Composition, application thereof and product with hair fixing effect after hair transplantation

Abstract

The invention provides a composition. The composition comprises genistein and formononetin, the content ratio of the genistein to the formononetin is (1): (3). According to the composition with the specific proportion, the hair fixing effect after hair transplantation is achieved, and after hair transplantation, hair can be subjected to a first-stage wound healing, a second-stage hair loss, a resting stage and a third-stage regrowth stage. In the process, formononetin mainly plays an anti-inflammatory role in the first stage, and promotes generation of Laminin in the second stage, so that the effects of stabilizing hair follicles and the like are achieved; genistein focuses on promoting hair to be converted from a resting stage to a growing stage by adjusting a GSK-3beta-beta-catenin signal channel in the second stage and inhibiting an AR receptor in the third stage so as to reduce sebum secretion. Due to different functions of the two components, the two components are combined together, so that a patient after hair transplantation can be more effectively nursed, and the hair fixing effect after hair transplantation is realized.

Description

Technical Field

[0001] The present invention relates to the field of extracting medicinal chemical components from plants, and particularly to a composition, its application, and a product with the effect of promoting hair growth and preventing hair loss after hair transplantation. Background Art

[0002] Currently, the postoperative care techniques after hair transplantation mainly include laser treatment technique, microneedle treatment technique, scalp massage technique, and hyperbaric oxygen treatment technique. However, it may cause adverse phenomena such as skin allergic reactions, bleeding, and ecchymosis.

[0003] At present, the postoperative care drugs after hair transplantation mainly include minoxidil, finasteride (only applicable to men), antibiotics, and glucocorticoids, etc., but there are some side effects: local redness, swelling, increased pain, dysfunction, etc.

[0004] The existing article "An herbal extract combination (biochanin A, acetyltetrapeptide-3, and ginseng extracts) versus 3% minoxidil solution for the treatment of androgenetic alopecia: A 24-week, prospective, randomized, triple-blind, controlled trial [J]" (Lueangarun and Panchaprateep, 2020) describes a combination including biochanin A, acetyltetrapeptide-3, and ginseng extracts, which can be used to treat androgenetic alopecia, but it is impossible to know whether it has the effect of promoting hair growth and preventing hair loss after hair transplantation. Therefore, it is very necessary to provide a composition with good effect of promoting hair growth and preventing hair loss after hair transplantation. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is that the provided composition has the effect of promoting hair growth and preventing hair loss after hair transplantation.

[0006] The present invention provides a composition, including genistein and formononetin; the content ratio of genistein to formononetin is (0.5~1.5):(1~12).

[0007] In some specific embodiments, the content ratio of genistein to formononetin is 1:3.

[0008] The present invention provides the application of the composition as described in any one of the above in the preparation of a product for promoting hair growth and preventing hair loss after hair transplantation.

[0009] In some specific embodiments, the hair strengthening includes: anti-inflammation, regulating the expression levels of genes or proteins related to hair strengthening, and / or regulating hormone levels.

[0010] In some specific embodiments, the anti-inflammation includes upregulating the level of Nrf2; the anti-inflammation includes inhibiting the level of inflammatory factors; the inflammatory factors include IL-6.

[0011] In some specific embodiments, the genes related to hair strengthening include β-catenin and GSK3β; the regulation of genes related to hair strengthening includes upregulating the phosphorylation levels of β-catenin and / or GSK3β; the proteins related to hair strengthening include laminin, and the regulation of proteins related to hair strengthening includes promoting the expression of laminin.

[0012] In some specific embodiments, the hormone includes androgen receptor, and the regulation of hormone levels includes inhibiting the level of androgen receptor.

[0013] The present invention provides a product for hair strengthening after hair transplantation, including the composition according to any one of the above technical solutions.

[0014] The present invention provides a hair strengthening method, applying the composition according to any one of the above technical solutions or the product according to the above technical solution to the scalp surface.

[0015] Compared with the prior art, the present invention provides a composition, including genistein and formononetin; the content ratio of genistein to formononetin is (0.5 - 1.5):(1 - 12). The present invention achieves the hair strengthening effect after hair transplantation through the composition with the above specific ratio: after hair transplantation, the hair will go through the stages of wound healing in the first stage, hair loss in the second stage, entering the resting phase and then regrowing in the third stage. During this process, formononetin mainly plays an anti-inflammatory role in the first stage and promotes the generation of Laminin in the second stage to stabilize hair follicles, etc.; genistein focuses on promoting the transformation of hair from the resting phase to the growth phase by regulating the GSK-3β-β-catenin signaling pathway in the second stage and inhibiting the AR receptor in the third stage to reduce sebum secretion. Since their functions are different, combining them together can more effectively care for patients after hair transplantation and achieve the hair strengthening effect after hair transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the quantitative analysis result of genistein and formononetin based on HPLC;

[0017] Figure 2 It is the influence of two phytoestrogen compositions on the relative viability of mouse macrophages RAW264.7;

[0018] Figure 3Effect of two phytoisoflavone compositions on the expression of Nrf2 in LPS-induced mouse macrophage RAW264.7 cells (left) Immunofluorescence imaging (right) Average fluorescence intensity comparison chart;

[0019] Figure 4 Inhibitory effect of two phytoisoflavone compositions on IL-6;

[0020] Figure 5 Effect of two phytoisoflavone compositions on the viability of 3T3 mouse embryonic fibroblasts;

[0021] Figure 6 Effect of two phytoisoflavone compositions on the expression of laminin in 3T3 cell layers (left) Immunofluorescence imaging (right) Average fluorescence intensity comparison chart;

[0022] Figure 7 Effect of two phytoisoflavone compositions on the viability of human dermal papilla cells (HDP);

[0023] Figure 8 Effect of two phytoisoflavone compositions on the expression level of p-GSK3β mRNA;

[0024] Figure 9 Effect of two phytoisoflavone compositions on the expression level of β-catenin mRNA;

[0025] Figure 10 Effect of two phytoisoflavone compositions on the viability of SZ95 cells;

[0026] Figure 11 Effect of two phytoisoflavone compositions on the expression level of AR mRNA;

[0027] Figure 12 . Molecular docking diagram of formononetin and Keap1;

[0028] Figure 13 . Molecular docking results of genistein and GSK-3β;

[0029] Figure 14 . Molecular docking results of genistein and AR;

[0030] Figure 15 Problems at different stages after hair transplantation and coping strategies of genistein and formononetin compositions. Detailed implementation methods

[0031] The present invention provides a composition, its applications, and a product with the effect of strengthening hair after hair transplantation. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0032] The present invention provides a composition, comprising genistein and formononetin; the content ratio of genistein to formononetin is (0.5 - 1.5):(1 - 12).

[0033] In some specific embodiments, the content ratio of genistein to formononetin is (0.5 - 1.5):(1 - 5);

[0034] In some specific embodiments, the content ratio of genistein to formononetin is 1:3.

[0035] The inventors of the present invention creatively discovered that the combination of genistein and formononetin in the present invention has a synergistic effect and has a good effect on strengthening hair after hair transplantation.

[0036] The effect of the composition of formononetin and genistein (3:1) on strengthening hair after hair transplantation is better than that of the above monomers. It can simultaneously achieve the enrichment of multiple targets and multiple pathways, improve the follicle survival rate, and promote the transformation of hair follicles to the growth phase. Compared with traditional single components or simple mixtures, the plant composition of the present invention can simultaneously achieve the enrichment of multiple targets and multiple pathways. By regulating multiple key signaling pathways such as the Nrf2 - IL - 6 pathway and the GSK3β - β - catenin pathway, as well as the synergistic effects on multiple targets such as androgen secretion, inflammatory response, and extracellular matrix, the regulatory ability of follicle physiological functions is comprehensively improved, overcoming the defects of single - component with single action and limited effect.

[0037] The above genistein and formononetin in the present invention can be commercially available or extracted according to the method of the present invention.

[0038] It can be: taking Trifolium pratense as the raw material and using ethanol reflux extraction method for extraction. The ethanol concentration is preferably 70%, the solid - liquid ratio is preferably 1:15; the extraction times are preferably 2 times, the first extraction time is preferably 2 h; the second extraction time is preferably 1 h. Then, macroporous resin purification is carried out, and the resin model is HPD - 600; the sample loading flow rate is preferably 2 BV / h; the water washing volume is preferably 6 BV; the water washing flow rate is preferably 4 BV / h; the volume of 70% ethanol is 4 BV; the ethanol washing flow rate is 4 BV / h.

[0039] The present invention provides the use of the composition described in any one of the above in the preparation of a hair loss prevention product after hair transplantation.

[0040] In some specific embodiments, the hair loss prevention includes: anti-inflammation, regulating the expression levels of genes or proteins related to hair loss prevention, and / or regulating hormone levels.

[0041] In some specific embodiments, the anti-inflammation includes up-regulating the level of Nrf2;

[0042] In a specific embodiment, the composition has an up-regulating effect on Nrf2 in LPS-induced macrophage Raw 264.7 cells.

[0043] In some specific embodiments, the anti-inflammation includes inhibiting the level of inflammatory factors; the inflammatory factors include IL-6.

[0044] In a specific embodiment, the composition of the present invention has an inhibitory effect on IL-6 secreted by LPS-induced macrophage Raw 264.7 cells.

[0045] In some specific embodiments, the genes related to hair loss prevention include β-catenin and GSK3β; the regulation of genes related to hair loss prevention includes up-regulating the phosphorylation (N-terminal Ser 9) levels of β-catenin and / or GSK3β;

[0046] In a specific embodiment, the composition of the present invention has a promoting effect on the phosphorylation levels of β-catenin and / or GSK3β in human dermal papilla cells.

[0047] The proteins related to hair loss prevention in the present invention include laminin, and the regulation of proteins related to hair loss prevention includes promoting the expression of laminin.

[0048] In a specific embodiment, the composition of the present invention has a promoting effect on laminin in 3T3 fibroblasts.

[0049] In some specific embodiments, the hormone includes androgen receptor, and the regulation of hormone levels includes inhibiting the level of androgen receptor.

[0050] In a specific embodiment, the composition of the present invention has an inhibitory effect on androgen receptor in sebaceous gland cells.

[0051] The inventor of the present invention has discovered that the composition of two phytoisoflavones in a specific ratio can exert an anti-inflammatory effect by regulating the Nrf2-IL-6 pathway at a lower concentration, reducing the inflammatory reaction around hair follicles, creating a good living environment for hair follicle cells. At the same time, it can promote the synthesis of laminin, enhance the stability of the extracellular matrix of hair follicle cells, thereby stabilizing the hair follicles, and regulate hair retention-related genes, including up-regulating the phosphorylation levels of β-catenin and / or GSK3β, promoting the transformation of hair from the telogen phase to the anagen phase. Finally, it inhibits androgen receptors (AR) to reduce sebum secretion and mitigate the adverse effects of androgens on hair follicles. Through the above pathways, the effect of hair retention after hair transplantation is achieved.

[0052] The present invention provides a product with the effect of hair retention after hair transplantation, comprising the composition according to any one of the above technical solutions.

[0053] The product with the effect of hair retention after hair transplantation according to the present invention further comprises excipients commonly used in cosmetics. It includes but is not limited to the following types and components:

[0054] Solvents: Water is the most common solvent. In addition, there are glycerol, propylene glycol, butylene glycol, etc.

[0055] Humectants: including panthenol, sodium hyaluronate, sodium polyglutamate, hydrolyzed wheat protein, hydrolyzed keratin, etc.

[0056] Emulsifiers: polysorbate-20, PEG-40 hydrogenated castor oil, cetearyl alcohol, trideceth-12, PEG-55 stearate, laureth-23, laureth-6, PEG-20 stearate, glyceryl stearate, etc.

[0057] Preservatives: such as DMDM hydantoin, phenoxyethanol, methylchloroisothiazolinone, methylisothiazolinone, methylparaben, propylparaben, etc.

[0058] Antistatic agents: behenyl trimethyl ammonium chloride, amino-terminated polydimethylsiloxane, cetrimonium chloride, guar hydroxypropyltrimonium chloride, behenamide propyl dimethylamine, etc.

[0059] Film-forming agents: polydimethylsiloxane, amino-terminated polydimethylsiloxane, etc.

[0060] Emollients: cetyl alcohol, shea butter, horse fat, phytosterol isostearate, glyceryl caprylate, geranium oil, frankincense (BOSWELLIA CARTERII) oil, etc.

[0061] Binders: hydroxyethyl cellulose.

[0062] Fragrances and flavorings: fragrances, (daily use) fragrances.

[0063] Other excipients: magnesium nitrate (emulsifying stabilizer), magnesium chloride (viscosity control), citric acid (pH regulator), disodium EDTA (chelating agent), CI 18965 (colorant), etc.

[0064] The dosage forms of the products with hair-fixing efficacy after hair transplantation provided by the present invention include aerosol types (without propellants), aerosol types (with propellants), gel types, aqueous solution types, etc. Common hair-fixing products such as hair mousse and styling hairspray, hair mousse corresponds to aerosol types (with propellants), and styling hairspray may also belong to the aerosol type dosage form; while the gel-like hair-fixing products belong to the gel type; some liquid hair-fixing sprays, etc. belong to the aqueous solution type.

[0065] In some specific embodiments, the content ratio of genistein and formononetin in the product is 1:3. The composition of the present invention can exert a significant hair-fixing effect at a lower concentration, which not only reduces the usage amount of raw materials, lowers the production cost, but also improves the safety and tolerance of the product. In practical applications, it is more conducive to developing various dosage forms of post-hair transplantation care products, such as shampoos, hair growth liquids, sprays, etc., for the convenience of patients.

[0066] The two plant compositions of the present invention are significantly superior to the monomer components in terms of hair-fixing after hair transplantation. It can comprehensively regulate hair follicles from multiple aspects, improve the survival rate of hair follicles and promote their transformation into the growth phase, so that the hair after hair transplantation grows more firmly, reduces the risk of hair loss, and improves the success rate and long-term effect of hair transplantation surgery.

[0067] The present invention provides a hair-fixing method, applying the composition described in any one of the above technical solutions or the product described in the above technical solution to the scalp surface.

[0068] The above usage method can be: applying it to the scalp and gently massaging until absorbed.

[0069] The present invention provides a composition, including genistein and formononetin; the content ratio of genistein and formononetin is (0.5 - 1.5):(1 - 12). The present invention achieves the hair-fixing efficacy after hair transplantation through the composition with the above specific ratio: first inhibiting inflammation to improve the survival rate of hair follicles; then promoting the expression of extracellular matrix laminin and regulating the GSK3β-β-Catenin signaling pathway to promote the transition of hair follicles from the resting phase to the growth phase, and finally inhibiting androgens to reduce sebum secretion.

[0070] It should be understood that the expression "one or more of..." includes each of the objects recited after the expression and various different combinations of two or more of the recited objects alone, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0071] The terms "comprising", "having", or "including", including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, without excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context otherwise.

[0072] In this application, the term "and / or" describes the relationship between associated objects and indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural.

[0073] It should be understood that as long as the present invention remains operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

[0074] The use of any and all examples or exemplary language in this document, such as "for example" or "including", is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0075] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0076] It should be understood that in various embodiments of this application, the magnitude of the serial numbers of the above processes does not mean the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0077] Some cases are described in the embodiments and comparative examples of the present invention, and some implementation manners of the present invention are shown in the embodiments. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0078] To further illustrate the present invention, the following describes in detail a composition provided by the present invention, its application, and a product for promoting hair growth after hair transplantation in combination with embodiments.

[0079] In the present invention, genistein and formononetin can be prepared by extraction or commercially available, and the present inventor does not limit this.

[0080] Example 1

[0081] Extraction of genistein and formononetin from Trifolium pratense L.:

[0082] 1.1 Reflux extraction of Trifolium pratense L. with ethanol

[0083] The reflux extraction of genistein and formononetin from Trifolium pratense L. was carried out according to the following table. The ethanol concentration, solid-liquid ratio, extraction time and extraction times described below were selected for the experiment, and a certain content of isoflavones was obtained.

[0084] Influencing factors Parameter Ethanol concentration (v%) 70 Ratio of material to liquid (g / mL) 1:15 First extraction time (h) 2 Second extraction time (h) 1

[0085] 1.2 Purification with macroporous resin

[0086] The crude extract was purified by macroporous resin after rotary evaporation. The experimental conditions are shown in the following table.

[0087] Resin model HPD-600 Resin dosage 1:3 Loading flow rate 2BV / h Volume of water washing 6BV Water washing flow rate 4BV / h Volume of 70% ethanol 4BV Alcohol washing flow rate 4BV / h

[0088] Take the 70% ethanol washing solution and carry out rotary evaporation, and then determine the contents of genistein and formononetin in each group of liquids respectively.

[0089] 1.3 Determination of the contents of formononetin and genistein

[0090] Using octadecylsilane chemically bonded silica gel as the filler; using acetonitrile - 0.1% formic acid water as the mobile phase; the detection wavelength is 255 nm.

[0091] Preparation of the standard solutions of genistein and formononetin: Take appropriate amounts of the reference substances of genistein and formononetin, weigh accurately, and dissolve them in methanol to prepare a mixed solution containing 0.1 mg of each per 1 ml, that is, obtain.

[0092] Preparation of the sample solution: Take an appropriate amount of the extract of Trifolium pratense L., weigh accurately, dilute it with methanol, shake well, filter, and take the continuous filtrate, that is, obtain. The results are as follows Figure 1 shown. Figure 1 The results showed that in the two kinds of plant isoflavone compositions derived from Trifolium pratense L. extracted by ethanol reflux and purified by macroporous resin HPD-600, the content ratio of genistein to formononetin was 1:3. This composition was selected for the subsequent efficacy experiments.

[0093] Anti-inflammatory experiment of the composition in Example 2

[0094] 2.1.1 Determination of the maximum safe concentration of the extract containing the two kinds of plant isoflavone compositions by MTT method

[0095] (1) Inoculation: When the cell density ≥ 70%, discard the old culture medium, add 5 mL of PBS to wash the cells, then digest with trypsin for 1 min. Pipette to resuspend the cells, add RPMI-1640 complete medium to terminate the digestion, mix well and transfer to a 2 mL centrifuge tube. Centrifuge at 1000 rpm for 4 minutes and discard the supernatant. Re-add 1 mL of complete medium, pipette to mix well, dilute by an appropriate multiple and count. Adjust the cell density to 8x10 4 cells / mL according to the counting result, and inoculate the cells into a 96-well plate at a volume of 200 μL per well, then place it back into the incubator for incubation (37 °C, 5% CO2).

[0096] (2) Experimental grouping: Set up a negative control group and an experimental group in the experiment, and set another 2 cell-free wells as zero-adjustment wells. Set 6 concentration gradients for the samples in the experimental group, and set 2 replicates for each concentration gradient;

[0097] (3) Preparation of solutions: According to the experimental design, prepare test substances with different concentrations using the basal medium;

[0098]

[0099] (4) Administration of drugs: After 24 h, take out the 96-well plate, discard the old culture medium, add 200 μL of basal medium to each well of the zero-adjustment wells and the blank control group, and add 200 μL of the sample prepared with the basal medium to each well of the experimental group. Plate according to 2 replicates for each concentration in each group. Then place it back into the incubator for culture (37 °C, 5% CO2);

[0100] (5) Detection: After 24 h of drug treatment, take out the 96-well plate, add 20 μL of MTT working solution (5 mg / mL) to each well, place it back into the incubator for continued culture for 4 h, then discard the liquid in the wells, re-add 150 μL of DMSO to each well, shake for 10 min and measure the absorbance (OD value) at a wavelength of 490 nm.

[0101] (6) Calculation of relative cell viability: Cell viability % = (OD value of the experimental group - OD value of the zero-adjustment wells) / (OD value of the blank control group - OD value of the zero-adjustment group) * 100%.

[0102] 2.1.2 Effects of a composition containing two phytoisoflavones on the regulation of Nrf2 and IL-6

[0103] (1) Cell incubation and drug administration

[0104] (2) Cell fixation and permeabilization: Discard the culture medium in the 24-well plate, wash three times with PBS, add 250 μL of 4% paraformaldehyde to each well, and fix at room temperature for 15 min. Recover the paraformaldehyde, wash three times with PBS, add 250 μL of 0.1% Triton to each well, and permeabilize at room temperature for 10 min.

[0105] (4) Blocking and primary antibody incubation: Discard the supernatant, wash three times with PBS, add 250 μL of 5% goat serum to each well, and incubate at room temperature for 1 h. Discard. Without washing, add the appropriate proportion of primary antibody to each well, and incubate overnight at 4 °C in the dark.

[0106] (5) Secondary antibody incubation and nuclear staining: Discard the supernatant, wash 3 times with PBS, add 250 μL of appropriately diluted secondary antibody to each well, and incubate at room temperature for 1 h. Discard the supernatant, wash 3 times with PBS, add 250 μL of Hochest33342 to each well, and incubate at room temperature for 5 min.

[0107] (6) Immunofluorescence photography

[0108] 2.1.3 Effects of two phytoisoflavone compositions on the secretion of IL-6 by LPS-induced mouse macrophage RAW264.7 cells

[0109] (1) Experimental grouping

[0110]

[0111] (2) Experimental procedures

[0112] The experiment was carried out according to the instructions of the ELISA kit from eBioscience.

[0113] 2.1.4 Experimental results

[0114] (1) Detection of safe concentration by MTT method

[0115] The results are as Figure 2 shown, Figure 2 which are the effects of two phytoisoflavone compositions on the relative viability of mouse macrophage RAW264.7 cells. When the concentration of the two phytoisoflavone compositions ≤ 1.5317 μg / mL, the cell viability was > 90%, and an appropriate concentration can be selected within the concentration range of ≤ 1.5317 μg / mL for determination.

[0116] (2) Regulation of Nrf2 in LPS-induced mouse macrophage RAW264.7 cells by two phytoisoflavone compositions

[0117] Figure 3 This is an immunofluorescence image of the effect of two phytoisoflavone compositions on the expression of Nrf2 in LPS-induced mouse macrophage RAW264.7 cells. Compared with the blank control group (BC), Nrf2 was significantly downregulated after LPS stimulation (NC) ( **** P < 0.0001), and vitamin E (VE, 20 μg / mL) could significantly upregulate the expression of Nrf2 ( ####P < 0.0001), indicating that the current stimulation conditions are effective; the concentrations of the composition at 0.7658 μg / mL and formononetin at 0.5866 μg / mL have an up-regulating effect on Nrf2 in LPS-induced macrophages Raw 264.7 cells ( #### P < 0.0001, ## P < 0.01), and the promotion rates are 77.74% and 40.35% respectively, with a statistically significant difference.

[0118] (3) Effects of two phytoestrogen compositions on the secretion of IL-6 by LPS-induced mouse macrophage RAW264.7 cells

[0119] The results are as Figure 4 shown, Figure 4 which are the inhibitory effects of two phytoestrogen compositions on IL-6. (Note: When performing statistical analysis using the T-test method, compared with the negative control group (NC), the significance is indicated by * for the blank control group (BC), and P < 0.0001 is indicated as **** P < 0.0001; compared with the negative control group (NC), the significance is indicated by # for the positive control group (PC), and P < 0.0001 is indicated as #### P < 0.0001; compared with the negative control group (NC), the significance is indicated by # for the experimental group, and P < 0.0001 is indicated as #### P < 0.0001).

[0120] Compared with the blank control group (BC), the concentration of IL-6 increased significantly after LPS stimulation (NC) ( **** P < 0.0001), and dexamethasone (PC, 100 μg / mL) could significantly inhibit the secretion of IL-6 ( #### P < 0.0001), indicating that the current stimulation conditions are effective; the concentrations of the composition (formononetin + genistein) at 0.7658 μg / mL and 0.3829 μg / mL have an inhibitory effect on the secretion of IL-6 by LPS-induced macrophages Raw 264.7 cells ( #### P < 0.0001, ## P < 0.01), and the inhibition rates are 24.93% and 12.97% respectively, with a statistically significant difference; the concentrations of formononetin at 0.58660 μg / mL and 0.29330 μg / mL have no inhibitory effect on the secretion of IL-6 by LPS-induced macrophages Raw264.7 cells.

[0121] 2.2 Laminin experiment of two phytoestrogen compositions

[0122] 2.2.1 Determination of the maximum safe concentration of the sample by the MTT method (3T3 fibroblasts)

[0123] 2.2.2 Immunofluorescence

[0124] The experimental method was the same as that in 2.1.2.

[0125] 2.2.3 Experimental Results

[0126] 2.2.3.1 MTT Assay

[0127] The results were as Figure 5 shown, Figure 5 showing the effects of two phytoestrogen compositions on the viability of 3T3 mouse embryonic fibroblasts. It can be seen that when the concentration of the two phytoestrogen compositions was ≤0.7658 μg / mL, the cell viability was >90%, and an appropriate concentration could be selected within the range of ≤0.7658 μg / mL for determination.

[0128] 2.2.3.1 Immunofluorescence

[0129] The results were as Figure 6 described, Figure 6 showing the immunofluorescence images of the effects of two phytoestrogen compositions on the expression of laminin in 3T3 cell layers. The two phytoestrogen compositions at a concentration of 0.7658 μg / mL had a promoting effect on the laminin of 3T3 fibroblasts, with a promotion rate of 132.07%, and the difference was statistically significant ( ## P < 0.01). The two phytoestrogen compositions at concentrations of 0.3829 μg / mL, formononetin at 0.5866 μg / mL and 0.2933 μg / mL had a promoting effect on the laminin of 3T3 fibroblasts, but there was no statistical significance.

[0130] 2.3 Regulation of GSK3β-β-catenin Experiment with Two Phytoestrogen Compositions

[0131] 2.3.1 Determination of the Maximum Safe Concentration of the Sample by MTT Assay (HDP Human Dermal Papilla Cells)

[0132] The method was the same as that in 2.1.1.

[0133] 2.3.2 RT-QPCR

[0134] The method was the same as that in 2.3.2.

[0135] 2.3.3 Experimental Results

[0136] (1) Detection of the Safe Concentration of the Sample in HDP Cells by MTT Assay

[0137] The results were as Figure 7 shown, Figure 7Effect of two phytoisoflavone compositions on the viability of human dermal papilla cells (HDP). It can be seen that when the concentration of the two phytoisoflavone compositions ≤ 3.0683 μg / mL, the cell viability is > 90%, and an appropriate concentration can be selected within the concentration range of ≤ 3.0683 μg / mL for determination.

[0138] (2) Detection of the regulation of GSK3β-β-catenin by the sample using RT-PCR

[0139] Table 1 Detection results of p-GSK3β mRNA expression level

[0140]

[0141]

[0142] v.s. BC, *p < 0.05, **p < 0.01; v.s. S2, #p < 0.05, ##p < 0.01.

[0143] Table 2 Detection results of β-catenin mRNA expression level

[0144]

[0145] v.s. BC, *p < 0.05, **p < 0.01; v.s. S2, #p < 0.05, ##p < 0.01.

[0146] The results are shown in Tables 1-2 and Figures 8 - 9 as follows Figure 8 Effect of two phytoisoflavone compositions on the expression level of p-GSK3β mRNA Figure 9 Effect of two phytoisoflavone compositions on the expression level of β-catenin mRNA. The results are the effects of two phytoisoflavone compositions on the expression level of p-GSK3β mRNA in human dermal papilla cells (HDP). It can be seen that compared with the blank control group (BC), the mRNA expression levels of p-GSK3β and β-catenin in the composition are both significantly up-regulated (p < 0.05). Except that the mRNA expression level of p-GSK3β is significantly up-regulated (p < 0.01) when the concentration of genistein is 0.1805 μg / mL, there is no significant difference in the other concentrations (p > 0.05). Compared with genistein, when the concentration is 3.0683 μg / mL, 1.5341 μg / mL and 0.7671 μg / mL, the mRNA expression levels are all significantly up-regulated (p < 0.05). It shows that the composition has the effect of up-regulating the mRNA expression levels of p-GSK3β and β-catenin, and under the same content of genistein, the up-regulation effect of the composition on p-GSK3β and β-catenin is better than that of genistein with the same content.

[0147] 2.4 Inhibition Experiment of AR with Two Kinds of Phytosterols Compositions

[0148] 2.4.1 Determination of the Maximum Safe Concentration of Samples by MTT Method (SZ95 Human Sebaceous Gland Cells)

[0149] The method is the same as that in 2.1.1

[0150] 2.4.2 RT-QPCR

[0151] (1) Cell Incubation: Inoculate the cell suspension into a 6-well cell culture plate and incubate

[0152] (2) Cell Administration and Collection

[0153]

[0154] Aspirate and discard the culture medium from the wells, and add different treatment groups to each well according to the above table. Place the 6-well plate back into the cell culture incubator for 24 hours and collect the cells

[0155] (3) RNA Extraction: Sample Lysis - Removal of Genomic DNA - Adsorption of RNA - Removal of Impurities - Elution of RNA - Detection of RNA Purity and Concentration

[0156] (4) RNA Reverse Transcription: Removal of Genomic DNA - Reverse Transcription - Obtaining cDNA

[0157] (5) Prepare primers, probes, and aliquot the multiplex PCR reaction system into each reaction tube, and add cDNA respectively

[0158] (6) Prepare primers, probes, and aliquot the multiplex PCR reaction system into each reaction tube, and add cDNA respectively

[0159] 2.4.3 Experimental Results

[0160] (1) Detection of the Safe Concentration of Samples in SZ95 Cells by MTT Method

[0161] The results are as Figure 10 shown Figure 10 for the effects of two kinds of phytosterols compositions on the viability of SZ95 cells. It can be seen from Figure 10 that when the concentration of the composition ≤ 1.5341 μg / mL, the cell viability is > 90%, and an appropriate concentration can be selected within the concentration range of ≤ 1.5341 μg / mL for determination

[0162] (2) Detection of the Inhibitory Effect of Samples on AR by RT-PCR Method

[0163] The results are as Figure 11 shown Figure 11The effects of two phytoestrogen compositions on the mRNA expression level of AR are as follows. Figure 11 As can be seen, the mRNA expression of the AR receptor in SZ95 cells is shown in the figure. Compared with the NC control group, when the concentrations of the two phytoestrogen compositions are 0.0962 μg / mL,

[0164] 0.3839 μg / mL, and 0.7668 μg / mL, the mRNA expression of the AR receptor is significantly decreased (p <

[0165] 0.05). Genistein also significantly decreases the mRNA expression of the AR receptor at concentrations of 0.0903 μg / mL and 0.1804 μg / mL. This indicates that the composition has the effect of reducing the mRNA expression level of the AR receptor. And when the content of genistein is the same, the down-regulation effect of the composition on the AR receptor at concentrations of 0.0962 μg / mL and

[0166] 0.3839 μg / mL is better than that of genistein with the same content (0.0226 μg / mL and 0.0903 μg / mL).

[0167] Example 3

[0168] For the above cell experiment results, molecular-level verification was carried out using AI molecular docking. The 2D structures of the compounds were obtained from the PubChem database, and the 3D crystal structures of Keap1 (Uniprot code: Q14145), GSK-3β (Uniprot code: P49841), and AR (Uniprot code: P10275) were downloaded from the Uniprot database (https: / / www.uniprot.org / ). The protein structures were initially cleaned by removing water molecules and non-critical ligands and further processed using the molecular docking software NPAIEngine. Subsequently, the target compounds (formononetin and genistein) were molecularly docked with their target proteins Keap1, GSK-3β, and AR, with default parameters used during the docking process. After docking, the ligand-protein complex with the lowest binding energy was selected, and the ligand-protein interaction was visually analyzed using PyMOL software.

[0169] Keap1 (Kelch-like ECH-associated protein 1) and Nrf2 (nuclear factor erythroid 2-related factor 2) are key molecules regulating cellular oxidative stress responses. Under steady-state conditions, Keap1, as an E3 ubiquitin ligase adaptor protein, promotes the ubiquitination and degradation of Nrf2, maintaining its low levels. When cells are stimulated by oxidative stress or electrophilic substances, the sensitive cysteine residues of Keap1 are modified, leading to conformational changes that prevent it from targeting Nrf2. As a result, Nrf2 becomes stable and translocates into the nucleus, activating gene transcription driven by the antioxidant response element (ARE). Therefore, Keap1 is a key protein regulating Nrf2. Thus, formononetin was docked with Keap1, and the docking results are as follows Figure 1 as shown Figure 12 . Molecular docking diagram of formononetin and Keap1

[0170] The Kelch domain of Keap1 (amino acid sequence 368–583) is the key region for its binding to Nrf2. As Figure 12 shown, the target compound can bind to the Kelch domain of Keap1, thereby competitively inhibiting the Keap1-Nrf2 interaction and further regulating the activity of Nrf2

[0171] In the classical Wnt / β-catenin signaling pathway, the activity of GSK-3β (glycogen synthase kinase-3β) plays a key regulatory role in the stability of β-catenin. When GSK-3β is phosphorylated at the Ser9 site (mediated by kinases such as Akt and PKA), its kinase activity is inhibited, thereby reducing the phosphorylation modification of β-catenin. Since phosphorylation mediated by GSK-3β usually promotes the ubiquitination and degradation of β-catenin, phosphorylation of GSK-3β (Ser9) inhibits the degradation of β-catenin, leading to enhanced stability and cytoplasmic accumulation. Genistein phosphorylates GSK-3β at the Ser9 site, and the molecular docking results are as follows Figure 13 as shown Figure 13 . Molecular docking results of genistein and GSK-3β

[0172] The androgen receptor (AR) is a nuclear transcription factor that can bind to androgens (such as testosterone and dihydrotestosterone DHT) and be activated. In the hair follicle sebaceous gland unit, after DHT (converted from testosterone by 5α-reductase) binds to AR, it activates downstream signaling pathways, promotes sebocyte proliferation and lipid synthesis, resulting in excessive sebum secretion. In addition, overactivation of AR shortens the growth phase of hair follicles and accelerates their entry into the regression and resting phases, thereby triggering hair loss again. Genistein can specifically bind to the ligand-binding domain (LBD, amino acids 665-919) of the androgen receptor (AR), and effectively block the transcriptional activation function of AR by competitively inhibiting the binding of endogenous androgens (such as DHT) to AR. The results of its molecular docking are as Figure 14 shown. Figure 14 . Results of the molecular docking of genistein with AR.

[0173] Figure 15 Problems at different stages after hair transplantation and coping strategies of the genistein and formononetin composition.

[0174] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A composition, characterized in that, It includes genistein and formononetin; the content ratio of genistein to formononetin is (0.5 - 1.5):(1 - 12).

2. The composition according to claim 1, wherein The content ratio of genistein to formononetin is 1:

3.

3. Use of the composition according to any one of claims 1 to 2 in the preparation of a hair-fixing product after hair transplantation.

4. The application according to claim 3, characterized in that, The hair fixing includes: anti-inflammation, regulating the expression levels of genes or proteins related to hair fixing and / or regulating hormone levels.

5. The application according to claim 4, characterized in that The anti-inflammation includes upregulating the level of Nrf2 and / or inhibiting the expression of IL-6.

6. The application according to claim 4, wherein The genes related to hair fixing include β-catenin and GSK3β; the regulation of genes related to hair fixing includes promoting the phosphorylation level of GSK-3β, upregulating β-catenin, and promoting the transformation of hair from the telogen phase to the anagen phase; the proteins related to hair fixing include laminin, and the regulation of proteins related to hair fixing includes promoting the expression of laminin.

7. The application according to claim 4, wherein The hormones include androgen receptors, and the regulation of hormone levels includes inhibiting the level of androgen receptors.

8. A product with the effect of strengthening hair after hair transplantation, characterized in that, It includes the composition according to any one of claims 1 to 2.

9. The product according to claim 8, wherein, The content ratio of the genistein and formononetin composition in the product is 1:

3.

10. A hair-fixing method, characterized in that, Apply the composition according to any one of claims 1 to 2 or the product according to claim 8 to the scalp surface.

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