A composition with hair growth and hair blackening effects and application thereof

Abstract

The present application belongs to the technical field of natural product daily chemical and scalp care, and discloses a composition with hair growth and black hair effects and application thereof. In view of the defects that existing chemical anti-hair loss ingredients have strong irritation, poor use experience and insufficient storage stability, and ordinary plant extracts have low active dissolution rate and weak efficacy, the present application selects six kinds of medicinal and edible plants to match specific lactic acid bacteria for directional fermentation to prepare core fermented plant extracts, and then matches eight kinds of auxiliary nourishing plant extracts to construct a multi-target synergistic anti-hair loss system in combination with daily chemical acceptable adjuvants. The composition has excellent anti-hair loss and hair growth effect, uses a whole aqueous phase matrix, is mild and non-irritating, and has a refreshing and non-sticky feeling. Fermentation modification significantly improves the stability of active ingredients, which can remain uniform and stable under normal temperature storage, light and cold and hot cycle conditions. The raw materials of the composition are safe and have no dependence, and the composition can be widely applied to various anti-hair loss and scalp care products.

Description

Technical Field

[0001] This invention belongs to the field of natural product daily chemical and scalp care technology, specifically relating to a plant fermentation composition with anti-hair loss effect and its application. Background Technology

[0002] With the accelerated pace of modern life, increased mental stress, irregular work and rest schedules, and intensified environmental pollution, the incidence of hair loss is rising year by year and showing a trend towards affecting younger people. Common types of hair loss in clinical practice include androgenetic alopecia (AGA), telogen effluvium, and seborrheic alopecia, whose pathogenesis involves multiple targets such as hair follicle cycle disorders, abnormal 5α-reductase activity, scalp microcirculation disorders, scalp inflammatory responses, and inhibition of dermal papilla cell proliferation. Currently, minoxidil is the only chemical ingredient approved by the FDA globally for topical use in hair loss prevention. It works by opening potassium ion channels, dilating scalp blood vessels, and prolonging the hair follicle growth phase, making it the core active ingredient in most hair loss prevention products on the market.

[0003] However, existing minoxidil formulations have significant drawbacks in practical applications: First, poor user experience. Commercially available minoxidil solutions often use high-concentration ethanol and propylene glycol as solvents and penetration enhancers, leaving a noticeable sticky and oily feeling on the scalp after use. This makes the scalp prone to dust accumulation and causes hair to become flat, resulting in low long-term user compliance. Second, strong scalp irritation. Propylene glycol and high-concentration ethanol can damage the scalp barrier, causing approximately 20%-30% of users to experience scalp itching, erythema, flaking, and even contact dermatitis. Sensitive skin individuals cannot tolerate these symptoms. Third, poor storage stability. Minoxidil's chemical structure is sensitive to light, easily undergoing oxidative degradation and crystallization under normal temperature and light conditions. Long-term storage significantly reduces the content of the active ingredient and diminishes its efficacy. Therefore, the product must be strictly protected from light, limiting its portability and usability.

[0004] Addressing the aforementioned pain points of minoxidil formulations, natural plant-derived anti-hair loss ingredients have become a research hotspot due to their high safety, mild effects, and minimal side effects. Traditional Chinese medicinal herbs such as Platycladus orientalis leaves, processed Polygonum multiflorum, and Eclipta prostrata have a long history of use in my country for hair growth and darkening. Modern research has also confirmed that active ingredients in various plants, such as flavonoids, saponins, alkaloids, and polysaccharides, can exert anti-hair loss effects by inhibiting 5α-reductase, improving scalp microcirculation, anti-inflammatory and antioxidant effects, and nourishing hair papilla cells. However, existing plant-based anti-hair loss compositions generally have the following shortcomings: First, they mostly use traditional water or alcohol extraction processes, and the barrier effect of plant cell walls leads to low dissolution rates and poor bioavailability of active ingredients, resulting in a weaker overall anti-hair loss effect that is difficult to match minoxidil; second, the formulation lacks multi-target synergistic design, often simply combining similar active ingredients with insignificant synergistic effects; third, plant extracts themselves have complex compositions, and polyphenols and flavonoids are prone to oxidation and discoloration, leading to stability issues such as turbidity, precipitation, and discoloration during long-term storage, with even faster quality degradation under light conditions.

[0005] Microbial fermentation technology is an effective means to enhance the activity and utilization rate of plant extracts. Through the enzyme system produced by microbial metabolism, plant cell wall cellulose, hemicellulose, and pectin can be decomposed, promoting the release of intracellular active ingredients. Simultaneously, it can transform large active molecules into smaller molecules that are more easily absorbed through the skin, and even generate new active metabolites, thus enhancing efficacy. Currently, there are related studies applying fermentation technology to hair loss prevention products, but these generally suffer from problems such as limited strain selection, poor matching between raw materials and strains, and lack of targeted fermentation design for hair loss prevention. Furthermore, they fail to simultaneously address the three core pain points of efficacy intensity, user experience, and storage stability, making it difficult to replace traditional minoxidil formulations.

[0006] Based on this, this invention uses 14 traditional medicinal and edible plants as raw materials, selects 6 core active ingredients, and matches them with exclusive lactic acid bacteria strains for targeted fermentation. Through precise matching of strains and raw materials, the active ingredients are targetedly enriched and enhanced. Then, it is scientifically compounded with 8 auxiliary nourishing ingredients to construct a multi-target synergistic hair loss prevention system. The resulting composition completely avoids irritating alcohol solvents, possesses excellent hair loss prevention and hair growth effects, a gentle and non-irritating user experience, and outstanding storage and light stability, effectively solving many shortcomings of existing technologies. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing anti-hair loss products and provide a plant-based fermented composition with anti-hair loss efficacy and its application. This composition uses all-plant-derived fermented ingredients as its core, and through a technical solution of targeted matching fermentation of microorganisms and raw materials and synergistic compounding of multiple components and multiple targets, it achieves a significant improvement in anti-hair loss efficacy. It also features a clean, non-greasy scalp, no irritation or itching, and excellent storage and light stability, and can be widely used in various scalp care and anti-hair loss shampoos and conditioners.

[0008] The objective of this invention can be achieved through the following technical solutions: A composition with hair growth and hair darkening effects, comprising, by weight, the following core fermented plant extracts: 5-15 parts fermented rose extract, 8-20 parts fermented Clematis chinensis extract, 3-12 parts fermented Panax notoginseng extract, 4-10 parts fermented Vitex trifolia extract, 6-18 parts fermented Acanthopanax senticosus extract, and 7-16 parts fermented mulberry twig extract. The fermented rose extract was prepared from rose medicinal materials through directed fermentation with Lactobacillus plantarum. The fermented *Clematis chinensis* extract was prepared by directed fermentation of *Clematis chinensis* herb by *Lactobacillus rhamnosus*. The fermented Panax notoginseng extract was prepared by directional fermentation of Panax notoginseng raw materials using Lactobacillus acidophilus. The fermented Vitex trifolia extract was prepared by directed fermentation of Vitex trifolia medicinal material using Lactobacillus rhamnosus. The fermented Acanthopanax senticosus extract was prepared by targeted fermentation of Acanthopanax senticosus medicinal material using Lactobacillus plantarum. The fermented mulberry twig extract is prepared by directional fermentation of mulberry twig medicinal material with Lactobacillus casei.

[0009] As a preferred embodiment of the present invention, the present invention further includes the following auxiliary plant extracts in parts by weight: 2-8 parts of Platycladus orientalis leaf extract, 1-6 parts of mulberry leaf extract, 1-5 parts of safflower extract, 2-7 parts of processed Polygonum multiflorum extract, 1-5 parts of walnut kernel extract, 3-9 parts of Eclipta prostrata extract, 2-6 parts of mulberry extract, and 1-5 parts of sesame extract.

[0010] As a preferred embodiment of the present invention, the preparation method of each core fermented plant extract includes the following steps: S1 Raw material pretreatment: Remove impurities from the corresponding dried medicinal materials, wash them, dry them at low temperature, pulverize them and pass them through a 40-60 mesh sieve to obtain medicinal material powder; S2 culture medium preparation: Add MRS liquid culture medium to the medicinal powder at a material-to-liquid ratio of 1g:10-20mL, stir thoroughly, autoclave at 121℃ and 0.1MPa for 20-30min, and cool naturally to below 35℃ to obtain the fermentation substrate; S3 Inoculation Fermentation: Inoculate the fermentation substrate with the activated seed liquid of the corresponding strain. The inoculation volume is 2%-5% of the total volume of the fermentation substrate. Ferment at 30-37℃ and 120-180r / min constant temperature shaking conditions for 48-72h. After fermentation, the fermentation stock solution is obtained. S4 inactivation post-processing: The fermentation broth was heated in a boiling water bath for 15-20 minutes to inactivate the bacteria. After cooling to room temperature, it was centrifuged at 8000 r / min for 15 minutes. The supernatant was filtered through a 0.45 μm microporous membrane. The filtrate was concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.20 at 60℃. After vacuum freeze-drying, it was pulverized to obtain the corresponding fermented plant extract powder.

[0011] 4. The composition with hair growth and hair darkening effects according to claim 3, characterized in that, in step S3, the concentration of the activated seed liquid of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus acidophilus*, and *Lactobacillus casei* is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

[0012] As a preferred technical solution of the present invention, the auxiliary plant extracts are all prepared by ethanol reflux extraction. The specific steps are as follows: the corresponding dried medicinal materials are pulverized and passed through a 40-mesh sieve, 10-15 times the volume of ethanol solution with a volume fraction of 65%-75% is added, soaked for 30 minutes, and then heated and refluxed for extraction 2-3 times, each extraction for 1-2 hours. The extracts are combined, filtered, and the ethanol is recovered under reduced pressure until there is no alcohol odor. The extract is then concentrated to a relative density of 1.15-1.20 and freeze-dried to obtain the corresponding auxiliary plant extract powder.

[0013] As a preferred embodiment of the present invention, the composition further includes, by weight, daily chemical acceptable excipients: 10-30 parts of moisturizer, 0.5-2 parts of solubilizer, and the balance being deionized water; the total weight of the composition is 100 parts.

[0014] As a preferred embodiment of the present invention, the humectant is any one or more combinations of butylene glycol, glycerin, and panthenol; the solubilizer is PEG-40 hydrogenated castor oil.

[0015] A method for preparing a composition having the above-mentioned hair growth and hair darkening effects includes the following steps: Y1 aqueous phase preparation: Weigh each core fermented plant extract and auxiliary plant extract according to the ratio, add 30%-40% of the total mass of deionized water, stir at low speed at 40-45℃ until the powder is completely dissolved, filter through a 0.45μm filter membrane to obtain the aqueous phase of the active ingredients; Y2 matrix phase preparation: Weigh out the humectant and solubilizer according to the ratio, add them to the remaining deionized water, heat to 45-50℃, and stir until completely dissolved and homogeneous to obtain the matrix phase; Y3 Mixing and Homogenization: Slowly add the aqueous phase of the active ingredient to the matrix phase, maintain a constant temperature of 40℃ and stir for 20-30 minutes until the system is completely homogeneous. Allow it to cool naturally to room temperature, and discharge the material after passing the inspection to obtain a composition with hair growth and hair darkening effects.

[0016] The application of a composition having hair growth and hair darkening effects as described above in the preparation of anti-hair loss and hair growth scalp care products, wherein the scalp care products are any one of anti-hair loss essence, anti-hair loss shampoo, scalp repair liquid, and leave-in scalp spray.

[0017] As a preferred embodiment of the present invention, the amount of the composition added to the scalp care product is 2-15 parts per 100 parts of the total product weight.

[0018] The beneficial effects of this invention are: (1) This invention uses 6 core fermented ingredients to act on different hair loss targets, and combines them with 8 auxiliary ingredients to form a three-dimensional anti-hair loss network. The targeted fermentation process effectively increases the content of characteristic active ingredients in each raw material and effectively improves the transdermal absorption rate. The overall anti-hair loss and hair growth effect is significantly better than that of unfermented formulas and single fermented ingredients. Animal experiments have confirmed that its hair growth promotion effect is comparable to that of commercially available 2% minoxidil, and there is no risk of rebound after discontinuation of the drug.

[0019] (2) This composition uses an all-aqueous matrix system, without the need to add irritating solvents such as ethanol and propylene glycol. The pH value is maintained at 5.5-6.0, which is highly compatible with the weakly acidic environment of the scalp. Human patch tests have confirmed that it is non-irritating and will not cause scalp itching, erythema or dermatitis. After use, it is refreshing and easily absorbed, without any sticky residue, and the hair does not become flat. Long-term user compliance is greatly improved.

[0020] (3) During the fermentation process, the stability of plant polyphenols and flavonoids that are easily oxidized is significantly enhanced after microbial modification. The composition remains clear and uniform after 6 months of storage at room temperature, 10 days of strong light irradiation, and 10 cycles of hot and cold. There is no layering, no precipitation, and no obvious discoloration. The retention rate of effective ingredients is over 95%. There is no need for strict light protection storage, which solves the industry pain point of light precipitation and poor stability of minoxidil preparations.

[0021] (4) All raw materials are medicinal and edible plants, and the fermentation strains are all food-grade lactic acid bacteria. There are no chemical drugs added, no hormones, no dependence. The production process is green and environmentally friendly, meets the safety requirements of daily chemical products, and is suitable for long-term use by all kinds of people. Detailed Implementation

[0022] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0023] I. Raw Material Preparation 1. Preparation of core fermented plant extracts In this embodiment, all strains were purchased from the China General Microbiological Culture Collection Center (CGMCC), and the concentration of the activation seed solution was adjusted to 5 × 10⁻⁶. 8 CFU / mL. All fermentation extracts were prepared according to the following process, with adjustments only made to the strains and raw materials: S1 Raw material pretreatment: Remove impurities from the corresponding dried medicinal materials, quickly wash them with clean water, dry them at a low temperature of 40℃, pulverize them with a high-speed pulverizer, and pass them through a 50-mesh sieve to obtain medicinal material powder; S2 culture medium preparation: Add MRS liquid culture medium to the medicinal powder at a material-to-liquid ratio of 1g:15mL, stir thoroughly, autoclave at 121℃ and 0.1MPa for 25min, and then cool naturally to 35℃ to obtain the fermentation substrate; S3 Inoculation Fermentation: Inoculate the fermentation substrate with the activated seed liquid of the corresponding strain. The inoculation amount is 3% of the fermentation substrate volume. Ferment at 35℃ and 150r / min for 60h with constant temperature shaking to obtain the fermentation stock solution. S4 inactivation post-processing: The fermentation broth was heated in a boiling water bath for 18 min to inactivate it. After cooling to room temperature, it was centrifuged at 8000 r / min for 15 min. The supernatant was filtered through a 0.45 μm microporous membrane. The filtrate was concentrated under reduced pressure at 60℃ to a clear extract with a relative density of 1.15 (measured at 60℃). The extract was then transferred to a vacuum freeze dryer and dried for 48 h. After drying, the extract was pulverized to obtain the corresponding fermented plant extract powder.

[0024] The following extracts were prepared using the above process: fermented rose extract, fermented *Clematis chinensis* extract, fermented *Panax notoginseng* extract, fermented *Vitex trifolia* extract, fermented *Acanthopanax senticosus* extract, and fermented mulberry twig extract.

[0025] 2. Assisting in the preparation of plant extracts The leaves of Platycladus orientalis, leaves of mulberry, safflower, processed Polygonum multiflorum, walnut kernels, Eclipta prostrata, mulberry, and sesame were pulverized and passed through a 40-mesh sieve. Each herb powder was added to 12 times its volume of 70% ethanol solution, soaked for 30 minutes, and then heated under reflux for extraction twice, 1.5 hours each time. The two extracts were combined and filtered through a 200-mesh filter cloth. The ethanol in the filtrate was recovered under reduced pressure until no alcohol odor was found. The extract was then concentrated to a relative density of 1.18, freeze-dried under vacuum, and pulverized to obtain powders of each auxiliary plant extract.

[0026] II. Preparation of Compositions in Examples and Comparative Examples The following examples and comparative examples were all prepared as scalp serum formulations, with a total mass of 100 parts. All components were measured in parts by mass, with the remainder being deionized water. The preparation method was uniformly as follows: (1) Weigh each plant extract powder according to the ratio, add 35 parts of deionized water, stir at 42°C until completely dissolved, filter through a 0.45μm filter membrane to obtain the effective aqueous phase; (2) Weigh out butanediol and PEG-40 hydrogenated castor oil according to the ratio, add them to the remaining deionized water, heat to 48°C and stir until completely dissolved to obtain the matrix phase; (3) Slowly add the effective aqueous phase to the matrix phase, keep warm at 40°C and stir for 25 minutes until the system is homogeneous, and cool naturally to room temperature to obtain the final product.

[0027] Example 1 5 parts fermented rose extract, 8 parts fermented Clematis chinensis extract, 3 parts fermented Panax notoginseng extract, 4 parts fermented Vitex trifolia extract, 6 parts fermented Acanthopanax senticosus extract, 7 parts fermented mulberry twig extract, 2 parts Platycladus orientalis leaf extract, 1 part mulberry leaf extract, 1 part safflower extract, 2 parts processed Polygonum multiflorum extract, 1 part walnut kernel extract, 3 parts Eclipta prostrata extract, 2 parts mulberry extract, 1 part sesame extract, 10 parts butylene glycol, 0.5 parts PEG-40 hydrogenated castor oil, and deionized water to make up to 100 parts.

[0028] Example 2 10 parts fermented rose extract, 14 parts fermented Clematis chinensis extract, 7 parts fermented Panax notoginseng extract, 7 parts fermented Vitex trifolia extract, 12 parts fermented Acanthopanax senticosus extract, 11 parts fermented mulberry twig extract, 5 parts Platycladus orientalis leaf extract, 3 parts mulberry leaf extract, 3 parts safflower extract, 4 parts processed Polygonum multiflorum extract, 3 parts walnut kernel extract, 6 parts Eclipta prostrata extract, 4 parts mulberry extract, 3 parts sesame extract, 15 parts butylene glycol, 1 part PEG-40 hydrogenated castor oil, and deionized water to make up to 100 parts.

[0029] Example 3 15 parts fermented rose extract, 20 parts fermented Clematis chinensis extract, 12 parts fermented Panax notoginseng extract, 10 parts fermented Vitex trifolia extract, 18 parts fermented Acanthopanax senticosus extract, 16 parts fermented mulberry twig extract, 8 parts Platycladus orientalis leaf extract, 6 parts mulberry leaf extract, 5 parts safflower extract, 7 parts processed Polygonum multiflorum extract, 5 parts walnut kernel extract, 9 parts Eclipta prostrata extract, 6 parts mulberry extract, 5 parts sesame extract, 30 parts butylene glycol, 2 parts PEG-40 hydrogenated castor oil, and deionized water to make up to 100 parts.

[0030] Comparative Example 1 In Example 2, all fermented plant extracts were replaced by conventional 70% ethanol reflux extracts of the corresponding raw materials, while the remaining components, dosages, and preparation methods were exactly the same as in Example 2.

[0031] Comparative Example 2 The fermented rose extract in Example 2 was removed, and the missing portion was made up with deionized water. The remaining components, dosages, and preparation methods were exactly the same as in Example 2.

[0032] Comparative Example 3 The fermented *Clematis chinensis* extract in Example 2 was removed, and the missing portion was made up with deionized water. The remaining components, dosages, and preparation methods were exactly the same as in Example 2.

[0033] Comparative Example 4 The fermented Panax notoginseng extract in Example 2 was removed, and the missing part was made up with deionized water. The remaining components, dosages and preparation methods were exactly the same as in Example 2.

[0034] Comparative Example 5 The fermented Vitex trifolia extract in Example 2 was removed, and the missing portion was made up with deionized water. The remaining components, dosages, and preparation methods were exactly the same as in Example 2.

[0035] Comparative Example 6 The fermented Acanthopanax senticosus extract in Example 2 was removed, and the missing part was made up with deionized water. The remaining components, dosages and preparation methods were exactly the same as in Example 2.

[0036] Comparative Example 7 The fermented mulberry twig extract in Example 2 was removed, and the missing portion was made up with deionized water. The remaining components, dosages, and preparation methods were exactly the same as in Example 2.

[0037] Comparative Example 8 In Example 2, all fermentation strains of fermented extracts were replaced with Saccharomyces cerevisiae, fermentation process parameters remained unchanged, and the remaining components, dosages and preparation methods were exactly the same as in Example 2.

[0038] Comparative Example 9 The fermented rose extract in Example 2 was replaced with fermented rose extract (prepared using the same fermentation process as Lactobacillus plantarum), and the remaining components, dosages, and preparation methods were exactly the same as in Example 2.

[0039] Comparative Example 10 The fermented *Clematis chinensis* extract in Example 2 was replaced with fermented *Leonurus japonicus* extract (prepared using the same fermentation process as *Lactobacillus rhamnosus*), and the remaining components, dosages, and preparation methods were exactly the same as in Example 2.

[0040] Comparative Example 11 Commercially available 2% minoxidil tincture (containing 50% ethanol and 10% propylene glycol) was used as a positive control.

[0041] III. Performance Testing 1. Test of efficacy in preventing hair loss and promoting hair growth (mouse hair removal model) Sixty SPF-grade male C57BL / 6 mice, 6-8 weeks old and weighing 20-22g, were selected. After acclimatization for 3 days, the backs of the mice were treated with 8% sodium sulfide solution to remove hair, covering an area of ​​approximately 3cm × 3cm. The mice were randomly divided into 15 groups of 4 mice each, corresponding to Examples 1-3, Comparative Examples 1-11, and the blank control group, respectively. The blank control group was treated with an equal volume of physiological saline, while the other groups were treated with the corresponding sample. 50μL of the sample was applied once daily for 28 consecutive days.

[0042] After the experiment, mice were euthanized by cervical dislocation. Skin tissue from the central area of ​​the shaved back was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (HE). The morphology of hair follicles was observed under an optical microscope, the number of hair follicles per unit field of view was counted, and the hair shaft length was measured. At the same time, the weight of hair from the same area of ​​skin was weighed using an electronic analytical balance, and the hair density was calculated. The results were taken as averages for each group and recorded in Table 1.

[0043] 2. Skin irritation test An acute skin irritation test was conducted on rabbits. Fifteen healthy adult rabbits were selected. Twenty-four hours prior to the test, hair was removed from both sides of the spine on the back of the rabbits, approximately 2.5cm × 2.5cm on each side, dividing them into an intact skin group and a damaged skin group. 0.5mL of the test sample was applied to the left side of the hairless area, while physiological saline was applied to the right side as a control. After application, the area was covered and fixed with sterile gauze. Application was repeated once daily for seven consecutive days. Skin erythema and edema were observed at 24h, 48h, and 72h after the last application. Scoring was performed according to the "Cosmetic Safety Technical Specifications," and the average score for each group was calculated to evaluate the intensity of irritation. The results are recorded in Table 2.

[0044] 3. Sensory evaluation of greasiness Thirty healthy participants, half male and half female, aged 18-45 years, with no history of scalp diseases, were recruited. A double-blind, controlled trial was conducted. Each test sample was randomly numbered, and participants applied the sample to their scalp in sequence. After leaving the sample on for 30 minutes, participants rated the oiliness of their scalp using the following criteria: 1 point = very refreshing, completely non-greasy; 3 points = slightly oily, acceptable; 5 points = moderately oily, noticeable discomfort; 7 points = very oily, hair sticky and flat; 10 points = extremely oily, unacceptable. The average score of the 30 participants was recorded in Table 2.

[0045] 4. Stability testing (1) Stability during room temperature storage Each test sample was sealed and stored in a constant temperature and humidity chamber at 25℃ and 60% relative humidity for 6 months. Samples were taken at 0 months and 6 months to observe the appearance (clarity, color, presence or absence of precipitation). The total flavonoid content in the samples was determined by ultraviolet spectrophotometry, and the total flavonoid retention rate after 6 months was calculated.

[0046] (2) Light stability Each test sample was sealed and placed in a light test chamber and continuously irradiated for 10 days at an illuminance of 4500 lx ± 500 lx and a temperature of 25°C. Samples were taken on the 10th day to observe their appearance, determine the total flavonoid content, and calculate the total flavonoid retention rate after light irradiation.

[0047] (3) Stability of hot and cold cycles Each test sample was sealed and placed in a thermal cycling test chamber. The cycling conditions were set as follows: -10℃ for 24 hours → 40℃ for 24 hours as one cycle. The cycle was repeated 10 times. After the cycle was completed, the samples were returned to room temperature. The appearance was observed to see if there were any phenomena such as layering, turbidity, precipitation, or discoloration.

[0048] The stability test results are recorded in Table 3.

[0049] 5. Hair darkening efficacy test (1) Tyrosinase activation test (in vitro) The activation effect of samples on tyrosinase was determined using the L-DOPA oxidation method. Phosphate buffer (pH 6.8) was used to add the test sample solution (final concentration 2 mg / mL) and 100 U / mL tyrosinase solution to each example, respectively. After thorough mixing, the mixture was incubated at 37°C for 10 min. Then, 0.5 mmol / L L-DOPA solution was added to initiate the reaction. The absorbance change was continuously measured at 475 nm to calculate the initial rate of the enzymatic reaction. The tyrosinase activity of the blank control group was taken as 100%, and the relative tyrosinase activity of each group was calculated. Each group was measured in triplicate, and the average value was taken.

[0050] (2) Determination of melanin content in mouse hair Using mouse back hair from the above-mentioned anti-hair loss efficacy test, 10 mg of hair was taken from each group, chopped, and 1 mL of 1 mol / L NaOH solution (containing 10% DMSO) was added. The solution was heated in an 80℃ water bath for 30 min until the hair was completely dissolved. After cooling, the absorbance was measured at a wavelength of 475 nm. The melanin content was calculated according to the melanin standard curve. The results were expressed as μg / mg hair, and the average value of each group was taken.

[0051] The results of the above hair-darkening efficacy test are recorded in Table 4.

[0052] IV. Test Results Table 1 Results of hair loss prevention and hair growth efficacy tests in mice of each group Group Number of hair follicles (per field of view) Hair shaft length (μm) Hair density (mg / cm²) Blank control 18.2±2.1 2150±180 12.3±1.5 Example 1 36.5±2.8 3820±210 28.7±2.2 Example 2 42.3±3.1 4260±240 33.5±2.6 Example 3 41.8±2.9 4190±230 32.9±2.4 Comparative Example 1 27.6±2.5 3010±200 21.4±2.0 Comparative Example 2 34.2±2.6 3580±220 26.8±2.1 Comparative Example 3 31.8±2.4 3360±210 24.9±2.3 Comparative Example 4 33.7±2.7 3510±200 26.2±2.2 Comparative Example 5 34.0±2.5 3540±210 26.5±2.0 Comparative Example 6 32.5±2.3 3420±220 25.3±2.1 Comparative Example 7 33.1±2.6 3470±200 25.7±2.3 Comparative Example 8 28.9±2.4 3150±190 22.6±1.9 Comparative Example 9 34.5±2.7 3610±220 27.1±2.2 Comparative Example 10 32.2±2.5 3390±210 25.1±2.0 Comparative Example 11 39.7±3.0 4080±230 31.2±2.5 As shown in Table 1: 1. The number of hair follicles, hair shaft length and hair density of Examples 1-3 were significantly higher than those of the blank control group, confirming that the composition of the present invention has a clear effect on preventing hair loss and promoting hair growth; among them, Example 2 had the best effect, with hair density 2.7 times that of the blank group, and the overall effect was slightly better than that of commercially available 2% minoxidil tincture (Comparative Example 11).

[0053] 2. The indicators of Comparative Example 1 (unfermented) decreased by 25%-35% compared with Example 2, indicating that the directional fermentation process can significantly improve the anti-hair loss activity of plant components, which is one of the core technical contributions of this invention.

[0054] 3. When one core fermentation component was missing from each of the Comparative Examples 2-7, all efficacy indicators decreased to varying degrees. The decrease was more pronounced when Clematis armandii, Acanthopanax senticosus, and mulberry twig were missing, confirming that the six core components are indispensable and together constitute a synergistic effect system, thus verifying the scientific nature and creativity of the formulation.

[0055] 4. The efficacy of Comparative Example 8 (yeast fermentation) was close to that of the unfermented group but significantly lower than that of the lactic acid bacteria fermentation group. This indicates that the lactic acid bacteria strains selected in this invention have specific compatibility with the raw materials, and not all strains can achieve synergistic effects. The selection of strains is creative.

[0056] 5. In Comparative Examples 9 and 10, the efficacy declined after the core ingredients were replaced with similar plants, which confirms that the core raw materials such as rose and Clematis chinensis selected in this invention are irreplaceable and that the formulation screening is creative.

[0057] Table 2. Evaluation results of skin irritation and oiliness in each group. Group Complete skin irritation score Broken skin irritation score Stimulating evaluation Greasiness rating Example 1 0.0 0.1 Non-irritating 2.3 Example 2 0.0 0.1 Non-irritating 2.1 Example 3 0.1 0.2 Non-irritating 2.5 Comparative Example 1 0.1 0.2 Non-irritating 3.2 Comparative Example 2 0.0 0.1 Non-irritating 2.4 Comparative Example 3 0.0 0.1 Non-irritating 2.3 Comparative Example 4 0.0 0.1 Non-irritating 2.2 Comparative Example 5 0.1 0.2 Non-irritating 2.6 Comparative Example 6 0.0 0.1 Non-irritating 2.3 Comparative Example 7 0.0 0.1 Non-irritating 2.2 Comparative Example 8 0.1 0.2 Non-irritating 3.0 Comparative Example 9 0.0 0.1 Non-irritating 2.5 Comparative Example 10 0.0 0.1 Non-irritating 2.4 Comparative Example 11 1.2 2.3 Mild stimulation 7.8 As shown in Table 2: 1. All plant-derived groups (Examples and Comparative Examples 1-10) had skin irritation scores below 0.5, which is considered non-irritating and significantly superior to commercially available minoxidil tincture (Comparative Example 11). This demonstrates that the composition of the present invention has good scalp tolerance and will not cause problems such as itching, erythema, or dermatitis.

[0058] The greasiness scores of Examples 1-3 were only 2.1-2.5 points, which is in the "very refreshing" range, while minoxidil tincture scored as high as 7.8 points, which is considered heavily greasy. This shows that the present invention has completely solved the pain point of greasiness in traditional anti-hair loss products and significantly improved the user experience.

[0059] Table 3 Stability test results for each group Group Appearance after 6 months at room temperature Total flavonoid retention rate at room temperature for 6 months Appearance after 10 days of light exposure Total flavonoid retention rate after 10 days of light exposure Appearance after 10 cycles of hot and cold water Example 1 Clear light brown color, no precipitation. 95.8% Clear light brown, slightly darker 94.2% Clarified and homogeneous, with no stratification or precipitation. Example 2 Clear brownish-yellow color, no precipitation. 97.1% Clear brownish-yellow, slightly darker 96.2% Clarified and homogeneous, with no stratification or precipitation. Example 3 Clear brownish-yellow color, no precipitation. 96.5% Clear brownish-yellow, slightly darker 95.7% Clarified and homogeneous, with no stratification or precipitation. Comparative Example 1 Slightly cloudy with a small amount of sediment 82.3% Noticeably cloudy, color deepened 71.5% Layering occurred, with sediment at the bottom. Comparative Example 2 Clarified, no precipitation 96.2% Clarify, slightly deepen 95.0% Clarification and uniformity Comparative Example 3 Clarified, no precipitation 95.7% Clarify, slightly deepen 94.6% Clarification and uniformity Comparative Example 4 Clarified, no precipitation 96.0% Clarify, slightly deepen 94.8% Clarification and uniformity Comparative Example 5 Clarified, no precipitation 95.5% Clarify, slightly deepen 94.1% Clarification and uniformity Comparative Example 6 Clarified, no precipitation 95.9% Clarify, slightly deepen 94.5% Clarification and uniformity Comparative Example 7 Clarified, no precipitation 95.6% Clarify, slightly deepen 94.3% Clarification and uniformity Comparative Example 8 Slightly cloudy 85.1% The cloudiness worsened and the color deepened. 74.3% A small amount of flocculent precipitate appeared Comparative Example 9 Clarified, no precipitation 96.3% Clarify, slightly deepen 95.1% Clarification and uniformity Comparative Example 10 Clarified, no precipitation 95.4% Clarify, slightly deepen 94.0% Clarification and uniformity Comparative Example 11 A small amount of crystals precipitated 70.2% Numerous needle-like crystals precipitated 58.6% Increased crystallization and distinct stratification As shown in Table 3: 1. Examples 1-3 remained clear and homogeneous after being stored at room temperature for 6 months, exposed to strong light for 10 days, and subjected to 10 cycles of hot and cold. There was no stratification or precipitation, and the total flavonoid retention rate was over 94%, demonstrating excellent storage stability and light stability.

[0060] 2. Comparative Example 1 (unfermented) showed a significant decrease in stability under both room temperature and light, with the appearance of turbidity and precipitation, and a low retention rate of effective ingredients. This indicates that fermentation modification can significantly improve the stability of plant active ingredients, which is the core mechanism of this invention to solve the stability problem.

[0061] 3. Comparative Example 11 (minoxidil tincture) showed crystallization after 6 months at room temperature, and a large amount of crystallization after 10 days of light exposure. The retention rate of effective ingredients was less than 60%. After cold and heat cycling, the crystallization increased further. Its stability was far worse than that of the composition of the present invention, which confirms that the present invention has achieved a breakthrough improvement in storage performance.

[0062] 4. Comparative Example 8 (yeast fermentation) showed slightly better stability than the unfermented group, but far worse stability than the lactic acid bacteria fermentation group, further verifying the scientific nature of the strain selection and the exclusive synergistic effect of the present invention.

[0063] Table 4 Results of hair-darkening efficacy tests for each group Group Relative tyrosinase activity (%) Hair melanin content (μg / mg) Blank control 100.0±2.5 3.2±0.3 Example 1 152.6±3.8 5.8±0.4 Example 2 178.3±4.2 7.1±0.5 Example 3 174.5±4.0 6.9±0.4 V. Typical Trial Cases Twenty volunteers aged 22-45 with mild to moderate androgenetic alopecia were recruited. They used the composition from Example 2 twice daily, morning and evening, for three consecutive months. After the trial, statistics showed that 17 volunteers reported a significant reduction in hair loss, with a reduction of over 50% in hair loss during combing; 15 volunteers observed new vellus hair growth at the hairline; and no adverse reactions such as scalp itching, erythema, or oiliness were reported among the volunteers, resulting in a satisfaction rate of 95%.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A composition having the effects of promoting hair growth and darkening hair, characterized in that, By weight, it includes the following core fermented plant extracts: 5-15 parts fermented rose extract, 8-20 parts fermented Clematis chinensis extract, 3-12 parts fermented Panax notoginseng extract, 4-10 parts fermented Vitex trifolia extract, 6-18 parts fermented Acanthopanax senticosus extract, and 7-16 parts fermented mulberry twig extract. The fermented rose extract was prepared from rose medicinal materials through directed fermentation with Lactobacillus plantarum. The fermented *Clematis chinensis* extract was prepared by directed fermentation of *Clematis chinensis* herb by *Lactobacillus rhamnosus*. The fermented Panax notoginseng extract was prepared by directional fermentation of Panax notoginseng raw materials using Lactobacillus acidophilus. The fermented Vitex trifolia extract was prepared by directed fermentation of Vitex trifolia medicinal material using Lactobacillus rhamnosus. The fermented Acanthopanax senticosus extract was prepared by targeted fermentation of Acanthopanax senticosus medicinal material using Lactobacillus plantarum. The fermented mulberry twig extract is prepared by directional fermentation of mulberry twig medicinal material with Lactobacillus casei.

2. The composition with hair growth and hair darkening effects according to claim 1, characterized in that, By weight, it also includes the following auxiliary plant extracts: 2-8 parts of Platycladus orientalis leaf extract, 1-6 parts of mulberry leaf extract, 1-5 parts of safflower extract, 2-7 parts of processed Polygonum multiflorum extract, 1-5 parts of walnut kernel extract, 3-9 parts of Eclipta prostrata extract, 2-6 parts of mulberry extract, and 1-5 parts of sesame extract.

3. The composition with hair growth and hair darkening effects according to claim 1, characterized in that, The preparation methods for the core fermented plant extracts include the following steps: S1 Raw material pretreatment: Remove impurities from the corresponding dried medicinal materials, wash them, dry them at low temperature, pulverize them and pass them through a 40-60 mesh sieve to obtain medicinal material powder; S2 culture medium preparation: Add MRS liquid culture medium to the medicinal powder at a material-to-liquid ratio of 1g:10-20mL, stir thoroughly, autoclave at 121℃ and 0.1MPa for 20-30min, and cool naturally to below 35℃ to obtain the fermentation substrate; S3 Inoculation Fermentation: Inoculate the fermentation substrate with the activated seed liquid of the corresponding strain. The inoculation volume is 2%-5% of the total volume of the fermentation substrate. Ferment at 30-37℃ and 120-180r / min constant temperature shaking conditions for 48-72h. After fermentation, the fermentation stock solution is obtained. S4 inactivation post-processing: The fermentation broth was heated in a boiling water bath for 15-20 minutes to inactivate the bacteria. After cooling to room temperature, it was centrifuged at 8000 r / min for 15 minutes. The supernatant was filtered through a 0.45 μm microporous membrane. The filtrate was concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.20 at 60℃. After vacuum freeze-drying, it was pulverized to obtain the corresponding fermented plant extract powder.

4. The composition with hair growth and hair darkening effects according to claim 3, characterized in that, In step S3, the concentration of the activated seed liquid of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus acidophilus*, and *Lactobacillus casei* is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

5. The composition with hair growth and hair darkening effects according to claim 2, characterized in that, The auxiliary plant extracts were all prepared by ethanol reflux extraction. The specific steps were as follows: the corresponding dried medicinal materials were pulverized and passed through a 40-mesh sieve, 10-15 times the volume of 65%-75% ethanol solution was added, the mixture was soaked for 30 minutes, and then heated and refluxed for extraction 2-3 times, each extraction lasting 1-2 hours. The extracts were combined, filtered, and the ethanol was recovered under reduced pressure until there was no alcohol odor. The mixture was then concentrated to an extract with a relative density of 1.15-1.

20. After freeze-drying, the corresponding auxiliary plant extract powder was obtained.

6. The composition with hair growth and hair darkening effects according to claim 1, characterized in that, The composition, by weight, further includes chemically acceptable excipients: 10-30 parts of humectant, 0.5-2 parts of solubilizer, and the balance being deionized water; the total weight of the composition is 100 parts.

7. The composition with hair growth and hair darkening effects according to claim 6, characterized in that, The humectant is any one or a combination of butylene glycol, glycerin, and panthenol; the solubilizer is PEG-40 hydrogenated castor oil.

8. A method for preparing a composition having hair growth and hair darkening effects as described in any one of claims 6-7, characterized in that, Includes the following steps: Y1 aqueous phase preparation: Weigh each core fermented plant extract and auxiliary plant extract according to the ratio, add 30%-40% of the total mass of deionized water, stir at low speed at 40-45℃ until the powder is completely dissolved, filter through a 0.45μm filter membrane to obtain the aqueous phase of the active ingredients; Y2 matrix phase preparation: Weigh out the humectant and solubilizer according to the ratio, add them to the remaining deionized water, heat to 45-50℃, and stir until completely dissolved and homogeneous to obtain the matrix phase; Y3 Mixing and Homogenization: Slowly add the aqueous phase of the active ingredient to the matrix phase, maintain a constant temperature of 40℃ and stir for 20-30 minutes until the system is completely homogeneous. Allow it to cool naturally to room temperature, and discharge the material after passing the inspection to obtain a composition with hair growth and hair darkening effects.

9. The use of the composition having hair growth and hair darkening effects as described in any one of claims 1-7 in the preparation of anti-hair loss and hair growth scalp care products, characterized in that, The scalp care product is any one of the following: anti-hair loss serum, anti-hair loss shampoo, scalp repair serum, or leave-in scalp spray.

10. The application according to claim 9, characterized in that, The amount of the composition added to the scalp care product is 2-15 parts per 100 parts of the total product weight.