A hair-darkening composition, its preparation method and application

Abstract

This invention belongs to the field of daily chemical products technology, and particularly relates to a hair-darkening composition, its preparation method, and its application. The hair-darkening composition provided by this invention is scientifically formulated with acetyl hexapeptide-1 and extracts of Polygonum multiflorum, Epimedium, and Aristolochia debilis root extract and Avena sativa seed extract. Through the synergistic effect of small molecule peptides and multiple plant active ingredients, it achieves multi-target and multi-pathway regulation of melanin production mechanisms. Each active ingredient, under specific ratios, exhibits significant synergistic effects, synergistically promoting melanin synthesis in hair follicles. Compared to single ingredients, it effectively enhances hair-darkening efficacy, providing a stable and efficient technical solution for improving gray hair and restoring its natural color, possessing outstanding practicality and application value.

Description

Technical Field

[0001] This invention belongs to the field of daily chemical products technology, and in particular relates to a hair-darkening composition, its preparation method and application. Background Technology

[0002] Hair is not only a highly recognizable visual identifier of the human body, but also a key symbol connecting social, cultural, and psychological dimensions. Hair color is essentially the result of complex regulation of pigment units in the hair follicle, and its hue and depth are determined by the ratio and total amount of eumelanin and pheomelanin. Graying hair (canities) is a direct manifestation of the decline in the function of the hair follicle pigment system, and its causes include physiological aging as well as various exogenous or pathological factors.

[0003] At the molecular biological level, hair melanin production is a process precisely regulated by multiple signaling pathways, with microphthalmia-associated transcription factor (MITF) acting as the master regulator. According to existing research, the main signaling pathways for melanin production include Wnt / β-catenin, α-MSH / MC1R / cAMP, SCF / c-KIT, EDN1 / EDNRB, TGF-β, and PI3K / AKT. These pathways ultimately converge on the regulation of MITF, thereby affecting melanocyte proliferation and differentiation, as well as the expression of melanin synthases, ultimately influencing melanin production. Furthermore, hair color is also related to melanosome maturation and transport, oxidative stress, and other processes.

[0004] Natural plant-based ingredients, due to their low toxicity, multiple targets, and mild effects, show promise for regulating melanin production and improving hair follicle function. Since melanin production is influenced by various factors, developing compositions with synergistic effects across multiple pathways and targets is of significant practical importance. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a hair-darkening composition, its preparation method, and its application. The hair-darkening composition is obtained by compounding small molecule peptides with various plant raw materials, enabling multi-target and multi-pathway action, thereby effectively enhancing melanin production and ultimately achieving a hair-darkening effect.

[0006] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides a hair-darkening composition comprising compound A and compound B; compound A comprises acetyl hexapeptide-1, Polygonum multiflorum extract and Epimedium extract, and compound B comprises red stalk flower root extract and rough oat seed extract.

[0007] This invention discloses a highly efficient and safe hair-darkening composition, which is composed of compound A and compound B in synergy. Through the scientific combination of the two compound compounds, it breaks through the bottleneck of traditional hair-darkening technology, realizes multi-target and multi-pathway synergistic regulation of melanin synthesis, and achieves a long-lasting and effective hair-darkening effect.

[0008] Among them, compound A, as the core hair-darkening component of the composition, is composed of acetyl hexapeptide-1, Polygonum multiflorum extract and Epimedium extract. The three components have clear division of labor and synergistic effect, jointly constructing a full-pathway regulation system for melanin synthesis, solving the technical limitation of traditional hair-darkening ingredients with single target, and providing an innovative solution for improving gray hair and restoring hair pigmentation.

[0009] Acetyl hexapeptide-1, as the core of upstream regulation, precisely targets melanocortin receptor 1 (MC1R) on the surface of melanocytes. By activating the MC1R signaling pathway, it initiates the upstream regulatory program of melanin synthesis, laying the foundation for melanin production. Polygonum multiflorum extract specifically acts on the MAPK / ERK signaling pathway. By precisely regulating the phosphorylation level of this pathway, it effectively promotes the proliferation and differentiation of melanocytes, significantly enhances the activity of melanocytes, and provides sufficient functional cells for melanin synthesis. Epimedium extract targets the PI3K / AKT signaling pathway. By activating this pathway, it enhances the survival ability of melanocytes and synergistically promotes the expression of melanin synthesis-related genes, providing continuous impetus for melanin synthesis.

[0010] The synergistic effect of the three core components can significantly upregulate the expression levels of key factors in the melanin synthesis process, including the mRNA expression of microphthalmia-associated transcription factor MITF (a core regulator of melanin synthesis), tyrosinase (TYR), tyrosinase-associated protein 1 (TRP-1), and tyrosinase-associated protein 2 (TRP-2 / DCT). This comprehensively activates the key pathways and functional enzyme systems of melanin production at the core regulatory level, accelerating the biosynthesis, transport, and deposition of melanin in hair, ultimately achieving a highly efficient and long-lasting hair darkening effect.

[0011] To further enhance the hair-darkening efficacy and stability of the composition, this invention introduces compound B, composed of extracts from the root of *Aristolochia debilis* and the seed of *Oatella simonii*, in addition to compound A. Compound B can effectively eliminate and reduce oxidative damage to melanocytes caused by reactive oxygen species (ROS) by activating the body's own antioxidant defense system, protecting the structural and functional integrity of melanocytes, and fundamentally improving problems such as insufficient melanin production and premature graying of hair caused by oxidative stress. Compound B and compound A form a synergistic and complementary effect, enabling the entire composition to achieve a synergistic effect on hair darkening, anti-oxidation, and cell protection, further enhancing the safety and efficacy of the hair-darkening effect, and ultimately achieving precise improvement of gray hair and comprehensive care for hair health.

[0012] Preferably, the mass ratio of compound A to compound B is (1-2):(1-2).

[0013] More preferably, the mass ratio of compound A to compound B is 1.5:1.

[0014] This invention, through experimental screening, determined that the optimal ratio of compound A to compound B is 1.5:1. This preferred ratio is not a simple choice of proportion, but rather a key conclusion based on the H2O2-induced oxidative stress model. At this ratio, the composition has the strongest promoting effect on melanin production under oxidative stress conditions, and the effect is significantly better than using any compound alone or other ratio combinations. It can maximize the synergistic effect of the compound, thereby significantly improving the hair-darkening efficacy of the composition.

[0015] Preferably, the mass ratio of acetyl hexapeptide-1, Polygonum multiflorum extract and Epimedium extract is 0.1:(5-20):(5-15).

[0016] More preferably, the mass ratio of acetyl hexapeptide-1, Polygonum multiflorum extract and Epimedium extract is 0.1:15:10.

[0017] Experimental studies have revealed that the optimal ratio of acetyl hexapeptide-1, Polygonum multiflorum extract, and Epimedium extract is crucial for ensuring their synergistic effect. Extensive testing has verified that when the ratio of the three core components is within the preferred range defined in this invention, the components achieve an optimal synergistic effect, resulting in a "1+1+1>3" synergistic effect. If the component ratio deviates from this preferred range, the synergistic effect between the components will be significantly weakened, the activation efficiency of key melanin synthesis pathways will decrease, and the activity of functional enzymes will be reduced, thus significantly diminishing the hair-darkening effect of the composition and failing to achieve the expected improvement in gray hair.

[0018] Preferably, the mass ratio of the root extract of *Aristolochia debilis* to the seed extract of *Oatella bursa-pastoris* is (1-2):(1-2).

[0019] More preferably, the mass ratio of the red stalk flower root extract to the rough oat seed extract is 1:1.

[0020] The preferred mass ratio of *Aristolochia debilis* root extract to *Oatella bursa-pastoris* seed extract in this invention is (1-2):(1-2), more preferably 1:1. In an H2O2-induced oxidative stress model, the two extracts exhibit a synergistic effect within the above-mentioned preferred ratio range, effectively inhibiting the decrease in melanin production caused by oxidative stress. The synergistic effect is strongest at a 1:1 ratio, while the synergistic effect weakens if the ratio deviates. This preferred ratio achieves the optimal synergistic protective effect of the two extracts, thereby enhancing the strength and stability of the composition against oxidative stress damage.

[0021] Preferably, the composition further includes a solvent, said solvent including 1,3-butanediol.

[0022] Based on the optimized ratio of active ingredients, this invention further adds a specific solvent, 1,3-butanediol, which can effectively improve the solubility and dispersion uniformity of active ingredients such as acetyl hexapeptide-1, Polygonum multiflorum extract, and Epimedium extract, reduce the aggregation or precipitation of active ingredients, and ensure the full effect of each functional substance.

[0023] Secondly, the present invention provides the application of the hair-darkening composition in the preparation of hair care products.

[0024] Preferably, the hair care products include one or more of the following: shampoo, conditioner, hair mask, hair serum, scalp serum, and hair darkening liquid.

[0025] Thirdly, the present invention provides a method for preparing the hair-darkening composition, comprising the following steps: Simply mix acetyl hexapeptide-1, Polygonum multiflorum extract, Epimedium extract, Aristolochia debilis root extract, and Oat seed extract with a solvent until homogeneous.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: The hair-darkening composition provided by this invention is scientifically formulated with acetyl hexapeptide-1 and extracts of Polygonum multiflorum, Epimedium, and Aristolochia debilis root extract and Avena sativa seed extract. This allows for the synergistic effect of small molecule peptides and multiple plant active ingredients, achieving multi-target and multi-pathway regulation of melanin production mechanisms. Each active ingredient, under specific ratios, exhibits significant synergistic effects, jointly promoting melanin synthesis in hair follicles. Compared to single-ingredient formulations, this composition effectively enhances hair-darkening efficacy, providing a stable and efficient technical solution for improving gray hair and restoring its natural color. It possesses outstanding practicality and application value. Attached Figure Description

[0027] Figure 1The left and right graphs show the effects of compound A on the mRNA expression of MC1R in the melanin production pathway in B16F10 cells, respectively. Compared with the control group, ***p<0.001. Figure 2 The figure shows the effect of compound A on the expression of MAPK protein in the melanin production pathway in B16F10 cells; compared with the control group, ***p<0.001; Figure 3 The figure shows the effect of compound A on the expression of AKT and β-catenin proteins in the melanin production pathway in B16F10 cells; compared with the control group, **p<0.01, ***p<0.001; Figure 4 The figure shows the effect of compound A on the mRNA expression of MITF, TYR, TRP-1, and TRP-2 in B16F10 cells; compared with the control group, **p<0.01, ***p<0.001; Figure 5 The figure shows the effect of compound B on H2O2-induced oxidative stress in B16F10 cells, specifically the effect on intracellular ROS levels. Figure 6 This is a graph showing the results of the new hair growth status and melanin deposition level observed in mouse experiments of the sample described in Example 14. Detailed Implementation

[0028] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0029] Unless otherwise specified, the experimental methods used below are conventional methods, and the raw materials used below are all available through conventional commercial channels.

[0030] The acetyl hexapeptide-1 described in the following examples is from Nanjing Spico Biotechnology Co., Ltd., and is a white to off-white powder with a molecular weight of 870.02±1, purity ≥98.0%, moisture ≤7.0%, and acetic acid ≤15.0%. The Polygonum multiflorum extract is from Huzhou Jiamei Biochemical Products Co., Ltd., and is a reddish-brown clear liquid with a characteristic odor, and a total solids content ≥2.5%. The Epimedium extract is from Huzhou Jiamei Biochemical Products Co., Ltd., and is a brown clear liquid with a characteristic odor. The total solids content is ≥2.5%; the root extract of *Aristolochia debilis* was purchased from Silab (distributor: Guangzhou Bisheng Biotechnology Co., Ltd.), and its appearance is a light brown liquid with a special odor, with a total solids content of (2.0-3.2)% and a total polyphenol content of 7.6-11.6 g / L; the seed extract of *Oatella bursa-pastoris* was purchased from Silab (distributor: Guangzhou Bisheng Biotechnology Co., Ltd.), and its appearance is a slightly yellow transparent liquid with a special odor; the total solids content is (2.0-3.2)% and the protein content is 14-21 g / L.

[0031] Examples 1-6, Comparative Examples 1-10: Examples 1-6 and Comparative Examples 1-10 respectively provide a compound A prepared by compounding one or more of acetyl hexapeptide-1, Polygonum multiflorum extract and Epimedium extract, and the formulation of compound A is shown in Table 1.

[0032] The preparation method of the compound A is as follows: According to the mass of Table 1, weigh acetyl hexapeptide-1, Polygonum multiflorum extract and Epimedium extract respectively. First, pre-dissolve acetyl hexapeptide-1 in a small amount of 1,3-butanediol. Then, mix Polygonum multiflorum extract and Epimedium extract evenly. Finally, mix the three together evenly and make up the difference with 1,3-butanediol to obtain the compound A.

[0033] Table 1. Formulation table (mass percentage) of compound A described in Examples 1-6 and Comparative Examples 1-10 Test Example 1: Using compound A described in Examples 1-6 and Comparative Examples 1-10 as test samples, their melanin production effect was tested. The specific test methods are as follows: B16F10 cells in the logarithmic growth phase were harvested and cultured at a rate of 1.2 × 10⁻⁶ cells / year. 5Cells were seeded at a density of [number] cells / mL in 12-well plates, with 1 mL of complete culture medium added to each well. Cells were incubated at 37°C for 24 hours. After incubation, the control group received only 1640 medium; the experimental group was treated with 1640 medium containing the test samples (samples were diluted 10-fold with 1640 medium before cell experiments). After another 24 hours of incubation, cells were collected by trypsin digestion and centrifuged at 1200 rpm for 5 minutes. The precipitate was washed twice with PBS, and 100 μL of 1.0 mol / L NaOH solution containing 10% DMSO was added. The cells were heated at 80°C for 1 hour to dissolve intracellular melanin. Finally, the melanin content was quantified by measuring absorbance at 405 nm.

[0034] Relative melanin content (%) = (ODT / ODB) × 100% Wherein, ODT and ODB represent the average OD values ​​at 405 nm for the experimental group and the control group, respectively. The specific test results are shown in Table 2.

[0035] Table 2 Results of melanin content test The synergistic effect of the three components in compound A is calculated using the following formula (1): S=E 123 / (E1+E2+E3)-1 (1) In the above formula, E1, E2, and E3 represent the percentage increase in melanin content (relative to the control group) when each component is used alone at its corresponding concentration. 123 The percentage increase in melanin content (relative to the control group) when the three components are combined. S>0 indicates synergistic enhancement; the larger the S value, the better the synergistic effect. Specific test results are shown in Table 3.

[0036] Table 3. Synergistic index results of the three components in compound A As shown in Table 2, the melanin production data indicate that single ingredients such as acetyl hexapeptide-1, Polygonum multiflorum extract solution, and Epimedium extract solution have a certain effect on promoting melanin production, and the effect increases with increasing concentration. The melanin-promoting effect of the two-component and three-component combinations is greater than that of the single ingredients. Based on the calculation results of the ternary synergistic index (Table 3), it is shown that when the mass ratio of acetyl hexapeptide-1:Polygonum multiflorum extract:Epimedium extract is in the range of 0.1:(5-20):(5-15), the three-component combinations all show a synergistic effect. Among them, the synergistic effect is strongest when the mass ratio of acetyl hexapeptide-1:Polygonum multiflorum extract:Epimedium extract is 0.1:15:10 (S value reaches 1.54).

[0037] Examples 7-9, Comparative Examples 11-18: Examples 7-9 and Comparative Examples 11-18 respectively provide a compound B prepared by combining one or two of the following: red stalk flower root extract and rough oat seed extract. The formulation of compound B is shown in Table 4.

[0038] The preparation method of the compound B is as follows: Weigh out the root extract of *Aristolochia debilis* and the seed extract of *Oryza sativa* according to the mass specified in Table 4, mix them evenly, and then make up the mass with 1,3-butanediol to obtain the compound B.

[0039] Table 4. Formulation table (mass percentage) of compound B described in Examples 7-9 and Comparative Examples 11-18 Test Example 2: Using compound B described in Examples 7-9 and Comparative Examples 11-18 as test samples, the melanin content under an oxidative stress model was tested. The specific test method is as follows: B16F10 cells in the logarithmic growth phase were harvested and cultured at a rate of 1.2 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of [number] cells / mL in 12-well plates, with 1 mL of complete culture medium added to each well, and incubated at 37°C for 24 hours. After incubation, the control group received only 1640 medium; the experimental group was pretreated with 1640 medium containing the test samples for 24 hours (samples were diluted 10-fold with 1640 medium before cell experiments); then stimulated with 100 μM H2O2 for 12 hours. Cells were collected by trypsin digestion and centrifuged at 1200 rpm for 5 minutes. The precipitate was washed twice with PBS, and 100 μL of 1.0 mol / L NaOH solution containing 10% DMSO was added, followed by heating at 80°C for 1 hour to dissolve intracellular melanin. Finally, the melanin content was determined by measuring the absorbance at 405 nm.

[0040] Relative melanin content (%) = (ODT / ODB) × 100% Wherein, ODT and ODB represent the average OD values ​​at 405 nm for the experimental group and the control group, respectively. The specific test results are shown in Table 5.

[0041] Table 5. Results of melanin content testing under oxidative stress model. The synergistic effect of the two components in compound B is calculated using the following formula (2): S=E 12 / (E1+E2)-1 (2) Where E1 and E2 represent the percentage increase in melanin content when each component is used alone at its corresponding concentration, respectively. 12The percentage increase in melanin content when the two components are combined. S>0 indicates synergistic enhancement, S>0.5 indicates significant synergy, and the larger the S value, the better the synergistic effect. Specific test results are shown in Table 6.

[0042] Table 6. Synergistic index results of the two components in compound B. In the H2O2-induced oxidative stress model, the root extract of *Amomum villosum* and the seed extract of *Ophiopogon japonicus* exhibited a significant synergistic effect in a mass ratio ranging from 2:1 to 1:2, effectively inhibiting the decrease in melanin production induced by oxidative stress. The synergistic effect was strongest at a mass ratio of 1:1, with a synergistic index S=2.65. The synergistic effect weakened when the proportion of *Amomum villosum* or *Ophiopogon japonicus* was too high. Therefore, the preferred mass ratio of *Amomum villosum* root extract to *Ophiopogon japonicus* seed extract is 1:1, at which ratio they exhibit the optimal synergistic protective effect against the decrease in melanin production induced by oxidative stress.

[0043] Examples 10-13: Examples 10-13 provide a composition prepared by compound A and compound B, the formulation of which is shown in Table 7. Compound A is the compound A described in Example 5 (acetyl hexapeptide-1: Polygonum multiflorum: Epimedium = 0.1:15:10), and compound B is the compound B described in Example 9 (Aristolochia debilis root extract: Oat seed extract = 1:1). The composition is obtained by directly mixing compound A and compound B until homogeneous.

[0044] Table 7 Formulation table of the compositions described in Examples 10-13 Test Example 3: The compositions described in Examples 10-13 were used as test samples to test their melanin production effect. Specific test methods are described in Test Example 2. The test results are shown in Table 8.

[0045] Table 8. Results of melanin content testing under oxidative stress model. Table 8 shows that, under the H2O2-induced oxidative stress model, when compound A and compound B are combined in a mass ratio of 1.5:1, the promoting effect on melanin production under oxidative stress is the strongest, and it is significantly better than using them alone or in other ratios.

[0046] Test Example 4: Using the compound A (acetyl hexapeptide-1: Polygonum multiflorum: Epimedium = 0.1:15:10) described in Example 5 as the test sample, its pathway and target were tested. The specific test method is as follows: B16F10 cells in the logarithmic growth phase were harvested and cultured at a rate of 1.2 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 1640 cells / mL in 6-well plates, with 2 mL of complete culture medium added to each well. The plates were incubated at 37°C for 24 hours. After incubation, the control group received only 1640 medium; the experimental group was treated with 1640 medium containing the test samples (samples were diluted 10-fold with 1640 medium before cell experiments). After another 24 hours of incubation, the medium was discarded, and 500 μL of trypsin was added to each well for 8 minutes of digestion. 2 mL of 1640 medium was added to stop the digestion, and the cells were gently pipetted until suspended. The cells were then aspirated into centrifuge tubes and centrifuged at 4°C at low speed (not exceeding 3000 rpm) for 5 minutes. The supernatant was discarded, and the cells were resuspended in PBS. The cells were centrifuged at 4°C at low speed (not exceeding 3000 rpm) for 5 minutes, the supernatant was discarded, and the cell pellet was collected.

[0047] Total RNA was extracted from cell pellet using a commercial RNA extraction kit, and RNA purity and concentration were determined using a Nanodrop 2000 spectrophotometer. In a 20 μL reverse transcription reaction system, 10 μL of RNA, 4 μL of reverse transcriptase, and 1 μL of genomic DNA removal agent were added, with the volume made up to RNase-free water. Reverse transcription was performed in a PCR system (Eastwin LifeSciences Inc, China). Real-time quantitative PCR was performed on a Bio-Rad system with the following program: 95°C pre-denaturation for 30 seconds; followed by 40 cycles (95°C for 15 seconds, 60°C for 1 minute); fluorescence signals were collected at 0.5°C intervals during the melting phase; and finally, the cells were incubated at 95°C for 15 seconds. Primer sequences and target genes used are detailed in Table 3. All reactions were performed in triplicate, with GAPDH as an internal control gene. The relative gene expression level was calculated using the ΔΔCt method. A = CT (target gene, test sample) - CT (internal standard, sample) B = CT (test sample, control sample) - CT (internal standard, quality control sample) K=AB Fold change = 2 -K Table 9 Primer sequences and target genes Western blotting was used to detect protein expression levels. After washing the treated B16F10 cell pellet with pre-chilled PBS, RIPA lysis buffer containing protease inhibitors was added. Cells were scraped off with a cell scraper and transferred to centrifuge tubes. After lysis on ice for 30 minutes, the supernatant was collected by centrifugation to obtain the total protein solution. Protein concentration was determined using a BCA kit. The protein solution was added to 5× reducing protein loading buffer at a 4:1 ratio, denatured in a boiling water bath for 15 minutes, and stored at -20°C for later use. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane, which was blocked with 5% skim milk. The membrane was then incubated overnight at 4°C with primary antibodies (AKT, p-AKT, β-catenin, p38, p-p38, CREB, p-CREB, β-actin). The next day, after washing with TBST, the membrane was incubated at room temperature for 30 minutes with horseradish peroxidase-labeled secondary antibody. The signal was detected by electrochemiluminescence immunoassay, and protein expression was quantified using ImageJ software.

[0048] Figure 1 The effect of compound A on MC1R mRNA expression in the melanin production pathway in B16F10 cells was determined by RT-PCR. The results showed that compound A alone upregulated MC1R by 1.88-fold compared to the control group. Furthermore, the effect of compound A on the PKA / CREB signaling pathway in B16F10 cells was examined by Western blot. The results showed that compound A alone significantly upregulated p-CREB / CREB by 1.66-fold compared to the control group. This indicates that compound A can promote melanin production-related pathways by activating the MC1R / PKA / CREB signaling pathway.

[0049] Figure 2 The effect of compound A on the p38 MAPK signaling pathway in B16F10 cells was examined by Western blot. The results showed that, compared with the control group, treatment with compound A alone significantly upregulated p-p38 / p38MAPK by 2.46 times. This indicates that compound A can regulate melanin production-related processes by activating the p38 MAPK signaling pathway.

[0050] Figure 3 The effects of compound A on the PI3K / AKT / GSK-3β / β-catenin signaling pathway in B16F10 cells were examined using Western blot. The results showed that, compared to the control group, compound A treatment alone significantly downregulated p-AKT / AKT by 0.44-fold and upregulated β-catenin by 2.19-fold. This indicates that compound A can participate in melanin production-related processes by regulating the PI3K / AKT / GSK-3β / β-catenin signaling pathway.

[0051] Figure 4 The effects of compound A on the mRNA expression of melanin-related transcription factors and enzymes, including MITF, TYR, TRP-1, and TRP-2, in B16F10 cells were detected by qRT PCR. The results showed that, compared with the control group, compound A significantly upregulated MITF by 2.55-fold, TYR by 2.89-fold, TRP-1 by 2.82-fold, and TRP-2 by 2.81-fold. This indicates that compound A, after being formulated, can participate in melanin-related processes by regulating relevant genes.

[0052] In summary, the combination exhibits activity at various target sites, such as the MC1R, MAPK, and PI3K / AKT pathways. Furthermore, compound A promotes the mRNA expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-associated protein 1 (TRP-1), and tyrosinase-associated protein 2 (TRP2 / DCT). MITF is well-known as a key "master regulator" in melanin production, activating a series of crucial enzyme genes related to melanin synthesis, including TYR, TRP1, and TRP2. It is also responsible for the survival, proliferation, and differentiation of melanocytes. TYR is the first and most important rate-limiting enzyme in melanin synthesis, while TYRP1 and TYRP2 participate in subsequent reactions, collectively determining melanin production. This demonstrates that the prepared compound A possesses multi-target and multi-pathway activity, exhibiting a strong melanin-promoting effect.

[0053] Test Example 5: The compound B described in Example 9 (red stalk flower root extract: rough oat seed extract = 1:1) was used as the test sample (the experimental concentrations were set at 0.1% and 0.2%, respectively) to test its effect on inhibiting oxidative stress.

[0054] Cell culture and treatment methods were the same as those used in the melanin content assay under the oxidative stress model in Test Example 2. After 12 hours of H2O2 stimulation, the DCFH-DA fluorescent probe was diluted to a final concentration of 10 μM with serum-free medium at a ratio of 1:1000, according to the instructions of the reactive oxygen species (ROS) detection kit. 1.0 mL of the diluted DCFH-DA working solution was added to each well, and the cells were incubated at 37°C in the dark for 20 minutes. After incubation, the cells were gently washed three times with pre-warmed serum-free DMEM medium. Images were acquired using an inverted fluorescence microscope. Subsequently, cells were collected by trypsin digestion, and the cell suspension density was adjusted to 5 × 10⁻⁶ cells / well. 4 The fractions were inoculated at a density of 1 / mL into black 96-well plates. Fluorescence intensity was detected using a multi-mode microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Each group had 3 replicates.

[0055] An oxidative stress model was established by treating B16F10 cells with H2O2, and the intracellular ROS level in B16F10 cells was detected using the DCFH-DA fluorescent probe. Figure 5 As shown, H2O2 treatment significantly increased intracellular ROS levels, manifested as increased fluorescence intensity; while treatment with compound B (mass ratio of 1:1) of 0.1% and 0.2% of the root extract of *Aristolochia debilis* and the seed extract of *Ophiopogon japonicus* significantly inhibited H2O2-induced ROS accumulation and decreased fluorescence intensity, indicating that compound B can inhibit oxidative stress.

[0056] Test Example 6: The sample for Example 14 was prepared according to the components described in Table 10 below, and the antagonistic and protective effects of the sample against hydroquinone-induced damage were evaluated using a mouse model.

[0057] Table 10 (1) Experimental methods and procedures: Six-week-old SPF-grade male C57BL / 6 mice were selected and acclimatized for one week in a standard environment [temperature (22±2)℃, humidity (50±10)%, 12h light / dark cycle alternation], with free access to food and water during the experiment. Before the start of the experiment (Day 0), hair was removed from a 2cm×3cm area on the back of the mice using an animal shaver, and residual hair shafts were removed with depilatory cream; when the mouse skin turned pink and was undamaged, the hair follicles were confirmed to be in the synchronous resting phase. The mice were randomly divided into three groups: a blank control group, a hydroquinone model group, and the sample administration group of Example 14. The blank control group was uniformly coated with distilled water, 200μL / time, once a day, for 28 consecutive days; the other two groups were uniformly coated with an equal amount of 4% hydroquinone aqueous solution on the test area of ​​the back every day to induce local pigment loss by inhibiting hair follicle tyrosinase activity, thus constructing a gray hair model. After the hydroquinone was absorbed, the sample of Example 14 was simultaneously applied to the same area for 28 consecutive days. During the experiment, the general signs of the mice and the integrity of the skin in the drug administration area were observed daily to ensure no inflammatory response. The test areas of the mice in each group were photographed and recorded before the start of modeling (D0) and on day 28 (D28) to observe the growth status of new hair and the level of melanin deposition, and to evaluate the antagonistic and protective effects of the samples on hydroquinone-induced damage.

[0058] (2) Experimental conclusions: Model group: After 28 consecutive days of topical application of 4% hydroquinone solution, depigmentation of hair follicles on the back of C57BL / 6 mice was successfully induced, resulting in large areas of white hair, which confirmed the effectiveness of chemical modeling.

[0059] Example 14 Sample Group: While subjected to hydroquinone damage, the sample intervention resulted in mice maintaining predominantly black fur by day 28, with only a few scattered gray-white hairs visible. This was a significant increase in the number of black hairs compared to the model group. The results indicate that the sample in Example 14 effectively antagonizes the inhibitory effect of hydroquinone on tyrosinase activity, and its protective effect on melanin production in hair follicles is significant, fully demonstrating its hair-darkening effect. Specific test results can be found in [link to specific test results]. Figure 6 .

[0060] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A hair restorer composition, characterized by comprising The composition comprises compound A and compound B; compound A comprises acetyl hexapeptide-1, Polygonum multiflorum extract and Epimedium extract, and compound B comprises red stalk flower root extract and rough oat seed extract.

2. The hair restorer composition of claim 1, wherein The mass ratio of compound A to compound B is (1-2):(1-2).

3. The hair restorer composition of claim 2, wherein The mass ratio of compound A to compound B is 1.5:

1.

4. The hair restorer composition of claim 1, wherein The mass ratio of the acetyl hexapeptide-1, Polygonum multiflorum extract and Epimedium extract is 0.1:(5-20):(5-15).

5. The hair-darkening composition according to claim 1, characterized in that, The mass ratio of the root extract of *Aristolochia debilis* and the seed extract of *Oatella bursa-pastoris* is (1-2):(1-2).

6. The hair-darkening composition according to claim 1, characterized in that, The composition further includes a solvent, said solvent including 1,3-butanediol.

7. The use of the hair-darkening composition according to any one of claims 1-6 in the preparation of hair care products.

8. The application as described in claim 7, characterized in that, The hair care products include one or more of the following: shampoo, conditioner, hair mask, hair serum, scalp serum, and hair darkening liquid.

9. The method for preparing the hair-darkening composition according to claim 6, characterized in that, Includes the following steps: Simply mix acetyl hexapeptide-1, Polygonum multiflorum extract, Epimedium extract, Aristolochia debilis root extract, and Oat seed extract with a solvent until homogeneous.

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