[0071] S2 Primary extraction: Take the soybean powder and black truffle powder in step S1, use phosphate buffer solution as the extraction solvent, under the conditions of pH value of 7 and temperature of 45°C, continuously stir and extract for 2 hours, filter to remove insoluble substances, and collect the supernatant;

[0072] S3 Composite protease hydrolysis: Take the supernatant in step S2, use a composite protease composed of trypsin, papain, and flavor protease with a mass ratio of 3:2:1, carry out hydrolysis under the conditions of pH value of 8 and temperature of 37°C for 6 hours to obtain a hydrolysis solution, inactivate the above hydrolysis solution, and then separate and purify it by high performance liquid chromatography to obtain extract A;

[0073] S4 Secondary extraction: Take the grape seed powder, cocoa bean powder, Chinese gallnut powder, and holly leaf powder in step S1 and mix them as raw materials, select an ethanol aqueous solution with a mass concentration of 70% as the solvent, the mass ratio of the raw materials to the solvent is 1:20, extract at 75°C for 6 hours, filter to remove solid residues, and concentrate the filtrate by vacuum distillation to remove most of the solvent to obtain an extract;

[0074] S5 Saccharomyces cerevisiae fermentation: Cool the extract in step S4 to 30 °C, adjust the pH value to 5.5, add Saccharomyces cerevisiae for fermentation, and continue for 18 hours to obtain fermentation broth B;

[0075] S6 Enzymatic reaction: Mix the extract A in step S3 with the fermentation broth B in step S5, control the temperature at 40 °C and the pH value at 7.5, add tyrosinase and polyphenol oxidase with a mass ratio of 3:2, continue the reaction for 6 hours, inactivate the enzyme, and obtain enzymolysis solution C;

[0076] S7 Post-treatment: Purify the enzymolysis solution C in step S6 by column chromatography, and finally obtain the melanin precursor composition in powder form by freeze-drying.

[0077] Comparative Example 1

[0078] This comparative example provides a melanin precursor composition without adding soybeans, where soybeans are replaced with black truffles in equal weight parts, and the rest is the same as in Example 2.

[0079] Comparative Example 2

[0080] This comparative example provides a melanin precursor composition without adding black truffles, where black truffles are replaced with soybeans in equal weight parts, and the rest is the same as in Example 2.

[0081] Comparative Example 3

[0082] This comparative example provides a melanin precursor composition without adding grape seeds, where grape seeds are replaced with cocoa beans in equal weight parts, and the rest is the same as in Example 2.

[0083] Comparative Example 4

[0084] This comparative example provides a melanin precursor composition without adding cocoa beans, where cocoa beans are replaced with grape seeds in equal weight parts, and the rest is the same as in Example 2.

[0085] Comparative Example 5

[0086] This comparative example provides a melanin precursor composition without adding Chinese gallnuts, where Chinese gallnuts are replaced with holly leaves in equal weight parts, and the rest is the same as in Example 2.

[0087] Comparative Example 6

[0088] This comparative example provides a melanin precursor composition without adding holly leaves, where holly leaves are replaced with Chinese gallnuts in equal weight parts, and the rest is the same as in Example 2.

[0089] Comparative Example 7

[0090] This comparative example provides a melanin precursor composition. The difference between this comparative example and Example 2 is that in the S3 complex protease hydrolysis, the complex protease consists of trypsin and flavor protease at a mass ratio of 2:1, and the rest is the same as in Example 2.

[0091] Comparative Example 8

[0092] This comparative example provides a melanin precursor composition. The difference between this comparative example and Example 2 is that in the S3 complex protease hydrolysis, the complex protease consists of papain and flavor protease at a mass ratio of 2:1, and the rest is the same as in Example 2.

[0093] Comparative Example 9

[0094] This comparative example provides a melanin precursor composition. The difference between this comparative example and Example 2 is that it does not include the S5 Saccharomyces cerevisiae fermentation step. In the S6 enzymatic reaction step, the extract A from the S3 step is mixed with the extract from the S4 step, and the rest is the same as in Example 2.

[0095] Comparative Example 10

[0096] This comparative example provides a melanin precursor composition. The difference between this comparative example and Example 2 is that it does not include the S6 enzymatic reaction step. The extract A from the S3 step is directly mixed with the fermentation broth B from the S5 step and then subjected to the S7 post-treatment step, and the rest is the same as in Example 2.

[0097] Application Example 1

[0098] The melanin precursor composition prepared in Example 1 was used as a raw material in a hair dye. The preparation method of the hair dye includes the following steps: Based on 100 parts by weight, an appropriate amount of water was added to an emulsifying pot and heated to 45°C. 2 parts of cocamidopropyl betaine, 2 parts of sodium cocoyl glutamate, 1 part of cetearyl glucoside, 5 parts of guar gum, 4 parts of xanthan gum, 0.5 part of vitamin E, 0.5 part of vitamin C, and 0.5 part of resveratrol were added to the emulsifying pot and stirred evenly. The temperature was lowered to 35°C, 12 parts of the melanin precursor composition prepared in Example 1 was added and stirred evenly. Then, an appropriate amount of pH regulator, fragrance, and the remaining water were added, and stirring was continued until evenly mixed. After cooling to room temperature, the hair dye was obtained.

[0099] Application Examples 2 - 3, Comparative Application Examples 1 - 10

[0100] The difference from Application Example 1 is that the melanin precursor composition prepared in Example 1 in Application Example 1 was replaced with the melanin precursor compositions prepared in Examples 2 - 3 and Comparative Examples 1 - 10 respectively, and the rest was the same as in Application Example 1. The corresponding hair dyes of Application Examples 2 - 3 and Comparative Application Examples 1 - 10 were prepared.

[0101] Test Example 1: Safety Test

[0102] Human Skin Patch Test

[0103] Test samples: Hair dyes prepared in Application Examples 1-3 and Comparative Application Examples 1-10

[0104] Test subjects: Volunteers aged 18-55 who met the test requirements were selected as the test subjects and randomly divided into 13 groups with 10 people in each group.

[0105] Test method: Qualified patch test devices with an area not exceeding 50 mm2 and a depth of about 1 mm were selected. The test sample was placed in the small chamber of the patch test device, and the dosage was 0.02 mL. The control hole was a blank control (without placing any substance). The patch test device with the test sample was applied to the back or the flexor side of the forearm of the subject with a low-allergenic tape, and gently pressed with the palm to make it evenly adhere to the skin for 24 h. At 30 min after removing the test patch device (after the indentation disappeared), the skin reaction was observed according to the standard in Table 1, and the observation results were recorded.

[0106] Table 1 Grading Criteria for Skin Reactions in Skin Occlusive Patch Test

[0107]

[0108]

[0109] Table 2 Results of Human Skin Patch Test

[0110]

[0111]

[0112] As can be seen from the results in Table 2, the results of the human skin patch test of the hair dyes prepared in Application Examples 1-3 were all negative reactions, indicating that the hair dyes prepared with the melanin precursor composition prepared in Examples 1-3 of the present invention have no potential adverse reactions to human skin, high safety, mildness and no irritation. Most of the test results of the hair dyes prepared in Comparative Application Examples 1-10 were negative, and only individual test subjects in Comparative Application Example 3, Comparative Application Example 9, and Comparative Application Example 10 showed suspicious reactions, only with weak erythema.

[0113] Test Example 2: Dyeing Effect Test

[0114] Standard undyed off-white hair bundles were selected for the experiment. There was no significant difference between groups. The temperature and humidity of the laboratory environment were kept relatively stable. The hair dyes prepared in Application Examples 1-3 and Comparative Application Examples 1-10 with the same dosage were evenly applied to the hair bundles, and the start time was recorded. The color change of the hair bundles was observed every 10 minutes, and the data was recorded. After 60 minutes, the hair dyes on the hair bundles were rinsed off, the hair bundles were dried, and the final color was visually evaluated.

[0115] Table 3 Test Results of Dyeing Effect

[0116]

[0117]

[0118] As can be seen from the results in Table 3, the dyeing effects of the hair dyes prepared in Application Examples 1-3 are significantly improved compared with those in Comparative Application Examples 1-10. The melanin precursor compositions in Application Examples 1-3 use specific plant raw materials, namely soybeans, black truffles, grape seeds, cocoa beans, Chinese gallnuts, and holly leaves, as raw materials for extracting melanin synthesis starting substances. They coordinate and cooperate with each other, and the prepared hair dyes have the effects of high dyeing efficiency and good dyeing effect. The hair dyes in Application Examples 1-3 can dye grayish-white hair bundles black after 20 minutes of dyeing, with a fast dyeing speed and high efficiency. The hair bundles obtained in Application Example 2 are smooth, black, shiny, and close to the color of natural hair after 30 minutes of dyeing, with a good dyeing effect. In contrast, in Comparative Application Examples 1-6, due to the lack of one of the plant raw materials, namely soybeans, black truffles, grape seeds, cocoa beans, Chinese gallnuts, and holly leaves, the dyeing effects of the prepared hair dyes become worse, the dyeing efficiency is relatively reduced, and the gloss of the hair bundles after dyeing is poor and not natural enough. In Comparative Application Examples 7-8, the composition of the compound protease is changed, and the dyeing effects of the prepared hair dyes are also not good, indicating that the compound protease composed of trypsin, papain, and flavor protease is beneficial to the synthesis of the melanin precursor composition under a specific ratio. In Comparative Application Example 9, the step of Saccharomyces cerevisiae fermentation is omitted, and in Comparative Application Example 10, the enzymatic reaction step is omitted. The hair dyes prepared can only turn grayish-black after 50 minutes of hair dyeing, and the hair bundles after dyeing have no luster, with a poor dyeing effect and low dyeing efficiency. It shows that the present invention uses natural raw materials and combines compound protease hydrolysis, Saccharomyces cerevisiae fermentation, and enzymatic reaction. Saccharomyces cerevisiae can help transform the components in the above plant extracts to produce intermediate products that can be used for melanin synthesis; tyrosinase and polyphenol oxidase are key enzymes in melanin synthesis, and they participate in the biosynthesis of melanin; tyrosinase catalyzes the conversion of tyrosine to dopa and further to melanin; while polyphenol oxidase can catalyze the oxidation polymerization of polyphenolic substances to form pigments. The melanin precursor composition prepared through the above steps can significantly improve the dyeing effect.

[0119] Test Example 3: Dyeing Durability Test

[0120] Select standard undyed hair bundles for the experiment. There is no significant difference between groups. Keep the temperature and humidity of the laboratory environment relatively stable. Evenly apply the same amount of hair dyes prepared in Application Examples 1-3 and Comparative Application Examples 1-10 on the hair bundles, and observe the color change of the hair bundles. After 60 minutes, rinse off the hair dyes on the hair bundles and dry the hair bundles. Then wash the hair bundles with the same colorless shampoo once a day, rinse them clean and dry the hair bundles. Record the starting fading time and complete fading time after washing. Repeat each sample three times and take the average value. In addition, expose the dyed hair bundles to ultraviolet light for irradiation once a day for 60 minutes each time to simulate the effect of sun exposure, record the starting fading time and complete fading time after irradiation, repeat each sample three times and take the average value.

[0121] Table 4 Results of Dyeing Durability Test

[0122]

[0123]

[0124] As can be seen from the results in Table 4, the dyeing durability of the hair dyes prepared in Application Examples 1-3 is significantly improved compared to that of Comparative Application Examples 1-10. The melanin precursor compositions in Application Examples 1-3 use specific plant raw materials, namely soybeans, black truffles, grape seeds, cocoa beans, Chinese gallnuts, and holly leaves, as raw materials for extracting melanin synthesis starting substances, and combine compound protease hydrolysis, Saccharomyces cerevisiae fermentation, and enzymatic reactions. Each raw material coordinates and cooperates with each other, resulting in a hair dye with good dyeing durability. In contrast, in Comparative Application Examples 1-6, due to the lack of one of the plant raw materials, soybeans, black truffles, grape seeds, cocoa beans, Chinese gallnuts, and holly leaves, the dyeing durability of the prepared hair dyes is relatively poor. The hair bundles fade after being washed with shampoo for about one month after dyeing and are basically completely faded after two months. In Comparative Application Examples 7-8, the composition of the compound protease was changed, and the dyeing durability of the prepared hair dyes was also not good, indicating that the compound protease composed of trypsin, papain, and flavor protease is beneficial to the synthesis of the melanin precursor composition under specific ratios. In Comparative Application Example 9, the Saccharomyces cerevisiae fermentation step was omitted, and in Comparative Application Example 10, the enzymatic reaction step was omitted. The durability of the prepared hair dyes becomes poor after dyeing, and fading occurs within less than one month of washing with shampoo and is completely faded within less than two months. Under ultraviolet lamp irradiation conditions, fading occurs within about half a month and is completely faded within one month. It shows that the present invention uses natural raw materials and combines compound protease hydrolysis, Saccharomyces cerevisiae fermentation, and enzymatic reactions. Saccharomyces cerevisiae can help transform the components in the above plant extracts to produce intermediate products that can be used for melanin synthesis; tyrosinase and polyphenol oxidase are key enzymes in melanin synthesis, and they participate in the biosynthesis of melanin; tyrosinase catalyzes the conversion of tyrosine to dopa and further to melanin; while polyphenol oxidase can catalyze the oxidation polymerization of polyphenolic substances to form pigments. The melanin precursor composition prepared through the above steps can significantly improve the dyeing durability and increase the retention time of the hair dyeing effect.

[0125] Test Example 4: Use Experience Test

[0126] Thirty-nine volunteers aged 18-55 with white hair who met the test requirements were selected as the test subjects and randomly divided into 13 groups, with 3 people in each group. There were no significant differences between the groups. After the volunteers washed and dried their hair, the hair dyes prepared in Application Examples 1-3 and Comparative Application Examples 1-10 with the same dosage were evenly applied to the hair. After 60 minutes, the hair dyes on the hair were rinsed off and the hair was dried. The dyeing effects were evaluated, and the evaluation indicators were as follows (taking the average value):

[0127] Odor Evaluation: The odor of the dyed hair was evaluated according to the acceptance degree, and the evaluation criteria were as follows:

[0128] 3 points: The odor is fragrant and very satisfactory

[0129] 2 points: The odor is acceptable

[0130] 1 point: Slight pungent odor

[0131] 0 point: Severe pungent odor, unacceptable

[0132] Evaluation of hair dyeing effect: The odor of the hair after dyeing is evaluated according to the satisfaction with the hair dyeing effect. The evaluation criteria are as follows:

[0133] 3 points: The color is full, natural, smooth, jet-black and shiny

[0134] 2 points: The color is jet-black, and the glossiness is slightly poor

[0135] 1 point: The black color is grayish, without luster

[0136] 0 point: No coloring

[0137] Evaluation of durability: The test subjects wash their hair once a day with the same colorless shampoo after hair dyeing. After one month, the change of hair color is evaluated. The evaluation criteria are as follows:

[0138] 3 points: Close to the original color after dyeing, with basically no change

[0139] 2 points: The color fades slightly, and the glossiness decreases

[0140] 1 point: The color fades significantly

[0141] 0 point: The color basically fades

[0142] Table 5 Test results of usage experience

[0143] Hair color after 60 minutes of dyeing Odor evaluation Evaluation of hair dyeing effect Durability evaluation Application Example 1 Smooth, black and shiny 3.0 3.0 3.0 Application Example 2 Smooth, black and shiny 3.0 3.0 3.0 Application Example 3 Smooth, black and shiny 3.0 3.0 3.0 Comparative Application Example 1 Poor black gloss 2.3 2.3 2.0 Comparative Application Example 2 Poor black gloss 2.7 2.0 2.0 Comparative Application Example 3 Poor black gloss 2.3 2.3 2.3 Comparative Application Example 4 Poor black gloss 3.0 2.0 1.7 Comparative Application Example 5 Poor black gloss 2.3 2.3 2.0 Comparative Application Example 6 Poor black gloss 2.7 2.3 1.7 Comparative Application Example 7 Poor black gloss 2.3 1.7 1.7 Comparative Application Example 8 Poor black gloss 2.3 1.7 1.7 Comparative Application Example 9 Greyish-black and dull 2.3 1.3 1.3 Comparative Application Example 10 Greyish-black and dull 2.0 1.0 1.0

[0144] It can be seen from the test results of the usage experience in Table 5 that compared with Comparative Application Examples 1-10, the hair dyes prepared in Application Examples 1-3 have significant improvements in terms of hair dyeing effect and durability, with a fragrant odor, being easy to accept, having a good usage experience, good hair dyeing effect and excellent durability, and good market prospects. It shows that the present invention uses natural raw materials and combines compound protease hydrolysis, Saccharomyces cerevisiae fermentation and enzymatic reaction. Saccharomyces cerevisiae can help transform the components in the above plant extracts to produce intermediate products that can be used for melanin synthesis; tyrosinase and polyphenol oxidase are key enzymes for melanin synthesis, and they participate in the biosynthesis of melanin; tyrosinase catalyzes the conversion of tyrosine into dopa and further into melanin; while polyphenol oxidase can catalyze the oxidative polymerization of polyphenolic substances to form pigments. The melanin precursor composition prepared through the above steps can significantly improve the dyeing durability, improve the hair dyeing effect and usage experience.

[0145] As described above, it is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. A melanin precursor composition, characterized in that The composition comprises the following raw materials by weight: 14-20 parts of soybeans, 12-18 parts of black truffles, 10-15 parts of grape seeds, 8-12 parts of cocoa beans, 6-10 parts of gallnuts, and 6-10 parts of holly leaves. The composition is prepared by pretreatment, primary extraction, composite protease hydrolysis, secondary extraction, brewer's yeast fermentation, and enzymatic reaction of the above raw materials, and comprises the following steps: S1 pretreatment: washing all raw materials to remove impurities, drying, sterilizing, and ultrafine grinding to obtain soybean powder, black truffle powder, grape seed powder, cocoa bean powder, gallnut powder, and holly leaf powder according to weight; S2: primary extraction: mix the soybean powder and black truffle powder obtained in step S1, use phosphate buffer solution as extraction solvent, continue stirring and extracting for 2-4 hours at a pH value of 4-7 and a temperature of 25-45°C, filter to remove insoluble substances, and collect the supernatant; S3: Compound protease hydrolysis: taking the supernatant of step S2, using compound protease, hydrolyzing under suitable pH and temperature conditions for 6-10 hours to obtain a hydrolyzate, inactivating the enzyme in the hydrolyzate, and then separating and purifying by high performance liquid chromatography to obtain extract A; S4 secondary extraction: taking the grape seed powder, cocoa bean powder, gallnut powder and holly leaf powder in step S1 as raw materials, selecting an ethanol aqueous solution with a mass concentration of 50-70% as a solvent, and the mass ratio of the raw material to the solvent is 1:10 to 2:10, extracting at 60-75°C for 6-8 hours, filtering to remove solid residues, and concentrating the filtrate by vacuum distillation to remove most of the solvent to obtain an extract; S5: fermentation with brewer's yeast: cooling the extract obtained in step S4 to 25-30° C., adjusting the pH value to 4.5-5.5, adding brewer's yeast for fermentation for 18-24 hours to obtain fermentation liquid B; S6 enzymatic reaction: the extract A in step S3 is mixed with the fermentation liquid B in step S5, the temperature is controlled at 30-40°C and the pH value is 6.5-7.5, tyrosinase and polyphenol oxidase are added, the reaction is continued for 6-8 hours, the enzyme is inactivated, and an enzymatic solution C is obtained; S7 post-treatment: purifying the enzymatic hydrolyzate C in step S6 by column chromatography, and finally freeze-drying to obtain a melanin precursor composition in powder form; In step S3, the composite protease is composed of trypsin, papain, and flavor protease in the mass ratio of (1-3): (2-3): (1-2); In step S3, the pH value is 7-8 and the temperature is 25-37°C; The mass ratio of tyrosinase to polyphenol oxidase in step S6 is (2-3): (1-2).

2. The melanin precursor composition according to claim 1, characterized in that The composition comprises the following raw materials by weight: 16-18 parts of soybeans, 14-16 parts of black truffles, 12-14 parts of grape seeds, 10-12 parts of cocoa beans, 8-10 parts of gallnuts, and 8-10 parts of holly leaves.

3. The melanin precursor composition according to claim 1, characterized in that The composition comprises the following raw materials by weight: 17 parts of soybeans, 15 parts of black truffles, 13 parts of grape seeds, 10 parts of cocoa beans, 8 parts of gallnuts, and 8 parts of holly leaves.

4. Use of a melanin precursor composition in the preparation of a hair dye, characterized in that: The melanin precursor composition is the melanin precursor composition according to any one of claims 1 to 3.

5. A hair dye, characterized in that: The invention comprises 8-15 wt % of a melanin precursor composition and auxiliary materials usable in cosmetics, wherein the melanin precursor composition is the melanin precursor composition according to any one of claims 1 to 3.

6. The hair dye according to claim 5, characterized in that The auxiliary materials include surfactant, thickener, antioxidant, pH regulator, essence and water.

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