Hair growth composition having melanin formation promoting effect, and method for preparing and use thereof

Abstract

The application discloses a hair-growing composition with melanin formation promotion function and a preparation method and application thereof, and the hair-growing composition comprises L-tyrosine, a urea-based compound, zinc ions, a plant extract and water; the composition of the application has high safety, and simultaneously acts on a hair growth cycle and a melanin generation process in multiple paths, multiple targets and multiple mechanisms such as providing raw materials, promoting enzyme activity, activating a path, improving microecology and the like, fundamentally cooperatively improves alopecia and canities problems, realizes integrated treatment of 'hair growth' and 'black hair', and avoids the limitation of a single target.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically, to a hair growth composition that promotes melanin formation, its preparation method, and its application. Background Technology

[0002] With the accelerated pace of modern life, increased work pressure, and more complex environmental factors, hair loss and premature graying are no longer exclusive to middle-aged and elderly people, but are showing a significant trend towards affecting younger individuals. These two problems often occur together, severely impacting personal image and causing immense psychological stress, social anxiety, and even depression, significantly reducing quality of life. Statistics show that hundreds of millions of people worldwide suffer from varying degrees of hair loss and graying, creating a huge market demand for efficient and safe solutions.

[0003] Current mainstream solutions for hair loss and gray hair have significant shortcomings. For example, drugs like minoxidil and finasteride primarily promote hair growth by dilating scalp blood vessels or inhibiting dihydrotestosterone (DHT). However, they are ineffective in improving gray hair and may be accompanied by side effects such as scalp irritation and sexual dysfunction, with a high relapse rate after discontinuation. Hair dyeing is the most direct way to cover gray hair, but it is essentially chemical coloring and cannot fundamentally restore the hair follicle's own pigment production ability. Frequent use of chemical hair dyes is not only cumbersome, but the paraphenylenediamine (PPD) and other ingredients they contain can also cause allergic reactions and even pose potential health risks. Traditional health supplements or hair care products mostly rely on traditional ingredients such as Polygonum multiflorum and black sesame, with unclear mechanisms of action, low concentrations of active ingredients, and difficulty in penetrating deep into the hair follicles due to the barrier effect of the scalp's stratum corneum, resulting in often unsatisfactory effects. Other emerging therapies may focus on a single target, such as providing only a certain peptide to attempt to activate melanocytes. This method ignores the complexity of hair follicle aging and gray hair formation, and often yields little result due to the "weakest link" effect.

[0004] In summary, existing technologies either treat hair growth and black hair separately or fail to address the problem at its physiological root, lacking a comprehensive solution that can efficiently and safely solve the coexisting problems of hair loss and gray hair.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] This invention proposes a hair growth composition that promotes melanin formation, its preparation method, and its application, aiming to provide a composition that is safe in ingredients and can effectively solve both gray hair and hair loss problems.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a hair growth composition that promotes melanin formation, the hair growth composition comprising L-tyrosine, a urea compound, zinc ions, plant extracts, and water; the plant extracts comprising Polygonum multiflorum extract, Platycladus orientalis leaf extract, Juglans regia extract, and Ligustrum lucidum extract; the urea compound being acetamide, dimethylurea, or hydantoin.

[0008] The composition provided by this invention contains L-tyrosine, a key substrate for melanin synthesis, zinc ions, a cofactor for various enzymatic reactions related to melanin formation, and specific plant extracts that can improve scalp microecology and promote tyrosinase activity. Through the combined multi-target action of providing key raw materials, enhancing enzyme activity, and improving microecology, it synergistically solves the problems of gray hair and hair loss, and the ingredients are safe.

[0009] In addition, urea compounds function as a solubilizer, preventing L-tyrosine from coordinating with zinc ions or other trace element ions to form precipitates. In terms of efficacy, dimethylurea has a moisturizing and softening effect; hydantoin has a broad-spectrum antibacterial effect and can be used as a preservative; acetamide has a moisturizing and conditioning effect.

[0010] The applicant's research found that the underlying physiological mechanisms of hair loss and graying hair are as follows: (1) Deterioration of hair follicle microecology and stem cell depletion: Hair follicle stem cells (HFSCs) located in the bulge area of ​​hair follicles are responsible for the periodic regeneration of hair; and melanocyte stem cells (McSCs) also located in the bulge area are the "reservoir" of hair follicle melanocytes. The depletion or dysfunction of McSCs leads to the inability to differentiate into mature melanocytes, which is the most upstream and fundamental cause of gray hair; the decrease in the activity of HFSCs leads to the shortening of the hair follicle growth phase and the prolongation of the resting phase, which eventually manifests as hair loss. (2) Melanocyte dysfunction and interruption of melanin synthesis pathway: Even if McSCs can successfully differentiate into mature melanocytes, the melanin synthesis pathway will be interrupted due to dysfunction such as decreased activity of key enzymes (tyrosinase), insufficient substrate supply, and melanosome transport disorders. (3) Oxidative stress: Excessive reactive oxygen species produced in vivo and in vitro can directly damage stem cells, leading to DNA damage and apoptosis in McSCs and HFSCs; they can also directly oxidize and inactivate key enzymes, thereby inhibiting melanin synthesis; chronic oxidative stress and inflammatory response caused by reactive oxygen species can damage the extracellular matrix around hair follicles, affecting the normal structure and function of hair follicles. (4) Dysregulation of key signaling pathways: The signaling pathway composed of melanocyte-stimulating hormone (α-MSH) and its receptor MC1R is crucial for hair follicle growth and pigment production. It can activate the intracellular cAMP (cyclic adenosine monophosphate) pathway, thereby upregulating the expression of the key transcription factor MITF (microphthalmia-associated transcription factor). MITF is the "master switch" that regulates melanin synthesis. It can initiate the transcription of a series of melanin synthesis-related genes such as tyrosinase (TYR), tyrosinase-associated protein 1 (TRP-1), and dopachrome tautomerase (DCT / TRP-2), thereby comprehensively initiating and accelerating melanin production.

[0011] This invention addresses the aforementioned physiological mechanisms by adding L-tyrosine to the composition, providing a key raw material for melanin synthesis and thus promoting melanin formation. Zinc ions are a core component of TRP-1 and an important component of melanosomes. The addition of zinc ions to the composition helps improve the expression activity and stability of TRP-1, enhances its catalytic activity in melanin synthesis, promotes melanin receptor signal transduction, and protects melanocytes from free radical damage. Specific plant extracts can improve the microecology, provide antioxidant, anti-inflammatory, and antibacterial effects, and enhance tyrosinase activity, thereby strengthening the hair follicle's resistance to oxidative stress, slowing stem cell depletion, and promoting its normal differentiation and migration. Among them, the main active components of Polygonum multiflorum extract are stilbene glycosides and anthraquinone compounds (especially emodin), which have strong antioxidant effects and promote melanin synthesis by activating MITF transcription factors to increase the expression of tyrosine-related proteins TRP-1, TRP-2 and tyrosinase. Two important components of Ligustrum lucidum extract, tyrosine and oleanolic acid, can promote the expression of c-kit receptor protein in melanocytes, thereby activating tyrosinase and promoting melanin synthesis. Wild walnut (Juglans regia) extract is rich in the mineral copper, juglone, phenolic compounds, fatty acids, and oils. Copper is a key component of tyrosinase, and the addition of copper... It is beneficial to the catalytic activity of tyrosinase and promotes melanin synthesis; juglone has significant antibacterial activity; phenolic compounds have excellent antioxidant, anti-inflammatory and antibacterial activities; fatty acids and oils can replenish lipids in the scalp and skin, repair damaged sebum film, enhance the skin's water-locking ability, and relieve dryness and flaking; Platycladus orientalis leaf extract is rich in flavonoids, terpenes and volatile oils and tannins, with extremely strong antioxidant, anti-inflammatory and broad-spectrum antibacterial activities, and can also promote hair follicle cycle transformation and activate growth signaling pathways. Cedarol (terpenoid) in it can inhibit the activity of 5α-reductase, thereby reducing the production of dihydrotestosterone (DHT) and thus reducing hair loss.

[0012] In some embodiments, the hair growth composition further includes at least one of European larch wood extract and copper peptides. European larch wood extract is rich in dihydroquercetin glucoside (a flavonoid compound) which has excellent antioxidant, soothing, anti-inflammatory, and sebum-regulating effects, effectively improving the scalp environment. Copper peptides can safely and efficiently replenish cells with bioavailable copper ions, directly enhancing the catalytic efficiency of tyrosinase. Additionally, it can prolong the hair follicle growth phase, increase hair follicle size, stimulate dermal papilla cell proliferation, and promote the production of vascular endothelial growth factor (VEGF), thereby improving scalp blood supply.

[0013] In some embodiments, the mass ratio of zinc to copper in the hair growth composition is (1:0.5) to (1:0.85), for example, it can be any value among 1:0.5, 1:0.55, 1:0.6, 1:0.65, 1:0.7, 1:0.75, 1:0.8, 1:0.85, or (1:0.5) to (1:0.85). Maintaining a zinc to copper mass ratio of (1:0.5) to (1:0.85) is beneficial for improving the transcriptional expression of tyrosinase and a series of melanin synthesis-related genes such as tyrosinase-associated protein 1 (TRP-1) and dopachrome tautomerase (DCT / TRP-2), avoiding the decrease in tyrosinase activity due to excessive zinc, or the decrease in TRP-1 expression due to excessive copper.

[0014] In some embodiments, the hair growth composition comprises, by weight percentage: 4%~36% L-tyrosine, 1.5%~16% urea compound, 0.7%~7% zinc ions, 10%~16% Polygonum multiflorum extract, 2%~5% Platycladus orientalis leaf extract, 5%~12% wild walnut extract and 5%~12% Ligustrum lucidum extract, with the balance being water.

[0015] In some embodiments, the hair growth composition comprises, by weight percentage: 8%–25% L-tyrosine, 3.5%–13% urea compounds, 1.5%–5% zinc ions, 10%–16% Polygonum multiflorum extract, 2%–5% Platycladus orientalis leaf extract, 5%–12% wild walnut extract and 5%–12% Ligustrum lucidum extract, with the balance being water.

[0016] In some embodiments, the hair growth composition having the function of promoting melanin formation comprises, by weight percentage: 8%~25% L-tyrosine, 3.5%~13% urea compound, 1.5%~5% zinc ions, 10%~16% Polygonum multiflorum extract, 2%~5% Platycladus orientalis leaf extract, 5%~12% Juglans regia extract, 5%~12% Ligustrum lucidum extract, 2%~8% Larch extract and 2%~5% copper peptide, with the balance being water.

[0017] Secondly, the present invention provides a method for preparing any of the above-described hair growth compositions that promote melanin formation, comprising the following steps: (1) Under stirring, a zinc salt solution and an L-tyrosine solution were mixed to carry out a precipitation reaction. After solid-liquid separation and washing, a tyrosine-zinc complex was obtained. (2) The tyrosine-zinc complex, urea compound and water are mixed in proportion and heated and stirred to obtain a eutectic solvent; (3) Add plant extract to the eutectic solvent, stir to dissolve, and separate solid and liquid to obtain the composition.

[0018] The preparation method provided by this invention first prepares a ternary eutectic solvent (DES) for L-tyrosine-zinc-urea compounds, and then uses it to dissolve plant extracts. On the one hand, the eutectic solvent has strong dissolving power, capable of simultaneously dissolving water-soluble and lipid-soluble substances, thereby improving the solubility and dispersion of active ingredients in plant extracts and avoiding precipitation. On the other hand, the eutectic solvent can promote the absorption of active substances through the scalp. This is because the excellent dissolving power of the eutectic solvent can improve the solubility of various active substances (including water-soluble and lipid-soluble substances), and the eutectic solvent can reversibly reduce the skin barrier function, opening channels for drugs. Therefore, it plays a dual role, and the preparation method is simple, the raw materials are widely available, and the process is short.

[0019] The preparation method provided by this invention can improve the compatibility of the components. The applicant found that when L-tyrosine and zinc ions are dissolved in water, an insoluble precipitate is formed. The precipitate can only dissolve when the pH is adjusted to below 3 or above 9 by adding alkali or acid, but obviously this pH is not suitable for cosmetics. In order to promote the compatibility of the components, the applicant introduced a urea compound and successfully synthesized a ternary eutectic solvent through the above-described embodiment. The components in this system have high compatibility and good storage stability. These implementation schemes first involve coordinating zinc salt with L-tyrosine, then mixing it with a urea compound and heating to prepare a stable eutectic solvent. Under pH 9-10 conditions, L-tyrosine forms a stable five-membered chelate ring with zinc ions through the nitrogen atom of the amino group and one oxygen atom of the carboxylate group, thus forming a 4-coordinated insoluble compound with one zinc ion and two tyrosine ions. This eliminates the zinc salt anions (chloride or sulfate ions) (which form HCl or H₂SO₄ and react with the base, remaining in the liquid phase). This avoids the influence of strong acids on the system and, by excluding the strongly polar ligands of chloride and sulfate ions, facilitates the successful formation of a eutectic solvent with the urea compound. Furthermore, the applicant also found that first forming a eutectic solvent with zinc salt and a urea compound, then adding a certain proportion of L-tyrosine for dissolution, also yields a clear and homogeneous mixed solution. However, the amount of L-tyrosine dissolved in this case is less than in the former method (first obtaining the tyrosine-zinc complex, then mixing it with the urea compound). When the amount of L-tyrosine added is too large, precipitation occurs.

[0020] In some embodiments, the preparation of the L-tyrosine solution includes: adding L-tyrosine to deionized water, then adding alkali to adjust the pH to 9-10, and stirring to dissolve.

[0021] In some embodiments, the zinc salt solution is a saturated zinc salt solution.

[0022] In some embodiments, the zinc salt in the zinc salt solution is one or more of zinc sulfate and zinc chloride.

[0023] In some embodiments, the zinc salt solution and the L-tyrosine solution are fed in a molar ratio of zinc ions to L-tyrosine of 1:2.5 to 4; for example, it can be any value among 1:2.5, 1:3, 1:3.5, 1:4 or 1:2.5 to 4.

[0024] In some implementations, the temperature of the heating and stirring reaction is 60-80°C, and the time is 2-4 hours.

[0025] In some embodiments, the tyrosine-zinc complex and the urea compound are fed in a molar ratio of zinc to urea of ​​1:1.5 to 3; for example, it can be any value among 1:1.5, 1:2, 1:2.5, 1:3, or 1:1.5 to 3. A molar ratio of zinc to urea of ​​1:1.5 to 3 is beneficial for preparing a clear and homogeneous eutectic solvent and for the storage stability of the eutectic solvent. Too high or too low a ratio will lead to turbidity of the solution or precipitation after storage.

[0026] In some embodiments, the water in step (2) accounts for 20% to 80% of the total mass of the tyrosine-zinc complex, urea compound, and water; for example, it can be any value among 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 20% to 80%. A water content percentage of 20% to 80% is beneficial for preparing a clear and homogeneous eutectic solvent, maintaining a suitable viscosity and solubility of the eutectic solvent, and promoting the storage stability of the eutectic solvent. Too high or too low a water content will result in turbidity of the solution or precipitation after storage. Too high a water content percentage will reduce the solubility of the eutectic solvent (especially for fat-soluble components), while too low a water content percentage will result in excessive viscosity, making it difficult to stir and dissolve plant extracts.

[0027] In some embodiments, the plant extracts include extracts of Polygonum multiflorum, Platycladus orientalis, wild walnut, and Ligustrum lucidum.

[0028] In some implementations, step (3) involves adding one or more of European larch wood extract and copper peptides to the eutectic solvent.

[0029] Thirdly, the present invention provides the application of any of the above-mentioned hair growth compositions that promote melanin formation, wherein the hair growth compositions that promote melanin formation are used in the preparation of hair and scalp cleaning and care products, including but not limited to: shampoo, scalp serum, scalp massage cream, conditioner, hair mask or hair treatment.

[0030] In some embodiments, the hair growth composition having melanin-promoting properties is used to prepare a scalp serum, which, by weight percentage, comprises the following components: 3%–8% of the hair growth composition having melanin-promoting properties, 5%–12% of a moisturizer, 0.5%–2.5% of a surfactant, 0%–2% of an antioxidant, 0.05%–0.1% of a preservative, 0.02%–0.1% of a pH adjuster, and the balance being water.

[0031] In some embodiments, the antioxidant is one or more of ascorbyl glucoside, resveratrol, vitamin E, bisabolol, and caffeine.

[0032] In some embodiments, the humectant is at least one of propylene glycol, butylene glycol, glycerin, and panthenol.

[0033] In some embodiments, the surfactant is selected from at least one of Olivem 1000, polysorbate-20, and PEG-40 hydrogenated castor oil.

[0034] In some embodiments, the preservative is selected from at least one of phenoxyethanol, ethylhexylglycerin, and p-hydroxyacetophenone.

[0035] In some embodiments, the pH adjuster is selected from at least one of citric acid and lactic acid.

[0036] The present invention has the following beneficial effects: (1) The composition of the present invention, through the scientific combination of L-tyrosine, zinc ions, urea compounds, specific plant extracts and copper peptides, acts on the hair growth cycle and melanin production process simultaneously through multiple pathways, multiple targets and multiple mechanisms, such as providing raw materials, promoting enzyme activity, activating pathways and improving microecology. It fundamentally and synergistically improves hair loss and gray hair problems, realizes the integrated treatment of "hair growth" and "black hair", avoids the limitations of a single target, and comprehensively regulates from the root cause. It is expected to achieve a relatively stable effect after discontinuation.

[0037] (2) The composition provided by the present invention has high safety. Its main components are derived from natural plants, the list of cosmetic raw materials already used, and biocompatible components, thus avoiding the systemic side effects of chemical drugs and the chemical irritation risk of hair dyes.

[0038] (3) The preparation method provided by the present invention simultaneously improves the permeability and compatibility of the components and enhances the storage stability of the composition; a low eutectic solvent is prepared by using active substances, and then plant extracts are dissolved in the low eutectic solvent. The system formed can serve as an excellent transdermal absorption carrier, significantly improving the dispersibility of active ingredients and enhancing their ability to penetrate the stratum corneum of the scalp, ensuring that the effective ingredients reach the hair follicle papilla and hair follicle melanocytes, thus solving the problem of difficult penetration of traditional products. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 To test the expression levels of tyrosinase-related proteins in Example 1 and Comparative Example 3 in Example 6.

[0041] Figure 2 This is a photograph of the hair of the subject in test case 9 after the experiment. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0043] In the description of the embodiments of this application, the technical terms "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly indicating the number, specific order, or primary and secondary relationship of the indicated technical features.

[0044] The term "embodiment" in this document means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments.

[0045] In the embodiments of this application, the term "or / and" is only a description of the relationship between related objects, indicating that there can be three kinds of relationships. For example, A or / and B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0046] Those skilled in the art can understand the specific meaning of the above terms in the embodiments of this application according to the specific circumstances.

[0047] Some ingredient descriptions: Polygonum multiflorum extract, Platycladus orientalis leaf extract, wild walnut extract, Ligustrum lucidum extract, and European larch wood extract were all purchased from Hunan Langlin Biological Resources Co., Ltd.; copper peptide was purchased from Wuhan Kemike Biomedical Technology Co., Ltd.

[0048] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0049] Example 1

[0050] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 15.59% L-tyrosine, 6.86% dimethylurea, 2.83% zinc ions, 14.15% Polygonum multiflorum extract, 3.77% Platycladus orientalis leaf extract, 9.43% wild walnut extract and 9.43% Ligustrum lucidum extract, with the balance being water.

[0051] This embodiment also provides a method for preparing the above-mentioned hair growth composition that promotes melanin formation, comprising the following steps: (1) Preparation of tyrosine-zinc complex Prepare a 0.12 mol / L L-tyrosine solution and adjust the pH to 9.2 with sodium hydroxide to obtain an L-tyrosine solution; prepare a saturated zinc chloride solution; add the zinc chloride solution to the L-tyrosine solution with stirring, and after the addition is complete, keep stirring for 1 hour and then filter. Take the filter cake, wash it three times with pure water, and then dry it in a 60℃ forced-air oven to obtain the tyrosine-zinc complex.

[0052] The zinc chloride solution and L-tyrosine solution were fed in at a molar ratio of zinc ions to L-tyrosine of 1:2.8.

[0053] (2) Preparation of eutectic solvent Tyrosine-zinc complex, dimethylurea and water were mixed in a certain proportion and heated to 70°C and stirred for 3 hours to obtain a eutectic solvent.

[0054] The tyrosine-zinc complex and dimethylurea were fed together at a molar ratio of zinc to dimethylurea of ​​1:1.8, with water accounting for 60% of the total weight of the tyrosine-zinc complex, dimethylurea and water.

[0055] The density of the eutectic solvent at 25°C was measured to be 1.34 g / cm³ using a DMA35 densitometer. 3 .

[0056] (3) Preparation of the composition The extracts of Polygonum multiflorum, Platycladus orientalis, Juglans regia, and Ligustrum lucidum were weighed and dissolved in the above-mentioned eutectic solvent. The mixture was heated to 40°C and stirred until dissolved. After centrifugation at 1000 rpm for 1 min, the supernatant was collected to obtain the composition. The yield of the supernatant was 98.54%. The eutectic solvent, Polygonum multiflorum extract, Platycladus orientalis extract, Juglans regia extract, and Ligustrum lucidum extract were added in a mass ratio of 134:30:8:20:20.

[0057] The mass ratio of zinc to copper in the composition was determined to be 1:0.612 using inductively coupled plasma atomic emission spectrometry.

[0058] Example 2

[0059] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 4.72% L-tyrosine, 14.83% dimethylurea, 5.73% zinc ions, 14.15% Polygonum multiflorum extract, 3.77% Platycladus orientalis leaf extract, 9.43% wild walnut extract and 9.43% Ligustrum lucidum extract, with the balance being water.

[0060] This embodiment also provides a method for preparing the above-mentioned hair growth composition that promotes melanin formation, comprising the following steps: (1) 24.32 g of zinc chloride and 62.88 g of dimethylurea were mixed and heated to 60 °C and stirred for 1 h to obtain a clear and homogeneous eutectic solution. The density of the eutectic solvent at 25 °C was measured to be 1.47 g / cm³ using a DMA35 densitometer. 3 .

[0061] (2) Heat the above eutectic solvent to 40°C and add 160.8g of water. Divide the mixture into two portions. Then, add 2g of L-tyrosine to each portion of the eutectic solvent every 5 minutes while stirring. Stop adding L-tyrosine when precipitation occurs (precipitation occurs when a total of 12g of the first portion of the eutectic solvent is added, so the second portion is stopped when 10g is added). The second portion of the eutectic solvent is used as a solvent to dissolve the plant extract to prepare the composition.

[0062] (3) Weigh out the extracts of Polygonum multiflorum, Platycladus orientalis, Juglans regia and Ligustrum lucidum and dissolve them in the second eutectic solvent. Heat to 40°C and stir to dissolve. Then centrifuge at 1000 rpm for 1 min and take the supernatant to obtain the composition. The yield of the supernatant is 96.22%. The eutectic solvent, Polygonum multiflorum extract, Platycladus orientalis extract, Juglans regia and Ligustrum lucidum extract are added in a mass ratio of 134:30:8:20:20.

[0063] The mass ratio of zinc to copper in the composition was determined to be 1:0.322 using inductively coupled plasma atomic emission spectrometry.

[0064] Example 3

[0065] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 15.15% L-tyrosine, 6.67% dimethylurea, 2.75% zinc ions, 13.75% Polygonum multiflorum extract, 3.67% Platycladus orientalis leaf extract, 9.16% wild walnut extract, 9.16% Ligustrum lucidum extract, and 2.86% copper peptide, with the balance being water.

[0066] The only difference between the preparation method and Example 1 is that in step (3), copper peptide is added to the supernatant after centrifugation (supernatant yield is 98.34%) and stirred evenly to obtain the composition.

[0067] The mass ratio of zinc to copper in the composition was determined to be 1:0.844 using inductively coupled plasma atomic emission spectrometry.

[0068] Example 4

[0069] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 15.02% L-tyrosine, 6.61% dimethylurea, 2.73% zinc ions, 13.63% Polygonum multiflorum extract, 3.63% Platycladus orientalis leaf extract, 9.09% wild walnut extract, 9.09% Ligustrum lucidum extract, and 3.68% copper peptide, with the balance being water.

[0070] The only difference between the preparation method and Example 1 is that in step (3), copper peptide is added to the supernatant after centrifugation (the yield of the supernatant is 98.27%) and stirred evenly to obtain the composition.

[0071] The mass ratio of zinc to copper in the composition was determined to be 1:0.891 using inductively coupled plasma atomic emission spectrometry.

[0072] Example 5

[0073] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 25.01% L-tyrosine, 12.30% acetaminophen, 4.54% zinc ions, 15.61% Polygonum multiflorum extract, 4.68% Platycladus orientalis leaf extract, 11.71% Juglans regia extract, 11.71% Ligustrum lucidum extract, and 3.99% copper peptide, with the balance being water.

[0074] This embodiment also provides a method for preparing the above-mentioned hair growth composition that promotes melanin formation, comprising the following steps: (1) Preparation of tyrosine-zinc complex Prepare a 2 mol / L L-tyrosine solution and adjust the pH to 9.5 with sodium hydroxide to obtain an L-tyrosine solution; prepare a saturated zinc sulfate solution; add the zinc sulfate solution to the L-tyrosine solution with stirring, and after the addition is complete, keep stirring for 1 hour and then filter. Take the filter cake, wash it three times with pure water, and then dry it in a 60℃ forced-air oven to obtain the tyrosine-zinc complex.

[0075] The zinc sulfate solution and L-tyrosine solution were fed in at a molar ratio of zinc ions to L-tyrosine of 1:2.5.

[0076] (2) Preparation of eutectic solvent Tyrosine-zinc complex, acetamide and water were mixed in a certain proportion and heated to 60°C and stirred for 2 hours to obtain a eutectic solvent.

[0077] The tyrosine-zinc complex and acetamide were fed in a molar ratio of zinc to acetamide of 1:3, with water accounting for 20% of the total weight of the tyrosine-zinc complex, acetamide and water.

[0078] The density of the eutectic solvent at 25°C was measured to be 1.25 g / cm³ using a DMA35 densitometer. 3 .

[0079] (3) Preparation of the composition The extracts of Polygonum multiflorum, Platycladus orientalis, Juglans regia, and Ligustrum lucidum were weighed and dissolved in the above-mentioned eutectic solvent. The mixture was heated to 40°C and stirred until dissolved. After centrifugation at 1000 rpm for 1 min, the supernatant was collected and copper peptides were added to obtain the composition. The yield of the supernatant was 99.38%. The eutectic solvent, Polygonum multiflorum extract, Platycladus orientalis extract, Juglans regia extract, and Ligustrum lucidum extract were added in a mass ratio of 134:40:12:30:30.

[0080] The mass ratio of zinc to copper in the composition was determined to be 1:0.65 using inductively coupled plasma atomic emission spectrometry.

[0081] Example 6

[0082] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 8.97% L-tyrosine, 3.74% hydantoin, 1.63% zinc ions, 10.7% Polygonum multiflorum extract, 2.14% Platycladus orientalis leaf extract, 5.35% Juglans regia extract, 5.35% Ligustrum lucidum extract, 2.14% copper peptide, and 2.67% Larch extract, with the balance being water.

[0083] This embodiment also provides a method for preparing the above-mentioned hair growth composition that promotes melanin formation, comprising the following steps: (1) Preparation of tyrosine-zinc complex Prepare a 0.1 mol / L L-tyrosine solution and adjust the pH to 10 with sodium hydroxide to obtain an L-tyrosine solution; prepare a saturated zinc chloride solution; add the zinc chloride solution to the L-tyrosine solution with stirring, and after the addition is complete, keep stirring for 1 hour and then filter. Take the filter cake, wash it three times with pure water, and then dry it in a 60℃ forced-air oven to obtain the tyrosine-zinc complex.

[0084] The zinc chloride solution and L-tyrosine solution were fed in a zinc ion to L-tyrosine molar ratio of 1:4.

[0085] (2) Preparation of eutectic solvent Tyrosine-zinc complex, hydantoin, and water were mixed in a certain proportion, heated to 80°C, and stirred for 4 hours to obtain a eutectic solvent.

[0086] The tyrosine-zinc complex and hydantoin were fed together at a molar ratio of zinc to acetamide of 1:1.5, with water accounting for 80% of the total weight of the tyrosine-zinc complex, hydantoin and water.

[0087] The density of the eutectic solvent at 25°C was measured to be 1.35 g / cm³ using a DMA35 densitometer. 3 .

[0088] (3) Preparation of the composition The extracts of Polygonum multiflorum, Platycladus orientalis, Juglans regia, and Ligustrum lucidum were weighed and dissolved in the above-mentioned eutectic solvent. The mixture was heated to 40°C and stirred until dissolved. After centrifugation at 1000 rpm for 1 min, the supernatant was collected and copper peptides were added to obtain the composition. The yield of the supernatant was 97.848%. The eutectic solvent, Polygonum multiflorum extract, Platycladus orientalis extract, Juglans regia extract, and Ligustrum lucidum extract were added in a mass ratio of 134:20:4:10:10.

[0089] The mass ratio of zinc to copper in the composition was determined to be 1:0.85 using inductively coupled plasma atomic emission spectrometry.

[0090] Example 7

[0091] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 16.86% L-tyrosine, 5.36% dimethylurea, 3.06% zinc ions, 14.15% Polygonum multiflorum extract, 3.77% Platycladus orientalis leaf extract, 9.43% wild walnut extract and 9.43% Ligustrum lucidum extract, with the balance being water.

[0092] The preparation method differs from Example 1 only in that, in step (2), the tyrosine-zinc complex and dimethylurea are fed in a molar ratio of zinc to dimethylurea of ​​1:1.3; the density of the eutectic solvent at 25°C is measured to be 1.38 g / cm³ using a DMA35 densitometer. 3 The supernatant yield in step (3) was 97.54%.

[0093] Example 8

[0094] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 12.87% L-tyrosine, 10.07% dimethylurea, 2.34% zinc ions, 14.15% Polygonum multiflorum extract, 3.77% Platycladus orientalis leaf extract, 9.43% wild walnut extract and 9.43% Ligustrum lucidum extract, with the balance being water.

[0095] The preparation method differs from Example 1 only in that, in step (2), the tyrosine-zinc complex and dimethylurea are fed in a molar ratio of zinc to dimethylurea of ​​1:3.2; the density of the eutectic solvent at 25°C is measured to be 1.27 g / cm³ using a DMA35 densitometer. 3 The supernatant yield in step (3) was 99.17%.

[0096] Example 9

[0097] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 35.08% L-tyrosine, 15.44% dimethylurea, 6.37% zinc ions, 14.15% Polygonum multiflorum extract, 3.77% Platycladus orientalis leaf extract, 9.43% wild walnut extract and 9.43% Ligustrum lucidum extract, with the balance being water.

[0098] The preparation method differs from Example 1 only in that, in step (2), water accounts for 10% of the total weight of the tyrosine-zinc complex, hydantoin, and water; the density of the eutectic solvent at 25°C was measured to be 1.38 g / cm³ using a DMA35 densitometer. 3 The supernatant yield in step (3) was 95.54%.

[0099] Example 10

[0100] This embodiment provides a hair growth composition that promotes melanin formation, comprising, by weight percentage: 3.90% L-tyrosine, 1.72% dimethylurea, 0.71% zinc ions, 14.15% Polygonum multiflorum extract, 3.77% Platycladus orientalis leaf extract, 9.43% wild walnut extract and 9.43% Ligustrum lucidum extract, with the balance being water.

[0101] The preparation method differs from Example 1 only in that, in step (2), water accounts for 90% of the total weight of the tyrosine-zinc complex, hydantoin, and water; the density of the eutectic solvent at 25°C was measured to be 1.25 g / cm³ using a DMA35 densitometer. 3 The supernatant yield in step (3) was 96.98%.

[0102] Comparative Example 1 This embodiment provides a composition comprising, by weight percentage: 17.07% L-tyrosine, 5.12% urea, 3.10% zinc ions, 14.15% Polygonum multiflorum extract, 3.77% Platycladus orientalis leaf extract, 9.43% Juglans regia extract and 9.43% Ligustrum lucidum extract, with the balance being water.

[0103] The preparation method differs from that in Example 1 only in that urea is used instead of dimethylurea in step (2); the density of the eutectic solvent at 25°C was measured to be 1.23 g / cm³ using a DMA35 densitometer. 3 The supernatant yield in step (3) was 95.74%.

[0104] Comparative Example 2 This embodiment provides a composition comprising, by weight percentage: 16.95% L-tyrosine, 5.26% ethylene glycol, 3.08% zinc ions, 14.15% Polygonum multiflorum extract, 3.77% Platycladus orientalis leaf extract, 9.43% Juglans regia extract and 9.43% Ligustrum lucidum extract, with the balance being water.

[0105] The preparation method differs from Example 1 only in that ethylene glycol is used instead of dimethylurea in step (2); the density of the eutectic solvent at 25°C was measured to be 1.18 g / cm³ using a DMA35 densitometer. 3 The supernatant yield in step (3) was 94.86%.

[0106] Comparative Example 3 This embodiment provides a composition comprising, by weight percentage: 16.05% L-tyrosine, 7.06% dimethylurea, 14.56% Polygonum multiflorum extract, 3.88% Platycladus orientalis leaf extract, 9.71% wild walnut extract and 9.71% Ligustrum lucidum extract, with the balance being water.

[0107] The preparation method is as follows: Dimethylurea was dissolved in deionized water, then L-tyrosine was added, the mixture was heated to 40°C and stirred to dissolve, and finally plant extracts were added to obtain the composition.

[0108] Test Example 1: Skin Irritation Test The test was conducted according to the relevant methods of human skin patch testing in the "Cosmetic Safety Technical Specifications" (2015 edition). Twenty volunteers aged 25-45 years who met the inclusion criteria were selected as subjects, including 10 males and 10 females. The test sample (8% aqueous solution of the composition) was added to the patch applicator, with the amount precisely controlled at 0.020 ± 0.005 g; the blank control group's patch applicator did not contain any added substance. Using non-irritating adhesive tape, the patch applicator containing the test sample was attached to the flexor surface of the subject's forearm, and then gently pressed with the palm to ensure even adhesion to the skin. The patch was applied every 24 hours. After each application, the patch was left to stand for 30 minutes until the indentation on the skin surface completely disappeared before observing and recording the skin's reaction.

[0109] If a subject shows a suspicious or positive skin reaction, the patch test for that subject must be terminated immediately. If the skin reaction is negative, the patch application procedure should be repeated on the same test site. The entire patch test needs to be repeated a total of 6 times. During the test, the skin reaction level and the corresponding number of subjects should be counted according to the judgment criteria in Table 1. Finally, the test results should be summarized in Table 2.

[0110] Table 1

[0111] Table 2

[0112] As can be seen from the table above, Examples 1 to 4 provided by the present invention do not cause irritation to human skin, while Examples 5 and 6 have slight irritation.

[0113] Test Example 2: Transdermal Transfusion Test Percutaneous permeation was performed using the Franz diffusion cell system, with fresh pig skin (after subcutaneous fat removal) as the model. The treated skin samples were fixed between the supply and receiving chambers, with the skin layer facing the supply chamber. An 8% aqueous solution of the composition or an aqueous solution of the same concentration of Polygonum multiflorum extract (control group) was added to the supply chamber, while PBS solution was added to the receiving chamber. The receiving fluid was collected after 12 hours. High-performance liquid chromatography (HPLC) was used to quantitatively analyze the concentrations of anthraquinone compounds and stilbene glycosides (both major active ingredients in Polygonum multiflorum extract) in the receiving fluid, and the cumulative permeation per unit area was calculated. All experiments were performed in triplicate, and the results are expressed as an arithmetic mean. The cumulative permeation per unit area Q was calculated as (c × V) / A, where c is the total protein concentration in the receiving fluid, V is the volume of the receiving fluid, and A is the effective permeation area. The cumulative permeation per unit area results for each experimental group are summarized in Table 3. The HPLC testing conditions for anthraquinone compounds were as follows: a Synergi Hydro-RP column was used, with a column temperature of 25℃, a detection wavelength of 254nm, an injection volume of 10μL, a mobile phase of chromatographic grade methanol:ultrapure water = 85:15, and a flow rate of 1.0mL / min. The HPLC testing conditions for stilbene glycosides were as follows: a Synergi Hydro-RP column was used, with a column temperature of 25℃, a detection wavelength of 320nm, an injection volume of 10μL, a mobile phase of chromatographic grade acetonitrile:ultrapure water = 25:75, and a flow rate of 1.0mL / min.

[0114] Table 3

[0115] As shown in Table 3, the compositions provided in the embodiments of the present invention are more effective at transdermal delivery of active substances compared to the control group. A comparison of Example 1 and Comparative Examples 1-3 shows that the compositions prepared using the components provided in the present invention are more conducive to transdermal delivery of active substances. Specifically, in Comparative Examples 1 and 2, urea and ethylene glycol were used instead of dimethylurea in Example 1, respectively. This change in the polarity of the eutectic solvent likely led to poorer solubility and dispersibility for lipid-soluble substances, and decreased compatibility with the stratum corneum of the skin, ultimately resulting in a decrease in the transdermal permeation of the active substances. In Comparative Example 3, the absence of a eutectic solvent resulted in lower compatibility among the components, making it difficult to uniformly disperse the active substances. Even when prepared as an 8% aqueous solution, there was still a significant amount of precipitate, leading to a decrease in the active substance content in the supply tank and thus a lower transdermal permeation. Furthermore, the supernatant yields of Examples 1 and Comparative Examples 1-2 also confirm this (i.e., poorer solubility and dispersibility for lipid-soluble substances).

[0116] Test Example 3 Storage Stability Test To investigate the storage stability of the eutectic solvent (DES) and the composition, the eutectic solvent and composition prepared in the specific embodiments were grouped and subjected to the following tests: (1) standing at 25°C for 30 days; (2) standing at 8°C for 2 days; (3) standing at 50°C for 2 days; and the appearance of the samples after standing was recorded. The results are shown in Table 4.

[0117] Table 4

[0118] As shown in Table 4, the DES and compositions provided in Examples 1 and 5 of this invention exhibit excellent storage stability and temperature resistance. The composition in Example 2 has poor low-temperature resistance; due to its poor solubility, cooling significantly affects its solubility, resulting in noticeable turbidity at low temperatures. The hydantoin used in Example 6 has a high density, high molecular rigidity, and poor mobility. When the temperature decreases, molecular motion weakens, the hydrogen bond network reorganizes, leading to supersaturation and precipitation of a certain component. Therefore, both the DES and the composition exhibit noticeable turbidity at low temperatures. In Examples 7 and 8, the molar ratio of tyrosine-zinc complex to zinc in dimethylurea and dimethylurea exceeds the preferred range (1:1.5~3). In Examples 9 and 10, the percentage of water content during DES preparation exceeds the preferred range (20%~80%), resulting in poor stability of the formed hydrogen bond network. Consequently, the temperature resistance of the DES decreases, and the storage stability and temperature resistance of the composition also decrease. The storage stability and temperature resistance of the composition have a significant impact on the stability of the prepared personal care products, thus affecting their efficacy.

[0119] Test Example 4: Tyrosinase Activity Test Mouse B16F10 melanoma cells were used at a rate of 5 × 10⁻⁶ 3 Cells were seeded at a density of 180 μL / mL in 96-well plates. After cell attachment, 20 μL of the test sample (5% composite solution (filtered through a 0.22 μm filter) was added to each well, with 5 replicates for each group. After 72 h, the culture medium was discarded, and the cells were washed twice with 100 μL of PBS. 100 μL of 1% Triton X-100 solution was added to each well, and the cells were lysed at -80 °C for 30 min. After thawing at room temperature, the cells were preheated at 37 °C for 10 min, and 100 μL of 10 mmol / L L-DOPA was added to each well. The cells were reacted at 37 °C for 2 h, and the absorbance at 475 nm was measured. An equal volume of PBS was added to the control group. The relative tyrosinase activity (%) was calculated as: (Experimental group absorbance / Control group absorbance) × 100%. The results are shown in Table 5.

[0120] Test Example 5: Melanin Synthesis Test Mouse B16F10 melanoma cells were injected at 2.5 × 10⁻⁶.4 Cells were seeded at a density of 10 cells / mL in 6-well plates. After cell adhesion, the test samples were added (5% composite solution (filtered through a 0.22 μm filter) for the experimental group and 200 ng / mL α-MSH solution for the positive control group). An equal volume of PBS solution was added to the blank group. Each sample was divided into two replicates. After incubation for 72 h, the culture medium was discarded, and the cells were washed twice with PBS solution. The cells were digested with 0.25% trypsin, pipetted to form a cell suspension, and then centrifuged at 1000 rpm for 5 min. After discarding the supernatant, 200 μL of 1 mol / L NaOH solution (containing 10% DMSO) was added to each sample. The cells were then lysed by incubating at 80 °C for 1 h to dissolve the melanin granules. The cell lysates were transferred to 96-well plates, 100 μL per well. The absorbance was measured at 405 nm using a microplate reader. The melanin synthesis rate was calculated as: (Experimental group absorbance / Blank group absorbance) × 100%. The results are listed in Table 5.

[0121] Table 5

[0122] Note: * indicates a statistically significant difference between the experimental group and the control group (p<0.05), ** indicates a significant difference between the experimental group and the control group (p<0.01), and *** indicates an extremely significant difference between the experimental group and the control group (p<0.001).

[0123] As shown in Table 5, the composition provided by this invention is beneficial for improving tyrosinase and melanin synthesis. In Example 2, compared to Example 1, the lower L-tyrosine content and higher zinc ion content led to decreased tyrosinase activity and a lower melanin synthesis rate. In Example 3, compared to Example 1, the addition of copper peptides improved tyrosinase activity, as tyrosinase is a copper ion enzyme. In Example 4, compared to Example 3, the addition of even more copper peptides increased the copper ion content, leading to a decrease in the expression of tyrosinase-related protein-1. Therefore, although tyrosinase activity increased, melanin synthesis decreased. In Example 5, the zinc ion content, copper ion content, and L-tyrosine content were all increased, but the excessively high zinc ion concentration led to a decrease in tyrosinase activity, thus reducing melanin synthesis. In Example 6, the zinc ion content, copper peptide content, and L-tyrosine content were all decreased, resulting in lower tyrosinase activity and a decrease in melanin synthesis. In Comparative Example 3, no zinc ions were added, and the composition was difficult to completely dissolve after being prepared into an 8% solution. As a result, the content of active substances decreased, and therefore the tyrosinase activity and melanin synthesis decreased compared to Example 1.

[0124] Test Example 6: Expression level test of tyrosinase-related proteins Mouse B16F10 melanoma cells were injected at 2.5 × 10⁻⁶. 4Cells were seeded at a density of 10 cells / mL in 6-well plates. After cell adhesion, the test samples were added (5% composite solution (filtered through a 0.22 μm filter) for the experimental group, and 200 ng / mL α-MSH solution for the positive control group, and an equal volume of PBS solution for the blank group). Each group was divided into 3 replicates. After 72 h of incubation, the culture medium was discarded, and the cells were washed twice with PBS solution. After digestion, centrifugation (5000 rpm, 5 min), and washing, the cells were obtained. Lysis buffer was added to lyse the cells to extract proteins. After centrifugation, the supernatant of the centrifuge tube was used to determine the protein expression level. The sample loading was 50 μg. After electrophoresis, transfer to a membrane, and blocking, primary antibody (dilution ratio 1:500) was added, and the cells were incubated overnight at 4 °C. After washing with PBS buffer, secondary antibody (horseradish peroxidase-labeled rabbit IgG, dilution ratio 1:2000) was added, and the cells were incubated at room temperature for 2 h. After washing with PBS buffer, ECL reaction was performed, with β-actin antibody as an internal control. The ratios of absorbance of the bands of TYR, TRP-1, and the corresponding β-actin fragments were compared as relative expression levels. Figure 1 As shown in Table 6. During electrophoresis, the stacking gel was kept at a constant voltage of 60V for 30 minutes, and the separating gel was kept at a constant voltage of 80V for 60 minutes; during membrane transfer, the current was 200mA for 60 minutes; and during blocking, 5% skim milk powder blocking solution was used for 120 minutes.

[0125] Table 6

[0126] Note: * indicates a statistically significant difference between the experimental group and the control group (p<0.05), ** indicates a significant difference between the experimental group and the control group (p<0.01), and *** indicates an extremely significant difference between the experimental group and the control group (p<0.001).

[0127] As shown in Table 6, the composition provided by this invention is beneficial for improving the expression of tyrosinase and TRP-1. In Example 2, compared to Example 1, the increased zinc ion content led to a decrease in tyrosinase expression due to inhibition, while the TRP-1 expression level increased. In Example 3, the addition of copper peptides, compared to Example 1, was beneficial for improving tyrosinase expression levels, as tyrosinase is a copper ion enzyme. In Example 4, the addition of more copper peptides, compared to Example 3, resulted in a decrease in TRP-1 expression due to the increased copper ion content. In Example 5, the zinc ion content, copper ion content, and plant extract content were all increased, thus exhibiting higher TYR and TRP-1 expression levels. In Example 6, the zinc ion content, copper peptide content, and plant extract content were all decreased, thus exhibiting lower TYR and TRP-1 expression levels. In Comparative Example 3, no zinc ions were added, and the composition was difficult to completely dissolve in an 8% solution, resulting in a decrease in the content of active substances, and therefore exhibiting lower TYR and TRP-1 expression levels compared to Example 1.

[0128] Test Example 7: In vitro inhibition of 5α-reductase activity test (1) Six male SD rats were selected, euthanized after overnight fasting, and prostate tissue was dissected and obtained. Tris-HCl buffer pre-cooled to 4°C and pH 7.0 was added to the tissue at a solid-liquid ratio of 1:5, and the mixture was centrifuged at 3000g for 10 min at 4°C. The supernatant was collected and centrifuged again at 10000g for 30 min at the same temperature to obtain crude 5α-reductase extract.

[0129] (2) The experiment was set up with four groups: blank group, experimental group, positive control group and reaction group. The reaction system and operation procedure of each group are as follows: Experimental group operation: 500 μL of pH5.5 PBS buffer, 200 μL of 5% (w / w) composition solution, 200 μL of 300 mg / L testosterone solution and 200 μL of 0.8 g / L reduced coenzyme II (NADPH) solution were added to the stoppered test tube in sequence. Finally, 0.5 mL of the above-mentioned crude 5α-reductase extract was added. The test tube was placed in a constant temperature environment of 37℃ for 30 min. When the reaction was terminated, 3 mL of dichloromethane was added, and then centrifuged at 5000 r / min for 10 min. The upper aqueous phase was discarded, and the residue after centrifugation was dissolved in 1 mL of methanol. 10 μL of the methanol solution was taken and the content of residual testosterone was determined by high performance liquid chromatography. The reaction system and operating procedures of the positive control group were completely consistent with those of the experimental group, except that the composition solution was replaced with an equal volume of 0.05 mg / L finasteride solution. The reaction conditions of the blank group were the same as those of the experimental group, except that an equal volume of PBS buffer was used instead of the crude 5α-reductase extract. The reaction setup of the reaction group was the same as that of the experimental group, except that an equal volume of PBS buffer was used instead of the composition solution. The relative peak area (S) corresponding to testosterone in each group was detected by high-performance liquid chromatography, and the 5α-reductase inhibitory activity was calculated according to the following formula: Inhibition rate (%) = (S / S) 实验组 -S 反应组 ) / (S 空白组 -S 反应组 The experimental results are shown in Table 7.

[0130] Table 7

[0131] As shown in Table 7, the composition provided by the present invention has good 5α-reductase inhibitory activity, which can reduce the conversion of testosterone to DHT and reduce hair follicle atrophy.

[0132] Test Example 8: In Vitro Antioxidant Test The DPPH free radical scavenging ability assay kit was operated according to the instructions as follows. The experiment included an experimental group (5% solution of the composition, filtered through a 0.22 μm filter), a positive control group (100 mg / L vitamin C solution), and a blank group. The absorbance of each tube was measured at a wavelength of 517 nm. DPPH free radical scavenging rate (%) = [1-(A1-A2)÷A] 空白组 ×100%, where A1 represents the absorbance value of the experimental group or positive control group, and A2 represents the absorbance value of the experimental group control or positive control group control. 空白组 The absorbance value of the blank tube is shown in Table 8.

[0133] Table 8

[0134] Note: * indicates a statistically significant difference between the experimental group and the model group (p<0.05), ** indicates a significant difference between the experimental group and the model group (p<0.01), and *** indicates an extremely significant difference between the experimental group and the model group (p<0.001).

[0135] Application examples A scalp serum, by weight percentage, comprises the following components: 5% hair growth composition that promotes melanin formation, 8% moisturizer, 1.5% surfactant, 0.084% preservative, 0.036% pH adjuster, and the balance being water.

[0136] The humectant is 1,3-propanediol and glycerin in a mass ratio of 1:5; the surfactant is Olivem 1000; the preservative is phenoxyethanol and p-hydroxyacetophenone in a mass ratio of 1:0.3; and the pH adjuster is citric acid.

[0137] The hair growth compositions that promote melanin formation in the components of Examples 1 and 2 correspond to the hair growth compositions of Examples 1 and 2, respectively; the hair growth compositions that promote melanin formation in the components of Comparative Examples 1 to 3 correspond to the compositions of Comparative Examples 1 to 3, respectively.

[0138] Test Example 9: Clinical Trial The human efficacy clinical trial includes two trials: one evaluating hair growth and moisturizing effects, and the other evaluating hair darkening effects.

[0139] I. Hair growth and moisturizing effects A total of 50 volunteers aged 25 to 35 years diagnosed with androgenetic alopecia (AGA) were recruited to participate in the trial and randomly divided into 5 groups, half male and half female, corresponding to Application Example 1, Application Example 2, Application Comparative Example 1, Application Comparative Example 2 and Application Comparative Example 3, with 10 people in each group.

[0140] Subjects applied 3 mL of scalp serum to their scalp using a scalp applicator each night, gently massaging for 3-5 minutes until absorbed, for 8 consecutive weeks. During the trial, subjects shampooed their hair at least three times a week, following their usual routine, but only using the designated shampoo (which contained no active ingredients). Before and after the trial, scalp stratum corneum moisture content was measured using a skin moisture meter. The mean value within each group and the rate of change in scalp stratum corneum moisture content were calculated as: (scalp stratum corneum moisture content after the trial - scalp stratum corneum moisture content before the trial) / scalp stratum corneum moisture content before the trial × 100%. Hair density (in roots / cm²) at specific locations on the subjects was measured using a dermoscopy before and after the trial. 2Measurements were taken, and the mean within each group and the rate of change in hair density were calculated as follows: (Hair density after the test - Hair density before the test) / Hair density before the test × 100%. Hair was not washed within 48 hours prior to each measurement, and the subject sat quietly for 30 minutes in an indoor environment with a temperature of 20℃ and relative humidity of 50% before the test. Skin moisture was measured at three random locations on the top of each subject's head, and the average value was taken. The dermoscopy test site was a 2cm diameter area centered on the Baihui acupoint. Before the test, the hair was trimmed to a residual length of no more than 1mm. The dermoscopy was placed in the center of the trimmed area to capture a local hair image. Image analysis software was used to count the number of local hairs and the number of terminal hairs (diameter greater than 30 micrometers). The results are listed in Table 9.

[0141] Table 9

[0142] As shown in Table 9, the composition provided in the application examples of this invention has good hair growth and moisturizing effects, and compared with the comparative application, it exhibits superior hair growth effect due to better transdermal permeability. It should be noted that hair density and terminal hair percentage are two important indicators for evaluating hair growth effect. Hair loss is essentially the miniaturization of hair follicles, i.e., hair becomes thinner and softer. The scalp essence provided by this invention can not only increase hair density but also increase the percentage of terminal hair, demonstrating excellent efficacy.

[0143] II. Hair Darkening Effect A total of 50 participants aged 25 to 35 with premature graying (average gray hair density ≥10 strands / cm² in the crown area) were recruited. 2 Male volunteers participated in the trial and were randomly divided into 5 groups, corresponding to Application Example 1, Application Example 2, Application Comparative Example 1, Application Comparative Example 2, and Application Comparative Example 3, with 10 participants in each group. Each night, participants applied 3 mL of scalp serum evenly to the scalp using a scalp applicator, gently massaging for 3-5 minutes until fully absorbed, for 12 consecutive weeks. During the trial, participants maintained their original shampooing habits, shampooing at least three times a week, and consistently using the designated shampoo without active ingredients. Before and after the trial, scalp serum was applied to selected locations (three 1 cm diameter spots on the top of the head). 2 The density of gray hair in the region was statistically analyzed, and the results are listed in Table 10. Figure 2 The images show actual photos of the hair of some participants after the experiment, as well as magnified images of the roots.

[0144] Table 10

[0145] As can be seen from the data in Table 10, the composition provided in the application examples of the present invention has good hair-darkening effect, and compared with the application comparison ratio, it exhibits a higher hair-darkening effect due to better transdermal permeability.

[0146] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a hair growth composition that promotes melanin formation, characterized in that, Includes the following steps: (1) Under stirring, a zinc salt solution and an L-tyrosine solution were mixed to carry out a precipitation reaction. After solid-liquid separation and washing, a tyrosine-zinc complex was obtained. (2) Mix the tyrosine-zinc complex, urea compound and water in a certain proportion, heat and stir to react, and obtain a eutectic solvent; or, mix the tyrosine-zinc complex, acetamide and water in a certain proportion, heat and stir to react, and obtain a eutectic solvent. (3) Add plant extract to the eutectic solvent, stir to dissolve, and separate the solid and liquid to obtain the composition; The plant extracts include Polygonum multiflorum extract, Platycladus orientalis leaf extract, wild walnut extract, and Ligustrum lucidum extract; the urea compound is dimethylurea or hydantoin; The hair growth composition comprises, by weight percentage: 4%~36% L-tyrosine, 1.5%~16% urea compound or acetamide, 0.7%~7% zinc ions, 10%~16% Polygonum multiflorum extract, 2%~5% Platycladus orientalis leaf extract, 5%~12% wild walnut extract and 5%~12% Ligustrum lucidum extract, with the balance being water; The zinc salt solution and the L-tyrosine solution are fed in a molar ratio of zinc ions to L-tyrosine of 1:2.5~4; the tyrosine-zinc complex and the urea compound or acetamide are fed in a molar ratio of zinc element to urea compound or acetamide of 1:1.5~3; in step (2), the water accounts for 20%~80% of the total mass of the tyrosine-zinc complex, urea compound or acetamide and water; the temperature of the heating and stirring reaction is 60~80℃ and the time is 2~4h.

2. A method for preparing a hair growth composition that promotes melanin formation, characterized in that, Includes the following steps: (1) Under stirring, a zinc salt solution and an L-tyrosine solution were mixed to carry out a precipitation reaction. After solid-liquid separation and washing, a tyrosine-zinc complex was obtained. (2) Mix the tyrosine-zinc complex, urea compound and water in a certain proportion, heat and stir to react, and obtain a eutectic solvent; or, mix the tyrosine-zinc complex, acetamide and water in a certain proportion, heat and stir to react, and obtain a eutectic solvent. (3) Add plant extract, European larch wood extract and copper peptide to the eutectic solvent, stir to dissolve and separate solid and liquid to obtain the composition; The plant extracts include Polygonum multiflorum extract, Platycladus orientalis leaf extract, wild walnut extract, and Ligustrum lucidum extract; the urea compound is dimethylurea or hydantoin; The hair growth composition comprises, by weight percentage: 8%~25% L-tyrosine, 3.5%~13% urea compound or acetamide, 1.5%~5% zinc ions, 10%~16% Polygonum multiflorum extract, 2%~5% Platycladus orientalis leaf extract, 5%~12% Juglans regia extract, 5%~12% Ligustrum lucidum extract, 2%~8% Larch extract and 2%~5% copper peptide, with the balance being water; The zinc salt solution and the L-tyrosine solution are fed in a molar ratio of zinc ions to L-tyrosine of 1:2.5~4; the tyrosine-zinc complex and the urea compound or acetamide are fed in a molar ratio of zinc element to urea compound or acetamide of 1:1.5~3; in step (2), the water accounts for 20%~80% of the total mass of the tyrosine-zinc complex, urea compound or acetamide and water; the temperature of the heating and stirring reaction is 60~80℃ and the time is 2~4h.

3. The method for preparing a hair growth composition that promotes melanin formation according to claim 1 or 2, characterized in that, The mass ratio of zinc to copper in the hair growth composition is (1:0.5) to (1:0.85).

4. The method for preparing the hair growth composition for promoting melanin formation according to claim 1, characterized in that, The hair growth composition comprises, by weight percentage: 8%~25% L-tyrosine, 3.5%~13% urea compound or acetamide, 1.5%~5% zinc ions, 10%~16% Polygonum multiflorum extract, 2%~5% Platycladus orientalis leaf extract, 5%~12% wild walnut extract and 5%~12% Ligustrum lucidum extract, with the balance being water.

5. A method for preparing a hair growth composition that promotes melanin formation according to claim 1 or 2, characterized in that, The preparation of the L-tyrosine solution includes: adding L-tyrosine to deionized water, then adding alkali to adjust the pH to 9-10, and stirring to dissolve; And / or, the zinc salt solution is a saturated zinc salt solution; And / or, the zinc salt in the zinc salt solution is one or more of zinc sulfate and zinc chloride.

6. The application of the hair growth composition prepared by the method of preparing a hair growth composition that promotes melanin formation according to any one of claims 1 to 5, characterized in that, The hair growth composition is used in the preparation of hair and scalp cleansing and care products.

7. The application of the hair growth composition prepared by the method for preparing the hair growth composition for promoting melanin formation according to claim 6, characterized in that, The hair growth composition that promotes melanin formation is used to prepare a scalp serum. By mass percentage, the scalp serum comprises the following components: 3%~8% of the hair growth composition that promotes melanin formation, 5%~12% of a moisturizer, 0.5%~2.5% of a surfactant, 0%~2% of an antioxidant, 0.05%~0.1% of a preservative, 0.02%~0.1% of a pH adjuster, and the balance being water.

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