Herbal composition for preventing hair loss, growing hair and blackening hair and preparation method thereof

Abstract

The invention provides a herbal anti-hair-loss, hair-growing and hair-blacking composition and a preparation method thereof.The composition comprises herbal extracts such as cacumen biotae, radix polygoni multiflori and amur corktree bark, and the herbal extracts cooperate with active ingredients such as a hair-growing peptide compound, a polyamino acid polysaccharide condensation compound and adenosine triphosphate; and the integrated effects of hair loss prevention, hair growth and hair blackening are realized through a multi-target action mechanism. According to the preparation method, an ultrasonic-reflux alternate extraction technology is adopted to efficiently enrich active ingredients of the herbal medicine, and the activity of thermosensitive ingredients is protected through a gradient temperature compounding technology. Experiments show that the composition can significantly promote hair follicle growth and inhibit DKK1 expression, and is safe and non-irritant; the composition is suitable for hair growth liquid, shampoo and other dosage forms, and has the advantages of high efficiency, synergy and stability.

Description

Technical Field

[0001] This invention relates to the field of cosmetics and daily chemical technology, and in particular to a herbal composition for preventing hair loss, promoting hair growth and darkening hair, and its preparation method. Background Technology

[0002] Hair health directly impacts a person's appearance and self-confidence. With the fast pace of modern life, increased stress, irregular sleep and diet, and genetic factors, problems such as hair loss, thinning hair, premature graying, and sensitive scalp are becoming increasingly prominent and affecting younger people. These issues have become significant health and beauty concerns for people of all ages. Sensitive hair loss caused by abnormal scalp sebum secretion and a damaged scalp barrier is also frequent, creating an urgent market demand for products that combine multiple benefits such as preventing hair loss, promoting hair growth, darkening hair, and scalp care.

[0003] Existing hair loss prevention, hair regrowth, and hair darkening products are mainly divided into three categories, but all of them have significant technical defects: First, chemically synthesized products, such as preparations with minoxidil and finasteride as core ingredients, can be effective against some hair loss targets, but have side effects such as scalp irritation, dryness, and itching. Long-term use may lead to dependence, and most of them only focus on the single effect of preventing hair loss or promoting hair growth, failing to address hair darkening and scalp barrier repair, thus limiting the suitable population; Second, single herbal extract products, which mostly rely on traditional hair-growth herbs such as arborvitae leaves and Polygonum multiflorum, have limited targets due to their single ingredients, only able to improve blood circulation or have simple anti-inflammatory effects, and are unable to address issues such as hormonal imbalance, hair follicle dormancy, and insufficient melanin synthesis. The overall effect is unsatisfactory due to multiple overlapping causes of hair problems; thirdly, although ordinary compound products attempt to combine multiple ingredients, there are problems such as unreasonable combination logic and weak synergistic effect. For example, they have not formed an effective combination targeting core targets such as 5α-reductase inhibition, hair follicle activation, and melanin production. In addition, some products use a single reflux extraction process, resulting in low dissolution rate of active ingredients in herbs and more impurities, which affects the efficacy and stability of the products; furthermore, most existing products ignore the connection between scalp barrier repair and hair nutrient supply, fail to solve the vicious cycle of barrier damage, inflammation, and hair loss, and lack targeted improvement solutions for gray hair problems, making it difficult to meet consumers' needs for comprehensive hair care. Summary of the Invention

[0004] In view of this, the present invention proposes a herbal composition for preventing hair loss, promoting hair growth and darkening hair, and a method for preparing the same, to solve the above problems.

[0005] The technical solution of this invention is achieved as follows: a herbal composition for preventing hair loss, promoting hair growth, and darkening hair, comprising the following raw materials in parts by weight: 5-15 parts of Platycladus orientalis leaf extract, 3-10 parts of Polygonum multiflorum extract, 2-8 parts of Phellodendron chinense bark extract, 3-9 parts of Angelica sinensis extract, 2-7 parts of Rehmannia glutinosa root extract, 3-8 parts of Sophora flavescens extract, 1-5 parts of Panax ginseng root extract, 2-6 parts of Rosmarinus officinalis leaf extract, 1-4 parts of Watercress extract, 2-5 parts of Saffron saffron extract, 0.5-3 parts of Rhus chinensis gall extract, 1-4 parts of Zanthoxylum bungeanum fruit extract, 0.8-3 parts of Cnidium monnieri fruit extract, 1-4 parts of Ligustrum lucidum extract, 0.8-3 parts of Ligusticum chuanxiong extract, 2-5 parts of Cornus officinalis extract, 0.3-2 parts of Arctium lappa root extract, and a carrier.

[0006] Preferably, it further comprises 0.5-3 parts by weight of a hair growth peptide complex and 1-5 parts by weight of a multi-amino acid polysaccharide condensate, wherein the hair growth peptide complex is composed of palmitoyl tripeptide-1, acetyl tetrapeptide-3, myristoyl pentapeptide-4 and hexapeptide-3 in a mass ratio of (1-3):(2-4):(1-3):1.

[0007] Preferably, the polyamino acid polysaccharide condensate is prepared by condensation reaction of complex amino acids and complex polysaccharides at a mass ratio of 1-3:1. The condensation reaction conditions are: pH 6.0-7.0, temperature 45-55℃, and reaction time 3-4 hours. The molecular weight of the polyamino acid polysaccharide condensate is 5000-50000 Da.

[0008] Preferably, the composite amino acid is composed of cysteine, serine, glycine, and lysine in a mass ratio of (0.5-1.2):(0.8-2.0):(1.5-1.8):(0.3-0.9); The complex polysaccharide is composed of Tremella fuciformis polysaccharide, hyaluronic acid, and oat β-glucan in a mass ratio of (1-2):(0.8-1.5):(2.3-2.5).

[0009] Preferably, it further comprises 0.1-2 parts by weight of adenosine triphosphate (ATP), 0.3-1.5 parts by weight of erythritol, and 1.8-2.5 parts by weight of betaine, wherein the purity of the adenosine triphosphate is ≥98%.

[0010] Preferably, the carrier comprises 1-5 parts panthenol, 0.01-0.1 parts vitamin H, and 5-15 parts 1,3-propanediol.

[0011] Preferably, the preparation method of the herbal composition for preventing hair loss, promoting hair growth, and darkening hair includes the following steps: S1. Raw material pretreatment: Platycladus orientalis leaves, Polygonum multiflorum, Phellodendron bark, Angelica sinensis, Rehmannia glutinosa root, Sophora flavescens, Rhus chinensis gall, Zanthoxylum bungeanum fruit, Cnidium monnieri fruit, Ligustrum lucidum, Ligusticum chuanxiong, Cornus officinalis, and Arctium lappa root are washed and dried at a low temperature of 40-50℃ until the moisture content is ≤8%. After pulverizing, they are passed through a 50-60 mesh sieve to obtain mixed herbal powder. Ginseng root extract, rosemary leaf extract, watercress extract, and saffron extract are filtered through a 100-200 mesh sieve to remove impurities and are ready for use. S2. Compound extraction: Add 8-12 times the volume of 55-65% ethanol aqueous solution to the mixed herbal powder for compound extraction to obtain crude extract. Concentrate and purify the crude extract to obtain herbal concentrate. S3, Compound Mixture: S3-1: Add panthenol, vitamin H, 1,3-propanediol, erythritol and betaine to the herbal concentrate in sequence, and stir at 35-40℃ and 250-300 rpm for 20-30 minutes to form the base solution; S3-2: Add ginseng root extract, rosemary leaf extract, watercress extract, and saffron extract to the base liquid, keep the temperature and stirring speed constant, and continue stirring for 25-35 minutes until completely dispersed; S3-3: Finally, cool to 30-35℃, add adenosine triphosphate, hair growth peptide complex, and polyamino acid polysaccharide condensate, and stir for 30-40 minutes until the system is homogeneous; S4. Homogenization and sterilization: Homogenize the above mixture at 12000-15000 rpm for 12-15 minutes, rapidly cool to 25-30℃, and filter through a 0.22μm filter membrane to obtain the composition.

[0012] Preferably, the compound extraction in step S2 is an alternating ultrasonic-reflux extraction, with an ultrasonic power of 200-250W, each ultrasonic extraction lasting 10-15 minutes, a reflux temperature of 65-75℃, each reflux lasting 20-30 minutes, and the alternating cycle repeated 2-3 times. The extracts are combined and filtered through a 300-400 mesh filter cloth to obtain a crude extract.

[0013] Preferably, the concentration in step S2 involves concentrating the crude extract under reduced pressure at 50-60°C and a vacuum of -0.08 to -0.09 MPa to a relative density of 1.15-1.20. The purification includes membrane filtration purification, wherein the pore size of the membrane filtration is in the range of 10-200 nm.

[0014] Herbal compositions for preventing hair loss, promoting hair growth, and darkening hair are used to prepare hair loss prevention and hair growth solutions, shampoos, or conditioners.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention utilizes the synergistic effects of multiple herbal extracts, combined with hair growth peptide complexes, multi-amino acid polysaccharide condensates, and bioactive ingredients such as ATP, to form a complete efficacy system for preventing hair loss, promoting hair growth, darkening hair, and protecting the scalp, exhibiting clear synergistic effects. Among them, the herbal extracts and bioactive ingredients target key targets such as 5α-reductase inhibition, RSPO1 secretion promotion, DKK1 secretion inhibition, and melanin production, while also possessing antibacterial, anti-inflammatory, and antioxidant effects. Crocus extract and other ingredients can promote the production of filaggrin, effectively repairing the scalp barrier and providing a healthy environment for hair growth.

[0016] The ultrasonic-reflux alternating extraction method used in this invention can efficiently and fully extract the active ingredients from the herbal raw materials; more importantly, the gradient temperature control process used in the compounding process can maximize the protection of the activity of heat-sensitive bioactive substances.

[0017] The composition of this invention has a scientific and controllable preparation process, with clear proportions of each component, making it suitable for large-scale production. The finished product has good stability and can meet the industrial production needs of daily chemical products. This composition can be easily prepared into various dosage forms such as hair growth liquid, shampoo, and conditioner to meet the usage habits and needs of different consumers, and has broad market application prospects. Detailed Implementation

[0018] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0019] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0021] In this invention Ginseng (PANAX GINSENG) root extract: Ginseng root was extracted twice at 60°C for 3 hours each time using a 60% (v / v) ethanol aqueous solution to obtain ginseng root extract.

[0022] Rosemary (ROSMARINUS OFFICINALIS) leaf extract: Dried rosemary leaves were extracted by heating and reflux with a 50% (v / v) ethanol aqueous solution. After filtration and concentration, an extract rich in antioxidant active ingredients such as rosmarinic acid and sauropic acid was obtained.

[0023] Watercress (NASTURTIUM OFFICINALE) flower / leaf / stem extract: The dried aerial parts (flowers, leaves, and stems) of watercress were extracted with water at 70℃ using ultrasound-assisted extraction. After fine filtration and concentration, the extract was obtained, which is rich in flavonoid active ingredients.

[0024] Saffron (CROCUS SATIVUS) flower extract: The dried stigmas of saffron flowers were percolated with an 80% ethanol solution at 28°C. The resulting extract was concentrated under reduced pressure at below 50°C to obtain an extract rich in active ingredients such as crocin.

[0025] Example 1 Composition and proportions of the composition: 5 parts of Platycladus orientalis leaf extract, 3 parts of Polygonum multiflorum extract, 2 parts of Phellodendron chinense bark extract, 3 parts of Angelica sinensis extract, 2 parts of Rehmannia glutinosa root extract, 3 parts of Sophora flavescens extract, 1 part of Panax ginseng root extract, 2 parts of Rosmarinus officinalis leaf extract, 1 part of Watercress extract, 2 parts of Saffron extract, 0.5 parts of Rhus chinensis gall extract, 1 part of Zanthoxylum bungeanum fruit extract, 0.8 parts of Cnidium monnieri fruit extract, 1 part of Ligustrum lucidum extract, 0.8 parts of Ligusticum chuanxiong extract, 2 parts of Cornus officinalis extract, and 0.3 parts of Arctium lappa root extract. Hair growth peptide complex 0.5 parts: palmitoyl tripeptide-1, acetyl tetrapeptide-3, myristoyl pentapeptide-4 and hexapeptide-3 = 1:2:1:1; One part of polyamino acid polysaccharide condensate: complex amino acids: complex polysaccharides 1:1, cysteine, serine, glycine, lysine = 0.5:0.8:1.5:0.3; tremella polysaccharide, hyaluronic acid, oat β-glucan = 1:0.8:2.3; The polyamino acid polysaccharide condensate is prepared by condensation reaction of complex amino acids and complex polysaccharides in a mass ratio of 1:1. The condensation reaction conditions are: pH=6.0, temperature 45℃, and reaction time 3 hours. The molecular weight of the polyamino acid polysaccharide condensate is 5000 Da.

[0026] 1 part panthenol, 0.01 part vitamin H, 5 parts 1,3-propanediol, and deionized water to make up to 100 parts.

[0027] Example 2 Composition and proportions of the composition: 15 parts of Platycladus orientalis leaf extract, 10 parts of Polygonum multiflorum extract, 8 parts of Phellodendron chinense bark extract, 9 parts of Angelica sinensis extract, 7 parts of Rehmannia glutinosa root extract, 8 parts of Sophora flavescens extract, 5 parts of Panax ginseng root extract, 6 parts of Rosmarinus officinalis leaf extract, 4 parts of Watercress extract, 5 parts of Saffron crocus extract, 3 parts of Rhus chinensis gall extract, 4 parts of Zanthoxylum bungeanum fruit extract, 3 parts of Cnidium monnieri fruit extract, 4 parts of Ligustrum lucidum extract, 3 parts of Ligusticum chuanxiong extract, 5 parts of Cornus officinalis extract, and 2 parts of Arctium lappa root extract. Hair growth peptide complex in 3 parts: palmitoyl tripeptide-1, acetyl tetrapeptide-3, myristoyl pentapeptide-4 and hexapeptide-3 = 3:4:3:1; Five parts of polyamino acid polysaccharide condensate: complex amino acids: complex polysaccharides 3:1, cysteine, serine, glycine, lysine = 1.2:2.0:1.8:0.9; tremella polysaccharide, hyaluronic acid, oat β-glucan = 2:1.5:2.5; The polyamino acid polysaccharide condensate is prepared by condensation reaction of complex amino acids and complex polysaccharides at a mass ratio of 3:1. The condensation reaction conditions are: pH 7.0, temperature 55℃, and reaction time 4 hours. The molecular weight of the polyamino acid polysaccharide condensate is 50,000 Da.

[0028] Panthenol 5 parts, Vitamin H 0.1 parts, 1,3-propanediol 15 parts, deionized water to make up to 100 parts.

[0029] Example 3 Composition and proportions of the composition: 10 parts of Platycladus orientalis leaf extract, 8 parts of Polygonum multiflorum extract, 5 parts of Phellodendron chinense bark extract, 6 parts of Angelica sinensis extract, 5 parts of Rehmannia glutinosa root extract, 5 parts of Sophora flavescens extract, 3 parts of Panax ginseng root extract, 4 parts of Rosmarinus officinalis leaf extract, 3 parts of Watercress extract, 3 parts of Saffron extract, 2 parts of Rhus chinensis gall extract, 2 parts of Zanthoxylum bungeanum fruit extract, 2 parts of Cnidium monnieri fruit extract, 2 parts of Ligustrum lucidum extract, 1.5 parts of Ligusticum chuanxiong extract, 3 parts of Cornus officinalis extract, and 1 part of Arctium lappa root extract. Two portions of hair growth peptide complex: palmitoyl tripeptide-1, acetyl tetrapeptide-3, myristoyl pentapeptide-4 and hexapeptide-3 in a ratio of 2:3:2:1; Three portions of polyamino acid polysaccharide condensate: complex amino acids: complex polysaccharides 2:1, cysteine, serine, glycine, lysine = 0.8:1.5:1.3:0.6; tremella polysaccharide, hyaluronic acid, oat β-glucan = 1.5:1.2:2.4; The polyamino acid polysaccharide condensate is prepared by condensation reaction of complex amino acids and complex polysaccharides at a mass ratio of 2:1. The condensation reaction conditions are: pH 6.5, temperature 50℃, and reaction time 3 hours. The molecular weight of the polyamino acid polysaccharide condensate is 30000 Da.

[0030] Panthenol 3 parts, Vitamin H 0.05 parts, 1,3-propanediol 10 parts, deionized water to make up to 100 parts.

[0031] The compositions of Examples 1-3 above were prepared using the following methods: S1. Raw material pretreatment: Platycladus orientalis leaves, Polygonum multiflorum, Phellodendron bark, Angelica sinensis, Rehmannia glutinosa root, Sophora flavescens, Rhus chinensis gall, Zanthoxylum bungeanum fruit, Cnidium monnieri fruit, Ligustrum lucidum, Ligusticum chuanxiong, Cornus officinalis, and Arctium lappa root are washed and dried at 45℃ until the moisture content is ≤8%. After pulverizing, they are passed through a 55-mesh sieve to obtain mixed herbal powder. Ginseng root extract, rosemary leaf extract, watercress leaf / stem extract, and saffron extract are filtered through a 150-mesh sieve to remove impurities and are ready for use. S2. Compound Extraction: Add 10 times the volume of 60% ethanol aqueous solution to the mixed herbal powder and perform alternating ultrasonic-reflux extraction. The ultrasonic power is 200W, and the ultrasonic time is 12 minutes each time. The reflux temperature is 70℃, and the reflux time is 25 minutes each time. The alternating cycle is repeated 3 times. The extracts are combined and filtered through a 400-mesh filter cloth to obtain a crude extract. The crude extract is concentrated under reduced pressure at 55℃ and a vacuum degree of -0.08MPa to a relative density of 1.18. It is then purified by filtration through a 100nm membrane, with a molecular weight cutoff of 8000Da. Impurities are removed and active ingredients are retained to obtain a concentrated herbal extract. S3, Compound Mixture: S3-1: Add panthenol, vitamin H, 1,3-propanediol, erythritol and betaine to the herbal concentrate in sequence, and stir at 40℃ and 300rpm for 25 minutes to form the base solution; S3-2: Add ginseng root extract, rosemary leaf extract, watercress extract, and saffron extract to the base liquid, keep the temperature and stirring speed constant, and continue stirring for 30 minutes until completely dispersed; S3-3: Finally, cool down to 32℃, add adenosine triphosphate, hair growth peptide complex, and polyamino acid polysaccharide condensate, and stir for 35 minutes until the system is homogeneous; S4. Homogenization and sterilization: The above mixture is homogenized at 13000 rpm for 15 minutes, rapidly cooled to 30°C, and filtered through a 0.22 μm filter membrane to obtain the composition.

[0032] Comparative Example 1 The difference between this comparative example and Example 3 is that it does not contain the hair growth peptide complex and ATP; otherwise, it is the same as Example 3.

[0033] Comparative Example 2 The difference between this comparative example and Example 3 is that it does not contain polyamino acid polysaccharide condensates and ATP; otherwise, it is the same as Example 3.

[0034] Comparative Example 3 The difference between this comparative example and Example 3 is that the active ingredients do not contain privet fruit extract, chuanxiong extract, cornus officinalis extract, or burdock root extract, while the proportions of the remaining core active ingredients, auxiliary active ingredients, and carriers are the same as in Example 3.

[0035] Comparative Example 4 The difference between this comparative example and Example 3 is that the active ingredients do not contain sumac gall extract, Sichuan pepper fruit extract, or saffron extract, while the proportions of the remaining core active ingredients, auxiliary active ingredients, and carriers are the same as in Example 3.

[0036] Comparative Example 5 The difference between this comparative example and Example 3 is that step S2 of the preparation method of the composition, which is complex extraction, is changed to single reflux extraction. 10 times the volume fraction of 60% ethanol aqueous solution is added to the mixed herbal powder, and the mixture is refluxed at 70°C for 1.5 hours. The crude extract is obtained by filtration. The remaining steps are the same as in Example 3.

[0037] I. In vitro efficacy test (a) Ex vivo mouse hair follicle growth test 1. Experimental materials: C57BL / 6 mice (6-8 weeks old), with hair follicles isolated from the dorsal whiskers; The compositions of each example and comparative example (diluted with serum-free DMEM to a final concentration of 0.01 mg / mL); Positive control (minoxidil 10 μM); Serum-free DMEM culture medium, CO2 incubator, microscope, and image analysis software.

[0038] 2. Experimental steps: Mouse whisker hair follicles were aseptically isolated, and intact hair follicles of uniform length (1.0-1.2 mm) were selected and seeded into 24-well plates, one hair follicle per well, with 1 mL of culture medium added.

[0039] Grouping: blank control group (culture medium only), positive control group, examples 1-3, comparative examples 1-5, with 6 replicates per group.

[0040] The culture medium containing the corresponding sample was changed daily, and the samples were cultured in a 37℃, 5% CO2 incubator for 14 days. The hair follicle growth length was measured by microscopic imaging every 2 days.

[0041] 3. Evaluation indicators: Total hair follicle growth length (mm) and relative growth rate (%, with the blank control group as 100%) after 14 days. 4. Test Results Table 1: Results of hair follicle growth test in isolated mice

[0042] (II) Test on the secretion-promoting effect of dermal papilla cells 1. Experimental Materials Cell model: Human dermal papilla cells (DPCs, purchased from the ATCC Biostandard Resource Center in the United States), passaged to 3-5 generations for experiments; Sample preparation: The compositions of each example and comparative example (diluted to a final concentration of 0.01 mg / mL with DMEM medium containing 10% fetal bovine serum). Control settings: blank control group (DMEM medium containing only 10% fetal bovine serum) and positive control group (recombinant human RSPO1 protein, final concentration 50 ng / mL). Reagents and instruments: DPCs-specific culture medium, fetal bovine serum, trypsin, RSPO1 enzyme-linked immunosorbent assay (ELISA) kit, CO2 incubator, microplate reader, and clean bench.

[0043] 2. Experimental Procedure Cell seeding: Logarithmic growth phase DPCs were digested with trypsin and the concentration was adjusted to 5 × 10⁻⁶. 4 Cells were seeded at a density of 1 mL / well in 24-well plates and cultured at 37°C with 5% CO2 for 24 hours until the cell adhesion and confluence rate reached 70%-80%. Sample intervention: Discard the original culture medium, add 1 mL of fresh culture medium to the blank control group, add culture medium containing recombinant human RSPO1 protein to the positive control group, and add the corresponding diluted sample culture medium to the groups of Examples 1-3 and Comparative Examples 1-5, with 6 replicates per group; Culture and collection: After culturing for another 48 hours, collect the cell supernatant, centrifuge at 1000 rpm for 5 minutes to remove debris, and keep the supernatant for later use; RSPO1 content detection: Follow the ELISA kit instructions, add standard and sample supernatant to the enzyme-labeled plate in sequence, and incubate at 37°C for 60 minutes; after washing, add enzyme-labeled secondary antibody and incubate at 37°C for 30 minutes; after washing, add chromogenic substrate and incubate in the dark for 15 minutes, add stop solution, and detect OD value at 450nm wavelength. Data calculation: Plot a standard curve using the OD values ​​of the standard, substitute the OD values ​​of the sample to calculate the RSPO1 concentration, and take the average value of each set of replicates.

[0044] 3. Evaluation Indicators RSPO1 secretion concentration (pg / mL); The relative increase rate of RSPO1 (%) = (RSPO1 concentration in the sample group - RSPO1 concentration in the blank control group) / RSPO1 concentration in the blank control group × 100%, which reflects the promoting effect of the sample on RSPO1 secretion.

[0045] 4. Test Results Table 2: RSPO1 Test Results

[0046] (III) 5α-Reductase Inhibition Rate Test 1. Experimental materials: Type II 5α-reductase (recombinant human), testosterone (substrate), NADPH (coenzyme), and compositions of each example and comparative example (diluted to a final concentration of 0.1 mg / mL with DMSO). Blank control group (DMSO), positive control (finasteride 1 μM); high performance liquid chromatography (HPLC).

[0047] 2. Experimental steps: Reaction system (200 μL): containing 0.5 U of 5α-reductase, 10 μM of testosterone, and 0.2 mM of NADPH. Add the sample solution or control solution and incubate at 37°C for 60 minutes.

[0048] The reaction was terminated by adding 1 mL of ethyl acetate. Dihydrotestosterone (DHT) was extracted, centrifuged, and the supernatant was evaporated to dryness. The supernatant was then redissolved in methanol, and the peak area of ​​DHT was detected by HPLC.

[0049] 3. Evaluation index: 5α-reductase inhibition rate (%) = (DHT peak area of ​​blank group - DHT peak area of ​​sample group) / DHT peak area of ​​blank group × 100%.

[0050] 4. Test Results Table 3: Results of 5α-reductase inhibition rate test

[0051] (iv) DKK1 inhibition rate test 1. Experimental Materials Cell model: Human dermal papilla cells (DPCs, purchased from ATCC Biostandard Resource Center, USA), passaged 3-5 times; Induction and Samples: Dihydrotestosterone (DHT, final concentration 10 nM, used to induce DKK1 secretion); compositions of each example and comparative example (diluted to 0.01 mg / mL with DMEM). Control settings: blank control group (no DHT, no sample), model control group (containing DHT ± no sample), positive control group (containing DHT and finasteride, final concentration 1 μM). Reagents and instruments: DPCs culture medium, fetal bovine serum, DKK1 ELISA kit (Abcam), CO2 incubator, ELISA reader.

[0052] 2. Experimental Procedure Cell seeding: After digestion of DPCs, adjust the concentration to 5 × 10⁻⁶. 4 The cells / mL were inoculated into 24-well plates (1 mL per well), and incubated at 37°C with 5% CO2 for 24 hours, resulting in a fusion rate of 80%. Induction and intervention: Discard the original culture medium, add culture medium containing 10 nMDHT to the model group, positive group and sample group, and add ordinary culture medium to the blank group; then add corresponding diluted sample to the sample group and finasteride to the positive group, with 6 replicates per group; Culture and collection: Continue culturing for 48 hours, collect the supernatant, centrifuge at 1000 rpm for 5 minutes to remove debris, and set aside for later use; DKK1 detection: Follow the ELISA kit procedure, add standard and sample supernatant to the microplate, incubate at 37°C for 60 minutes; after washing, add enzyme-labeled secondary antibody and incubate for 30 minutes; incubate the chromogenic substrate in the dark for 15 minutes, add stop solution, and measure the OD value at 450 nm. 3. Data calculation: The concentration of DKK1 was calculated using the standard curve method, and the average value of the replicates was taken; DKK1 inhibition rate (%) = (Model group concentration - Sample group concentration) / (Model group concentration - Blank group concentration) × 100%.

[0053] 4. Test Results Table 4: Results of DKK1 Inhibition Rate Test

[0054] (v) B16 melanocyte melanin production test 1. Experimental materials: B16 mouse melanoma cells; compositions of each example and comparative example (diluted to a final concentration of 0.02 mg / mL with DMEM containing 10% fetal bovine serum); blank control group (culture medium only), positive control (arbutin 50 μM); trypsin, microplate reader, cuvette.

[0055] 2. Experimental steps: B16 cells were used at a rate of 1×10 4 Inoculate one sample per well into a 96-well plate. After 24 hours of culture, allow the sample to adhere to the plate. Then, replace the medium with the medium containing the corresponding sample and continue culturing for another 72 hours.

[0056] Collect cells, wash twice with PBS, add 1 mol / L NaOH solution (containing 10% dimethyl sulfoxide), and incubate at 80°C for 30 minutes to dissolve melanin.

[0057] The microplate reader measures absorbance (OD value) at a wavelength of 475 nm.

[0058] 3. Evaluation index: Melanin production promotion rate (%) = (OD value of sample group - OD value of blank group) / OD value of blank group × 100%.

[0059] 4. Test Results Table 5: Results of melanin production test in B16 melanocytes

[0060] (vi) HaCaT cell filament polymerase generation test 1. Experimental materials: HaCaT human keratinocytes; compositions of each example and comparative example (diluted to a final concentration of 0.01 mg / mL with DMEM containing 10% fetal bovine serum); blank control group (culture medium only); filaggrin ELISA kit and microplate reader.

[0061] 2. Experimental steps: HaCaT cells at 2×10 4 Inoculate one sample per well into a 24-well plate. After 24 hours of culture and adhesion, replace the medium with the medium containing the corresponding sample and continue culturing for another 48 hours.

[0062] Collect the cell culture supernatant and perform the ELISA kit procedure according to the instructions to detect the filaggrin content.

[0063] 3. Evaluation indicators: filamentous polymer content (ng / mL) and relative increase rate (%, with the blank control group as 100%).

[0064] 4. Test Results Table 6: Results of HaCaT Cell Filament Polymerization Test

[0065] II. Results Analysis: Tables 1 and 2 show that the relative growth rate of hair follicles and the relative increase rate of RSPO1 secretion in Examples 1-3 were significantly higher than those in the comparative examples, with Example 3 showing the best effect and being superior to the positive control. Comparative examples 1-2 lacked hair growth peptide complexes or multi-amino acid polysaccharide condensates and ATP; comparative examples 3-4 lacked some innovative herbal components; and comparative example 5 used a single extraction process, all resulting in decreased hair follicle activation and growth effects. This confirms the crucial role of synergistic effects of various components and optimized processes in hair growth.

[0066] Tables 3 and 4 show that Examples 1-3 exhibited excellent 5α-reductase inhibition and DKK1 inhibition rates, with Example 3 showing a better DKK1 inhibition rate than the positive control. Comparative Examples 1-2, lacking the energy-supplying factor ATP, and Comparative Examples 3-4, lacking anti-inflammatory and blood-activating herbal components, showed weakened anti-hair loss target inhibition effects, indicating that the composition can block hair loss pathways at multiple targets.

[0067] Table 5 shows that the melanin production promotion rate of Examples 1-3 was higher than that of the comparative example, and Example 3 was superior to the positive control. Comparative Example 3, lacking synergistic hair-darkening components such as Ligustrum lucidum and Ligusticum chuanxiong, had a promotion rate of only 22.1%, highlighting the enhancing effect of the herbal combination on melanin synthesis.

[0068] Table 6 shows that the relative increase rate of filaggrin in Examples 1-3 was significantly higher than that in the comparative examples, indicating that the compositions can effectively repair the scalp barrier and provide a good environment for healthy hair growth. Meanwhile, Comparative Example 4 shows that the extracts of sumac galls, zanthoxylum bungeanum fruit, and saffron effectively and synergistically promote the synthesis of filaggrin.

[0069] III. Typical Cases 1. Patient Information Mr. Zhang, male, 35 years old, date of consultation: January 5, 2025, type of hair loss: seborrheic alopecia 2. Chief complaint: Progressive thinning of hair on the crown for 2 years, accompanied by oily scalp and itching, with approximately 50-60 hairs falling out daily. In the past six months, the patient has noticed a receding hairline and increasingly visible scalp on the crown, affecting appearance, hence the consultation. The patient has previously used several commercially available anti-hair loss shampoos with limited effectiveness, and the parting has gradually widened.

[0070] 3. Past medical history: Good health, no history of any special diseases, and no family history of hair loss.

[0071] 4. Usage Plan After a definitive diagnosis, the patient decided to undergo a 6-month comprehensive hair regrowth treatment. The treatment plan was as follows: twice daily, morning and evening, apply approximately 1 mL of the herbal hair loss prevention, hair growth, and darkening composition (Formula 3 of Example 3) evenly to the sparse areas of the scalp, gently massaging until absorbed. No rinsing is required. During the treatment, maintain a normal diet and lifestyle, use a mild, neutral shampoo, and have regular monthly follow-up appointments to record the efficacy and any adverse reactions.

[0072] 5. Treatment outcomes and follow-up Based on a comparison of baseline and post-treatment clinical photographs and professional hair analysis, the efficacy was assessed as follows: After 3 months of treatment: The patient reported a significant reduction in hair loss during shampooing. Clinical observation revealed a decrease in the exposed scalp area on the top of the head, an increase in new, fine, soft hair growth, and a slight increase in hair density. Results showed that hair density in the target area increased by approximately 15% compared to before treatment. The patient also reported an improvement in scalp oiliness.

[0073] After 6 months of treatment: hair loss has essentially stopped. New hair growth on the top of the head has increased, become thicker and darker, and some has already transformed into terminal hair. Scalp coverage has significantly improved, resulting in a noticeable improvement in appearance. Results show that hair density in the target area has increased significantly by approximately 29% compared to before treatment. Fine new hair growth has also been observed at the hairline.

[0074] Safety assessment: Throughout the entire 6-month treatment period, the patient did not experience any adverse reactions such as scalp itching, redness, stinging, or contact dermatitis, and the condition was well tolerated. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A herbal composition for preventing hair loss, promoting hair growth, and darkening hair, characterized in that, The product comprises the following raw materials in parts by weight: 5-15 parts of Platycladus orientalis leaf extract, 3-10 parts of Polygonum multiflorum extract, 2-8 parts of Phellodendron chinense bark extract, 3-9 parts of Angelica sinensis extract, 2-7 parts of Rehmannia glutinosa root extract, 3-8 parts of Sophora flavescens extract, 1-5 parts of Panax ginseng root extract, 2-6 parts of Rosmarinus officinalis leaf extract, 1-4 parts of Watercress extract, 2-5 parts of Saffron crocus extract, 0.5-3 parts of Rhus chinensis gall extract, 1-4 parts of Zanthoxylum bungeanum fruit extract, 0.8-3 parts of Cnidium monnieri fruit extract, 1-4 parts of Ligustrum lucidum fruit extract, 0.8-3 parts of Ligusticum chuanxiong extract, 2-5 parts of Cornus officinalis extract, 0.3-2 parts of Arctium lappa root extract, and a carrier.

2. The herbal composition for preventing hair loss, promoting hair growth, and darkening hair as described in claim 1, characterized in that, It also contains 0.5-3 parts by weight of a hair growth peptide complex and 1-5 parts by weight of a multi-amino acid polysaccharide condensate, wherein the hair growth peptide complex is composed of palmitoyl tripeptide-1, acetyl tetrapeptide-3, myristoyl pentapeptide-4 and hexapeptide-3 in a mass ratio of (1-3):(2-4):(1-3):

1.

3. The herbal composition for preventing hair loss, promoting hair growth, and darkening hair as described in claim 2, characterized in that, The polyamino acid polysaccharide condensate is prepared by condensation reaction of complex amino acids and complex polysaccharides at a mass ratio of 1-3:

1. The condensation reaction conditions are: pH 6.0-7.0, temperature 45-55℃, and reaction time 3-4 hours. The molecular weight of the polyamino acid polysaccharide condensate is 5000-50000 Da.

4. The herbal composition for preventing hair loss, promoting hair growth, and darkening hair as described in claim 1, characterized in that, The complex amino acid is composed of cysteine, serine, glycine, and lysine in a mass ratio of (0.5-1.2):(0.8-2.0):(1.5-1.8):(0.3-0.9). The complex polysaccharide is composed of Tremella fuciformis polysaccharide, hyaluronic acid, and oat β-glucan in a mass ratio of (1-2):(0.8-1.5):(2.3-2.5).

5. The herbal composition for preventing hair loss, promoting hair growth, and darkening hair as described in claim 1, characterized in that, It also contains 0.1-2 parts by weight of adenosine triphosphate, 0.3-1.5 parts by weight of erythritol, and 1.8-2.5 parts by weight of betaine.

6. The herbal composition for preventing hair loss, promoting hair growth, and darkening hair as described in claim 1, characterized in that, The carrier comprises 1-5 parts panthenol, 0.01-0.1 parts vitamin H, and 5-15 parts 1,3-propanediol.

7. The method for preparing the herbal composition for preventing hair loss, promoting hair growth, and darkening hair as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Raw material pretreatment: Platycladus orientalis leaves, Polygonum multiflorum, Phellodendron bark, Angelica sinensis, Rehmannia glutinosa root, Sophora flavescens, Rhus chinensis gall, Zanthoxylum bungeanum fruit, Cnidium monnieri fruit, Ligustrum lucidum, Ligusticum chuanxiong, Cornus officinalis, and Arctium lappa root are washed and dried at a low temperature of 40-50℃ until the moisture content is ≤8%. After pulverizing, they are passed through a 50-60 mesh sieve to obtain mixed herbal powder. Ginseng root extract, rosemary leaf extract, watercress leaf / stem extract, and saffron extract are filtered through a 100-200 mesh sieve to remove impurities and are ready for use. S2. Compound extraction: Add 8-12 times the volume of 55-65% ethanol aqueous solution to the mixed herbal powder for compound extraction to obtain crude extract. Concentrate and purify the crude extract to obtain herbal concentrate. S3, Compound Mixture: S3-1: Add panthenol, vitamin H, 1,3-propanediol, erythritol and betaine to the herbal concentrate in sequence, and stir at 35-40℃ and 250-300 rpm for 20-30 minutes to form the base solution; S3-2: Add ginseng root extract, rosemary leaf extract, watercress extract, and saffron extract to the base liquid, keep the temperature and stirring speed constant, and continue stirring for 25-35 minutes until completely dispersed; S3-3: Finally, cool to 30-35℃, add adenosine triphosphate, hair growth peptide complex, and polyamino acid polysaccharide condensate, and stir for 30-40 minutes until the system is homogeneous; S4. Homogenization and sterilization: Homogenize the above mixture at 12000-15000 rpm for 12-15 minutes, rapidly cool to 25-30°C, and filter to obtain the composition.

8. The preparation method according to claim 7, characterized in that, The compound extraction described in step S2 is an alternating ultrasonic-reflux extraction. The ultrasonic power is 200-250W, and each ultrasonic extraction lasts for 10-15 minutes. The reflux temperature is 65-75℃, and each reflux lasts for 20-30 minutes. The extraction is repeated 2-3 times. The extracts are combined and filtered through a 300-400 mesh filter cloth to obtain a crude extract.

9. The preparation method according to claim 7, characterized in that, The concentration in step S2 involves concentrating the crude extract under reduced pressure at 50-60°C and a vacuum of -0.08 to -0.09 MPa to a relative density of 1.15-1.

20. The purification includes membrane filtration purification, wherein the pore size of the membrane filtration is in the range of 10-200 nm.

10. The herbal hair loss prevention, hair growth and hair darkening composition as described in claims 1-6 is used to prepare hair loss prevention and hair growth liquid, shampoo or conditioner.

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