A shampoo composition with anti-hair loss effect and a preparation method thereof

Abstract

The application discloses a shampoo composition with anti-hair loss and hair growth effects and a preparation method thereof, and belongs to the technical field of daily chemical products. The shampoo composition comprises anti-hair loss active substances, cleaning agents, suspension stabilizing phases, moisturizing agents, pH regulators, preservatives, fragrances, anti-inflammatory soothing agents, scalp conditioning agents, chelating agents, thickening and foam stabilizing agents and deionized water. The anti-hair loss active substances are obtained by sequentially subjecting plant raw materials to aerobic glycosylation, short-time enzyme inactivation treatment at 75-85 DEG C and anaerobic conversion, and the plant raw materials comprise red ginseng, radix falcaliae, female privet fruit, cypress leaves, wax chrysanthemum, white chrysanthemum, coix seed, rehmannia, euphorbia humifusa, angelica and salvia miltiorrhiza. The directional conversion of saponins and flavonoid glycosides is realized through a staged enzyme and bacteria synergistic conversion process, and a multi-target synergistic network and a scalp micro-ecological conditioning system are fused, so that the prepared shampoo composition has an inhibitory effect on type II 5 alpha-reductase, has the effects of anti-inflammation and soothing and improvement of scalp microcirculation, and is suitable for daily anti-hair loss and hair growth care.

Description

Technical Field

[0001] This invention belongs to the field of daily chemical products technology, specifically relating to a shampoo composition with anti-hair loss and hair growth effects and its preparation method. Background Technology

[0002] Androgenetic alopecia (AGA) is the most common type of hair loss. Its pathological mechanism primarily involves type II 5α-reductase in the hair follicle converting testosterone to dihydrotestosterone (DHT). DHT binds to androgen receptors in the papillary cells of the hair follicle epithelium, activating downstream signaling pathways, leading to a shortened hair follicle growth phase, follicle miniaturization, and ultimately, progressive hair loss. Currently, clinical treatments for AGA mainly include oral medications and hair transplantation surgery. However, oral medications have side effects and relapse issues after discontinuation, while hair transplantation surgery has limitations in terms of trauma and cost.

[0003] Traditional Chinese medicine (TCM) has a long history of application in the prevention of hair loss and hair growth. Current research indicates that saponins, stilbene glycosides, flavonoids, iridoid glycosides, and phenolic acids in TCM herbs such as red ginseng, Polygonum multiflorum, Platycladus orientalis leaves, Ligustrum lucidum, Rehmannia glutinosa, Eclipta prostrata, Angelica sinensis, and Salvia miltiorrhiza have effects such as inhibiting 5α-reductase activity, promoting hair follicle growth, improving scalp microcirculation, and anti-oxidation. Based on this research, some anti-hair loss shampoos have directly added these TCM extracts to their formulas through physical compounding. However, this simple mixing method has significant shortcomings: firstly, the active ingredients in the extracts are mostly in the form of glycosides, with large molecular weights and high polarity, resulting in low transdermal absorption; secondly, the content of active ingredients in different batches of extracts fluctuates greatly, leading to unstable 5α-reductase inhibition rates and affecting product efficacy.

[0004] Microbial fermentation is one of the technical means to improve the bioavailability of active ingredients in traditional Chinese medicine. Chinese patent CN112494418A discloses an anti-hair loss shampoo containing natural fermentation products of Aspergillus, yeast, and Lactobacillus. Chinese patent CN121550076B discloses a method for preparing a hair care composition through stepwise aerobic and anaerobic fermentation. However, none of the above-mentioned prior art achieves precise timing control of the fermentation process, nor does it achieve directional transformation.

[0005] Therefore, how to achieve temporal control of enzyme activity during fermentation to directionally transform saponins and flavonoids in traditional Chinese medicine and improve the stability of 5α-reductase inhibitory activity is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a shampoo composition with anti-hair loss and hair growth effects, and its preparation method. The composition employs a staged enzyme-bacterial synergistic transformation process (i.e., a staged fermentation process) to prepare anti-hair loss active ingredients, and integrates a multi-target scalp care system. The specific technical solution is as follows: In a first aspect, the present invention provides a shampoo composition with anti-hair loss and hair growth effects, comprising the following components in parts by weight: The formula contains 5-15 parts anti-hair loss active ingredient, 15-25 parts detergent, 1-5 parts suspension stabilizer, 2-5 parts moisturizer, 0.1-0.5 parts pH adjuster, 0.3-0.8 parts preservative, 0.2-0.6 parts fragrance, 0.1-0.3 parts anti-inflammatory soothing agent, 0.3-0.8 parts scalp conditioner, 0.05-0.15 parts chelating agent, 0.5-2 parts thickener and foam stabilizer, and 50-75 parts deionized water. The plant raw materials include the following components by weight: 5-10 parts red ginseng, 5-10 parts Polygonum multiflorum, 5-10 parts Ligustrum lucidum, 5-10 parts Platycladus orientalis leaves, 3-8 parts Helichrysum fusiforme, 3-8 parts white chrysanthemum, 10-20 parts Coix lacryma-jobi, 3-5 parts Rehmannia glutinosa, 3-6 parts Eclipta prostrata, 3-5 parts Angelica sinensis, and 2-4 parts Salvia miltiorrhiza. The anti-hair loss active ingredient is obtained by sequentially subjecting the plant raw material to aerobic saccharification, short-term enzyme inactivation treatment at 75-85℃, and anaerobic transformation.

[0007] Preferably, the amount of Job's tears used is 10-20 parts. Job's tears are rich in amylopectin, and its compact granular structure necessitates microbial enzymatic hydrolysis to effectively release fermentable sugars. If the amount of Job's tears is less than 10 parts, the sugar production during the aerobic saccharification stage will be insufficient, failing to provide a sufficient carbon source for subsequent lactic acid bacteria acid production; if it is more than 20 parts, excessive hydrolysis of starch in Job's tears will lead to an increase in the osmotic pressure of the system, inhibiting the metabolic activity of Bacillus and affecting fermentation efficiency.

[0008] Preferably, the amount of white chrysanthemum used is 3-8 parts. The traditional efficacy of white chrysanthemum is to prevent hair loss caused by excessive sex hormones. Its flavonoid components (luteolin, farnesin-7-O-glucoside) are converted into highly active aglycones by enzymes activated in an acidic environment during the anaerobic transformation stage, which inhibits the activity of 5α-reductase.

[0009] Preferably, the dosage of Rehmannia glutinosa is 3-5 parts. Rehmannia glutinosa is rich in iridoid glycosides (such as catalpol), which undergo hydrolysis and transformation under the action of matrix-bound enzymes such as β-glucosidase in the anaerobic acidic environment, generating secondary glycosides or aglycones with stronger permeability and higher biological activity, which synergistically enhance the nourishing effect on hair follicle papilla cells and promote scalp microcirculation.

[0010] Preferably, the dosage of *Eclipta prostrata* is 3-6 parts. *Eclipta prostrata* is a traditional Chinese medicine for darkening hair, containing flavonoid glycosides, triterpenoid saponins, and ecliptaside. The structures of its saponins and flavonoid glycosides are consistent with the target components of this invention. After staged fermentation, the aglycone conversion rate is increased, which can enhance the dual effects of preventing hair loss and darkening hair.

[0011] Preferably, the dosage of Angelica sinensis is 3-5 parts. Angelica sinensis contains ferulic acid, flavonoid glycosides, and polysaccharides. Among them, the flavonoid glycosides can be hydrolyzed into more active aglycones by the residual enzyme system during the anaerobic stage; at the same time, the blood-activating effect of Angelica sinensis can improve the microcirculation of the scalp.

[0012] Preferably, the dosage of tanshinone is 2-4 parts. Tanshinone and salvianolic acid in tanshinone have antioxidant and microcirculation-improving effects. The water-soluble phenolic acids in tanshinone can dissolve in the fermentation broth, and their antioxidant properties can protect easily oxidized flavonoid aglycones during fermentation, thus improving the stability of the active ingredients.

[0013] Preferably, the mass ratio of red ginseng, Polygonum multiflorum, Ligustrum lucidum, and Platycladus orientalis leaves in the plant raw materials is 1:1:1:1.

[0014] Preferably, the cleaning agent comprises sodium lauryl ether sulfate and cocamidopropyl betaine in a mass ratio of 2-4:1.

[0015] More preferably, the detergent further includes sodium lauroyl sarcosinate and / or sodium lauroyl glutamate, in an amount of 8%-20% of the total mass of the detergent. Amino acid surfactants have excellent mildness and can reduce the irritation of sodium lauryl ether sulfate to the scalp, making the system more suitable for sensitive scalps.

[0016] Preferably, the suspended stable phase comprises guar hydroxypropyltrimethylammonium chloride, hydrogenated castor oil, and poloxamer 407, with a mass ratio of 1:0.5-0.8:0.2-0.5. The positively charged quaternary ammonium groups on the guar hydroxypropyltrimethylammonium chloride molecular chain can adsorb onto the surface of negatively charged solid particles through electrostatic interaction, forming an electric double layer and preventing particle aggregation and sedimentation. Hydrogenated castor oil, as a non-ionic suspending agent, can effectively encapsulate the oil-soluble active ingredients in the system. When the temperature rises above the critical gelation temperature (approximately 25-30°C), poloxamer 407 undergoes hydrophobic block dehydration to form micelles and physically crosslinks into a three-dimensional gel network, providing suitable viscosity support near scalp temperature. Together, these three components construct a stable suspension system.

[0017] Preferably, the moisturizer is selected from at least one of glycerin, butylene glycol, and 1,3-propanediol; the pH adjuster is citric acid, used to adjust the pH of the shampoo composition to 5.0-6.5; and the preservative is sodium benzoate.

[0018] Preferably, the anti-inflammatory and soothing agent is dipotassium glycyrrhizate. Dipotassium glycyrrhizate has a potent anti-inflammatory and soothing effect, which can inhibit scalp micro-inflammatory responses. Scalp micro-inflammatory responses are an important factor in the aggravation of hair loss, and it works synergistically with the DHT inhibitory effect of the fermented active ingredient of this invention.

[0019] Preferably, the scalp conditioner is hydrolyzed silk protein. Hydrolyzed silk protein can form a protective film on the hair surface, reducing mechanical hair loss during shampooing.

[0020] Preferably, the chelating agent is disodium EDTA or tetrasodium EDTA. EDTA can chelate calcium in water. 2+ / Mg 2+ This prevents it from precipitating with fatty acid salts, thereby improving the product's heat and cold resistance stability and preservative efficacy.

[0021] Preferably, the thickening and foam stabilizing agent is cocamidomethyl MEA. As a nonionic thickening and foam stabilizing agent, cocamidomethyl MEA works synergistically with suspension stabilizers to further improve the viscosity and foam stability of the shampoo composition.

[0022] Secondly, the present invention provides a method for preparing an anti-hair loss active ingredient, comprising the following steps: (1) Aerobic saccharification stage: After the plant raw material is crushed, it is mixed with deionized water at a mass ratio of 1:3-1:5, and inoculated with Bacillus amyloliquefaciens and Bacillus licheniformis. Fermentation is carried out under aerobic conditions to obtain aerobic saccharification liquid. (2) 75-85℃ short-term enzyme inactivation stage: The aerobic saccharification solution obtained in step (1) is heated to inactivate the live Bacillus bacteria while retaining its matrix-bound enzyme system to obtain the enzyme inactivation treatment solution; (3) Anaerobic conversion stage: Inoculate Lactobacillus corni and Lactobacillus casei into the enzyme-inactivating treatment solution obtained in step (2), and carry out fermentation under anaerobic conditions to reduce the pH of the system to 3.5-4.5 to obtain the fermentation conversion solution; (4) Post-processing stage: After fermentation is completed, the fermentation conversion liquid is centrifuged and filtered, and the filtrate is collected to obtain the anti-dehydration active substance.

[0023] Preferably, in step (1), the viable counts of both *Bacillus amyloliquefaciens* and *Bacillus licheniformis* are ≥1.0 × 10⁻⁶. 8 CFU / g, the total inoculum amount is 2%-6% of the total mass of the plant material described in step (1).

[0024] Preferably, in step (1), the temperature of the aerobic fermentation is 28-37℃; if the temperature is below 28℃, the metabolic activity of Bacillus is insufficient and the starch degradation efficiency is reduced; if the temperature is above 37℃, the strain will enter the decline period too early and the enzyme production capacity will decrease.

[0025] Preferably, in step (1), the aerobic fermentation time is 12-24h; if the time is less than 12h, the starch degradation will be insufficient; if the time is more than 24h, the small molecule sugars in the system will be excessively consumed, which is not conducive to the subsequent anaerobic stage.

[0026] Preferably, in step (1), the aeration rate is 0.5-1.5 vvm. If the aeration rate is less than 0.5 vvm, the dissolved oxygen is insufficient, the aerobic metabolism of Bacillus amyloliquefaciens and Bacillus licheniformis is inhibited, and the sugar production efficiency is reduced. If the aeration rate is greater than 1.5 vvm, there will be too much foam, which will increase the risk of fermentation liquid overflow.

[0027] Preferably, in step (2), the heating treatment temperature is 75-85℃ and the time is 5-15 min. If the temperature is below 75℃, the Bacillus will not be completely inactivated and may continue to multiply and consume small molecule sugars in the anaerobic stage; if the temperature is above 85℃, the matrix-bound enzyme system (including β-glucosidase, cellulase, etc.) will undergo irreversible denaturation and inactivation and cannot be activated in the subsequent acidic environment.

[0028] Preferably, in step (3), the viable counts of both *Lactobacillus zeatus* and *Lactobacillus casei* are ≥1.0 × 10⁻⁶. 8 The inoculum ratio of CFU / g to Lactobacillus cornii and Lactobacillus casei is 1:0.8-1.2, and the total inoculum is 3%-5% of the volume of the enzyme-inactivating liquid in step (2). If the inoculum is less than 3%, the lactic acid production will be insufficient, the pH of the system will not be able to drop to the target range, and the enzyme system will not be fully activated; if the inoculum is greater than 5%, the acid production will be too fast, the pH will drop below 3.0, and the enzyme activity will be inhibited.

[0029] Preferably, in step (3), the anaerobic fermentation temperature is 30-37℃ and the time is 24-48h. If the temperature is below 30℃, the lactic acid bacteria will metabolize slowly and produce low acid efficiency; if the temperature is above 37℃, the lactic acid bacteria will die prematurely.

[0030] Preferably, in step (3), the endpoint pH of the anaerobic fermentation is 3.5-4.5. When the pH is higher than 4.5, the β-glucosidase activity is insufficient, and the conversion rate of saponins and flavonoids is insufficient; when the pH is lower than 3.5, the enzyme system is inactivated in an overly acidic environment, and the pH of the product is too low, requiring additional adjustment, which increases the complexity of the process.

[0031] Preferably, in step (4), the centrifugation speed is 4000-8000 rpm and the time is 10-20 min; after centrifugation, the supernatant is collected and filtered and sterilized using a sterile filter membrane with a pore size of 0.22 μm.

[0032] Thirdly, the present invention provides a method for preparing a shampoo composition with anti-hair loss and hair growth effects, comprising the following steps: S1. Heat deionized water to 60-65℃, add guar gum hydroxypropyltrimethylammonium chloride, disperse evenly, and obtain phase A pregel; S2. Heat the A-phase pregel obtained in step S1 to 80-85℃, add detergent and thickener / foam stabilizer, keep warm and stir for 15-25 minutes to obtain phase B; S3. After heating the hydrogenated castor oil to complete melting, mix and disperse it with poloxamer 407 at 80-85℃ to obtain phase C, and keep it at the temperature for later use; S4. Keep the B phase obtained in step S2 at 80-85℃, add the C phase obtained in step S3, and homogenize at 1000-1500 rpm for 3-5 min. Then cool down to 55-60℃ to obtain a mixture. S5. Continue to cool the mixture obtained in step S4 to 40-45°C, add the anti-hair loss active ingredient, moisturizer, pH adjuster, preservative, fragrance, anti-inflammatory soothing agent, scalp conditioner and chelating agent prepared by the method described in the second aspect, and stir evenly to obtain the shampoo composition with the anti-hair loss and hair growth effect.

[0033] In this invention, both *Bacillus amyloliquefaciens* and *Bacillus licheniformis* are aerobic or facultative anaerobic bacteria capable of secreting extracellular enzyme systems such as α-amylase, saccharifying enzymes, cellulases, and β-glucosidase. Under aerobic conditions, they efficiently degrade starch and polysaccharides in plant materials such as Job's tears, generating small-molecule sugars (mainly maltose and glucose). After fermentation, a short-term enzyme inactivation treatment at 75-85℃ inactivates the live *Bacillus* bacteria. However, the portion of the secreted enzyme system adsorbed onto the cell wall and extracellular polymers (matrix-bound enzymes) retains its activity due to the protection of the polymers. In the subsequent anaerobic conversion stage, *Lactobacillus zei* and *Lactobacillus casei* utilize the small-molecule sugars produced in the aerobic stage for lactic acid fermentation, gradually reducing the pH of the system to 3.5-4.5. This acidic environment activates the remaining β-glucosidase and other enzyme systems, synergistically completing the directional conversion of saponins to deglycosylates and flavonoid aglycones.

[0034] The short-time enzyme inactivation treatment at 75-85℃ is a key step in the timing control of this invention. Specifically, the design principle of the 75-85℃ short-time enzyme inactivation treatment is as follows: treatment at 75-85℃ can completely inactivate live Bacillus bacteria within 5-15 minutes (verified by plate counting method, the number of live bacteria decreased by ≥6 log), thereby avoiding competition between Bacillus bacteria and small molecule sugars and lactic acid bacteria in the anaerobic stage; at the same time, based on the principle of enzyme thermal deactivation mechanics, the retention rate of matrix-bound enzymes such as β-glucosidase can reach 65%-85% under the short-time enzyme inactivation treatment at 75-85℃. These surviving enzyme systems are activated in the subsequent acidic environment to achieve the targeted transformation of the target component. If conventional high-temperature sterilization (≥95℃) is used, the surviving enzyme systems will be inactivated, and targeted transformation cannot be achieved; if this enzyme inactivation treatment is not performed, Bacillus bacteria will continue to multiply and consume carbon sources in the anaerobic stage, and the growth of lactic acid bacteria will be inhibited. Therefore, short-term enzyme inactivation treatment at 75-85℃ is a necessary and precise time-sequence control method to connect the aerobic saccharification stage and the anaerobic conversion stage and realize the staged fermentation process.

[0035] The present invention has the following beneficial effects: 1. This invention uses coix seed as the fermentation substrate in the aerobic saccharification stage, and utilizes α-amylase and saccharifying enzyme secreted by Bacillus amyloliquefaciens and Bacillus licheniformis to degrade its starch into small molecule sugars, providing a carbon source basis for sugar production in the aerobic stage and the subsequent anaerobic conversion stage.

[0036] 2. This invention utilizes white chrysanthemum as the active ingredient targeting androgenetic alopecia. Its flavonoids, during the anaerobic transformation stage, are activated by β-glucosidase in an acidic environment, transforming into highly active aglycones. These aglycones, along with other herbs such as Platycladus orientalis, Rehmannia glutinosa, Eclipta prostrata, Angelica sinensis, and Salvia miltiorrhiza, form a four-dimensional synergistic anti-hair loss network that inhibits dihydrotestosterone (DHT) production, promotes hair follicle nourishment, improves scalp microcirculation, and provides antioxidant protection. Rehmannia glutinosa's iridoid glycosides, after targeted transformation, enhance hair follicle nourishment; Eclipta prostrata's flavonoid glycosides and saponins, after transformation, strengthen hair darkening and anti-hair loss; Angelica sinensis's blood-activating components improve microcirculation and form a dual pathway with DHT inhibition; and Salvia miltiorrhiza's antioxidant components protect the stability of the active ingredients.

[0037] 3. This invention achieves staged enzyme-microbe synergistic directional fermentation through a three-stage sequential control: aerobic saccharification, short-term enzyme inactivation at 75-85℃ to retain substrate-bound enzymes, and anaerobic acid production to activate enzymes. It can inactivate live Bacillus bacteria while retaining most of the activity of substrate-bound enzymes, and activate the retained enzymes under acidic conditions in the anaerobic stage to complete the directional conversion of saponins and flavonoids. This effectively avoids the problems of nutrient competition and uncontrollable metabolic direction among strains in conventional mixed fermentation, and significantly improves the conversion efficiency.

[0038] 4. The present invention integrates amino acid surfactants, dipotassium glycyrrhizate, hydrolyzed silk protein, 1,3-propanediol, EDTA and cocamide methyl MEA into the shampoo composition to construct a multi-dimensional scalp care system that provides gentle cleansing, anti-inflammatory soothing, mechanical protection and stable suspension.

[0039] 5. The anti-hair loss active ingredient obtained in this invention has a significant inhibitory effect on type II 5α-reductase, and its IC50 value is [missing information]. 50 The pH value is significantly lower than that of conventional alcohol extracts, and the fermentation product naturally falls within the slightly acidic range of the scalp, thus possessing the effects of inhibiting DHT production and regulating the scalp's microecology. Furthermore, this invention eliminates the need for chemical anti-hair loss agents such as piroctone olamine salt, avoiding potential irritation to the scalp from chemical components and ensuring higher safety. Detailed Implementation

[0040] To more clearly illustrate the present invention, the following description, in conjunction with embodiments and comparative examples, further explains the invention. Those skilled in the art should understand that the following description represents preferred embodiments of the invention, but the invention is not limited to these embodiments. The raw materials and reagents used in this invention are all commercially available.

[0041] I. Preparation of Anti-hair Loss Active Ingredients Example 1 This embodiment provides a method for preparing an anti-hair loss active ingredient.

[0042] (1) Crush 10g of red ginseng, 10g of Polygonum multiflorum, 10g of Ligustrum lucidum, 10g of Platycladus orientalis leaves, 5g of Helichrysum fusiforme, 5g of Chrysanthemum morifolium, 15g of Coix lacryma-jobi, 4g of Rehmannia glutinosa, 4g of Eclipta prostrata, 4g of Angelica sinensis, and 3g of Salvia miltiorrhiza, mix them together, add 320g of deionized water (4 times the total mass of the plant materials), and stir evenly. Inoculate with Bacillus amyloliquefaciens and Bacillus licheniformis powder (both with a viable count ≥1.0×10⁻⁶). 8 (CFU / g, with a total inoculum of 4% of the total mass of plant material), aerobic fermentation was carried out at 32℃, with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm for 18 h to obtain an aerobic saccharified liquid.

[0043] (2) Heat the aerobic saccharification solution at 80°C for 10 min to inactivate live Bacillus bacteria and obtain an enzyme-inactivated solution.

[0044] (3) Inoculate the enzyme-inactivating solution with a suspension of Lactobacillus zeatus and Lactobacillus casei (both viable count ≥ 1.0 × 10⁻⁶). 8 (CFU / mL, inoculum ratio of 1:1, total inoculum amount of 4% of enzyme-inactivating liquid volume), anaerobic fermentation was carried out at 35℃ and stirring speed of 100 rpm for 36 h until the pH of the system dropped to 3.8, and the fermentation conversion broth was obtained.

[0045] (4) Centrifuge the fermentation conversion broth at 6000 rpm for 15 min, collect the supernatant, filter it with a sterile filter membrane with a pore size of 0.22 μm to remove bacteria, collect the filtrate, and obtain the anti-desorption active ingredient.

[0046] Example 2 This embodiment provides a method for preparing an anti-hair loss active ingredient.

[0047] (1) Crush 8g of red ginseng, 8g of Polygonum multiflorum, 8g of Ligustrum lucidum, 8g of Platycladus orientalis leaves, 4g of Helichrysum fusiforme, 4g of Chrysanthemum morifolium, 12g of Coix lacryma-jobi, 3g of Rehmannia glutinosa, 3g of Eclipta prostrata, 3g of Angelica sinensis, and 2g of Salvia miltiorrhiza, mix them together, add 252g of deionized water (4 times the total mass of the plant materials), and stir evenly. Inoculate with Bacillus amyloliquefaciens and Bacillus licheniformis powder (both with a viable count ≥1.0×10⁻⁶). 8 (CFU / g, with a total inoculum of 3% of the total mass of plant material), aerobic fermentation was carried out at 30℃, with an aeration rate of 0.8 vvm and a stirring speed of 150 rpm for 16 h to obtain an aerobic saccharified liquid.

[0048] (2) Heat the aerobic saccharification solution at 78°C for 12 min to inactivate the live Bacillus bacteria and obtain the enzyme-inactivated solution.

[0049] (3) Inoculate the enzyme-inactivating solution with a suspension of Lactobacillus zeatus and Lactobacillus casei (both viable count ≥ 1.0 × 10⁻⁶). 8 The inoculum was prepared at CFU / mL, with an inoculum ratio of 1:0.9 and a total inoculum of 3% of the enzyme-inactivating liquid volume. The mixture was then anaerobic fermented at 32℃ and 80 rpm for 30 h until the pH of the system dropped to 4.0, yielding the fermentation conversion broth.

[0050] (4) Centrifuge the fermentation conversion broth at 5000 rpm for 15 min, collect the supernatant, filter it with a sterile filter membrane with a pore size of 0.22 μm to remove bacteria, collect the filtrate, and obtain the anti-desorption active ingredient.

[0051] Example 3 This embodiment provides a method for preparing an anti-hair loss active ingredient.

[0052] (1) Crush 10g of red ginseng, 10g of Polygonum multiflorum, 10g of Ligustrum lucidum, 10g of Platycladus orientalis leaves, 6g of Helichrysum fusiforme, 6g of Chrysanthemum morifolium, 18g of Coix lacryma-jobi, 5g of Rehmannia glutinosa, 5g of Eclipta prostrata, 5g of Angelica sinensis, and 4g of Salvia miltiorrhiza, mix them together, add 356g of deionized water (4 times the total mass of the plant materials), and stir evenly. Inoculate with Bacillus amyloliquefaciens and Bacillus licheniformis powder (both with a viable count ≥1.0×10⁻⁶). 8 (CFU / g, with a total inoculum of 5% of the total mass of plant material), aerobic fermentation was carried out at 35℃, with an aeration rate of 1.2 vvm and a stirring speed of 250 rpm for 20 h to obtain an aerobic saccharified liquid.

[0053] (2) Heat the aerobic saccharification solution at 82°C for 8 min to inactivate the live Bacillus bacteria and obtain the enzyme-inactivated solution.

[0054] (3) Inoculate the enzyme-inactivating solution with a suspension of Lactobacillus zeatus and Lactobacillus casei (both viable count ≥ 1.0 × 10⁻⁶). 8 The inoculum was prepared at CFU / mL with an inoculum ratio of 1:1.1 and a total inoculum of 5% of the enzyme-inactivating liquid volume. The mixture was then anaerobic fermented at 37°C and 120 rpm for 40 h until the pH of the system dropped to 4.2, yielding the fermentation conversion broth.

[0055] (4) Centrifuge the fermentation conversion broth at 7000 rpm for 15 min, collect the supernatant, filter it with a sterile filter membrane with a pore size of 0.22 μm to remove bacteria, collect the filtrate, and obtain the anti-desorption active ingredient.

[0056] Example 4 This embodiment provides a method for preparing an anti-hair loss active ingredient.

[0057] The only difference between this embodiment and Example 1 is that: the amount of coix seed used is 10g; the aerobic fermentation temperature is 28℃ and the time is 12h; the aeration rate is 0.8 vvm; the stirring speed is 200rpm; the enzyme inactivation temperature is 75℃ and the time is 15min; the anaerobic fermentation temperature is 30℃ and the time is 24h; and the final pH is 3.5. All other operations are the same as in Example 1.

[0058] Example 5 This embodiment provides a method for preparing an anti-hair loss active ingredient.

[0059] The only difference between this embodiment and Embodiment 1 is that: the amount of coix seed used is 20g; the aerobic fermentation temperature is 37℃ and the time is 24h; the aeration rate is 1.5 vvm; the stirring speed is 250rpm; the enzyme inactivation temperature is 85℃ and the time is 5min; the anaerobic fermentation temperature is 37℃ and the time is 48h; and the final pH is 4.5. All other operations are the same as in Embodiment 1.

[0060] Example 6 This embodiment provides a method for preparing an anti-hair loss active ingredient.

[0061] The only differences between this embodiment and Example 1 are: the amount of white chrysanthemum is 3g, the amount of immortelle is 3g, the aerobic fermentation temperature is 28℃, the time is 12h, the aeration rate is 0.8 vvm, and the stirring speed is 200rpm; the enzyme inactivation temperature is 75℃, and the time is 15min; the anaerobic fermentation temperature is 30℃, the time is 24h, and the final pH is 3.5. All other operations are the same as in Example 1.

[0062] Comparative Example 1 The only difference between this comparative example and Example 1 is that the coix seed is replaced with an equal amount of yam, and the rest of the operation is the same as in Example 1.

[0063] Comparative Example 2 The only difference between this comparative example and Example 1 is that the white chrysanthemums are replaced with an equal amount of wild chrysanthemums; all other operations are the same as in Example 1.

[0064] Comparative Example 3 The only difference between this comparative example and Example 1 is that it lacks the immortelle; all other operations are the same as in Example 1.

[0065] Comparative Example 4 The only difference between this comparative example and Example 1 is that the amount of coix seed used is 8g, and the rest of the operation is the same as in Example 1.

[0066] Comparative Example 5 The only difference between this comparative example and Example 1 is that the amount of coix seed used is 25g, and the rest of the operation is the same as in Example 1.

[0067] Comparative Example 6 The only difference between this comparative example and Example 1 is that: 10g of red ginseng, 5g of Polygonum multiflorum, 5g of Ligustrum lucidum, and 5g of Platycladus orientalis leaves are used, while the rest of the operation is the same as in Example 1.

[0068] Comparative Example 7 The only difference between this comparative example and Example 1 is that after the aerobic saccharification stage, no enzyme inactivation treatment was performed, and Lactobacillus cornii and Lactobacillus casei were directly inoculated for anaerobic fermentation. The remaining operations were the same as in Example 1.

[0069] Comparative Example 8 The only difference between this comparative example and Example 1 is that after the aerobic saccharification stage, it is sterilized at 121°C for 15 minutes, then cooled and inoculated with Lactobacillus cornii and Lactobacillus casei for anaerobic fermentation. The rest of the operation is the same as in Example 1.

[0070] Comparative Example 9 The only difference between this comparative example and Example 1 is that: 10g of red ginseng, 10g of Polygonum multiflorum, 10g of Ligustrum lucidum, 10g of Platycladus orientalis leaves, 5g of Helichrysum fusiforme, 5g of Chrysanthemum morifolium, 15g of Coix lacryma-jobi, 4g of Rehmannia glutinosa, 4g of Eclipta prostrata, 4g of Angelica sinensis, and 3g of Salvia miltiorrhiza are pulverized and mixed, 320g of deionized water is added, and four bacteria, Bacillus amyloliquefaciens, Bacillus licheniformis, Lactobacillus zei, and Lactobacillus casei, are inoculated simultaneously and fermented under aerobic conditions (aeration rate of 1.0 vvm) for 54 hours. The remaining operations are the same as in Example 1.

[0071] Comparative Example 10 The only difference between this comparative example and Example 1 is that the aerobic saccharification stage is omitted. The plant raw materials are directly mixed with deionized water after being crushed. Instead of inoculating with Bacillus amyloliquefaciens and Bacillus licheniformis, Lactobacillus cornii and Lactobacillus casei are directly inoculated for anaerobic conversion (35°C, 36h). The rest of the operation is the same as in Example 1.

[0072] Comparative Example 11 The only difference between this comparative example and Example 1 is that the anaerobic conversion stage is omitted. After the aerobic saccharification liquid is treated with enzyme inactivation at 80℃ for 10min, it is not inoculated with Lactobacillus zei and Lactobacillus casei, but is directly centrifuged and filtered, and the filtrate is collected as the active substance.

[0073] Comparative Example 12 The only difference between this comparative example and Example 1 is the interchange of the order of the aerobic saccharification stage and the anaerobic conversion stage: first, the plant raw materials are mixed with deionized water, inoculated with Lactobacillus cornii and Lactobacillus casei, and fermented at 35°C under anaerobic conditions for 36 hours; then, Bacillus amyloliquefaciens and Bacillus licheniformis are inoculated, and aerobic fermentation is carried out at 32°C and an aeration rate of 1.0 vvm for 18 hours. The remaining operations are the same as in Example 1.

[0074] Comparative Example 13 This comparative example provides a conventional alcohol extract.

[0075] 10g of red ginseng, 10g of Polygonum multiflorum, 10g of Ligustrum lucidum, 10g of Platycladus orientalis leaves, 5g of Helichrysanthemum morifolium, 5g of Chrysanthemum morifolium, 15g of Coix lacryma-jobi, 4g of Rehmannia glutinosa, 4g of Eclipta prostrata, 4g of Angelica sinensis, and 3g of Salvia miltiorrhiza are pulverized and mixed. 320g of 70% ethanol solution is added, and the mixture is refluxed at 60℃ for 2 hours. After filtration, the filtrate is collected to obtain the conventional ethanol extract.

[0076] Comparative Example 14 The only difference between this comparative example and Example 1 is that the amount of white chrysanthemum is 2g and the amount of immortelle is 2g. The rest of the operation is the same as in Example 1.

[0077] Comparative Example 15 The only difference between this comparative example and Example 1 is that the amount of white chrysanthemum is 9g and the amount of immortelle is 9g, while the rest of the operation is the same as in Example 1.

[0078] Comparative Example 16 The only difference between this comparative example and Example 1 is that the plant materials are 10g of red ginseng, 10g of Polygonum multiflorum, 10g of Ligustrum lucidum, 10g of Platycladus orientalis leaves, 5g of Helichrysum fusiforme, 5g of Chrysanthemum morifolium, and 15g of Coix lacryma-jobi (excluding Rehmannia glutinosa, Eclipta prostrata, Angelica sinensis, and Salvia miltiorrhiza), with 260g of deionized water added. The rest of the preparation steps are the same as in Example 1.

[0079] II. Formulation of Shampoo Composition Application Example 1 This application example provides a shampoo composition with anti-hair loss and hair growth effects.

[0080] Take 5g of the anti-hair loss active ingredient prepared in Example 1, and add 12g of sodium lauryl ether sulfate (SLES), 4g of cocamidopropyl betaine (CAPB), 2g of sodium lauroyl sarcosinate, 0.5g of sodium lauroyl glutamate (total detergent 18.5g, SLES to CAPB mass ratio 3:1), 0.33g of guar hydroxypropyl trimethylammonium chloride, 0.33g of hydrogenated castor oil, 0.34g of poloxamer 407 (total suspension stabilizer 1g), 2g of 1,3-propanediol, 1g of glycerin (total moisturizer 3g), 1g of cocamidomethyl MEA, 0.1g of tetrasodium EDTA, 0.2g of dipotassium glycyrrhizate, 0.5g of hydrolyzed silk protein, 0.3g of citric acid, 0.5g of sodium benzoate, 0.4g of fragrance, and 69.5g of deionized water.

[0081] Prepare according to the following steps: S1. Heat deionized water to 62°C, add guar gum hydroxypropyltrimethylammonium chloride, disperse evenly, and obtain phase A pregel; S2. Heat the pregel of phase A to 82°C, add sodium lauryl ether sulfate, cocamidopropyl betaine, sodium lauroyl sarcosinate, sodium lauroyl glutamate and cocamidomethyl MEA, keep warm and stir for 20 min to obtain phase B; S3. After heating hydrogenated castor oil to complete melting, mix and disperse it with poloxamer 407 at 82°C to obtain phase C, and keep it at this temperature for later use; S4. Keep phase B at 82℃, add phase C, homogenize at 1200 rpm for 4 min, and then cool to 58℃ to obtain a mixture; S5. Continue cooling the mixture to 42°C, add the anti-hair loss active ingredient, 1,3-propanediol, glycerin, tetrasodium EDTA, dipotassium glycyrrhizate, hydrolyzed silk protein, citric acid, sodium benzoate and fragrance, and stir well to obtain the shampoo composition.

[0082] Application Example 2 This application example provides a shampoo composition with anti-hair loss and hair growth effects.

[0083] The only difference between this application example and application example 1 is that: 10g of the anti-hair loss active ingredient obtained in example 1 is used; the suspension stabilizing phase consists of 1g of guar gum hydroxypropyltrimethylammonium chloride, 1g of hydrogenated castor oil, and 1g of poloxamer 407 (total suspension stabilizing phase 3g); and 62.5g of deionized water is used. The remaining operations are the same as in application example 1.

[0084] Application Example 3 This application example provides a shampoo composition with anti-hair loss and hair growth effects.

[0085] The only difference between this application example and application example 1 is that: 15g of the anti-hair loss active ingredient obtained in example 1 is used; the suspension stabilizing phase consists of 1.67g of guar gum hydroxypropyltrimethylammonium chloride, 1.67g of hydrogenated castor oil, and 1.66g of poloxamer 407 (total suspension stabilizing phase 5g); and 55.5g of deionized water is used. The remaining operations are the same as in application example 1.

[0086] Compare with application example 1-16 Take 10g of each of the control active ingredients obtained from Comparative Examples 1-16, and prepare control shampoo compositions according to the same excipient ratio and preparation steps as in Application Example 2. Application Examples 8, 11, and 13 were used for the comparative efficacy test of the shampoo compositions in Test Example 4 and the comparative efficacy test of the anti-hair loss and hair strengthening effects in Test Example 5.

[0087] Compare with application example 17 Take 10g of the anti-hair loss active ingredient obtained in Example 1, and add 15g of sodium lauryl ether sulfate, 5g of cocamidopropyl betaine (total detergent 20g, mass ratio 3:1), 1g of guar hydroxypropyltrimethylammonium chloride, 1g of hydrogenated castor oil, 1g of poloxamer 407 (total suspension stabilizer 3g), 3g of glycerin, 0.3g of citric acid, 0.5g of sodium benzoate, 0.4g of fragrance, and 62.5g of deionized water; do not add sodium lauroyl sarcosinate, sodium lauroyl glutamate, dipotassium glycyrrhizate, hydrolyzed silk fibroin, tetrasodium EDTA, or cocamidomethyl MEA. The preparation steps are the same as in Application Example 2.

[0088] Test Example 1: 5α-Reductase Inhibitory Activity Test (Anti-Dehydration Active Ingredient) A type II 5α-reductase inhibitor screening kit was used for testing. Each test sample was dissolved in DMSO and prepared into a series of concentration gradients, with finasteride as a positive control and an equal volume of solvent as a negative control. Following the kit instructions, enzyme reaction buffer, type II 5α-reductase, cofactor, and substrate were added sequentially to 96-well plates. The plates were incubated at 37°C in the dark for 30 min, followed by the addition of stop solution. The consumption of cofactor was detected using a fluorescence microplate reader, and the inhibition rate and IC50 at each concentration were calculated. 50 value.

[0089] The test results are shown in Table 1.

[0090] Table 1. Results of 5α-reductase inhibitory activity tests in the examples and comparative examples.

[0091] As shown in Table 1, the ICs in Examples 1-6 50The values ​​were between 65-78 μg / mL, significantly lower than the 122 μg / mL of the conventional ethanol extract in Comparative Example 13, indicating that the anti-degradation active ingredient obtained by the staged fermentation process of this invention has excellent inhibitory effect on type II 5α-reductase. Specifically, the IC50 value of Example 1... 50 The value was as low as 65 μg / mL, demonstrating the multi-target synergistic effect between eleven plant ingredients: red ginseng, Polygonum multiflorum, Ligustrum lucidum, Platycladus orientalis leaves, Helichrysanthemum morifolium, Chrysanthemum morifolium, Coix lacryma-jobi, Rehmannia glutinosa, Eclipta prostrata, Angelica sinensis, and Salvia miltiorrhiza. In contrast, Comparative Example 16, lacking Rehmannia glutinosa, Eclipta prostrata, Angelica sinensis, and Salvia miltiorrhiza, showed a significantly lower IC50 value. 50 The value rose to 88 μg / mL, further verifying that Rehmannia glutinosa, Eclipta prostrata, Angelica sinensis, and Salvia miltiorrhiza have a synergistic effect.

[0092] In Comparative Example 1, after replacing Job's tears with yam, IC 50 The value increased to 110 μg / mL; in Comparative Example 2, after replacing white chrysanthemum with wild chrysanthemum, the IC50 value increased. 50 The values ​​increased to 106 μg / mL, both significantly higher than the 65 μg / mL in Example 1, indicating that coix seed and white chrysanthemum have irreplaceable substrate functions in the fermentation system of this invention. In Comparative Example 4, the IC50 value increased when the amount of coix seed was reduced to 8 parts or increased to 25 parts. 50 The values ​​were 116 μg / mL and 113 μg / mL, respectively, both higher than the 78 μg / mL in Example 4 and the 76 μg / mL in Example 5, confirming the rationality of the dosage range of 10-20 parts of Coix seed. Comparative Example 6 adjusted the ratio of red ginseng, Polygonum multiflorum, Ligustrum lucidum, and Platycladus orientalis leaves to 1:0.5:0.5:0.5, with an IC50 value of 103 μg / mL, indicating that the four plant ingredients (red ginseng, Polygonum multiflorum, Ligustrum lucidum, and Platycladus orientalis leaves) had the best synergistic effect when used in an equal ratio of 1:1:1:1.

[0093] Comparative Example 7: IC50 was lower than that of enzyme inactivation treatment 50 The value was 130 μg / mL; in Comparative Example 8, the IC50 value was 130 μg / mL when sterilized at 121 °C. 50 The value reached as high as 143 μg / mL, indicating that short-term enzyme inactivation at 75-85℃ significantly preserved the enzyme system from the substrate, which was significantly better than neither enzyme inactivation nor high-temperature sterilization. The IC50 values ​​for the simultaneous mixed fermentation of the four strains in Comparative Example 9 were also analyzed. 50 The value was 126 μg / mL. In Comparative Example 12, after the aerobic saccharification stage and the anaerobic conversion stage were interchanged, the IC50 value was 126 μg / mL. 50 The value was 136 μg / mL, indicating that the sequential order of the staged fermentation, including the aerobic saccharification stage, the short-term enzyme inactivation stage at 75-85℃, and the anaerobic conversion stage, was irreversible. In Comparative Examples 10 and 11, the aerobic saccharification stage and the anaerobic conversion stage were omitted, respectively. 50 The values ​​were as high as 153 μg / mL and 160 μg / mL, indicating that both are indispensable and necessary stages.

[0094] In Comparative Example 14, when both white chrysanthemum and immortelle decreased to 2 parts, the IC 50 The value was 133 μg / mL. In Comparative Example 15, when both white chrysanthemum and immortelle rose to 9 parts, the IC50 value was 133 μg / mL. 50 The value was 128 μg / mL, indicating that the dosage of white chrysanthemum and immortelle must be in the range of 3-8 parts to achieve a synergistic effect.

[0095] Test Example 2: Determination of Active Ingredient Content by HPLC (Anti-hair Loss Active Ingredient) The contents of ginsenoside Rb1, ginsenoside Rg3, luteolin and luteolin in each sample were determined by high performance liquid chromatography (HPLC). The conversion rates of Rb1 to Rg3 and luteolin to luteolin were calculated.

[0096] Chromatographic conditions: C 18 Chromatographic column (250 mm × 4.6 mm, 5 μm); mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid aqueous solution, gradient elution; flow rate 1.0 mL / min; detection wavelengths 203 nm (ginsenosides) and 350 nm (flavonoids); column temperature 30 ℃; injection volume 10 μL.

[0097] Sample preparation: Each sample was filtered through a 0.22 μm microporous membrane and then directly injected. Quantification was performed using the external standard method, and the content of each component was calculated based on the peak area.

[0098] The formula for calculating conversion rate is: Conversion rate (%) of Rb1 to Rg3 = Rg3 content / (Rb1 content + Rg3 content) × 100% Conversion rate (%) of luteolin to luteolin glycosides = (luteolin content / (luteolin glycoside content + luteolin content)) × 100% Table 2. HPLC conversion rate determination results of active ingredients in each example and comparative example.

[0099] As shown in Table 2, the conversion rate of Rb1 to Rg3 in Example 1 reached 80.5%, and the conversion rate of luteolin to luteolinin reached 77.2%, which were significantly higher than the 13.5% and 16.8% of the conventional alcohol extract in Comparative Example 13, indicating that the present invention achieved efficient and targeted conversion of saponins and flavonoids in plant raw materials through a staged fermentation process.

[0100] In Comparative Example 1, replacing Coix seed with Dioscorea opposita reduced the conversion rate of Rb1 to Rg3 to 53.4%, while in Comparative Example 2, replacing white chrysanthemum with wild chrysanthemum reduced the conversion rate of luteolin to luteolin to 34.5%. This comparison shows that Coix seed and white chrysanthemum play irreplaceable substrate functions in saponin and flavonoid conversion, respectively. In Comparative Example 3, the absence of Helichrysum slightly reduced the conversion rates of Rb1 to Rg3 and luteolin to luteolin, indicating that Helichrysum has a synergistic effect. In Comparative Example 16, the conversion rate of Rb1 to Rg3 was 64.5%, and the conversion rate of luteolin to luteolin was 68.6%, both lower than the 80.5% and 77.2% in Example 1, respectively; indicating that Rehmannia glutinosa, Eclipta prostrata, Angelica sinensis, and Salvia miltiorrhiza have a synergistic enhancing effect on the directional conversion efficiency of saponins and flavonoids.

[0101] Regarding the amount of plant materials used, in Comparative Example 4, when the amount of Coix seed was 8 parts, and in Comparative Example 5, when the amount was 25 parts, the conversion rates of Rb1 to Rg3 were 56.6% and 55.8%, respectively. In Comparative Example 6, when the ratio of the four ingredients, including red ginseng, deviated from 1:1:1:1, the conversion rate of Rb1 to Rg3 was 63.3%. In Comparative Examples 14 and 15, when both white chrysanthemum and immortelle deviated from the range of 3-8 parts, the conversion rates of Rb1 to Rg3 were 49.5% and 51.2%, respectively, and the conversion rates of luteolin to luteolinin decreased to 37.2% and 40.5%, respectively. The above results indicate that deviations in the amount and ratio of each plant material from the range defined in this invention lead to a significant decrease in the directional conversion efficiency, confirming the rationality of the plant material ratio range of this invention.

[0102] Regarding the fermentation process, in Comparative Example 7, lacking short-term enzyme inactivation treatment at 75-85℃, the conversion rate of Rb1 to Rg3 was 36.6%, and the conversion rate of luteolin to luteolin was 40.4%. In Comparative Example 8, when high-temperature sterilization at 121℃ was used, the two conversion rates decreased to 29.5% and 32.2%, respectively. In Comparative Example 9, when four strains were fermented simultaneously, the two conversion rates were 43.8% and 47.6%, respectively. In Comparative Example 12, when the aerobic saccharification stage and the anaerobic conversion stage were interchanged, the two conversion rates were 39.5% and 44.3%, respectively. In contrast, in Comparative Example 10, lacking the aerobic saccharification stage, the two conversion rates were only 16.2% and 30.5%, respectively; in Comparative Example 11, lacking the anaerobic conversion stage, the two conversion rates were only 6.8% and 9.2%, respectively. The above comparisons indicate that the enzyme inactivation treatment method, the timing control of staged fermentation, and the aerobic saccharification and anaerobic conversion stages all have a significant impact on the directional conversion of saponins and flavonoids.

[0103] Test Example 3: Transdermal Absorption Test The anti-hair loss active ingredient prepared in Example 1 and the conventional ethanol extract prepared in Comparative Example 13 were used for in vitro transdermal absorption experiments using the Franz diffusion cell method. Using isolated pig skin as a transdermal barrier, equal amounts of samples (based on the same amount of crude drug) were placed in a supply cell and stirred at a constant temperature of 32°C. The receiving liquid was collected at 2, 4, 6, 8, 12, and 24 hours, respectively. The cumulative permeation of luteolin and ginsenoside Rg3 in the receiving liquid was determined by HPLC, and the cumulative permeation rate of each component over 24 hours was calculated.

[0104] The test results are shown in Table 3.

[0105] Table 3 Comparison of transdermal absorption rates of different samples

[0106] As shown in Table 3, the 24-hour cumulative permeation rate of luteolin in the anti-hair loss active ingredient of Example 1 was 40.2%, significantly higher than the 12.3% of the conventional ethanol extract of Comparative Example 13; the 24-hour cumulative permeation rate of ginsenoside Rg3 was 30.1%, significantly higher than the 8.7% of the ethanol extract of Comparative Example 13. This indicates that the transdermal absorption rate of the anti-hair loss active ingredient is significantly improved after the present invention hydrolyzes flavonoid glycosides in plant raw materials into aglycones and converts ginsenoside Rb1 into Rg3 through a staged fermentation process.

[0107] Test Example 4: Physicochemical Properties Test of Shampoo Composition The shampoo compositions prepared in Application Examples 1-3 and Control Application Example 17 were tested for appearance, pH value, viscosity, centrifugal stability, heat resistance and cold resistance in accordance with the relevant requirements of QB / T 1974-2004 "Shampoo".

[0108] Appearance: Visually observe and record the sample color, uniformity, and presence of stratification or precipitation.

[0109] pH value: Take an appropriate amount of sample and measure it directly at 25℃ using a pH meter.

[0110] Viscosity: Take an appropriate amount of sample and measure it using a rotational viscometer (No. 3 rotor, 12 rpm, 25℃).

[0111] Centrifugation stability: Take an appropriate amount of sample and centrifuge at 3000 rpm for 30 min, then observe whether stratification or precipitation occurs.

[0112] Heat resistance stability: Take an appropriate amount of sample, seal it and store it in a 45℃ constant temperature oven for 1 month. After taking it out and restoring it to room temperature, observe whether there is any layering, precipitation or viscosity change.

[0113] Cold resistance stability: Take an appropriate amount of sample, seal it and store it in a -5℃ refrigerator for 7 days. After taking it out and restoring it to room temperature, observe whether stratification or precipitation occurs.

[0114] The test results are shown in Table 4.

[0115] Table 4. Test results of the physicochemical properties of the shampoo composition

[0116] As shown in Table 4, the shampoo compositions prepared in Examples 1-3 have a uniform appearance, a pH value between 5.5 and 5.8, a viscosity between 4500 and 7600 mPa·s, and all meet the requirements for centrifugal stability, heat resistance, and cold resistance.

[0117] In comparison application example 17, slight flocculent precipitation appeared in the centrifugal stability test, and slight precipitation also appeared after returning to room temperature in the cold stability test. This indicates that the system's chelating ability for metal ions is insufficient without tetrasodium EDTA, leading to the precipitation of fatty acid salts under low temperature or centrifugal conditions. Without cocamidomethyl MEA, the system viscosity decreased (from 6200 mPa·s to 5800 mPa·s), and foam stability was also weakened. These results demonstrate that tetrasodium EDTA and cocamidomethyl MEA play an important role in maintaining the ionic and physical stability of the shampoo composition.

[0118] Test Example 5: 5α-Reductase Inhibitory Activity Test of Shampoo Composition Shampoo compositions prepared in Application Examples 1-3 and Control Application Examples 8, 11, 13, and 16 were diluted with an equal volume of deionized water and centrifuged at 10,000 rpm for 10 min. The supernatant was used as the test sample. The inhibition rate and IC50 of each sample against type II 5α-reductase were determined according to the method in Test Example 1. 50 value.

[0119] Pretreatment before testing: Each shampoo composition sample was diluted with an equal volume of deionized water, centrifuged at 10,000 rpm for 10 min, and the supernatant was taken for testing to remove insoluble excipients (suspended stable phase, wax, etc.) and retain water-soluble active ingredients.

[0120] The test results are shown in Table 5.

[0121] Table 5 Results of 5α-reductase inhibitory activity test of shampoo composition samples

[0122] As shown in Table 5, the ICs used in Examples 1-3... 50 The values ​​ranged from 65 to 70 μg / mL, which was basically consistent with the data for the corresponding anti-hair loss active ingredient in Test Example 1, indicating that the anti-hair loss active ingredient retained 5α-reductase inhibitory activity after being formulated into a shampoo composition. In Control Application Example 8, when sterilized at 121°C, the IC50 value was... 50The value was 146 μg / mL; in the control application example 11, which lacked the anaerobic conversion stage, the IC50 value was 146 μg / mL. 50 The value was 163 μg / mL, consistent with the trend of the corresponding anti-hair loss active ingredient in Test Example 1, further verifying the influence of enzyme inactivation method and anaerobic conversion stage on the efficacy of the shampoo composition. In Control Application Example 13, when a conventional alcohol extract was used instead of the anti-hair loss active ingredient obtained by staged fermentation in this invention, the IC50 value was [not specified]. 50 The value was 122 μg / mL, indicating that the efficacy of the shampoo composition of the present invention is significantly superior to that of shampoo compositions formulated with conventional alcohol extracts. (IC50 value was used in Comparative Application Example 16.) 50 The value was 94 μg / mL, further verifying that Rehmannia glutinosa, Eclipta prostrata, Angelica sinensis, and Salvia miltiorrhiza have the effect of inhibiting 5α-reductase in the shampoo composition.

[0123] Test Example 6: Comprehensive Evaluation of Human Efficacy and Safety In accordance with the relevant requirements of the "Cosmetic Safety Technical Specifications" (2015 edition), the anti-hair loss and hair strengthening efficacy of the shampoo composition prepared in this invention was tested.

[0124] Subjects: Volunteers aged 25-55 years who met the diagnostic criteria for androgenetic alopecia were recruited. There were no statistically significant differences in age and gender distribution among the groups, and each group had at least 20 participants. Exclusion criteria included: pregnant or lactating women, those with scalp infections or damage, and those with a history of allergies to cosmetics.

[0125] Test samples: Application Example 2, Control Application Example 13 and Control Application Examples 16-17, and a blank control (a base shampoo composition without anti-hair loss active ingredients).

[0126] (1) Test method for anti-hair loss and hair strengthening efficacy: Subjects used the test sample once every two days for 12 consecutive weeks. At the beginning of use, and at 4, 8, and 12 weeks, local hair density was measured at the same location using dermoscopy or hair microscopy, and changes in the amount of hair loss were recorded. Evaluation index: Increase in hair density (roots / cm²) 2 (and the rate of reduction in the number of hairs lost.) When calculating the effectiveness rate, it is expressed as "improvement" (an increase in hair density of ≥5 hairs / cm²). 2 A reduction of ≥20% in the number of hair loss is a valid criterion.

[0127] (2) Skin irritation test method: Apply 1.0 g of the test sample to the filter paper of the spot tester and apply it to the flexor side of the subject's forearm for 24 hours. Observe the skin reaction at 0.5 h, 24 h and 48 h after removal. Record the erythema and edema scores according to the skin reaction grading standard of the Cosmetic Safety Technical Specification and calculate the irritation index.

[0128] (3) Combing breakage index test: Referring to T / TTGA 001-2022 "Evaluation Method for Anti-Hair Breakage Efficacy of Hair Products", the in vitro hair bundle test method was adopted. Undyed natural black hair was taken and made into standardized hair bundles (15cm in length and 2g in weight). Each shampoo composition sample was treated in a uniform manner. After natural drying, a universal testing machine (such as Instron 3343) equipped with a standard comb was used to comb from the root to the tip of the hair at a constant speed of 100mm / min. The force-displacement curve during the combing process was recorded, and the combing breakage index was calculated. Evaluation index: Combing breakage index. The lower the value, the less mechanical hair loss during the shampooing process.

[0129] The test results are shown in Table 6.

[0130] Table 6 Results of the test on the efficacy of human hair loss prevention and hair strengthening

[0131] As shown in Table 6, the increase in hair density in Application Example 2 was 40.5 hairs / cm². 2 The hair loss reduction rate was 64.3%, and the effectiveness rate was 90.0%, indicating that the shampoo composition prepared by the present invention has excellent anti-hair loss and hair strengthening effects.

[0132] Compared with the control example 16, the effectiveness rate was 50.0%, and the increase in hair density was 16.8 hairs / cm². 2 The number of hair loss cases decreased by 32.5%. The results showed that the effectiveness decreased when four of the herbal ingredients—Rehmannia glutinosa, Eclipta prostrata, Angelica sinensis, and Salvia miltiorrhiza—were missing, demonstrating the synergistic effect of the eleven-herb formula.

[0133] Compared with the effective rate of 70.0% in application example 17, the decrease in efficacy was not due to insufficient efficacy of anti-hair loss active ingredients, but rather due to the lack of excipient system, which led to a significant increase in irritation (irritation index increased from 0.05 to 0.27) and an increase in comb breakage index (from 38.5% to 43.2%). This shows the indispensability of the multi-maintenance excipient system in improving the overall care effect of the product and its long-term use.

[0134] 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 shampoo composition with anti-hair loss and hair growth effects, characterized in that, It contains the following components by weight: The formula contains 5-15 parts anti-hair loss active ingredient, 15-25 parts detergent, 1-5 parts suspension stabilizer, 2-5 parts moisturizer, 0.1-0.5 parts pH adjuster, 0.3-0.8 parts preservative, 0.2-0.6 parts fragrance, 0.1-0.3 parts anti-inflammatory soothing agent, 0.3-0.8 parts scalp conditioner, 0.05-0.15 parts chelating agent, 0.5-2 parts thickener and foam stabilizer, and 50-75 parts deionized water. The plant raw materials include the following components by weight: 5-10 parts red ginseng, 5-10 parts Polygonum multiflorum, 5-10 parts Ligustrum lucidum, 5-10 parts Platycladus orientalis leaves, 3-8 parts Helichrysum fusiforme, 3-8 parts white chrysanthemum, 10-20 parts Coix lacryma-jobi, 3-5 parts Rehmannia glutinosa, 3-6 parts Eclipta prostrata, 3-5 parts Angelica sinensis, and 2-4 parts Salvia miltiorrhiza. The anti-hair loss active ingredient is obtained by sequentially subjecting the plant raw material to aerobic saccharification, short-term enzyme inactivation treatment at 75-85℃, and anaerobic transformation.

2. The shampoo composition according to claim 1, characterized in that, The mass ratio of red ginseng, Polygonum multiflorum, Ligustrum lucidum, and Platycladus orientalis leaves in the plant raw materials is 1:1:1:

1.

3. The shampoo composition according to claim 1, characterized in that, The cleaning agent comprises sodium lauryl ether sulfate and cocamidopropyl betaine in a mass ratio of 2-4:1; the cleaning agent also comprises sodium lauroyl sarcosinate and / or sodium lauroyl glutamate, which are used in amounts of 8%-20% of the total mass of the cleaning agent.

4. The shampoo composition according to claim 1, characterized in that, The suspended stable phase comprises guar hydroxypropyltrimethylammonium chloride, hydrogenated castor oil, and poloxamer 407 in a mass ratio of 1:0.5-0.8:0.2-0.

5.

5. The shampoo composition according to claim 1, characterized in that, The moisturizer is selected from at least one of glycerin, butylene glycol, and 1,3-propanediol; the pH adjuster is citric acid; the preservative is sodium benzoate; the anti-inflammatory and soothing agent is dipotassium glycyrrhizate; the scalp conditioning agent is hydrolyzed silk protein; the chelating agent is disodium EDTA or tetrasodium EDTA; and the thickening and foaming agent is cocamidomethyl MEA.

6. A method for preparing an anti-hair loss active ingredient, characterized in that, Includes the following steps: (1) Aerobic saccharification stage: After crushing the plant raw materials, mix them with deionized water at a mass ratio of 1:3-1:5, inoculate with Bacillus amyloliquefaciens and Bacillus licheniformis, and ferment under aerobic conditions to obtain aerobic saccharification liquid; (2) 75-85℃ short-term enzyme inactivation stage: The aerobic saccharification solution obtained in step (1) is heated at 75-85℃ for 5-15 minutes to inactivate the live Bacillus bacteria while retaining its matrix-bound enzyme system to obtain the enzyme inactivation treatment solution; (3) Anaerobic conversion stage: Inoculate Lactobacillus corni and Lactobacillus casei into the enzyme-inactivating treatment solution obtained in step (2), and carry out fermentation under anaerobic conditions to reduce the pH of the system to 3.5-4.5 to obtain the fermentation conversion solution; (4) Post-processing stage: After fermentation is completed, the fermentation conversion liquid is centrifuged and filtered, and the filtrate is collected to obtain the anti-dehydration active substance.

7. The preparation method according to claim 6, characterized in that, In step (1), the viable counts of both *Bacillus amyloliquefaciens* and *Bacillus licheniformis* are ≥1.0 × 10⁻⁶. 8 The total inoculum amount is 2%-6% of the total mass of the plant material described in step (1); the temperature of the aerobic fermentation is 28-37℃, the aeration rate is 0.5-1.5 vvm, and the time is 12-24h.

8. The preparation method according to claim 6, characterized in that, In step (3), the viable counts of both *Lactobacillus zeatus* and *Lactobacillus casei* are ≥1.0 × 10⁻⁶. 8 The inoculum ratio of CFU / g to Lactobacillus cornis and Lactobacillus casei is 1:0.8-1.2, and the total inoculum is 3%-5% of the volume of the enzyme-inactivating liquid in step (2); the anaerobic fermentation temperature is 30-37℃ and the time is 24-48h.

9. The preparation method according to claim 6, characterized in that, In step (4), the centrifugation speed is 4000-8000 rpm and the time is 10-20 min; after centrifugation, the supernatant is collected and filtered and sterilized using a sterile filter membrane with a pore size of 0.22 μm.

10. A method for preparing a shampoo composition with anti-hair loss and hair growth effects, characterized in that, Includes the following steps: S1. Heat deionized water to 60-65℃, add guar gum hydroxypropyltrimethylammonium chloride, disperse evenly, and obtain phase A pregel; S2. Heat the A-phase pregel obtained in step S1 to 80-85℃, add detergent and thickener / foam stabilizer, keep warm and stir for 15-25 minutes to obtain phase B; S3. After heating hydrogenated castor oil to complete melting, mix and disperse it with poloxamer 407 at 80-85℃ to obtain phase C, and keep it at the temperature for later use; S4. Keep the B phase obtained in step S2 at 80-85℃, add the C phase obtained in step S3, and homogenize at 1000-1500 rpm for 3-5 min. Then cool down to 55-60℃ to obtain a mixture. S5. Continue to cool the mixture obtained in step S4 to 40-45°C, add the anti-hair loss active ingredient, moisturizer, pH adjuster, preservative, fragrance, anti-inflammatory soothing agent, scalp conditioner and chelating agent prepared by the method of claim 6, and stir evenly to obtain the shampoo composition with anti-hair loss and hair growth effects.

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