Hair-blacking cosmetic and preparation method thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a compound with a hair-blacking effect, an external cosmetic containing the compound and a preparation process of the external cosmetic. The tripeptide VNP (Val-Asn-Pro) and the cosmetic containing the VNP (Val-Asn-Pro) are firstly disclosed to be applied through skin (especially head skin) and have a good hair-blacking promoting effect, and compared with a control group, the hair-blacking effect of the tripeptide VNP and the cosmetic containing the VNP are enhanced along with increase of the concentration of the VNP. When the concentration reaches 10%, the in-vitro tyrosinase activation effect exceeds 40%. The blackening of the hair, especially the blackening of the hair, can be effectively promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and particularly relates to a compound with hair-blackening effect, an external cosmetic containing the compound and a preparation process thereof. Background Art

[0002] Everyone loves beauty. Chinese people believe that black hair is the most beautiful. A head full of thick, black and shiny hair can give people a feeling of vigor and vitality, and can make people radiant and more charming. Graying of hair is a characteristic of aging, while premature graying of hair is a sign of premature aging or illness, which can make people sad when looking in the mirror. As an appendage of human skin, hair also has physiological functions such as protecting the head, reducing or avoiding external mechanical and chemical damage, buffering damage to the head, preventing or reducing ultraviolet damage to the scalp and tissues and organs in the scalp, moisturizing and preventing freezing.

[0003] The reason why hair appears in different colors is due to the melanin content and melanin particles in the hair. The more melanin particles there are and the greater the density, the darker the hair is, and vice versa. Hair melanin is synthesized by melanocytes in hair follicles, and its anabolism can be divided into three stages: melanogenesis, transfer, and "degradation". Obstacles in any of these stages will affect melanin metabolism, leading to changes in hair color. Tyrosinase is a key enzyme in melanin synthesis. The functional impairment or loss of the tyrosinase system in the melanocytes of the skin and hair follicles will cause a reduction or loss of skin pigmentation, ultimately leading to corresponding diseases, such as dry, dull, and easily broken hair.

[0004] Grey hair refers to the partial or complete graying of hair. With the increase of age, it is an inevitable physiological trend for people's hair to turn from black to white. Graying or whitening of hair in old age is a normal physiological phenomenon. The number of melanocytes in gray hair is normal but the melanin is reduced, while the number of melanocytes in white hair is also reduced. Adolescent grey hair refers to the premature graying of hair, commonly known as "premature greying", which is one of the diseases of skin appendages. Due to the complex causes of adolescent grey hair, it has a greater psychological impact on adolescent patients and is a risk factor for coronary heart disease with a high incidence rate (15%-32%).

[0005] According to traditional Chinese medicine, the following factors are related to gray hair: First, deficiency of essence and weak blood: insufficient kidney essence cannot transform into yin blood, and the deficiency of yin blood causes the hair to lose its nourishment, so it turns gray. Second, excessive blood heat: emotional excitement causes water to fail to contain wood, liver hyperactivity and blood dryness, excessive blood heat, and hair roots lose nourishment, so the hair turns gray early. Third, liver depression and spleen dampness: liver qi stagnation damages the heart and spleen, spleen damages the function of transportation and transformation, and qi and blood have no source, so the hair turns gray.

[0006] Classical records show that black sesame seeds, processed fleece-flower roots, and angelica sinensis nourish blood and benefit essence; cypress leaves and eclipta prostrata cool the blood, blacken the beard and hair; semen platycladi nourishes the heart and soothes the mind, and is good at treating blood deficiency, palpitation, and insomnia. Modern medicine has proven that angelica sinensis can promote the proliferation of melanocytes, melanin production, and activate the activity of tyrosinase. The extract of processed fleece-flower root has a significant promoting effect on the in vitro proliferation and migration of melanocytes. The extract of scutellaria baicalensis can promote the proliferation of mouse melanoma B16 cells, activate tyrosinase, and increase the melanin content. Black sesame seeds, mint, fleece-flower root, scutellaria baicalensis, dodder seeds, and angelica pubescens have the effect of activating tyrosinase, which can cause varying degrees of pigmentation on the skin: increased pigmentation in the basal layer of the epidermis and the middle and lower layers of some spinous cells, and an increase in dopa-positive cells.

[0007] Modern medicine believes that the main cause of white hair is the weakened function of melanocytes to form melanin, reduced formation of hair melanin, and decreased activity of tyrosinase, resulting in the disappearance of pigment in the hair shaft. The mechanisms of white hair formation include:

[0008] (1) The number of hair matrix melanocytes decreases or disappears.

[0009] (2) Melanin growth disorder. Some scholars have also found through experiments on transgenic mice with white hair using LacZ as a reporter gene that during the process of melanin synthesis, excessive oxygen free radicals lead to functional defects in hair follicle stem cells. Therefore, the most likely main cause of white hair is abnormalities in melanocytes.

[0010] (3) The production of tyrosinase decreases or disappears.

[0011] (4) There are tyrosine enzyme inhibitors in the body. It is now known that the production of melanin is closely related to tyrosinase. Tyrosinase is commonly found in humans, animals, and plants. It is a metalloenzyme formed by the binding of divalent copper ions and enzyme proteins, and this enzyme catalyzes the formation of melanin from tyrosine. If the content or activity of copper ions in the human body decreases, local depigmentation will occur, leading to the occurrence of white hair.

[0012] (5) The migration of melanin from hair matrix melanocytes to hair cortex cells is impaired.

[0013] (6) Trace elements such as copper, iron, and cobalt are lacking or cannot be normally transported to the roots of the hair.

[0014] At present, there is no effective treatment for white hair. To meet the needs of beauty, the vast majority of patients have to cover it up with hair dyes. However, excessive hair dyeing can not only damage the protective film of the hair, harm the scalp and hair follicles, accelerate follicular keratinization, and cause the hair to become thinner, but also lead to a large amount of hair loss and even serious diseases. Hair dyes generally cause redox reactions in the hair, and bleaching agents are even more chemical decolorants that change the hair color while decolorizing it. Oxidants have an obvious oxidizing effect on certain keratin proteins and pigment components in the hair. After the hair is treated with a decolorant, the content of each amino acid can change significantly. For example, the content of cystine, methionine, and tyrosine decreases significantly, which has a great negative impact on the hair quality and color.

[0015] In 1975, AMES et al. first used the Salmonella typhimurium reverse mutation method to detect 169 oxidative hair dyes produced by 8 major companies in the US market. The results showed that 150 of them were mutagenic, with a positive rate as high as 89%. This research result attracted the attention of professionals and government departments. Subsequently, studies proved that in addition to the positive mutagenicity test of Salmonella (Ames test), mutagenic hair dye chromogen oxides were also analyzed and determined by methods such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and proton nuclear magnetic resonance spectrum (NMR Spectrum) in human lymphocytes, yeast, mammalian cells, etc. It was also confirmed that the urine of experimental animals and humans using hair dyes contained mutagens.

[0016] Therefore, developing more effective and safe compounds to restore gray or even white hair to black hair and fundamentally solve the problem of hair turning white has far-reaching significance for humans. Thus, it can also change the current situation of using hair dyes to make hair black, making hair care products better meet people's needs.

[0017] Shampoos have a long history of development. Before the 1930s, people used soap, washing powder, etc. to wash their hair. The emergence of hair conditioners in the 1980s changed people's hair washing habits. People no longer only focused on hair cleaning, but used hair conditioners in combination, paying attention to hair softness. Using hair conditioner requires doing so after shampooing, and it is not easy to rinse off completely, requiring multiple rinses, wasting time and resources. Although ordinary shampoos have strong degreasing power and can effectively remove dirt, their strong degreasing power can stimulate the scalp and hair during the process of removing dirt, causing side effects such as hair damage, scalp itching, and increased dandruff. Therefore, adding hair care ingredients to ordinary shampoos can not only remove dirt but also reduce such damage, nourish the hair, and improve the combability of hair in both wet and dry states.

[0018] Among the many hypotheses about the formation of white hair, it is very likely that there is a disorder in the periodic correct activation, differentiation, migration, and pigment synthesis of melanocyte lineage cells.

[0019] In recent years, promoting the production of hair melanin by activating tyrosinase to darken hair has been an important research direction. By various means, the medicament is deeply penetrated into the hair follicles to activate the activity of tyrosinase, which can promote the production of hair melanin, thus achieving the purpose of blackening hair.

[0020] In 2018, Zhang Limei and other scholars from the Beijing Technology and Business University's Beijing Key Laboratory of Food Flavor Chemistry published an academic article in RSC Advances, Isolation and evaluation of two angiotensin-I converting enzyme inhibitory peptides from fermented grains (Jiupei) used in Chinese Baijiu production (Separation and evaluation of two angiotensin-I converting enzyme inhibitory peptides from fermented grains (Jiupei) used in Chinese Baijiu production).

[0021] In this study, fermented grains (Jiupei, the raw material for Baijiu distillation) were used to isolate and identify low-molecular-weight peptides with angiotensin-converting enzyme (ACE) inhibitory activity. The extraction methods for Jiupei polypeptides included ultrasonic treatment, centrifugation, and filtration. Peptide purification was performed by adsorption on macroporous resin, gel chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC) through ultrafiltration. HPLC-QTOF-MS / MS was used to identify the peptides and measure their ACE inhibitory activity. Finally, the optimal parameters for the extraction and separation of Jiupei polypeptides were determined as a liquid / solid (ultrapure water / turbid powder) ratio of 15 mL / g, an extraction temperature of 57 °C, and an ultrasonic time of 33 minutes. XAD-16 resin was used to remove sugars and salts. Under these conditions, the total yield of the extracted peptides was 57.682 mg / 1 g of Jiupei. The identified peptides were Val-Asn-Pro and Tyr-Gly-Asp. Val-Asn-Pro exhibited some ACE inhibitory activity (IC 50 = 38.02 μM), while Tyr-Gly-Asp showed higher ACE inhibitory activity (IC 50 = 5.21 μM). These results provide an important basis for the study of Jiupei polypeptides, indicating the generation of trace amounts of polypeptides during the Baijiu production process.

[0022] In 2021, Jiang Yunsong et al. from the School of Food Science and Engineering, South China University of Technology, published an academic article in Food and Chemical Toxicology: Antioxidant mechanism exploration of the tripeptide Val-Asn-Pro generated from Jiuzao and its potential application in baijiu.

[0023] The article pointed out that the tripeptide Val-Asn-Pro (VNP) extracted from the raw materials of baijiu distillation (Jiuzao, called fermented grains in red wine and yellow rice wine fermentation) has in vitro antioxidant activity. The method in previous literature was used to isolate and extract VNP from fermented grains, and its potential antioxidant mechanism in vivo was further evaluated.

[0024] The content of VNP was identified as 5.25 mg / g Jiuzao. Through the NRF2 / Keap1-P38MAPK / PI3K-MAFK signaling pathway, activating downstream antioxidant enzymes, VNP significantly alleviated excessive oxidative stress. In addition, the addition of VNP to baijiu had no obvious effect on the flavor of baijiu, and its content remained stable during storage. These results indicate that VNP is an effective antioxidant component that can be used in baijiu to enhance its antioxidant effect without affecting the main flavor. The utilization of these functional components can also increase the added value of Jiuzao.

[0025] However, the existing literature has not disclosed any other activities of VNP (Val-Asn-Pro) or its effects on other proteins or enzymes. Summary of the Invention

[0026] In view of the strong demand of the general public, especially beauty lovers, for hair nourishing and blackening, the applicant has conducted in-depth research on existing substances with potential hair blackening effects. Unexpectedly, it was found that VNP (Val-Asn-Pro) has a significant tyrosinase activation effect, can significantly enhance the tyrosinase activity in the skin and hair follicles, promote the proliferation of melanocytes and melanin formation, can improve the hair color, and play a role in blackening brown or even white hair.

[0027] VNP (Val-Asn-Pro), with a relative molecular mass of 328.37 Da, and the SMILES code is O=C(N[C@@H](CC(N)=O)C(N1[C@@H](CCC1)C(O)=O)=O)[C@H](C(C)C)N, and the chemical structure is as follows:

[0028]

[0029] It has been found through research that VNP (Val-Asn-Pro) can be administered transdermally.

[0030] VNP (Val-Asn-Pro) is highly soluble in water, safe, non-irritating, and has good skin topical safety. It can be further prepared into skin topical cosmetics such as shampoos and hair conditioners.

[0031] The present invention first discloses the use of VNP (Val-Asn-Pro) for preparing cosmetics that promote black hair growth.

[0032] The present invention further provides cosmetics comprising VNP (Val-Asn-Pro), which can be hair care products for promoting black hair growth and nourishing hair, such as shampoos, hair conditioners, hair masks, and hair creams.

[0033] The VNP (Val-Asn-Pro) described in this application can be prepared according to the extraction process or synthesis process provided by the prior art, or can be directly purchased.

[0034] VNP (Val-Asn-Pro) is usually prepared into topical agents, topical pharmaceuticals, quasi-pharmaceuticals, or skin cosmetics in combination with additives.

[0035] The additives described above include surfactants, anti-dandruff and anti-itching agents, thickeners, hair-conditioning oils, preservatives, nutritional components, anti-inflammatory agents, cooling agents, moisturizers, antioxidants, metal ion chelators, fragrances, pearlescent agents, and pH regulators, etc.

[0036] As the surfactant, there is no particular limitation, and non-ionic, anionic, cationic, and amphoteric surfactants can be appropriately used.

[0037] Surfactants that can be selected include, but are not limited to, sodium lauryl sulfate or ammonium sulfate, octadecyl trimethyl ammonium chloride (abbreviated as 1831), coconut fatty acid diethanolamide (abbreviated as 6501), alkyl polyglycoside (AVG), cocoamidopropyl hydroxysultaine (abbreviated as CHSB), cocoamidopropyl betaine (abbreviated as CAB-35), polyquaternium, and cationic guar gum, one or more of them.

[0038] The anti-dandruff and anti-itching agents include, but are not limited to, clotrimazole, ketoconazole, piroctone olamine, zinc pyrithione (ZPT), diazolidinyl urea, borax, methylisothiazolinone, triclosan, hinokitiol, triclosan, chlorhexidine gluconate, phenoxyethanol, salicylic acid, one or more of them.

[0039] The thickeners include, but are not limited to, inorganic salts such as sodium chloride, cetearyl alcohol, guar gum, hydroxypropyl cellulose, one or more of them.

[0040] The hair-conditioning oils include, but are not limited to, one or more of isopropyl palmitate, castor oil, glyceryl tristearate, and cyclohexasiloxane.

[0041] The preservatives include, but are not limited to, Kathon, ethylparaben, resorcinol, isopropylmethylphenol, and benzalkonium chloride.

[0042] The nutritional components include, but are not limited to, one or more of wheat protein, vitamins, and amino acids.

[0043] The vitamins include, but are not limited to, one or more of vitamin E, vitamin C, niacinamide, D-panthenol, panthenol ethyl ether, biotin, pyridoxine hydrochloride, and riboflavin.

[0044] The anti-inflammatory agents include, but are not limited to, one or more of dipotassium glycyrrhizinate, β-glycyrrhetinic acid, allantoin, diphenhydramine hydrochloride, guaifenesin, and L-menthol.

[0045] The cooling agents include, but are not limited to, one or more of L-menthol and camphor.

[0046] The moisturizing agents include, but are not limited to, one or more of L-pyrrolidone carboxylic acid, sodium hyaluronate, chondroitin sulfate, Cordyceps sinensis extract, and Crocus sativus extract.

[0047] The antioxidants include, but are not limited to, one or more of butylated hydroxyanisole, isopropyl gallate, propyl gallate, and erythorbic acid.

[0048] Examples of the metal ion chelating agents include ethylenediaminetetraacetic acid or its salts.

[0049] Examples of the fragrances include natural fragrances such as orange oil, lemon oil, bergamot oil, lime oil, lemongrass oil, and lavender oil, and synthetic fragrances such as menthol, rose oxide, linalool, citral, and linalyl acetate.

[0050] The pearlescent agent is one of pearlescent flakes or pearlescent pastes.

[0051] As the pH regulator, citric acid or its salts, or tartaric acid or its salts can be selected.

[0052] The VNP (Val-Asn-Pro) described in the present application and the cosmetics containing VNP (Val-Asn-Pro) are applied through the skin (especially the scalp), and have a good effect of promoting hair blackening. Compared with the control group, the hair blackening effect is enhanced with the increase of the concentration. When the concentration reaches 10%, the in vitro tyrosinase activation effect can exceed 40%. It can effectively promote the hair, especially the hair, to turn black. Detailed implementation manners

[0053] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0054] Example 1 Repeated Skin Irritation Test on Experimental Animals (Refer to the 2015 Edition of the Cosmetics Safety and Technology Specifications)

[0055] I. Materials and Methods

[0056] 1. The test substance is a 10% aqueous solution of VNP (Val-Asn-Pro).

[0057] 2. Test animals: 4 ordinary-grade white New Zealand rabbits

[0058] 3. Test method: 24 hours before the test, the hair on both sides of the animal's back spine was shaved off, and the shaving area was about 3 cm × 3 cm on each side. During the test, 0.5 mL of the test substance was evenly applied to the 3 cm × 3 cm skin on the left side, and distilled water was used as a control on the right side. It was applied once a day for 7 consecutive days. Starting from the second day, the hair was removed before each application, washed with warm water, and the skin reaction was observed 1 hour later and the skin irritation reaction was scored. After the test, the total score for 14 days, the average score per animal for 14 days, and the average score per animal per day were calculated respectively, and the skin irritation intensity was classified.

[0059] II. Scoring Criteria

[0060]

[0061] III. Classification of Skin Irritation Intensity

[0062] Integral mean value Strength 0-0.5 Non-irritating 0.5-2.0 Slightly irritating 2.0-6.0 Moderately irritating 6.0-8.0 Strongly irritating

[0063] IV. Test Results

[0064] Table 1 Results of Repeated Skin Irritation Test

[0065]

[0066] As shown in the data in Table 1, repeated application of a 10% aqueous solution of VNP has no irritation to the skin of rabbits, proving that an aqueous solution of VNP at a concentration of 10% or less has good skin application safety and tolerance.

[0067] Example 2 Allergic Reaction Test (Refer to the 2015 Edition of the Cosmetics Safety and Technology Specifications, Local Occlusive Patch Test)

[0068] I. Materials and Methods

[0069] 1. Preparation method of the test substance: 10% aqueous solution of VNP (Val-Asn-Pro)

[0070] 2. Test animals: Albino guinea pigs of the ordinary grade British breed were divided into three groups: the test substance experimental group, the positive substance experimental group, and the test substance control group, with 20 animals in each group.

[0071] 3. Test method:

[0072] Inductive contact: Apply 0.2 ml of the test substance for induction on the skin of the shaved area (2 cm × 2 cm) on the left side of the animals in the test substance experimental group, cover it with two layers of gauze and one layer of cellophane, and fix it with adhesive tape for 6 hours of occlusion. Repeat the same method on the 7th and 14th days. In the positive substance experimental group, use a 4.0% 2,4-dinitrochlorobenzene acetone solution, and in the test substance control group, use the solvent for the same treatment.

[0073] Eliciting contact: 14 days after the last induction, apply 0.2 ml of the test substance for elicitation on the skin of the shaved area (2 cm × 2 cm) on the right side of the animals in the test substance experimental group and the test substance control group, which was shaved 24 hours ago, cover it with two layers of gauze and one layer of cellophane, and fix it with adhesive tape for 6 hours of occlusion. In the positive substance experimental group, use a 1.2% 2,4-dinitrochlorobenzene acetone solution for the same eliciting contact, and in the test substance control group, use the solvent for the same treatment. Observe the skin reaction at 24 and 48 hours after the eliciting contact, and conduct skin reaction scoring and sensitization intensity grading.

[0074] 4. Test results: The skin reaction conditions of the animals in each group were observed at 24 and 48 hours, as shown in Table 2:

[0075] Table 2 Results of the local skin allergy test in guinea pigs

[0076]

[0077] As shown in the data in Table 2, the sensitization rate of the animals in the positive substance test group was 65% at 24 hours and as high as 90% at 48 hours, proving that the model animals and the test method can be used to detect the skin allergy caused by the test substance. However, when the test substance was applied, no allergic reaction occurred in the test animals, and the sensitization rate was 0, proving that the 10% VNP solution has no sensitizing property.

[0078] Example 3 Activation effect of VNP (Val-Asn-Pro) aqueous solution on tyrosinase (Refer to the literature: Zhang Jian, In vitro experimental study on the hair blackening effect of different traditional Chinese medicine prescriptions, Master's thesis of Southern Medical University, Class of 2007)

[0079] The color of the skin mainly depends on the content of melanin in it, and the formation of melanin mainly goes through the following three-step reaction. First, tyrosine is oxidized to dopa, then dopa is oxidized to dopaquinone, and finally dopaquinone is converted into melanin through a series of complex reactions. The formation of dopaquinone is the key. It is produced under the catalysis of tyrosinase, the key enzyme in melanin formation. In this experiment, an in vitro simulation method was used to make the reaction of tyrosinase catalyzing L-dopa to produce dopaquinone proceed in a test tube. Dopaquinone has an absorption peak at a wavelength of 475 nm. By measuring the OD value of the mixed reaction solution at 475 nm, the concentration of dopaquinone can be reflected, and the role of the added reagent can be determined by comparison.

[0080] I. Reagent preparation:

[0081] PBS solution (phosphate buffer): Take 8 g of sodium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium chloride, and 0.2 g of potassium dihydrogen phosphate, dissolve them in 800 ml of deionized water, make up the volume to 1000 ml, and filter with a 0.22-μm microporous membrane to obtain it.

[0082] Preparation of tyrosinase solution: Accurately weigh 25 KU of tyrosinase, dissolve it in 125 mL of PBS, that is, the enzyme activity unit is 200 u / mL. Shake it to dissolve and set aside.

[0083] Preparation of levodopa solution: Precisely weigh 20 mg of levodopa 1 h before the experiment, dissolve it in 50 ml of PBS, that is, the concentration is 0.4 mg / mL. Shake it and fully dissolve it before use.

[0084] Preparation of the test substance:

[0085] Dissolve VNP (Val-Asn-Pro) in freshly prepared PBS solution to form aqueous solutions with high, medium, and low three-dose concentrations, which are 10 mg / mL, 50 mg / mL, and 100 mg / mL respectively.

[0086] II. Experimental method

[0087] Mix 100 μL of the above-prepared aqueous solutions of VNP (Val-Asn-Pro) with different concentrations or PBS solution with 50 μL of tyrosinase, incubate in a 37 °C constant temperature water bath for 5 min, then add or not add 50 μL of levodopa (L-dopa) and continue to incubate for 2 min. Immediately measure the optical density OD value at 475 nm with an enzyme-labeling instrument. Each sample is measured 6 times repeatedly, and the average value is obtained, and then the activation rate of tyrosinase is calculated.

[0088] The activation rate of tyrosinase activity is calculated according to the following formula:

[0089] Activation rate % = [(C - D) - (A - B)] / (A - B) × 100%

[0090] In the formula: A = A 475-药+酶

[0091] B = A 475-药-酶

[0092] C = A 475+药+酶

[0093] D = A 475+药-酶

[0094] Wherein,

[0095] The value of A is the OD value of 100 μL of PBS + 50 μL of tyrosine solution at 500 u / mL + 50 μL of 0.4% L-dopa solution.

[0096] The value of B is the OD value of 100 μL of PBS + 50 μL of PBS solution + 50 μL of 0.4% L-dopa solution.

[0097] The value of C is the OD value of 100 μL of the liquid medicine + 50 μL of tyrosine solution at 500 u / mL + 50 μL of 0.4% L-dopa solution.

[0098] The value of D is the OD value of 100 μL of the liquid medicine + 50 μL of PBS solution + 50 μL of 0.4% L-dopa solution.

[0099] Table 3 Effects of VNP (Val-Asn-Pro) aqueous solution on tyrosinase (n = 6)

[0100] Concentration <![CDATA[Value of C: A 475+药+酶 > <![CDATA[Value of A: A 475-药+酶 > Enzyme activation rate (%) 10mg / mL 0.263 0.238 10.5％ 50mg / mL 0.313 0.238 31.5％ 100mg / mL 0.346 0.238 45.4％

[0101] Experimental results: As shown in the above table, VNP (Val-Asn-Pro) aqueous solutions at high, medium, and low concentrations all have the effect of activating tyrosinase. And with the increase of the added concentration of VNP, the activation effect is enhanced, and the sample with the highest activation rate is the one at 100 mg / mL, with an enzyme activation rate of 45.4%.

[0102] Example 4 Effects of VNP (Val-Asn-Pro) aqueous solution on melanin production and tyrosinase activity

[0103] The mouse B16 melanoma cell line with the function of producing melanin has been widely used in research at home and abroad. This experiment uses the mouse B16 melanoma cell line to carry out the experiment.

[0104] I. Preparation of main working solutions:

[0105] PBS solution: The same as in Example 3.

[0106] Bispecific antibody stock solution: Take 0.625 g of sodium penicillin and 1.000 g of streptomycin, add about 80 ml of PBS, dissolve thoroughly, make up the volume to 100 mL, filter and sterilize with a 0.22 μm microporous membrane, and dilute 100 times before use.

[0107] Cell digestive solution (0.25% Trypsin - 0.02% EDTA-2Na): Weigh 0.25 g of trypsin and 0.02 g of EDTA-2Na, add about 80 ml of PBS, stir and dissolve thoroughly, make up the volume to 100 ml, place at 4 °C overnight, adjust the pH value to 8.0, filter and sterilize with a 0.22 μm filter, and set aside.

[0108] MTT working solution:

[0109] Weigh 1 g of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide powder (MTT), dissolve it in 200 ml to make a concentration of 5 mg / mL, filter and sterilize with a 0.22 μm filter, then aliquot into 1.5 ml EP tubes (avoid repeated freezing and thawing), wrap with tin foil to avoid light, and set aside.

[0110] II. B16 cell culture:

[0111] The mouse melanoma B16 cell line is cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) (containing 100 U / mL penicillin and 100 μg / mL streptomycin) until it is nearly confluent. Digest with 0.25% trypsin and 0.02% EDTA, centrifuge, resuspend the cells with the above medium, and adjust the cell density to 1×10 5 / mL, and inoculate into 96-well plates (for cell proliferation assay) and 6-well plates (for melanin content determination) respectively. 24 hours after cell inoculation, replace the medium with fresh medium containing different concentrations of the test substance, and set up a well without the test substance as a blank control. Continue to incubate for 48 h (cell proliferation assay) and 72 h (melanin content determination) and then conduct the determination.

[0112] III. Detection of cell proliferation by MTT method:

[0113] Take B16 cells in the logarithmic growth phase and inoculate them into 96-well plates, 100 μL per well (inoculation density is 1×10 4 / Well). After 24 h, aspirate the culture medium, and replace it with fresh culture medium containing different concentrations of the test substance (100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1 mg / mL, preparation method: dissolve the test substance in fresh culture medium). Set the well without the test substance as the blank control. After continuing to culture for 48 h, aspirate the culture medium, wash with PBS, add fresh culture medium, add 10 μL of 5 mg / mL MTT solution to each well in the dark, incubate in the incubator for 4 h, aspirate the culture medium, add 150 μL of DMSO to each well, shake well, and measure the absorbance at 475 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Each experiment is repeated 4 times. Calculate the proliferation rate according to the following formula:

[0114] Proliferation rate (%) = (OD 浓度 - OD0) / OD0 × 100%

[0115] The experimental results are as follows:

[0116] Table 4 Effects of aqueous solutions of different concentrations of VNP (Val-Asn-Pro) on the proliferation of melanoma cells (n = 4)

[0117] Concentration (mg / mL) Average absorbance Proliferation rate (%) Concentration (mg / mL) Average absorbance Proliferation rate (%) 100 1.601 38.73％ 1 1.197 3.73％ 50 1.514 31.20％ 0.5 1.176 1.91％ 20 1.486 28.77％ 0.2 1.168 1.21％ 10 1.305 13.08％ 0.1 1.163 0.78％ 5 1.268 9.88％ 0 1.154 0 2 1.212 5.03％ / / /

[0118] By comparing with the blank control, aqueous solutions of different concentrations of VNP (Val-Asn-Pro) have the effect of promoting the proliferation of B16 cells (P < 0.05); and it increases with the increase in the concentration of VNP (Val-Asn-Pro), with the highest being 38.73%.

[0119] IV. Determination of melanin content:

[0120] Inoculate B16 cells in the logarithmic growth phase into a 6-well plate, 1000 μL per well (inoculation density is 1 × 10 5 / mL). After 24 h, aspirate the culture medium, and replace it with fresh culture medium containing different concentrations of the test substance (100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1 mg / mL), 2000 μL per well. Set the well without the test substance as the blank control. After continuing to culture for 72 h, aspirate the culture medium, wash with PBS, digest with trypsin, terminate the digestion, and collect the digestion solution. Transfer the digestion solution in each well to a 1.5 ml EP tube, centrifuge, discard the supernatant, add 10% trichloroacetic acid, centrifuge, discard the supernatant, add 1 M NaOH solution, heat in a water bath at 80 °C until completely dissolved, transfer to an ELISA plate, and immediately measure the absorbance at 475 nm of each well with an ELISA reader. Each experiment is repeated 4 times. Calculate the melanin increase rate according to the following formula:

[0121] Melanin increase rate (%) = (OD 浓度 - OD0) / OD0 × 100%

[0122] The test results are as follows:

[0123] Table 5 Effects of VNP (Val-Asn-Pro) aqueous solutions at different concentrations on melanogenesis in B16 melanoma cells (n = 4)

[0124] Concentration (mg / mL) Average absorbance Melanin increase rate (%) Concentration (mg / mL) Average absorbance Melanin increase rate (%) 100 0.102 37.8％ 1 0.076 2.7％ 50 0.098 32.4％ 0.5 0.075 1.4％ 20 0.094 27.0％ 0.2 0.075 1.4％ 10 0.086 16.2％ 0.1 0.074 0 5 0.081 9.5％ 0 0.074 0 2 0.078 5.4％ /

[0125] After culturing for 72 h, the changes in the melanin content of cells in the blank control group and the VNP (Val-Asn-Pro) aqueous solution groups at different concentrations are shown in the above table. By comparison with the blank control, VNP (Val-Asn-Pro) aqueous solutions at different concentrations all had the effect of promoting melanogenesis in B16 melanoma cells (P < 0.05). When the concentration was relatively low, the increase rate was not obvious, but as the concentration increased, the melanin increase rate increased significantly. When the concentration reached 100 mg / mL, it reached a maximum of 37.8%.

[0126] Example 5 Hyperpigmentation test of VNP (Val-Asn-Pro) aqueous solutions at different concentrations on the skin of brown guinea pigs

[0127] I. Experimental animals:

[0128] 9 healthy brownish-yellow guinea pigs, both males and females.

[0129] II. Davidson fixative

[0130] 330 ml of 95% ethanol; 220 ml of formaldehyde; 115 ml of glacial acetic acid; 335 ml of distilled water;

[0131] III. Preparation of test samples: The preparation method was the same as that in Example 3.

[0132] IV. Experimental method:

[0133] The 9 guinea pigs were randomly divided into 3 groups and were respectively given VNP (Val-Asn-Pro) aqueous solutions at low, medium, and high doses (5 mg / mL, 10 mg / mL, 50 mg / mL). The brown areas on the backs of each guinea pig were shaved to expose the corresponding brown skin, which was randomly divided into 4 brown areas as different experimental groups for the experiment: blank control area (I), self-control area (II), positive control area (III), and sample area (IV). The normal skin on the back of the guinea pig without applying the test substance was used as the blank control (I), the skin coated with normal saline was used as the self-control (II), and 0.1% 8-methoxypsoralen (8-MOP) tincture was used as the positive control (III). VNP (Val-Asn-Pro) aqueous solutions at low, medium, and high doses were used as samples (IV), and the operation was carried out three times a day.

[0134] During the period of administering the test substance, the guinea pigs were shaved every three days. The test substance was continuously applied for 30 days, and tissue biopsies were taken after 30 days. The therapeutic effects and adverse reactions of the dorsal skin of the guinea pigs were observed and recorded. After the administration of the test substance was completed, a 4-cm area centered on the test site of the guinea pig was used as an observation unit to evaluate the therapeutic effect of the test substance and skin reactions (scales, erythema).

[0135] After the administration of the test substance was completed, a 4-cm area centered on the test site of the guinea pig was used as an observation unit to observe the therapeutic effect and skin reactions (scales, erythema).

[0136] Therapeutic effect judgment criteria:

[0137] Excellent means that the skin of the test area (4 cm) is normal, smooth, and without dandruff;

[0138] Good means that the skin of the test area is basically normal, without dandruff and hair loss (<5%);

[0139] Moderate means that dandruff and hair loss occur in the test area (<30%);

[0140] Poor means that dandruff and large-area hair loss occur in the test area (the area of hair loss >30%);

[0141] The total effective rate is calculated as excellent plus good.

[0142] The statistics of the appearance and health status of the animals are shown in Table 6. The skin color and hair color of the control group remained unchanged; the skin of the VNP (Val-Asn-Pro) group was light brownish-yellow, the skin of the test area was normal, without dandruff and erythema; the skin of the 8-MOP group was light brownish-yellow, the skin color was slightly darker than that of the control group, and scaling and hair loss reactions occurred.

[0143] Table 6 Observation of adverse reactions of the skin of test guinea pigs to each test substance (n = 3)

[0144] Group Excellent Good Medium Poor Adverse reaction rate Control group 9 0 0 0 0 Self-control group 8 1 0 0 0 8-MOP control group 0 7 1 1 22.2％ VNP (Val-Asn-Pro) high dose 3 0 0 0 0 VNP (Val-Asn-Pro) medium dose 3 0 0 0 0 VNP (Val-Asn-Pro) low dose 3 0 0 0 0

[0145] There were no adverse reaction phenomena in the test animals of the control group and the self-control group. 22.2% of the test animals in the 8-MOP administration group had certain adverse reactions on the skin. The skin of the guinea pigs in the VNP (Val-Asn-Pro) sample group was normal, without dandruff and erythema reactions, and no hair loss occurred.

[0146] V. Experimental method for histological examination:

[0147] After the experiment was completed, 1 cm × 1 cm skin was taken from the center of different test sites, fixed with 10% formaldehyde, and routinely paraffin-sectioned for standby.

[0148] Staining by Schmorl method: melanin granule cells

[0149] Schmorl staining is a special staining method for observing melanin. The specific staining method and experimental steps are as follows:

[0150] 1) Transfer the sections to running water for washing;

[0151] 2) Treat with a mixture of 30 ml of freshly prepared 0.037 mol / L (1%) FeCl3, 4 ml of freshly prepared 0.03 mol / L (1%) potassium ferricyanide, and 6 ml of distilled water for 10 min.

[0152] 3) Rinse with running water.

[0153] 4) Counterstain the cell nuclei with 1% safranin for 15 - 30 min.

[0154] 5) Wash with water, dehydrate, clear, and mount routinely.

[0155] The melanin content was statistically analyzed using the BI - 2000 medical image analysis system. The experimental data are expressed as and data processing was performed using Excel software. Statistical analysis was carried out using the t - test.

[0156] VI. Histological observation results:

[0157] (1) Melanin granule cell counting: The specimens stained by the Schmorl method were counted by a single - blind method using a reticular ocular micrometer. The number of melanin - granule - containing cells in the basal layer of the epidermis per mm was counted one by one under a high - power microscope. The results are as follows: 2 The results are as follows:

[0158] Table 7 Effects of different concentrations of VNP (Val - Asn - Pro) on guinea pig skin melanocytes and melanogenesis ( )

[0159] Group <![CDATA[Melanin granule cell content / mm 2 > Control group 1612.4±418 Self-control group 1676.2±339 8-MOP control group 2976.6±375 VNP (Val-Asn-Pro) high dose 3512.4±424 VNP (Val-Asn-Pro) medium dose 3200.9±482 VNP (Val-Asn-Pro) low dose 2867.7±361

[0160] Note: In Table 7, the number of samples in the control group, self - control group, and 8 - MOP control group is 9, that is, the mean and standard deviation data measured from 9 experimental animal samples. The number of samples in the high, medium, and low dose groups of VNP (Val - Asn - Pro) is 3.

[0161] The more the number of cells containing melanin granules, the more the number of cells with melanin - generating ability. From the data analysis in Table 7, it can be seen that there is a significant difference in the number of cells containing melanin granules in the epidermis of guinea pigs in the 8 - MOP group compared with the control group, indicating that 8 - MOP can increase the number of cells containing melanin granules per unit area of guinea pig skin. There are also significant differences in the number of cells containing melanin granules in the epidermis of guinea pigs in the low - dose, medium - dose, and high - dose groups of VNP (Val - Asn - Pro) compared with the control group, which also indicates that low - dose, medium - dose, and high - dose VNP (Val - Asn - Pro) can increase the number of cells containing melanin granules per unit area of guinea pig skin. There is no significant difference between the low - dose VNP (Val - Asn - Pro) group and the 8 - MOP group, indicating that the effect of low - dose VNP (Val - Asn - Pro) on increasing the number of cells containing melanin granules per unit area of guinea pig skin is similar to that of 8 - MOP.

[0162] VII. Melanin content index (MCI);

[0163] Use the BI - 2000 medical image analysis system to calculate the MCI of the Schmorl - stained sections. The statistics and calculations of the melanin content index are carried out from two aspects:

[0164] ① Melanin content index in melanocytes (MCI - 1): Select 10 basal cells containing melanin granules and measure the average percentage of the area of melanin granules in the examined cells accounting for the total area of the cells.

[0165] ② Melanin content index per unit area of the epidermis (MCI - 2): Select 10 areas of the epidermis and measure the average percentage of the area of melanin granules accounting for the total area of the epidermis at that place. The statistical data of the melanin content index are shown in Table 8.

[0166] Table 8 Effects of VNP (Val - Asn - Pro) on the melanin content index of the dorsal skin epidermis of brown guinea pigs

[0167] Group MCI-1 (%) MCI-2 (%) Control group 16.6±7.8 2.9±2.1 Self-control group 18.1±8.1 2.6±3.0 8-MOP control group 27.8±8.6 5.4±2.2 VNP (Val-Asn-Pro) high dose 38.4±6.4 8.5±1.8 VNP (Val-Asn-Pro) medium dose 33.6±7.1 6.3±1.4 VNP (Val-Asn-Pro) low dose 26.4±6.6 5.5±1.6

[0168] Note: In Table 8, the sample numbers of the control group, self - control group, and 8 - MOP control group are 9, that is, the mean and standard deviation data measured from 9 experimental animal samples. The sample numbers of the high - dose, medium - dose, and low - dose groups of VNP (Val - Asn - Pro) are 3.

[0169] Comparing the data of each group in Table 8, it can be seen that the intracellular melanin content index MCI - 1 of the specimens in the 8 - MOP group, low - dose, medium - dose, and high - dose groups of VNP (Val - Asn - Pro) has a significant increase compared with the control group, with statistical differences.

[0170] In the 8-MOP group and the low, medium, and high-dose test groups of VNP (Val-Asn-Pro), the epidermal melanin content index MCI-2 of the specimens was significantly increased compared with the control group, showing statistical differences, indicating that each test substance had the effect of promoting melanogenesis.

[0171] Example 6 Hair Conditioner Containing VNP (Val-Asn-Pro) (Unit: g)

[0172]

[0173]

[0174] Preparation Method:

[0175] (1) Weigh the raw materials according to the formula.

[0176] (2) Heat 70% deionized water, citric acid, glycerol, D-panthenol, polyquaternium-10, lauryl trimethyl ammonium chloride, guar gum hydroxypropyl trimethyl ammonium chloride, coconut oil acyl propyl dimethyl tertiary amine, EDTA-2Na, and VNP (Val-Asn-Pro) in the water phase pot to 60°C, stir evenly, and pump into the vacuum emulsifying pot.

[0177] (3) Add glycabene, cyclohexasiloxane, cetearyl alcohol, and coconut oil fatty acid monoethanolamide to the oil phase pot and heat to 60°C. Stir evenly and pump into the vacuum emulsifying pot.

[0178] (4) Start the vacuum device. Homogenize for 5 - 15 minutes under vacuum, make up the volume to the full amount with deionized water, and cool down while stirring.

[0179] (5) When the temperature of the vacuum emulsifying pot drops to 40°C, add vitamin E, essence, and methylisothiazolinone, and stir evenly.

[0180] (6) Continue to cool to room temperature and discharge the material.

[0181] Example 7 VNP (Val-Asn-Pro) Hair Cream (Unit: g)

[0182]

[0183]

[0184] Preparation Method: Take white oil, stearyl alcohol, monoglyceride, glycerol, polysorbate-80, ethylparaben, VNP (Val-Asn-Pro), add deionized water and mix. Heat to 90°C, stir, emulsify for 1 hour, then cool to 50°C, adjust the pH value to about 7 with 1M citric acid aqueous solution, add essence, and stir. Continuously cool to room temperature, discharge the material, and fill it for use.

[0185] Effect of Hair Conditioner and Hair Cream Containing VNP (Val-Asn-Pro) on Pigmentation in Guinea Pigs

[0186] Six healthy guinea pigs with brownish-yellow fur were randomly divided into two groups of three. After intraperitoneal anesthesia with 1% pentobarbital, the hair on the back of the guinea pigs was cut short with ophthalmic scissors to form a hairless area of 5 cm × 5 cm, taking care not to damage the epidermis. Four 2 cm × 2 cm areas were marked on the back of each guinea pig, with a 1 cm interval between the edges of each area. The hair conditioners or hair creams containing VNP (Val-Asn-Pro) prepared in Examples 6 and 7 were applied, that is, the cosmetics of Formulas 1-4 in Examples 6 or 7 were applied to the four areas on the back of each guinea pig. They were evenly coated with cotton swabs three times a day, about 0.2 g each time, and continuously coated for 20 days.

[0187] After coating, the animals were continued to be raised, and the skin and hair color changes in each area on the back of each guinea pig were observed.

[0188] 8 days 12 days 20 days Example 6 Formula 1 No abnormal change Grow brown hair, no abnormal change in skin Brown hair becomes longer, no abnormal change in skin Example 7 Formula 1 No abnormal change Grow brown hair, no abnormal change in skin Brown hair becomes longer, no abnormal change in skin Example 6 Formula 2 Skin is grayish black Skin is grayish black, grow black hair Skin is grayish black, black hair becomes longer Example 7 Formula 2 Skin is grayish black Skin is grayish black, grow black hair Skin is grayish black, black hair becomes longer Example 6 Formula 3 Skin is grayish black Skin is grayish black, grow black hair Skin is grayish black, black hair becomes longer Example 7 Formula 3 Skin is grayish black Skin is grayish black, grow black hair Skin is grayish black, black hair becomes longer Example 6 Formula 4 Skin is grayish black Skin is grayish black, grow black hair Skin is grayish black, black hair becomes longer Example 7 Formula 4 Skin is grayish black Skin is grayish black, grow black hair Skin is grayish black, black hair becomes longer

[0189] Through the above experimental observations, among the cosmetics of each formula in Examples 6 and 7, compared with Formula 1 without VNP (Val-Asn-Pro), the skin of guinea pigs given the cosmetics of Formulas 2-4 in Examples 6 and 7 with added VNP (Val-Asn-Pro) turned grayish-black on the 8th day of the experiment, and then began to grow black hair, which continued to grow. For the guinea pigs given Formula 1 in Examples 6 and 7 without added VNP (Val-Asn-Pro), the skin color did not change, and only brown hair grew.

[0190] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of the tripeptide Val-Asn-Pro for preparing cosmetics for improving hair color, characterized in that: The chemical structure of the tripeptide Val-Asn-Pro is as follows:

2. The use according to claim 1, characterized in that The cosmetic is used for promoting the proliferation of melanocytes and the formation of melanin.

3. The use according to claim 1, characterized in that The cosmetic product is used to promote the transformation of brown or even white hair into black hair.

4. The use according to claim 1, characterized in that The cosmetic works by enhancing tyrosinase activity in the skin and hair follicles.

5. The use according to claim 1, characterized in that The cosmetic is administered transdermally.

6. The use according to claim 1, characterized in that The cosmetics are shampoo, conditioner, hair cream, hair lotion or other cosmetics for external use on the skin.

7. The use according to claim 6, characterized in that The formula of the conditioner is as follows: Lauryltrimethylammonium chloride: 5.0g Cocoyl propyl dimethyl tertiary amine: 5.0g Glycerin: 3.0g Cetearyl alcohol: 2.0g D-Panthenol: 1.0g Polyquaternium-10: 0.2g Guar Hydroxypropyltrimonium Chloride: 0.2g Coconut oil fatty acid monoethanolamide: 1.0g EDTA-2Na: 0.1g Climbazole: 0.3g Flavor: 0.2g Citric acid: 0.5g Val-Asn-Pro: 0.5-5.0g Vitamin E: 0.5g Cyclohexasiloxane: 1.0g Methylisothiazolinone: 0.001g Deionized water to make up to 100ml Preparation method: (1) Weigh the raw materials according to the formula; (2) 70% deionized water, citric acid, glycerin, D-panthenol, polyquaternium-10, lauryl trimethyl ammonium chloride, guar hydroxypropyl trimethyl ammonium chloride, cocoyl propyl dimethyl tertiary amine, EDTA-2Na, and Val-Asn-Pro were heated to 60° C. in an aqueous phase pot, stirred evenly, and pumped into a vacuum emulsification pot; (3) adding climbazole, cyclohexasiloxane, cetearyl alcohol and coconut fatty acid monoethanolamide into the oil phase pot and heating to 60° C., stirring evenly, and pumping into the vacuum emulsification pot; (4) Start the vacuum device, homogenize under vacuum for 5-15 minutes, make up to the full volume with deionized water, and cool down with stirring; (5) When the temperature of the vacuum emulsifying pot drops to 40°C, add vitamin E, flavor and methylisothiazolinone and stir evenly; (6) Continue cooling to room temperature and discharging the product.

8. The use according to claim 6, characterized in that The formula of the hair cream is as follows: White oil: 20.0g Octadecanol: 5.0g Monoglyceride: 3.0g Glycerol: 8.0g Polysorbate 80: 1.4 g Ethyl paraben: 0.2g Ginseng extract: 0.5g 1M citric acid aqueous solution: appropriate amount Flavor: 0.5g Val-Asn-Pro: 0.5-5.0g Deionized water volume: 100ml Preparation method: Take white oil, octadecyl alcohol, monoglyceride, glycerol, polysorbate 80, ethylparaben, and Val-Asn-Pro, add deionized water to mix, heat to 90°C, stir, emulsify for 1 hour, then cool to 50°C, adjust the pH value to about 7 with 1M citric acid aqueous solution, add flavor, stir, continue to cool to room temperature, discharge, and fill.

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