Preparation method and application of traditional Chinese medicine hair blacking essence

Abstract

The present application relates to a kind of traditional Chinese medicine hair blacking essence preparation method, belong to the field of traditional Chinese medicine external preparation.The existing product component dissolution is little, color is light, easy to stratify, lack standard evaluation model.Scheme: taking gallnut 3 parts, raw land 3 parts, radix polygoni multiflori 3 parts, angelica 2 parts, caulis 2 parts, ligustrum lucidum 1 part, euphorbia humifusa 1 part, mulberry leaf 1 part and taxodium 1 part are mixed and decocted to obtain stock solution;Ferrous sulfate is dissolved in hot water to prepare mordant solution;Stock solution and mordant solution are mixed, heated and placed, then heated again, according to 10g xanthan gum is added per 1000ml mixed solution, and stirred to be paste-like.The essence is significantly melanin production in zebra fish model, safety is good, can be used for hair blacking cosmetics or drugs.

Description

Technical Field

[0001] This invention relates to the field of external preparations of traditional Chinese medicine and cosmetics, specifically to the preparation method and application of hair-darkening essence made from traditional Chinese medicine. Background Technology

[0002] With the accelerating pace of life and the aging population, the proportion of people with gray hair is increasing year by year and showing a trend towards younger ages. Traditional Chinese medicine theory believes that gray hair is related to liver and kidney deficiency and qi and blood deficiency; modern medical research shows that gray hair is related to factors such as reduced activity of hair follicle melanocytes, decreased tyrosinase activity, and trace element deficiency. Based on this, the preparation of topical hair-darkening products using Chinese herbal extracts has become a hot topic in research and market development.

[0003] Currently, most existing Chinese herbal hair-darkening products are formulated into topical preparations by directly compounding aqueous extracts of medicinal materials, or by compounding medicinal materials after ethanol extraction. While these methods allow the application of Chinese herbal ingredients to the scalp, they have the following shortcomings in practical use: First, the extraction solvent is singular, making it difficult to ensure the dissolution of different polar active ingredients in the medicinal materials, resulting in insufficient variety and total amount of effective ingredients and limited hair-darkening effects; Second, the extraction process often involves high-temperature and prolonged heating, which can easily cause degradation of heat-sensitive components, and the resulting liquid preparations have poor stability, easily leading to stratification or oxidation and inactivation of active ingredients; Third, existing preparations lack targeted penetration-enhancing methods, making it difficult for active ingredients to effectively reach the hair follicle target, resulting in low bioavailability; Fourth, current technologies rely heavily on subjective observation or simple in vitro experiments to evaluate the hair-darkening effects, lacking standardized in vivo animal model evaluation methods, leading to inaccurate product efficacy verification and difficulty in accurately reflecting its true impact on melanin metabolism.

[0004] Therefore, it is necessary to provide a method for preparing hair-darkening products and an application evaluation scheme that can improve the dissolution of active ingredients in traditional Chinese medicine, improve the stability of the preparation, facilitate external application, and be verified by standardized animal models. Summary of the Invention

[0005] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages that will be described later.

[0006] To achieve these objectives and other advantages of the present invention, a method for preparing a traditional Chinese medicine hair-darkening essence is provided, characterized by comprising the following steps: Weigh out 3 parts by weight of Gallnut, 3 parts by weight of Rehmannia glutinosa, 3 parts by weight of Polygonum multiflorum, 2 parts by weight of Angelica sinensis, 2 parts by weight of Spatholobus suberectus, 1 part by weight of Ligustrum lucidum, 1 part by weight of Eclipta prostrata, 1 part by weight of Morus alba leaf, and 1 part by weight of Platycladus orientalis leaf, and mix them to obtain the first Chinese herbal mixture formula.

[0007] Add 50 g of the Chinese herbal mixture formula one to 1500 ml of pure water, stir evenly, and then extract with ultrasonic assistance at 40℃ and 200W power for 20 minutes. Then heat to boiling and simmer for 40 minutes. After cooling, filter and collect the filtrate to obtain the original Chinese herbal liquid formula one.

[0008] Dissolve 4 g to 8 g of ferrous sulfate, 0.1 g to 0.2 g of L-ascorbic acid, and 0.04 g to 0.08 g of disodium EDTA in 40 ml to 80 ml of hot water at 90°C to 100°C to obtain a mordant solution.

[0009] Mix 1500 ml of the herbal extract formula one with 40 ml to 80 ml of mordant solution, stir well, and then add a citric acid-sodium citrate buffer pair with a mass ratio of 1:2 to stabilize the pH of the mixed solution between 5.0 and 5.5. Heat to 55°C to 60°C and let stand for 45 to 60 minutes, stirring at 100 rpm to 150 rpm during this time. Cool the mixture and keep it at 50°C. Add 15 g of propylene glycol and 5 g of azone and stir well to form a composite penetration-enhancing system. Then add xanthan gum in three equal portions, at a rate of 10 g per 1000 ml of mixed solution, while stirring rapidly at 200 rpm until a gel-like consistency is formed, thus obtaining the hair-darkening essence formula one.

[0010] Preferably, another method for preparing a hair-darkening essence from traditional Chinese medicine is provided, comprising the following steps: Traditional Chinese medicine mixture formula one is replaced with traditional Chinese medicine mixture formula two, which is composed of 3 parts by weight of Gallnut, 3 parts by weight of Terminalia chebula, 2 parts by weight of Rubus idaeus, 2 parts by weight of Cuscuta chinensis, 2 parts by weight of Rosa laevigata, 2 parts by weight of Lycium barbarum, 2 parts by weight of Morus alba, 2 parts by weight of Cyperus rotundus, 2 parts by weight of Cinnamomum cassia, 2 parts by weight of Longan aril, 2 parts by weight of Spatholobus suberectus, 2 parts by weight of Platycladus orientalis seed, 2 parts by weight of black sesame, 0.4 parts by weight of ginger extract, and 1 part by weight of Vitex trifolia; 50 g of traditional Chinese medicine mixture formula two is added to 1500 ml of pure water, stirred evenly, and then extracted with ultrasound at 40℃ and 200W for 20 minutes, then heated to boiling and simmered for 40 minutes, cooled, filtered, and the filtrate is collected to obtain traditional Chinese medicine stock solution formula two; 4 g to 8 g of ferrous sulfate, 0.1 g to 0.2 g L... - Dissolve ascorbic acid and 0.04 g to 0.08 g disodium EDTA in 40 ml to 80 ml of hot water at 90°C to 100°C to obtain a mordant solution; mix 1500 ml of the herbal extract formula two with 40 ml to 80 ml of the mordant solution, stir evenly, and then add a citrate-sodium citrate buffer pair with a mass ratio of 1:2 to stabilize the pH of the mixed solution between 5.0 and 5.5. Heat to 55°C to 60°C, let stand for 45 to 60 minutes, stirring at 100 rpm to 150 rpm during this time. Cool the mixture and keep it at 50°C. Add 15 g of propylene glycol and 5 g of azone and stir evenly to form a composite penetration-enhancing system. Then add xanthan gum in three equal portions, with the amount of xanthan gum added being 10 g per 1000 ml of mixed solution. At the same time, stir rapidly at 200 rpm until a gel-like consistency is formed to obtain hair-darkening essence formula two.

[0011] Preferably, the addition of xanthan gum in stages specifically includes: mixing 10 g of xanthan gum with 3 g to 5 g of glycerin for every 1000 ml of mixed solution, stirring at 300 rpm for 10 minutes to form a uniform pre-dispersion slurry, and after the mixed solution has complexed and stood for a period of time, cooling and keeping it at 40°C to 50°C, adding the pre-dispersion slurry in three equal portions with an interval of 5 minutes between each addition, while stirring rapidly at 200 rpm until a uniform, particle-free gel-like paste is formed.

[0012] Preferably, the predispersed slurry also includes gellan gum, with the amount of gellan gum added being 0.1 g to 0.3 g per 1000 ml of mixed solution. The gellan gum is mixed simultaneously with xanthan gum and glycerol to prepare the predispersed slurry.

[0013] Application of traditional Chinese medicine hair-darkening essence in the preparation of drugs or cosmetics that promote melanin production.

[0014] Preferably, the herbal hair-darkening essence is applied at a concentration of 1.6% by volume, in at least two consecutive application cycles. The first cycle is a low-frequency application period, with one application every 72 to 96 hours for 14 days. From the second cycle onwards, it is a high-frequency application period, with the application interval shortened to 48 to 72 hours within each cycle, and each cycle lasting 14 days. Each application within any cycle is performed after cleaning the scalp, and the dosage is 0.05 to 0.1 grams of herbal hair-darkening essence per square centimeter of scalp.

[0015] Preferably, the drug or cosmetic is in the form of a gel or ointment for external application to the scalp.

[0016] Preferably, the gel formulation is a thixotropic water-based semi-solid preparation. The specific application method is as follows: after the gel is evenly applied to the clean, dry scalp and hair root area, gently massage in circular motions with fingertips for 1 to 3 minutes to induce thixotropy. After the massage stops, a non-drip film is formed on the scalp surface and remains for 5 to 15 minutes.

[0017] The present invention has at least the following beneficial effects: 1. This invention solves the problems of shallow coloring, easy fading, and easy separation of traditional Chinese medicines such as gallnut, rehmannia root, and polygonum multiflorum in a precise ratio. After ultrasonic-assisted low-temperature extraction, these are mixed with ferrous sulfate mordant in an antioxidant and weakly acidic buffer system and heated. Xanthan gum is then added in a pre-dispersed glycerol manner under stirring to form a gel. This addresses the issues of shallow coloring, easy fading, and easy separation of formulations caused by traditional aqueous extracts. Tannins and ferrous ions fully complex during heating and settling, forming a dark-colored complex that firmly adheres to the hair. L-ascorbic acid and disodium EDTA synergistically prevent the oxidation of ferrous ions. The citrate-sodium citrate buffer ensures the complexation reaction takes place in an optimal pH microenvironment. Xanthan gum imparts a suitable consistency and smooth application, achieving a natural and long-lasting hair-darkening effect, convenient use, and stable formulation. Simultaneously, by precisely controlling the ferrous ion concentration in the mordant to below 0.5% in the final product and optimizing the complexation reaction conditions, the product meets cosmetic safety standards while effectively promoting hair darkening.

[0018] 2. The above solution addresses the issue of single-formula compatibility and individual differences affecting efficacy by providing Formula 2, composed of herbs such as Terminalia chebula, Raspberry, and Cuscuta, as an alternative combination within the same preparation process framework. Formula 2, while maintaining the source of tannins, adds more herbs that nourish the liver and kidneys and replenish essence and blood, giving the hair-darkening essence a stronger internal conditioning effect in addition to its mordant coloring properties. This achieves the beneficial effects of expanding its applicability and enhancing the synergistic effects of hair darkening and nourishing.

[0019] 3. The above solution solves the problems of clumping and insufficient hydration leading to a rough texture in the gel by pre-mixing xanthan gum with glycerin to form a pre-dispersion slurry and slowly adding it when the mixed solution is cooled to 40℃ to 50℃. The wetting effect of glycerin pre-wets the surface of the xanthan gum particles, preventing the formation of an outer gel film upon contact with water. Uniform hydration and expansion at a suitable temperature result in a fine, particle-free gel texture, improving the application experience and product quality.

[0020] 4. The above solution, by adding a trace amount of gellan gum to the xanthan gum-glycerol predispersed paste, utilizes the moderate synergistic effect of gellan gum and xanthan gum to solve the problem of hardening and decreased extensibility of the gel paste due to excessive cross-linking during long-term storage. Within a preset dosage range, gellan gum participates in the formation of a moderately dense three-dimensional network, maintaining the morphological stability of the gel paste while preventing excessive network shrinkage. This achieves the beneficial effect of maintaining good softness and consistent application performance throughout the shelf life.

[0021] 5. The above-described solution applies the hair-darkening essence prepared by the specific method to pharmaceutical or cosmetic applications that promote melanin production, precisely defining a volume concentration of 1.6% as the usage concentration. This solves the problem of existing products lacking precise usage parameters validated by standardized animal models. This concentration is determined based on concentration gradient experiments using a zebrafish embryo model, maximizing the melanin-promoting effect while ensuring 100% survival rate, achieving a beneficial balance between product safety and efficacy.

[0022] 6. The above-mentioned approach addresses the issues of conventional hair-darkening products, such as the lack of scientific basis for application frequency, initial scalp irritation, or insufficient long-term effects, by applying a 1.6% concentration of hair-darkening essence over at least two consecutive application cycles. The first cycle is a low-frequency application period, followed by a high-frequency intensive intervention in hair follicle melanin metabolism, achieving a balance between safety and long-lasting hair-darkening effects.

[0023] 7. The above solution addresses the problems of ordinary liquid formulations, such as easy dripping and short scalp retention time of active ingredients, by limiting the gel formulation to a thixotropic, water-based semi-solid preparation and specifying its application method as thixotropic application followed by massage, followed by the formation of a residual film that remains for 5 to 15 minutes after massage is stopped. This application method utilizes the thixotropic properties of the gel, allowing it to spread smoothly during application and form a non-dripping film upon standing, creating a closed, penetration-enhancing microenvironment for the active ingredients. This achieves the beneficial effect of simultaneously improving ease of use and efficiency in delivering active ingredients. Attached Figure Description

[0024] Figure 1This is a graph showing the melanin synthesis in zebrafish at a drug concentration of 1.6% at 72 hpf according to the present invention. Figure 2 This is a graph showing the effect of different drug concentrations (1.6%) of the present invention on melanin synthesis in zebrafish. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0026] Example 1 The preparation method of the hair-darkening essence made from traditional Chinese medicine includes the following steps: Step 1: Weigh out 30g of Gallnut, 30g of Rehmannia glutinosa, 30g of Polygonum multiflorum, 20g of Angelica sinensis, 20g of Spatholobus suberectus, 10g of Ligustrum lucidum, 10g of Eclipta prostrata, 10g of Morus alba leaves, and 10g of Platycladus orientalis leaves, and mix them to obtain the first Chinese herbal mixture formula.

[0027] Step 2: Add 50g of the herbal mixture formula 1 to 1500ml of pure water and stir well. Place the mixture in an ultrasonic extractor and extract with ultrasonic assistance at 40℃ and 200W power for 20 minutes. Then heat to boiling and maintain a simmer for 40 minutes. After cooling, filter and collect the filtrate to obtain the original herbal extract formula 1.

[0028] Step 3: Weigh 4g of ferrous sulfate, 0.1g of L-ascorbic acid, and 0.05g of disodium EDTA, add them to 50ml of hot water at 95℃, and stir until completely dissolved to obtain a mordant solution.

[0029] Step 4: Measure 1500ml of the original Chinese medicine extract (Formula 1), add 50ml of mordant solution, and stir to mix. Add a 1:2 citrate-sodium citrate buffer solution to the mixture, adjust and monitor the pH value, and stabilize it between 5.2±0.2. Heat to 60℃, let stand for 45 minutes, stirring at 100 to 150 rpm during this time, and then cool the mixture down to 50℃.

[0030] Step 5: Cool the mixed solution obtained in Step 4 to 50°C, add 15g of propylene glycol and 5g of azone, and stir until homogeneous to form a composite penetration-enhancing system. Weigh 16g of xanthan gum, mix it with 4g of glycerin, and stir at 300rpm for 10 minutes to form a uniform pre-dispersed paste. While stirring rapidly at 200rpm, add the pre-dispersed paste to the mixed solution in Step 4 in three equal portions, with a 5-minute interval between each addition, and continue stirring until a uniform, particle-free gel-like consistency is formed, obtaining the first formula for hair-darkening essence.

[0031] Example 2 The preparation method of the hair-darkening essence made from traditional Chinese medicine includes the following steps: Step 1: Weigh out 30g of Gallnut, 30g of Terminalia chebula, 20g of Rubus idaeus, 20g of Cuscuta chinensis, 20g of Rosa laevigata, 20g of Lycium barbarum, 20g of Morus alba, 20g of Cyperus rotundus, 20g of Cinnamomum cassia, 20g of Longan aril, 20g of Spatholobus suberectus, 20g of Platycladus orientalis seed, 20g of black sesame, 4g of ginger extract, and 10g of Vitex trifolia, and mix them to obtain Formula 2 of the Chinese herbal mixture.

[0032] Step 2: Add 50g of the herbal mixture (Formula 2) to 1500ml of pure water and stir well. Place the mixture in an ultrasonic extractor and extract with ultrasonic assistance at 40℃ and 200W for 20 minutes. Then heat to boiling and maintain a gentle boil for 40 minutes. After cooling, filter and collect the filtrate to obtain the original herbal extract (Formula 2).

[0033] Step 3: Weigh 4g of ferrous sulfate, 0.1g of L-ascorbic acid, and 0.05g of disodium EDTA, add them to 50ml of hot water at 98℃, and stir until completely dissolved to obtain a mordant solution.

[0034] Step 4: Measure 1500ml of the original Chinese medicine extract (Formula 2), add 50ml of mordant solution, and stir to mix. Add a 1:2 citrate-sodium citrate buffer solution to the mixture, adjust and monitor the pH value, and stabilize it between 5.3±0.2. Heat to 60℃, let stand for 45 minutes, stirring at 100 to 150 rpm during this time, and then cool the mixture down to 50℃.

[0035] Step 5: Cool the mixed solution obtained in Step 4 to 50°C, add 15g propylene glycol and 5g azone, and stir until homogeneous. Weigh 16g xanthan gum, mix it with 5g glycerin, and stir at 300rpm for 10 minutes to form a uniform pre-dispersed paste. While stirring rapidly at 200rpm, add the pre-dispersed paste to the mixed solution in Step 4 in three equal portions, with a 5-minute interval between each addition, and continue stirring until a uniform, particle-free gel-like paste is formed, obtaining Hair Darkening Essence Formula 2.

[0036] Example 3 A method for using a hair-darkening gel, employing the hair-darkening essence formula one or two prepared according to Example 1 or Example 2, includes the following steps: Step 1: Cleanse and dry the scalp and hair roots.

[0037] Step 2: Take an appropriate amount of the gel-like hair darkening essence and apply it evenly to the clean, dry scalp and hair roots at a dosage of 0.05 to 0.1 grams per square centimeter of scalp.

[0038] Step 3: Gently massage in circular motions with your fingertips for 1 to 3 minutes to allow the gel to thin and spread smoothly under shear force, promoting the penetration and absorption of active ingredients.

[0039] Step 4: After the massage stops, allow the gel to naturally regain its high viscosity on the scalp surface, forming a non-dripping film. Leave it on for 5 to 15 minutes, then rinse thoroughly with warm water.

[0040] Example 4 A method for the periodic application of a hair-darkening essence, using the hair-darkening essence formula one or two prepared in Example 1 or Example 2, at a usage concentration of 1.6% by volume, is applied in at least two consecutive application cycles, including the following steps: Step 1: The first cycle is a low-frequency application period, applied once every 72 to 96 hours for 14 days. During this cycle, each application should be done after cleansing the scalp, with a dosage of 0.05 to 0.1 grams of hair-darkening essence per square centimeter of scalp.

[0041] Step Two: Immediately after the first cycle, begin the second cycle. The second cycle is a high-frequency application period, with the application interval shortened to once every 48 to 72 hours, lasting for 14 days. During this cycle, each application should be performed after cleansing the scalp, with a dosage of 0.05 to 0.1 grams of hair-darkening essence per square centimeter of scalp.

[0042] Step 3: If necessary, after the second cycle ends, continue with the application frequency and dosage of the second cycle for a third and subsequent cycles, each lasting 14 days, until the desired hair darkening effect is achieved.

[0043] Example 5 An evaluation method for the efficacy of hair-darkening essence in promoting melanin production, using a zebrafish embryo model, includes the following steps: Step 1: Collect AB strain zebrafish eggs 9 hours after fertilization, place them in a 48-well plate, add 1 ml of 0.2 mM 1-phenyl-2-thiourea (PTU) culture medium to each well, and treat for 26 hours.

[0044] Step 2: Wash the zebrafish eggs treated with PTU culture medium twice with embryonic water, then add culture medium containing 1.6% by volume of the hair darkening essence formula one or two prepared in Example 1 or Example 2, and continue culturing for 37 hours until the embryos develop to 72 hpf.

[0045] Step 3: Observe and photograph the head region of the zebrafish from between its eyes to the back of its head under a microscope, and use ImageJ software to calculate the optical density value of the head region and calculate the relative optical density.

[0046] Comparative Example 1 The preparation method of the hair-darkening essence made from traditional Chinese medicine, based on Example 1, includes the following steps: Step 1 is the same as Step 1 in Example 1.

[0047] Step 2 is the same as Step 2 in Example 1.

[0048] Step 3: Measure 1500ml of the original Chinese herbal extract formula one, cool and keep it warm to 50℃, add 15g of propylene glycol and 5g of azone, and stir well. Weigh 15g of xanthan gum, first mix it with 4g of glycerin to form a pre-dispersion slurry, then add it in batches while stirring rapidly at 200rpm until the solution turns into a gel-like consistency, obtaining the first hair-darkening essence formula without ferrous sulfate mordant.

[0049] Comparative Example 2 The preparation method of the hair-darkening essence made from traditional Chinese medicine, based on Example 2, includes the following steps: Step 1 is the same as Step 1 in Example 2.

[0050] Step 2 is the same as Step 2 in Example 2.

[0051] Step 3: Measure 1500ml of the original Chinese medicine extract formula 2, cool and keep warm to 50℃, add 15g of propylene glycol and 5g of azone, and stir well. Weigh 15g of xanthan gum, first mix it with 5g of glycerin to form a pre-dispersion slurry, then add it in batches while stirring rapidly at 200 rpm until the solution turns into a gel-like consistency, thus obtaining the hair-darkening essence formula 2 without ferrous sulfate mordant.

[0052] Comparative Example 3 Step 1: Weigh 4g of ferrous sulfate, 0.1g of L-ascorbic acid, and 0.05g of disodium EDTA, add them to 50ml of hot water at 95℃, and stir until completely dissolved to obtain a ferrous sulfate solution.

[0053] Step 2: Take 1500ml of pure water, add 50ml of the above ferrous sulfate solution, stir and mix, add citrate-sodium citrate buffer to adjust the pH to 5.2±0.2, then cool and keep warm to 50℃. Weigh 15.5g of xanthan gum, pre-disperse it with glycerol, and slowly add it in portions while stirring rapidly at 200 rpm until a homogeneous gel is formed, obtaining a blank gel containing only the stabilizing mordant and gel matrix.

[0054] Experiments and Analysis 1. Experimental animals: Adult zebrafish of strain AB, embryos obtained through natural mating.

[0055] 2. Main reagents: 1-Phenyl-2-thiourea (PTU), 3-isobutyl-1-methylxanthine (IBMX), embryo culture water (containing CaCl2, MgSO4, NaHCO3, KCl).

[0056] 3. Instruments: Stereo microscope (equipped with an image acquisition system), 48-well cell culture plate, incubator, ImageJ image analysis software.

[0057] 4. Drug Concentration Determination Experiment: Zebrafish eggs were collected 9 hours post-fertilization (9 hpf) and randomly divided into groups of 20 eggs per group (10 eggs / well, 2 replicates). The groups included: a blank control group (ordinary embryonic water), a PTU group (0.2 mM PTU culture medium), a PTU Washing group (treated with 0.2 mM PTU for 26 h, then washed and replaced with embryonic water), and a drug-treated group. The drug-treated group was first treated with 0.2 mM PTU for 26 h, then washed and added with the culture medium prepared for Examples 1, 2, Comparative Example 1, and Comparative Example 2, respectively, at concentration gradients of 1.5625%, 3.125%, 6.25%, 12.5%, 25%, and 50% (v / v), and cultured until 72 hpf. Hatching rate and mortality rate were recorded to determine the safe drug concentration; the hatching rate and survival rate of zebrafish embryos at a concentration of 1.6% were further verified, showing that both hatching rate and survival rate were 100% at this concentration.

[0058] 5. Drug efficacy evaluation experiment: Zebrafish eggs were collected 9 hours after fertilization (9 hpf) and randomly divided into 9 groups, with 20 eggs in each group (10 eggs / well, 2 replicates). The treatment methods for each group are as follows: Control group (NC): embryos were cultured in water until 72 hpf.

[0059] PTU group: 0.2 mM PTU culture medium was continuously treated until 72 hpf.

[0060] PTU Washing group: After treatment with 0.2mM PTU for 26 hours, the embryos were washed twice with embryo fluid and then replaced with regular embryo fluid for continued culture until 72 hpf.

[0061] Positive control group (IBMX): After treatment with 0.2mM PTU for 26h, the embryos were washed twice with embryonic water and cultured with embryonic water containing 100μg / mlIBMX until 72hpf.

[0062] Example 1 group (1.6% iron): After treatment with 0.2mM PTU for 26h, the embryos were washed twice with water and cultured in culture medium containing 1.6% of the sample from Example 1 by volume until 72hpf.

[0063] Example 2 group (1.6% iron sulfadiazine): After treatment with 0.2mM PTU for 26h, the embryos were washed twice with water and cultured with culture medium containing 1.6% of the sample from Example 2 by volume until 72hpf.

[0064] Comparative Example 1 (1.6% Formula 1): After treatment with 0.2 mM PTU for 26 h, the embryos were washed twice with water and cultured in culture medium containing 1.6% of Comparative Example 1 sample by volume until 72 hpf.

[0065] Comparative Example 2 (1.6% Formula 2): After treatment with 0.2mM PTU for 26h, the embryos were washed twice with water and cultured in culture medium containing 1.6% of Comparative Example 2 sample by volume until 72hpf.

[0066] Comparative Example 3 (1.6% blank gel): After treatment with 0.2 mM PTU for 26 h, the embryos were washed twice with water and cultured in culture medium containing 1.6% of the Comparative Example 3 sample by volume until 72 hpf.

[0067] 6. Observation and Data Collection: At 72 hpf, 10 zebrafish juveniles were selected from each group, and the melanin deposition in the head was observed and images were taken under a stereomicroscope (4× and 10× objectives). The optical density (OD) value of a specific area of ​​the zebrafish head (from between the eyes to the back of the head) was measured using ImageJ software, and the average optical density was calculated.

[0068] 7. Data Processing and Analysis Relative optical density (%) = (average optical density of the drug-treated group / average optical density of the PTU group) × 100%.

[0069] Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS software. LSD-t test was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0070] 8. The experimental results are shown in Tables 1-3 and... Figures 1-2 .

[0071] Table 1: Effects of each group on zebrafish hatching rate Table 2: Effects of each group on the survival rate of zebrafish As shown in Tables 1 and 2, the hatching rate and survival rate of the NC, PTU, and PTU-Washing groups were all 100%. At a concentration of 1.5625%, the hatching rate and survival rate of all drug groups were 100%. With increasing concentration, the hatching rate and survival rate decreased. Specifically, the survival rates of the iron-added groups (Formula 1 and Formula 2) dropped to 45% and 35% respectively at a concentration of 3.125%, and to 0% at a concentration of 6.25%; while the survival rates of the unadded groups (Formula 1 and Formula 2) at a concentration of 3.125% were 50% and 0%, respectively. Based on the combined hatching rate and survival rate data, a concentration of 1.6% was selected for the next efficacy evaluation experiment. This concentration was confirmed in the supplementary validation experiment to have a hatching rate and survival rate of 100%.

[0072] Table 3: Effects of 1.6% drug concentration on melanin synthesis in zebrafish (n=10) Note: Compared with the control group, ****P < 0.0001; compared with the PTU washing group, IBMX group &&&&P < 0.0001; compared with the PTU washing group, ## < 0.01, ####P < 0.0001. Compared with the PTU washing group, the blank gel group P > 0.05, indicating no statistically significant difference.

[0073] 9. Analysis and Conclusion Safety analysis: At a concentration of 1.6%, the survival rate of zebrafish in both Example 1 and Example 2 was 100%, indicating that the hair-darkening essence of the present invention has no obvious acute toxicity to zebrafish embryos at this concentration and has good safety.

[0074] Efficacy analysis: Compared with the PTU Washing group (110.10%), the relative optical density of Example 1 was 145.45% and that of Example 2 was 142.59%, both of which were significantly increased (P<0.0001), indicating that the hair darkening essences of Formula 1 and Formula 2 with added ferrous sulfate mordant can significantly promote melanin synthesis in zebrafish embryos.

[0075] The relative optical density of Comparative Example 1 was 125.32% (P < 0.01), and that of Comparative Example 2 was 120.37% (no statistical difference). Both were lower than the iron-added group in the examples, proving that ferrous sulfate mordant is a key component for enhancing hair darkening effect. Its complexation with tannins in the Chinese herbal extract can significantly improve the melanin production-promoting effect.

[0076] The relative optical density of Comparative Example 3 (blank gel) was 111.35±9.08%, which was not significantly different from that of the PTU Washing group (110.104±11.4279%) (P>0.05). This indicates that ferrous sulfate and xanthan gum gel alone do not promote melanin production, and the efficacy comes from the synergistic effect of the active ingredients of traditional Chinese medicine and iron ions.

[0077] The relative optical density of Examples 1 and 2 both exceeded that of the positive control IBMX group (137.83%), indicating that the hair-darkening essence of the present invention has the potential to promote melanin synthesis better than conventional chemical inducers.

[0078] In summary, the preparation method of the herbal hair-darkening essence of this invention involves ultrasonic-assisted extraction of a specific herbal composition, followed by complexation with a stabilized ferrous sulfate mordant in a weakly acidic buffer system. A composite permeation-enhancing system is used to strengthen transdermal absorption, and glycerin is used to pre-disperse xanthan gum to form a thixotropic gel. The resulting product, verified using a zebrafish embryo model, significantly promotes melanin synthesis and exhibits good safety. Both Formula 1 and Formula 2 demonstrate excellent hair-darkening efficacy and possess industrialization value for development into natural hair-darkening products.

[0079] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for preparing a traditional Chinese medicine hair-darkening essence, characterized in that, Includes the following steps: Weigh out 3 parts by weight of Gallnut, 3 parts by weight of Rehmannia glutinosa, 3 parts by weight of Polygonum multiflorum, 2 parts by weight of Angelica sinensis, 2 parts by weight of Spatholobus suberectus, 1 part by weight of Ligustrum lucidum, 1 part by weight of Eclipta prostrata, 1 part by weight of Morus alba leaf, and 1 part by weight of Platycladus orientalis leaf, and mix them to obtain the first Chinese herbal mixture formula. Add 50 g of the Chinese herbal mixture formula one to 1500 ml of pure water, stir evenly, and then extract with ultrasonic assistance at 40℃ and 200W power for 20 minutes. Then heat to boiling and maintain a gentle boil for 40 minutes. After cooling, filter and take the filtrate to obtain the original Chinese herbal liquid formula one. Dissolve 4 g to 8 g of ferrous sulfate, 0.1 g to 0.2 g of L-ascorbic acid, and 0.04 g to 0.08 g of disodium EDTA in 40 ml to 80 ml of hot water at 90°C to 100°C to obtain a mordant solution. Mix 1500 ml of the herbal extract formula one with 40 ml to 80 ml of mordant solution, stir well, and then add a citric acid-sodium citrate buffer pair with a mass ratio of 1:2 to stabilize the pH of the mixed solution between 5.0 and 5.

5. Heat to 55°C to 60°C and let stand for 45 to 60 minutes, stirring at 100 rpm to 150 rpm during this time. Cool the mixture and keep it at 50°C. Add 15 g of propylene glycol and 5 g of azone and stir well to form a composite penetration-enhancing system. Then add xanthan gum in three equal portions, at a rate of 10 g per 1000 ml of mixed solution, while stirring rapidly at 200 rpm until a gel-like consistency is formed, thus obtaining the hair-darkening essence formula one.

2. The method for preparing the hair-darkening essence of traditional Chinese medicine as described in claim 1, characterized in that, The first herbal mixture formula is replaced with the second herbal mixture formula, which is composed of 3 parts by weight of Gallnut, 3 parts by weight of Terminalia chebula, 2 parts by weight of Rubus idaeus, 2 parts by weight of Cuscuta chinensis, 2 parts by weight of Rosa laevigata, 2 parts by weight of Lycium barbarum, 2 parts by weight of Morus alba, 2 parts by weight of Cyperus rotundus, 2 parts by weight of Cinnamomum cassia, 2 parts by weight of Longan aril, 2 parts by weight of Spatholobus suberectus, 2 parts by weight of Platycladus orientalis seed, 2 parts by weight of black sesame, 0.4 parts by weight of ginger extract, and 1 part by weight of Vitex trifolia. Add 50 g of the Chinese herbal mixture Formula 2 to 1500 ml of pure water, stir evenly, and then extract with ultrasonic assistance at 40℃ and 200W power for 20 minutes. Then heat to boiling and simmer for 40 minutes. After cooling, filter and take the filtrate to obtain the original Chinese herbal liquid Formula 2. Dissolve 4 g to 8 g of ferrous sulfate, 0.1 g to 0.2 g of L-ascorbic acid, and 0.04 g to 0.08 g of disodium EDTA in 40 ml to 80 ml of hot water at 90°C to 100°C to obtain a mordant solution. Mix 1500 ml of the herbal extract formula 2 with 40 ml to 80 ml of mordant solution, stir evenly, and then add a citric acid-sodium citrate buffer pair with a mass ratio of 1:2 to stabilize the pH of the mixed solution between 5.0 and 5.

5. Heat to 55°C to 60°C and let stand for 45 to 60 minutes, stirring at 100 rpm to 150 rpm during this time. Cool the mixture and keep it at 50°C. Add 15 g of propylene glycol and 5 g of azone and stir evenly to form a composite penetration-enhancing system. Then add xanthan gum in three equal portions, with the amount of xanthan gum added being 10 g per 1000 ml of mixed solution. Stir rapidly at 200 rpm until a gel-like consistency is formed to obtain hair-darkening essence formula 2.

3. The method for preparing the hair-darkening essence of traditional Chinese medicine as described in any one of claims 1 to 2, characterized in that, The specific steps for adding xanthan gum in stages include: For every 1000 ml of mixed solution, add 10 g of xanthan gum and 3 g to 5 g of glycerin. Stir at 300 rpm for 10 minutes to form a uniform pre-dispersed slurry. After the mixed solution has complexed and stood, cool it down and keep it at 40°C to 50°C. Add the pre-dispersed slurry in three equal portions, with an interval of 5 minutes between each addition. Stir rapidly at 200 rpm until a uniform, particle-free gel-like paste is formed.

4. The method for preparing the hair-darkening essence of traditional Chinese medicine as described in claim 3, characterized in that, The predispersed slurry also includes gellan gum, with 0.1 g to 0.3 g added per 1000 ml of mixed solution. The gellan gum is mixed with xanthan gum and glycerol simultaneously to prepare the predispersed slurry.

5. The application of the traditional Chinese medicine hair-darkening essence as described in any one of claims 1 to 4 in the preparation of drugs or cosmetics that promote melanin production.

6. The application as described in claim 5, characterized in that, The herbal hair-darkening essence is applied at a concentration of 1.6% by volume, over at least two consecutive application cycles, wherein: The first cycle is a low-frequency application period, with one application every 72 to 96 hours, lasting for 14 days; From the second cycle onwards, the application period will be high-frequency, with the application interval shortened to 48 to 72 hours within each cycle, and each cycle lasting 14 days. Each application within any cycle should be performed after cleaning the scalp, with a dosage of 0.05 to 0.1 grams of herbal hair-darkening essence per square centimeter of scalp.

7. The application as described in claim 5, characterized in that, The medicine or cosmetic is in gel form and is applied topically to the scalp.

8. The application as described in claim 7, characterized in that, The gel-like formulation is a thixotropic, water-based semi-solid preparation, and its specific application method is as follows: Apply the gel evenly to the cleansed, dry scalp and hair roots. Gently massage in circular motions with your fingertips for 1 to 3 minutes to induce thixotropy. After the massage stops, a non-drip film will form on the scalp surface and remain for 5 to 15 minutes.

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