Sophora flavescens fermentation extract, preparation method and application in anti-hair loss products

Abstract

The invention provides a lightyellow sophora root fermentation extract, a preparation method and application in an anti-hair loss product, the preparation method comprises the following steps: step 1, soaking lightyellow sophora root in water, cutting up, then inoculating a paecilomyces cicadae seed solution for fermentation culture, and after paecilomyces cicadae mycelium overgrows the lightyellow sophora root, drying to obtain a paecilomyces cicadae-lightyellow sophora root bidirectional fermentation product; and 2, crushing the paecilomyces cicadae-radix sophorae flavescentis bidirectional fermentation product, extracting by adopting a bifidobacterium liquid fermentation extraction method, or a hot water extraction method or an absolute ethyl alcohol extraction method, and drying to obtain the radix sophorae flavescentis fermentation extract. The sophora flavescens fermentation extract prepared by the method has good II type 5 alpha reductase inhibition capability, and the ethanol extract CH-KS-A of the penicillium-sophora flavescens bidirectional fermentation product particularly has broad-spectrum antibacterial activity on staphylococcus aureus, propionibacterium acnes and malassezia furfur, and has good anti-inflammatory and antioxidant activity; the composition can be used in anti-hair loss products.

Description

Technical Field

[0001] This disclosure belongs to the field of cosmetic preparation technology, specifically relating to a fermented extract of Sophora flavescens, its preparation method, and its application in anti-hair loss products. Background Technology

[0002] Androgenetic alopecia (AGA) is closely related to mechanisms such as elevated local dihydrotestosterone (DHT) levels in hair follicles, abnormal activation of androgen receptor (AR) signaling, inflammatory responses, and hair follicle cell apoptosis. Currently used treatments such as minoxidil (MXD) and finasteride (FS) have limitations, high relapse rates after discontinuation, and significant side effects. Therefore, developing a multi-target, highly effective, and safe regimen, especially one derived from natural products, has become an important research direction in this field.

[0003] Currently, commonly used plant extracts with hair loss prevention and hair growth-promoting effects include: Platycladus orientalis leaf extract, ginger extract, ginseng extract, Polygonum multiflorum extract, Eclipta prostrata extract, Rhodiola rosea extract, Angelica sinensis extract, Ligusticum chuanxiong extract, and mulberry leaf extract. Sophora flavescens is a legume plant... Sophora flavescens The dried root of *Sophora flavescens* (Ait.) is a traditional Chinese medicine with the effects of clearing heat and drying dampness, killing parasites and relieving itching. It is commonly used in TCM clinical practice to treat inflammatory skin diseases. Modern pharmacological studies have confirmed that *Sophora flavescens* is rich in various bioactive components, mainly including alkaloids, flavonoids, and phenolic acids. Previous studies have reported that *Sophora flavescens* extract has the potential to inhibit 5α-reductase, has antibacterial (e.g., *Propionibacterium acnes*) activity, and anti-inflammatory properties in vitro. These properties are highly correlated with improving the scalp microenvironment and intervening in potential AGA (acne-associated dermatitis). However, current technologies mostly focus on general activity observations of *Sophora flavescens* extract, and it is not yet clear whether and how it can exert its role in preventing and treating AGA by regulating the core AR signaling network, inflammation, and apoptosis pathways within hair follicle cells. In particular, how to further enhance the content and bioefficacy of its active ingredients through modern processes (such as directed fermentation) and systematically elucidate its multi-target mechanism of action remains an unresolved technological gap. Summary of the Invention

[0004] A brief overview of this disclosure is given below to provide a basic understanding of certain aspects of it. It should be understood that this overview is not an exhaustive summary of this disclosure. It is not intended to identify key or essential parts of this disclosure, nor is it intended to limit its scope. Its purpose is merely to present certain concepts in a simplified form as a prelude to the more detailed description that follows.

[0005] To solve the above-mentioned technical problems, the technical solution provided in this disclosure is as follows:

[0006] In a first aspect, this disclosure provides a method for preparing a fermented extract of Sophora flavescens, comprising: Step 1: Soak Sophora flavescens in water, then chop it up and inoculate it with Penicillium cicadae seed liquid for fermentation culture. After the Penicillium cicadae mycelium has fully grown Sophora flavescens, dry it to obtain Penicillium cicadae-Sophora flavescens bidirectional fermentation product. Step 2: The Penicillium cicadae-Sophora flavescens bidirectional fermentation product is crushed, and then extracted using Bifidobacterium liquid fermentation extraction, hot water extraction, or anhydrous ethanol extraction. After drying, the Sophora flavescens fermentation extract is obtained.

[0007] In the above-mentioned method for preparing the fermented extract of Sophora flavescens, as a preferred embodiment, in step one, the Penicillium cicadae is Cordyceps militaris CH2347, with the preservation number CGMCC No.40399.

[0008] In the above-mentioned method for preparing Sophora flavescens fermentation extract, as a preferred embodiment, in step one, the volume ratio of the mycelium of Cordyceps militaris CH2347 in the Penicillium cicadae seed liquid is 50-80% of the total liquid culture medium.

[0009] In a preferred embodiment of the above-mentioned method for preparing the fermented extract of Sophora flavescens, step one of the method for preparing the Penicillium cicadae seed liquid includes: (1) Take 2-3 solid strains of Cordyceps CH2347 with a diameter of about 0.5 cm and grow on PDA plates using a punch, inoculate them into 300 mL of potato glucose water culture medium, and culture at 180 rpm for 3 days at 28℃ to obtain Cordyceps CH2347 seed liquid 1. (2) Homogenize the seed liquid 1 of Cordyceps militaris CH2347, and continue to use it as seed liquid. Take 20 mL and inoculate it into 300 mL potato glucose water culture medium. Culture at 28℃ and 180 rpm for 4 days to obtain Cordyceps militaris CH2347 seed liquid 2, that is, the seed liquid of Cordyceps militaris.

[0010] In the above-mentioned method for preparing the fermented extract of Sophora flavescens, as a preferred embodiment, in step one, soaking Sophora flavescens in water before inoculation is to provide the necessary moisture for the growth of Penicillium cicadae and soften the medicinal tissue, which is conducive to the infection and enzymatic hydrolysis of Penicillium cicadae. It is preferred to soak it in water at room temperature (e.g., 25±2℃) for 2 hours.

[0011] In the preferred embodiment of the above-mentioned method for preparing the fermented extract of Sophora flavescens, in step one, the particle size of the chopped material is 0.3-1 cm, such as 0.3-0.5 cm or 0.6-0.8 cm, approximately the size of a soybean. Directly pulverizing dried Sophora flavescens results in fine particles that easily clump together after adding liquid, leading to poor aeration in the fermentation system and hindering the aerobic mycelial growth of *Penicillium cicadae*. This application uses Sophora flavescens that has been soaked in water, which softens and maintains a loose, porous structure, allowing the mycelium to spread rapidly along the pores and resulting in a more uniform contact area with the substrate. Simultaneously, the aeration and water retention are easier to control, maintaining the aerobic environment required for the metabolism of *Penicillium cicadae*.

[0012] In the above-mentioned method for preparing the fermented extract of Sophora flavescens, as a preferred embodiment, in step one, the inoculation ratio is 5-20 mL of the Penicillium cicadae seed liquid per 60 g of Sophora flavescens soaked in water, more preferably 15 mL of the Penicillium cicadae seed liquid per 60 g of Sophora flavescens soaked in water.

[0013] In the above-mentioned method for preparing Sophora flavescens fermentation extract, as a preferred embodiment, in step one, the fermentation culture temperature is 20~28℃ and the time is 8~20 days; more preferably, the fermentation culture temperature is 27~28℃ and the time is 12 days.

[0014] In the above-mentioned method for preparing Sophora flavescens extract, as a preferred embodiment, the drying in step one is freeze drying.

[0015] In the above-mentioned method for preparing the fermented extract of Sophora flavescens, as a preferred embodiment, step two, the liquid fermentation extraction method of Bifidobacterium includes: crushing the Penicillium cicadae-Sophora flavescens bidirectional fermentation product, passing it through a 50-mesh sieve, homogenizing the Penicillium cicadae-Sophora flavescens bidirectional fermentation product and water at a mass ratio of 1:9, adding 1% by mass of lyophilized Bifidobacterium powder, anaerobic fermenting at 37°C for 24 hours, centrifuging to collect the supernatant, and then freeze-drying to obtain the fermented extract of Sophora flavescens, namely CH-KS-LA.

[0016] In the above-mentioned method for preparing the fermented extract of Sophora flavescens, as a preferred embodiment, step two, the hot water extraction method includes: crushing the Penicillium cicadae-Sophora flavescens bidirectional fermentation product, passing it through a 50-mesh sieve, and extracting it in hot water at 85°C for 3 hours at a mass ratio of 1:99. After centrifugation, the supernatant is collected and freeze-dried to obtain the fermented extract of Sophora flavescens, namely CH-KS-W.

[0017] In the above-mentioned method for preparing the fermented extract of Sophora flavescens, as a preferred embodiment, in step two, the anhydrous ethanol extraction method is anhydrous ethanol Soxhlet reflux extraction; further, the anhydrous ethanol Soxhlet reflux extraction method includes: crushing the Penicillium cicadae-Sophora flavescens bidirectional fermentation product, passing it through a 50-mesh sieve, extracting it with anhydrous ethanol by Soxhlet reflux at a mass ratio of 1:25 for 3 hours, and then extracting it with anhydrous ethanol by Soxhlet reflux at a mass ratio of 1:15 for 3 hours, combining the two filtrates, rotary evaporating at 50°C, and then freeze-drying to obtain the fermented extract of Sophora flavescens, namely CH-KS-A.

[0018] Secondly, this disclosure provides a fermented extract of Sophora flavescens prepared by the above-described preparation method.

[0019] Thirdly, this disclosure provides the application of the above-mentioned Sophora flavescens fermentation extract in hair loss prevention products.

[0020] Fourthly, this disclosure provides a hair loss prevention product, comprising the above-mentioned Sophora flavescens fermented extract, preferably comprising 1 wt% of the Sophora flavescens fermented extract; more preferably, the hair loss prevention product is an essence, formulated by weight percentage from the following raw materials: propylene glycol 3%, glycerin 3%, panthenol 1%, carbomer 940 0.6%, Sophora flavescens fermented extract 1%, triethanolamine 0.35%, p-hydroxyacetophenone 0.5%, 1,2-hexanediol 0.5%, with the balance being deionized water.

[0021] The two-way fermentation technology of traditional Chinese medicine uses Chinese herbal medicines containing active ingredients as the fermentation substrate and beneficial medicinal fungi as the fermentation strain. During the growth of medicinal fungi on the substrate, a series of complex metabolic reactions produce new active ingredients and physiological functions. Therefore, two-way fermentation involves the Chinese herbal medicine substrate providing the necessary carbon and nitrogen sources for fungal growth, while the growth activity of the medicinal fungi drives the transformation of effective components in the Chinese herbal medicine substrate, resulting in a symbiotic and mutually beneficial relationship. This application utilizes two-way fermentation technology to obtain a high-performance Sophora flavescens extract.

[0022] Compared to existing technologies, the beneficial effects of this disclosure include, but are not limited to: 1. This disclosure discloses the preparation of a series of Sophora flavescens extracts and their fermented extracts from Cordyceps militaris CH2347, and evaluates their bioactivity and mechanism of action at the extracellular and intracellular levels, exploring their potential in the intervention of androgenetic alopecia. The ethanol extracts showed significantly higher total flavonoid and total phenolic content than the water extracts, especially the ethanol extract CH-KS-A from the fermentation of Sophora flavescens and Cordyceps militaris, with contents of (142.41±1.81) mg / g and (119.72±0.73) mg / g, respectively. All samples exhibited good type II 5α-reductase inhibition ability, with inhibition rates ranging from 40% to 70%. The ethanol extract CH-KS-A showed broad-spectrum antibacterial activity against Staphylococcus aureus, Propionibacterium acnes, and Malassezia furfur. In addition, the samples exhibited good anti-inflammatory and antioxidant activities.

[0023] 2. Safety evaluation of the ethanol extracts KS-A and CH-KS-A provided in this application showed that the samples met the biocompatibility standards at low concentrations, had no obvious irritant effect, and could effectively inhibit DHT-induced excessive activation of AR signaling at a safe concentration (7.81 μg / mL). Among them, CH-KS-A was significantly better than KS-A in inhibiting excessive activation of cellular AR signaling (##, P<0.01).

[0024] 3. The ethanol extract KS-A and CH-KS-A provided in this application regulate the expression of downstream target genes through multi-pathway synergistic effects: upregulating functional genes related to hair follicle growth such as ALP, IGF-1, and FGF-7, and downregulating inhibitory factors such as DKK-1 and TGF-β. Among them, the upregulation of ALP by CH-KS-A is significantly greater than that by KS-A (###, P<0.001).

[0025] 4. The ethanol extracts KS-A and CH-KS-A provided in this application significantly inhibited the expression of inflammatory factors IL-6 and TNF-α, demonstrating a clear anti-inflammatory effect and restoring the balance between Caspase-3 and Bcl-2. In these two key apoptosis factors, the effect of CH-KS-A was significantly stronger than that of KS-A (###, P<0.001), further demonstrating its strong potential in protecting the survival of dermal papilla cells. Attached Figure Description

[0026] Figure 1 The type II 5α reductase inhibition capacity of the five samples in Example 2 is shown; Figure 2 The results of in vitro anti-inflammatory ability testing of five samples in Effect Example 4 are shown. Figure A shows the results of the hyaluronidase inhibition experiment, and Figure B shows the results of the cyclooxygenase 2 (COX-2) inhibition experiment. Figure 3The results of in vitro antioxidant capacity testing in Example 5 are shown. Figures A and C show the DPPH free radical scavenging effect, Figures B and D show the hydroxyl free radical scavenging effect, Figure E shows the ABTS free radical scavenging effect, and Figure F shows the iron ion reducing capacity test effect. Figure 4 The diagram shows the results of cytotoxicity and cell proliferation detection of the samples in Example 6. Figure A shows the effect of KS-A and CH-KS-A on HDPC cell viability, Figure B shows the effect of minoxidil (MXD) on HDPC cell viability, and Figures C, D and E show the cell protective effects of KS-A, CH-KS-A and minoxidil after DHT model intervention, respectively. Figure 5 The effects of samples KS-A, CH-KS-A, and the positive control minoxidil (MXD) on the expression of genes such as AR are shown in Example 7. The AF diagram shows the effects of the samples on the gene expression levels of AR, TGF-β, ALP, DKK-1, IGF-1, and FGF-7 in HDPCs cells, respectively. Figure 6 The effects of samples KS-A, CH-KS-A, and the positive control minoxidil (MXD) in Effect Example 7 on the mRNA expression of inflammatory factors (IL-6, TNF-α) and apoptosis-related genes (Caspase-3, Bcl-2) are shown. The AD diagram shows the effects of the samples on the gene expression levels of IL-6, TNF-α, Caspase-3, and Bcl-2 in HDPCs cells, respectively. Figure 7 A photograph of the mouse experiment in Example 8 is shown. Detailed Implementation

[0027] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present disclosure. Any modifications or substitutions made to the methods, steps, or conditions of the present disclosure without departing from the spirit and substance of the present disclosure are within the scope of the present invention.

[0028] The technical solutions of this disclosure will be described below with reference to exemplary embodiments. Unless otherwise specified, the experimental methods used in the following embodiments and comparative examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples and comparative examples are commercially available.

[0030] The strains used in the following examples or comparative examples are as follows: *Cicada nymph* CH2347, accession number CGMCCNo.40399, previously disclosed in patent document CN118853418A, "New fungal strain *Cicada nymph* CH2347, *Cicada nymph* fermentation product and its preparation method, cosmetics, *Cicada nymph* mycelial extract and purified polysaccharide". *Bifidobacterium* HH-LP56, purchased from Zhengzhou Hehe Biotechnology Co., Ltd.

[0031] Preparation Example 1: Preparation of Sophora flavescens extract and fermentation products 1. Preparation of Penicillium cicadae seed liquid: 1) Inoculate the CH2347 solid inoculum of Cordyceps militaris grown on PDA plates (with culture medium; use a sterilized punch to take 2-3 solid inoculum cells with a diameter of about 0.5 cm) onto sterilized plates (115℃, 20°C). 1) In 300 mL of potato glucose water medium (Haibo Biotechnology Co., Ltd., model HB0233-4), the mycelium was cultured at 28℃ and 180 rpm for 3 days to obtain *Cicada spores* CH2347 seed liquid 1. At this time, the mycelial balls of *Cicada spores* CH2347 seed liquid 1 were of different sizes. 2) *Cicada spores* CH2347 seed liquid 1 was homogenized using a sterile glass homogenizer, and 20 mL was taken as seed liquid and inoculated into 300 mL of potato glucose water medium (Haibo Biotechnology Co., Ltd., model HB0233-4). It was cultured at 28℃ and 180 rpm for 4 days to obtain *Cicada spores* CH2347 seed liquid 2. At this time, the volume of *Cicada spores* CH2347 mycelium accounted for about 80% of the total liquid medium volume, which is the *Penicillium ciliata* seed liquid used for inoculation.

[0032] 2. Two-way fermentation of *Penicillium cicadae* and *Sophora flavescens*: Soak Sophora flavescens in water at room temperature (25±2℃) for 2 hours, then chop it into soybean-sized pieces. Take 60g and put it into a fermentation bottle. Sterilize at 121℃ for 30 minutes, add 15ml of Penicillium cicadae liquid (seed liquid), and incubate in a 28℃ incubator for 12 days. After the Penicillium cicadae mycelium has fully grown on Sophora flavescens, freeze-dry the fermentation product to obtain Penicillium cicadae-Sophora flavescens bidirectional fermentation product.

[0033] 3. Preparation of Sophora flavescens fermentation extract samples: 3.1 The Penicillium ciliata-Sophora flavescens bidirectional fermentation product was crushed with a pulverizer and passed through a 50-mesh sieve. Then, the Penicillium ciliata-Sophora flavescens bidirectional fermentation product and water were homogenized at a mass ratio of 1:9. 1% Bifidobacterium lyophilized powder was added, and the mixture was anaerobic fermented at 37℃ for 24 hours. After centrifugation, the supernatant was collected and freeze-dried to obtain the sample CH-KS-LA.

[0034] 3.2 The Sophora flavescens and Penicillium cicadae-Sophora flavescens bidirectional fermentation products were crushed by a pulverizer, passed through a 50-mesh sieve, and extracted with deionized water at 85℃ for 3 hours at a mass ratio of 1:99. After centrifugation, the supernatant was collected and freeze-dried to obtain samples KS-W and CH-KS-W, respectively.

[0035] 3.3 The *Sophora flavescens* and *Penicillium cicadae*-*Sophora flavescens* bidirectional fermentation products were crushed separately using a pulverizer, passed through a 50-mesh sieve, and extracted with anhydrous ethanol by Soxhlet reflux at a mass ratio of 1:25 for 3 hours, followed by extraction with anhydrous ethanol by Soxhlet reflux at a mass ratio of 1:15 for 3 hours. The two filtrates were combined, rotary evaporated at 50℃, and then freeze-dried to obtain samples KS-A and CH-KS-A, respectively. Here, the mass ratio refers to the ratio of the mass of *Sophora flavescens* / *Penicillium cicadae*-*Sophora flavescens* bidirectional fermentation product to the mass of anhydrous ethanol.

[0036] Example 1: Determination of the content of active substances The content of active substances in the secondary fermentation product CH-KS-LA, water extracts KS-W and CH-KS-W, and ethanol extracts KS-A and CH-KS-A of the five samples obtained in Preparation Example 1 was determined.

[0037] 1.1 Determination of total flavonoid content: Prepare 10 mg / mL sample solutions, 5% NaNO2, 10% Al(NO3)3, 1 mol / L NaOH and rutin standard solutions (0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, 0.015625 mg / mL). Take 400 μL of sample or standard solution into a 1.5 mL EP tube, add 50 μL of 5% NaNO2, mix well, and let stand for 6 min. Then add 50 μL of 10% Al(NO3)3, mix well, and let stand for 6 min. Finally, add 500 μL of 1 mol / L NaOH, mix well, and let stand for 15 min. Measure the absorbance at 510 nm using a microplate reader. Set up three replicates for each sample. Plot the rutin standard curve with rutin concentration as the x-axis and absorbance as the y-axis: y = 2.3317x + 0.0403, R0. 2 =0.9964. See Table 1 for the results.

[0038] 1.2 Determination of total phenol content: Prepare 5 mg / mL sample solutions, 10% Folin-phenol solution, 700 mmol / L Na₂CO₃ solution, and gallic acid standard solutions (0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL), respectively. Take 100 μL of sample or standard solution into a 1.5 mL EP tube, add 200 μL of 10% Folin-phenol solution, vortex to mix, then add 800 μL of 700 mM Na₂CO₃ solution. Incubate at room temperature (25°C) for 2 h. Measure the absorbance at 765 nm using a microplate reader. Each sample is tested in triplicate. Plot the gallic acid standard curve with gallic acid concentration on the x-axis and absorbance on the y-axis as y = 3.3477x + 0.0667, R0. 2 =0.9904. See Table 1 for the results.

[0039] 1.3 Determination of total sugar content: Prepared sample solutions of 0.125 mg / mL, 6% phenol solution, and D-glucose standard solutions at concentrations of 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, and 0 mg / mL. In a 1.5 mL centrifuge tube, sequentially add 200 μL of sample, 100 μL of phenol solution, and 400 μL of concentrated sulfuric acid solution, mix well, and incubate in a boiling water bath for 30 min. Three sample groups were set up: the standard curve group consisted of diluted glucose solution; the sample group consisted of sample diluted to a concentration of 0.125 mg / mL; and the blank control group consisted of distilled water. 200 μL of each sample was pipetted into a 96-well plate, and the plate was zeroed using a blank well. The absorbance was measured at 490 nm using a microplate reader. Each sample was prepared in triplicate. A glucose standard curve was plotted with D-glucose concentration on the x-axis and absorbance on the y-axis as y = 7.2567x + 0.1315, R0. 2 =0.997. See Table 1 for the results.

[0040] 1.4 Determination of total protein content: The total protein content of the samples was determined using the Bradford method protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the instructions. The results are shown in Table 1.

[0041] Table 1 Content of active substances

[0042] The content of active substances is shown in Table 1. The total flavonoid content of each sample ranged from 33.44 to 142.41 mg / g DW. The total flavonoid content of the ethanol extracts KS-A and CH-KS-A was significantly higher than that of the corresponding water extracts KS-W and CH-KS-W, with CH-KS-A having the highest total flavonoid content. CH-KS-A had the highest total phenolic content (119.72 ± 0.73 mg / g DW), followed by KS-A and CH-KS-W. The secondary fermentation product CH-KS-LA had a relatively low total phenolic content but the highest total sugar content. The total protein content was higher in CH-KS-W and CH-KS-LA.

[0043] Example 2: Test of type II 5α reductase inhibition ability 5α-Reductase (5αR) is a key enzyme in the metabolism of the androgen testosterone (T). Under electron-donating conditions provided by reduced coenzyme II (NADPH), it converts testosterone into dihydrotestosterone (DHT). Overexpression of 5αR leads to high levels of DHT. DHT binds to the androgen receptor (AR) five times more readily than T. High levels of DHT binding to the androgen receptor can easily cause androgen-dependent diseases. Type II 5α-reductase (5αR2, encoded by the SRD5A2 gene) is mainly expressed in tissues such as the prostate and hair follicles, and is prone to causing androgenetic alopecia.

[0044] This experiment was conducted according to the instructions for the Type II (SRD5A2) 5α-reductase inhibition rate assessment kit (Huizhiheyuan Biotechnology (Suzhou) Co., Ltd.). Test results are shown below. Figure 1 As shown in Table 2, the inhibitory activity of five samples against 5αR2 at concentrations of 2 mg / mL and 1 mg / mL was determined. The results showed that all samples had a certain degree of inhibitory activity against 5αR2. Finasteride (FS) was used as a positive control and showed a 5αR2 clearance rate of approximately (91.18±6.45)% at a concentration of 0.025 mg / mL, demonstrating strong clearance activity. The clearance rates of each sample were between 40% and 70%, and CH-KS-A also showed good clearance ability at a lower concentration (1 mg / mL).

[0045] Table 25 Results of αR2 Inhibition Ability Test

[0046] Example 3: Antibacterial Ability Test In the pathogenesis of AGA, scalp microbial imbalance is an important synergistic pathogenic factor. Therefore, targeting Staphylococcus aureus (S. aureus) is crucial. S. aureus ), Propionibacterium acnes ( C. acnes Malassezia furfur ( ), M. furfur The antibacterial experiment of these bacteria is of key significance for the study of their efficacy in preventing hair loss. These bacteria secrete metabolites such as lipases and proteases, which damage the scalp barrier, induce the release of pro-inflammatory factors such as IL-6 and TNF-α, and thus damage hair follicles (especially hair papilla cells) and accelerate hair follicle miniaturization. M. furfur Bacterial breakdown can produce free fatty acids from scalp sebum, stimulating excessive sebum secretion. Sebum buildup further promotes bacterial proliferation, creating a vicious cycle of "microbial proliferation - sebum dysbiosis - inflammatory damage," thus exacerbating hair loss. Therefore, assessing the inhibitory effect of test substances on these key pathogens is an important indicator for evaluating their potential to alleviate hair loss by regulating the scalp microecology and improving hair follicle health.

[0047] 3.1 Staphylococcus aureus culture: Weigh Luria-Bertani broth / agar (OXOID, UK) and prepare according to the instructions. After preparation, autoclave at 121℃ for 15 minutes. Use a sterile inoculation loop to pick up the frozen bacterial culture and streak it in three zones on the surface of a solid agar plate. Then invert the plate and incubate at 37℃ for 18-24 hours. Pick typical single or isolated colonies from the plate and inoculate them into liquid culture medium. Incubate at 37℃ and 180 rpm with shaking for 12 hours to obtain the seed culture. Then subculture at an inoculation rate of 1% (v / v).

[0048] 3.2 Propionibacterium acnes culture: Weigh Brain Heart Infusion (BHI) broth / agar (Beijing Aoboxing Biotechnology Co., Ltd.), prepare according to the instructions, and autoclave at 121℃ for 15 minutes. Use a sterile inoculation loop to pick up the frozen bacterial culture and streak it in three zones on the surface of a solid agar plate. Then, invert the plate and anaerobically incubate at 37℃ for 3-5 days. Pick typical single or isolated colonies from the plate and inoculate them into liquid culture medium. Incubate anaerobically at 37℃ for 3-5 days to obtain the seed culture. Then, subculture at an inoculation rate of 5% (v / v).

[0049] 3.3 Malassezia furfur culture: Weigh 2963 modified Dixon medium and Tween 40 (Qingdao Haibo Biotechnology Co., Ltd.), prepare according to the instructions, and autoclave at 121℃ for 15 minutes. Use a sterile inoculation loop to pick up the frozen inoculum and streak it in three zones on the surface of a solid agar plate, then invert it and incubate at 30℃ for 5-7 days. Pick typical single or isolated colonies from the plate and inoculate them into liquid medium, incubate at 30℃ and 180 rpm for 5-7 days as seed culture, and then subculture at an inoculation rate of 5% (v / v).

[0050] 3.4 Inhibition zone diameter test The sample pair was determined using the plate punching method. S. aureus , C. acnes and M. furfur The diameter of the inhibition zone. The specific method is as follows: Take 200 μL of a concentration of 1×10⁻⁶... 6 CFU / mL bacterial suspensions were evenly spread onto solid agar plates using a spreader method, and wells were punched using a 10 mm diameter sterile punch. 100 μL of sample was added to each well, and incubation was continued: Staphylococcus aureus was incubated at 37°C for 16 h, Propionibacterium acnes for 48 h anaerobic incubation at 37°C, and Malassezia furfur for 72 h. The diameter (d) of the inhibition zone was then measured. All test samples were prepared to a concentration of 20 mg / mL, with the negative control being the sample solvent. S. aureus and C. acnes The positive control was 5 μg / mL erythromycin. M. furfur The positive control was 0.5 mg / mL ketoconazole.

[0051] The results are shown in Table 3. Among the tested samples, KS-A and CH-KS-A exhibited superior broad-spectrum antibacterial activity. S. aureus , C. acnes The inhibition zone is approximately 22.4 mm in diameter, and has an inhibitory effect on... M. furfur The inhibition zone diameter reached 20.00~21.13 mm, among which... M. furfur The antibacterial effect was close to that of the positive control ketoconazole (22.33 mm). KS-W and CH-KS-W showed weaker antibacterial activity and species selectivity, only effective against certain bacteria. S. aureus , C. acnes It exhibits weak inhibitory activity (inhibition zone diameter 12.5~13.87 mm), and has a relatively low inhibitory effect on bacteria. M. furfur The inhibition zone diameter was only about 3 mm, indicating extremely weak activity. CH-KS-LA showed no antibacterial activity against any of the tested pathogens (inhibition zone diameter was 0 mm).

[0052] Table 3 Results of the inhibition zone diameter test

[0053] 3.5 Minimum Inhibitory Concentration (MIC) Test The micro-broth method was used to determine the sample's effect. S. aureus , C. acnes and M. furfurThe minimum inhibitory concentration (MIC) was determined. The specific method was as follows: the sample was diluted twofold to different concentrations ranging from 0.15625 to 20 mg / mL. In a 96-well plate, 100 μL of a 1×10⁻⁶ mg / mL solution was added to each well. 6 The bacterial suspension at CFU / mL and 100 μL of sample solution were cultured for a further period of time. Staphylococcus aureus was cultured at 37℃ for 16 h, Propionibacterium acnes was cultured anaerobically at 37℃ for 48 h, and Malassezia furfur was cultured at 30℃ for 72 h. Finally, the minimum inhibitory concentration was determined by visual observation. S.aureus and C.acnes The positive control was erythromycin. M.furfur The positive control was ketoconazole.

[0054] The results are shown in Table 4. The antibacterial activity of the tested samples was negatively correlated with the MIC value. Specifically, KS-A and CH-KS-A exhibited excellent antibacterial activity. C. acnes The MIC values ​​for all samples were 0.15625 mg / mL, consistent with the positive control erythromycin; S. aureus Its MIC value is 0.15625~0.3125 mg / mL, which is close to the activity of erythromycin; for M. furfur The MIC values ​​were 1.25–2.5 mg / mL, significantly better than other tested samples. Among them, CH-KS-A exhibited the best overall antibacterial activity.

[0055] Table 4 Results of Minimum Inhibitory Concentration Test

[0056] Example 4: Extracellular anti-inflammatory efficacy test 4.1 Hyaluronidase Inhibition Experiment Methods: Reagent preparation: (1) Acetic acid buffer (pH=5.6): Mix 4.8 mL of 0.2 mol / L acetic acid solution and 45.2 mL of 0.2 mol / L sodium acetate solution, then add water to 100 mL; (2) P-DAB colorimetric reagent: Mix 0.8 g of p-dimethylaminobenzaldehyde with 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol; (3) 0.5 mg / mL sodium hyaluronate solution; (4) 500 U / mL hyaluronidase solution; (5) 2.5 mol / L CaCl2 solution; (6) 5.0 mol / L NaOH solution; (7) Acetylacetone solution: Dissolve 3.5 mL of acetylacetone in 50 mL of sodium carbonate solution (1.0 mol / L), prepare fresh before use. Sample preparation: Prepare samples using PBS solution at a concentration of 4 mg / mL. Add samples according to the following table and measure the OD value, and calculate the inhibition rate according to the following formula: Inhibition rate (%) = [ (A - B) - (C - D) ] / (A - B) × 100%.

[0057] Table 5 Grouping of the hyaluronidase inhibition experiment

[0058] See results Figure 2 Figure A in the diagram. (As shown in the image) Figure 2 As shown in Figure A, using 1 mg / mL dipotassium glycyrrhizinate (DG) as a positive control, the inhibitory activities of the various samples showed different results. Among them, CH-KS-A had an inhibition rate of approximately 55%, which was significantly better than the positive control. (P<0.001); KS-A had an inhibition rate of about 40%, while KS-W, CH-KS-W, and CH-KS-LA had an inhibition rate of only about 20%, which was significantly lower than the positive control.

[0059] 4.2 Cyclooxygenase 2 (COX-2) Inhibition Experiment Follow the instructions for use of the cyclooxygenase-2 (COX-2) inhibitor screening kit (Shanghai Beyotime Biotechnology Co., Ltd.). Sample preparation: Prepare samples using the COX-2 Assay Buffer solution provided in the kit at a concentration of 4 mg / mL.

[0060] See results Figure 2 Figure B in the diagram. (For example...) Figure 2As shown in B, with 100 μM celecoxib as the positive control, the COX-2 inhibitory activities of each sample were as follows: CH-KS-A (approximately 95%) and KS-A (approximately 90%) showed relatively high inhibitory activities, close to the positive control level; CH-KS-W (approximately 60%) and KS-W (approximately 50%) showed moderate inhibitory activities; and CH-KS-LA (approximately 40%) showed the lowest inhibitory activity.

[0061] Example 5: Extracellular Antioxidant Efficacy Test 5.1 DPPH free radical scavenging experiment Methods: Take 400 μL of sample solutions with different mass concentrations (0.125-4 mg / mL), add 400 μL of 0.2 mmol / L DPPH solution respectively, mix well, and let stand in the dark for 30 min. Measure the absorbance (OD) of the mixture at a wavelength of 517 nm. 517 ), denoted as Ax; similarly, take 400 μL of sample solutions of different mass concentrations, add an equal volume of anhydrous ethanol, mix well, react in the dark for 30 min, and then measure OD. 517 Let A1 be the absorbance value. Take 400 μL of 0.2 mmol / L DPPH solution, add 400 μL of anhydrous ethanol, and record the absorbance value as A0 using the same method. Calculate the DPPH free radical scavenging rate using the following formula: Scavenging rate (%) = [(A0 + A1 - Ax) / A0] × 100%. See the results below. Figure 3 Figures A and C in the diagram.

[0062] 5.2 Hydroxyl radical scavenging experiment The hydroxyl radical scavenging capacity was determined using the salicylic acid method. The specific method was as follows: Prepare 3.0 mmol / L ethanol-salicylic acid solution, 8.0 mmol / L FeSO4 solution, and 0.02 mmol / L hydrogen peroxide solution. Take 500 μL of sample solutions with different mass concentrations (0.125–4 mg / mL), add 150 μL of FeSO4 solution, 500 μL of salicylic acid, 225 μL of deionized water, and 125 μL of hydrogen peroxide solution, respectively. Mix thoroughly and react in a 37°C water bath for 1 h at 4000 rpm·min. -1 Centrifuge for 10 min, and measure the absorbance of the mixture at a wavelength of 517 nm, denoted as Ax; the absorbance measured without hydrogen peroxide solution is denoted as Ax0. Following the same method, replace the sample solution with deionized water and measure its absorbance, denoted as A0. The hydroxyl radical scavenging rate is calculated using the following formula: Scavenging rate (%) = [(A0 - (Ax - Ax0)) / A0] × 100%. See results below. Figure 3 Figures B and D in the diagram.

[0063] 5.3 ABTS Free Radical Scavenging Experiment All samples were dissolved in PBS to prepare a sample solution with a concentration of 4 mg / mL. The positive control was vitamin C at a concentration of 50 μg / mL. The procedure was performed according to the instructions of the Total Antioxidant Capacity Assay Kit (ABTS method) (Shanghai Beyotime Biotechnology Co., Ltd.). Results are shown below. Figure 3 Figure E in the diagram.

[0064] 5.4 Experiment on the reducing power of iron ions All samples were dissolved in PBS to prepare a sample solution with a concentration of 4 mg / mL. The positive control was vitamin C at a concentration of 50 μg / mL. The procedure was performed according to the instructions of the Total Antioxidant Capacity Assay Kit (FRAP method) (Shanghai Beyotime Biotechnology Co., Ltd.). Results are shown below. Figure 3 The F-graph in the diagram.

[0065] See the experimental results. Figure 3 .from Figure 3 As shown in Figures A, B, C, and D, with increasing concentration, the scavenging rates of DPPH and hydroxyl radicals in all samples gradually increased and tended to saturate. CH-KS-A exhibited a significantly lower half-maximum concentration (EC50) than the other samples, demonstrating the strongest activity, while CH-KS-LA had the highest EC50 value and the weakest activity. Figure 3 In Figure E, CH-KS-W showed the strongest total antioxidant capacity and the highest equivalent value, while CH-KS-A had a significantly lower equivalent value. From... Figure 3 As can be seen from the F-plot, CH-KS-W has the strongest FeSO4 equivalent reducing power, CH-KS-LA has a slightly better FeSO4 equivalent reducing power than the positive control VC (ascorbic acid), and CH-KS-A has a weaker reducing power.

[0066] Example 6: Detection of sample cytotoxicity and cell proliferation Cytotoxicity assay of samples: HDPCs cells in good growth condition were selected and subjected to a concentration of 1.5 × 10⁻⁶. 4 Cells were added at a density of 100 cells per well to 96-well culture plates. After culturing for 24 hours, the supernatant was discarded, and samples of different concentrations (7.81-61.25 μg / mL) were added, followed by another 24 hours of culturing. Detection was performed using the Cell Counting Kit-8 (CCK-8) kit (Beijing Bairui Biotechnology Co., Ltd.).

[0067] Cell proliferation assay of samples: First, select HDPCs cells in good growth condition, and then... 4Cells were added to 96-well plates at a density of 100 cells per well. After 24 h of stimulation with 31.25 μg / mL DHT solution, the supernatant was discarded, and samples of different concentrations were added. Cells were cultured for another 24 h, and cell viability was then assessed.

[0068] The results are as follows Figure 4 As shown in the figure, Figure A illustrates the effects of KS-A and CH-KS-A on HDPC cell viability, Figure B shows the effect of minoxidil (MXD) on HDPC cell viability, and Figures C, D, and E show the cytoprotective effects of KS-A, CH-KS-A, and minoxidil after DHT model intervention, respectively. In the absence of model intervention, the effects of KS-A and CH-KS-A on cell viability decreased in a concentration-dependent manner; at low concentrations (15.63 μg / mL), cell viability remained above 80%; however, cell viability decreased significantly with increasing concentrations. Minoxidil (MXD) maintained cell viability above 90% within the concentration range of 0–50 μg / mL, without significant cytotoxicity.

[0069] After constructing the cell model using DHT, the cell viability in the Model group was significantly reduced. Within the concentration range of 3.91–15.63 μg / mL, the cell viability of KS-A increased with increasing concentration, significantly alleviating the cytotoxicity of the model. Within the concentration range of 1.95–15.63 μg / mL, the cell viability of CH-KS-A increased in a concentration-dependent manner, and a concentration of 7.81 μg / mL significantly restored cell viability. Therefore, 7.81 μg / mL of KS-A and CH-KS-A was selected for subsequent experiments. Minoxidil significantly improved cell viability within the concentration range of 3.13–25 μg / mL; subsequently, 5 μg / mL was chosen as the effective concentration.

[0070] Example 7: Detection of gene expression related to qRT-PCR (1) RNA extraction: First, discard the supernatant in the six-well plate, wash with PBS 2-3 times, add 1 mL of total RNA extraction reagent (Beijing Solarbio Science & Technology Co., Ltd.) to lyse the cells, then transfer to an enzyme-free centrifuge tube, add 200 μL of pre-chilled chloroform, mix thoroughly, allow to separate into layers, and centrifuge at 12000 xg for 15 min. Aspirate 400 μL of the upper aqueous phase into a new enzyme-free centrifuge tube, add 400 μL of pre-chilled isopropanol, invert to mix, precipitate on ice for 10 minutes, centrifuge again, and discard the supernatant. Add 1 mL of 75% ethanol, mix well, centrifuge, and discard the supernatant. Repeat this step twice. Finally, open the cap, invert, and air dry the centrifuge tube.

[0071] (2) cDNA conversion: Add 30 μL of DEPC water to the dried RNA, mix gently, detect the RNA content, and use the UEIris II RT-PCR System for First-Strand cDNA Synthesis (with dsDNase) kit (Beijing Bairui Biotechnology Co., Ltd.) for reverse transcription to obtain cDNA.

[0072] (3) Finally, RT-PCR experiments were performed using the Universal SYBR Green qPCR Supermix kit (Beijing Bairui Biotechnology Co., Ltd.). A three-step amplification method was used for detection; specific operating procedures are detailed in the manufacturer's instructions. Gene information and primer sequence design were completed using the NCBI platform, as shown in Table 6.

[0073] Table 6 Real-Time PCR Primer Sequences

[0074] 7.1 Regulatory effects on androgen receptors and related signaling pathways Alkaline phosphatase (ALP), a key marker enzyme for the differentiation and functional activity of dermal papilla cells, is negatively regulated by androgen receptor (AR) signaling. Upon binding to AR, DHT recruits transcriptional repressors (such as histone deacetylase HDAC), thereby inhibiting ALP promoter activity and leading to downregulation of ALP expression. In dermal papilla cells, reduced ALP activity hinders normal extracellular matrix mineralization, affecting the maintenance of the hair follicle cycle and contributing significantly to hair follicle miniaturization. Conversely, DKK-1, a classic inhibitor of the Wnt signaling pathway, is directly positively regulated by AR signaling. Wnt signaling is crucial for hair follicle stem cell proliferation and regeneration; increased DKK-1 expression inhibits this pathway, leading to quiescent hair follicle stem cells and follicle atrophy. Dermal papilla cells play a central role in hair follicle development and cycle regulation, maintaining normal hair follicle growth through the secretion of insulin-like growth factor (IGF-1) and vascular endothelial growth factor (VEGF). IGF-1 and fibroblast growth factor-7 (FGF-7) are key growth factors regulating cell proliferation and differentiation, and both are transcriptionally repressed by the DHT-AR complex. In dermal papilla cells, decreased expression of IGF-1 and FGF-7 weakens cell proliferation and follicle nutritional support, directly contributing to the progression of follicle miniaturization in androgenetic alopecia.

[0075] Based on a DHT-induced cell model, this study examined the effects of KS-A, CH-KS-A, and the positive control minoxidil (MXD) on the expression of genes such as AR, ALP, DKK-1, IGF-1, FGF-7, and TGF-β (see [link to study]. Figure 5 Figures A, B, C, E, and F in the diagram show that both KS-A and CH-KS-A significantly inhibited AR mRNA expression, with a superior inhibitory effect compared to MXD, indicating that both extracts can effectively antagonize DHT-induced excessive activation of AR signaling. ALP expression was significantly decreased in the model group, but after CH-KS-A treatment, ALP expression was significantly upregulated, with a recovery rate comparable to the MXD-treated group. Furthermore, IGF-1 and FGF-7 expression were significantly decreased in the model group, and KS-A and CH-KS-A significantly increased IGF-1 expression levels, with better effects than MXD, suggesting that the samples can reverse the transcriptional repression of growth factor-related genes by DHT.

[0076] Regarding DKK-1, its expression was significantly increased in the model group. Treatment with KS-A and CH-KS-A significantly downregulated DKK-1 expression, with stronger effects than MXD. Simultaneously, TGF-β expression increased in the model group, and KS-A and CH-KS-A significantly inhibited its expression, with inhibitory effects similar to MXD. These results indicate that both KS-A and CH-KS-A extracts from *Paecilomyces pubescens* can effectively inhibit the abnormally high expression of DHT-induced fibrosis-related genes, thereby alleviating the pathological process related to androgenetic alopecia at the molecular level. The ethanol extract CH-KS-A from the bidirectional fermentation of *Paecilomyces pubescens* showed significantly better performance.

[0077] 7.2 Anti-inflammatory and anti-apoptotic effects IL-6 and TNF-α are key pro-inflammatory cytokines. In the process of androgenetic alopecia, DHT binding to AR can activate the NF-κB signaling pathway (AR can directly interact with the p65 subunit of NF-κB and enhance its transcriptional activity), thereby promoting the transcriptional expression of IL-6 and TNF-α. In dermal papilla cells, overexpression of IL-6 and TNF-α induces cellular inflammatory stress, disrupts the homeostasis of the hair follicle microenvironment, and inhibits the activity of hair follicle stem cells through paracrine mechanisms.

[0078] The balance of apoptosis is mainly regulated by the pro-apoptotic gene Caspase-3 and the anti-apoptotic gene Bcl-2, both of which are influenced by the DHT-AR signaling pathway. Studies have shown that the DHT-AR complex can upregulate the transcription and activation of Caspase-3 by activating the JNK signaling pathway; simultaneously, excessive activation of AR signaling inhibits Bcl-2 promoter activity (e.g., through recruitment of histone deacetylases), thereby downregulating its expression. In hair follicles, increased Caspase-3 expression and decreased Bcl-2 expression can disrupt the apoptotic balance, inducing apoptosis in hair follicle epithelial cells and directly participating in hair follicle degeneration. This study confirms that DHT can upregulate Caspase-3 and downregulate Bcl-2, further clarifying the key molecular targets of androgen-induced hair follicle cell apoptosis.

[0079] Based on a DHT-induced cell model, this study further examined the effects of KS-A, CH-KS-A, and the positive control MXD on the mRNA expression of inflammatory factors (IL-6, TNF-α) and apoptosis-related genes (Caspase-3, Bcl-2). The results are shown below. Figure 6 Regarding inflammatory factors, both KS-A and CH-KS-A significantly inhibited IL-6 mRNA expression, with significantly better effects than MXD (##, P < 0.01) (see [link to relevant documentation]). Figure 6 (See Figure A in the table). Both also significantly reduced TNF-α mRNA expression, with CH-KS-A showing a significantly better effect than MXD (###, P < 0.001) (see Figure A in the table). Figure 6 Figure B in the diagram shows that the sample can effectively alleviate DHT-induced cellular inflammatory responses.

[0080] See Figure 6 Figures C and D in the diagram show that, regarding apoptosis-related genes, Caspase-3 expression was significantly increased and Bcl-2 expression was significantly decreased in the DHT model group. After treatment with KS-A and CH-KS-A, Caspase-3 expression was significantly downregulated, with CH-KS-A showing a significantly stronger effect than KS-A (###, P<0.001) and MXD (#, P<0.05). Simultaneously, Bcl-2 expression significantly increased, with CH-KS-A showing a significantly better regulatory effect than KS-A (###, P<0.001) and also better than MXD (#, P<0.05). These results indicate that KS-A and CH-KS-A can reverse the DHT-induced imbalance in apoptosis gene expression, thereby inhibiting the apoptosis process in hair follicle cells, with CH-KS-A showing a significantly superior effect.

[0081] Example 8: Mouse experiments were conducted using the ethanol extract sample CH-KS-A solution of the bidirectional fermentation product of Paecilomyces muscardinae obtained in Preparation Example 1 and the essence prepared therefrom.

[0082] 8.1 Preparation method of serum: Referring to the formulation shown in Table 7, weigh 80% of the total deionized water shown in Table 7 into a beaker. While stirring slowly and continuously at 300 rpm, evenly sprinkle Carbomer 940 onto the liquid surface in portions, continuing stirring for 20-30 minutes to allow it to fully swell. Then, slowly add pre-diluted triethanolamine; the system will then transform into a viscous matrix. In another beaker, add glycerol, propylene glycol, panthenol, and CH-KS-A sequentially, dissolving them with a small amount of deionized water and then transferring the solution to the main gel system. Finally, dissolve p-hydroxyacetophenone in a mixture of 1,2-hexanediol, add it to the system, and stir until homogeneous.

[0083] Table 7 Serum Formula

[0084] 8.2 Mouse treatment methods: SPF-grade male C57BL / 6J mice, weighing 19.86g-21.64g before the experiment, were provided by Spiford (Suzhou) Biotechnology Co., Ltd. After a 7-day acclimatization period, all mice were housed in the same SPF-grade animal facility. All animals underwent necessary systemic health checks before exposure; healthy animals were selected for this experiment. All animals were fed standard formulated feed and had unrestricted water intake. After anesthesia, the mice were shaved in a rectangular area slightly larger than 2cm × 2cm on both sides of the spine. Depilatory cream was evenly applied to the shaved area, left for 3-5 minutes, then wiped off and washed to remove any remaining cream. Except for the control group, all mice in each group received an intraperitoneal injection of 25mg / mL testosterone propionate solution at a dose of 80mg / kg, once daily for 16 consecutive days. Drug intervention was administered 30 minutes after modeling each day.

[0085] Mice were randomly divided into 5 groups: blank group, model group, positive control group, sample group, and essence group, with 3 mice in each group. The mice were given samples for 16 days, and the first day of sample administration was defined as D1.

[0086] (1) Control group: Hair removal on the back, with 100μL of physiological saline applied daily. (2) Model group: After hair removal on the back, physiological saline was applied externally 30 min after modeling, 100μL / animal / day.

[0087] (3) Positive control group: After hair removal on the back, 5% minoxidil solution was applied externally 30 min after modeling, 100 μL / animal / day.

[0088] (4) Sample group (CH-KS-A group): Dissolve the lyophilized CH-KS-A in a mixed solution of 1,2-propylene glycol:water = 1:9 (volume ratio) at a concentration of 10 mg / mL; after hair removal on the back, apply the sample solution of 10 mg / mL to the skin 30 min after modeling, 100 μL / animal / day.

[0089] (5) Essence group: After hair removal on the back, apply essence 30 minutes after modeling, 100μL / each / day.

[0090] On day 16, after anesthesia, photos were taken to observe changes in mouse skin color and hair growth.

[0091] See results Figure 7As can be seen, at the end of the experiment, the hair growth on the backs of mice in the sample group and the essence group was significantly better than that in the model group, showing a good hair growth promoting effect. In summary, the fermented extract of Sophora flavescens provided in this disclosure, especially the ethanol extract of Sophora flavescens after fermentation by Penicillium cicadae, has multi-target and multi-pathway bioregulatory functions. It can not only inhibit enzyme activity and microbial growth exogenously, but also antagonize androgen signals in cells, promote the expression of hair follicle-related functional genes, and inhibit inflammation and apoptosis, showing good potential for the prevention and treatment of androgenetic alopecia.

[0092] Finally, it should be noted that, if any, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0093] Although this disclosure has been described above through specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this disclosure within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this disclosure.

Claims

1. A method for preparing a fermented extract of Sophora flavescens, characterized in that, include: Step 1: Soak Sophora flavescens in water, then chop it up and inoculate it with Penicillium cicadae seed liquid for fermentation culture. After the Penicillium cicadae mycelium has fully grown Sophora flavescens, dry it to obtain Penicillium cicadae-Sophora flavescens bidirectional fermentation product. Step 2: The Penicillium cicadae-Sophora flavescens bidirectional fermentation product is crushed, and then extracted using Bifidobacterium liquid fermentation extraction, hot water extraction, or anhydrous ethanol extraction. After drying, the Sophora flavescens fermentation extract is obtained.

2. The method for preparing the fermented extract of Sophora flavescens according to claim 1, characterized in that, In step one, the *Penicillium cicadae* is *Cicadae* CH2347, with accession number CGMCC No. 40399; Preferably, the volume percentage of *Penicillium cicadae* CH2347 mycelium in the *Penicillium cicadae* seed solution is 50-80% of the total liquid culture medium. More preferably, the method for preparing the *Penicillium cicadae* seed liquid includes: (1) Take 2-3 solid strains of Cordyceps CH2347 with a diameter of about 0.5 cm and grow on PDA plates using a punch, inoculate them into 300 mL of potato glucose water culture medium, and culture at 180 rpm for 3 days at 28℃ to obtain Cordyceps CH2347 seed liquid 1. (2) Homogenize the seed liquid 1 of Cordyceps militaris CH2347, and continue to use it as seed liquid. Take 20 mL and inoculate it into 300 mL potato glucose water culture medium. Culture at 28℃ and 180 rpm for 4 days to obtain Cordyceps militaris CH2347 seed liquid 2, that is, the seed liquid of Cordyceps militaris.

3. The method for preparing the fermented extract of Sophora flavescens according to claim 1 or 2, characterized in that, In step one, the soaking treatment involves soaking in water at room temperature for 2 hours.

4. The method for preparing the fermented extract of Sophora flavescens according to any one of claims 1-3, characterized in that, In step one, the particle size of the chopped material is 0.3-1 cm.

5. The method for preparing the fermented extract of Sophora flavescens according to any one of claims 1-4, characterized in that, In step one, the inoculation ratio is 5-20 mL of the Penicillium cicadae seed solution per 60 g of soaked Sophora flavescens, more preferably 15 mL of the Penicillium cicadae seed solution per 60 g of soaked Sophora flavescens.

6. The method for preparing the fermented extract of Sophora flavescens according to any one of claims 1-5, characterized in that, In step one, the fermentation culture temperature is 20~28℃ and the time is 8~20 days; more preferably, the fermentation culture temperature is 27~28℃ and the time is 12 days. Optionally, in step one, the drying process is freeze drying.

7. The method for preparing the fermented extract of Sophora flavescens according to any one of claims 1-6, characterized in that, In step two, the liquid fermentation extraction method of Bifidobacterium includes: crushing the Penicillium cicadae-Sophora flavescens bidirectional fermentation product, passing it through a 50-mesh sieve, homogenizing the Penicillium cicadae-Sophora flavescens bidirectional fermentation product and water at a mass ratio of 1:9, adding 1% by mass of Bifidobacterium freeze-dried powder, anaerobic fermenting at 37°C for 24 hours, centrifuging to collect the supernatant, and then freeze-drying to obtain the Sophora flavescens fermentation extract; Alternatively, the hot water extraction method includes: crushing the Penicillium cicadae-Sophora flavescens bidirectional fermentation product, passing it through a 50-mesh sieve, extracting it in hot water at 85°C for 3 hours at a mass ratio of 1:99, centrifuging it, taking the supernatant and freeze-drying it to obtain the Sophora flavescens fermentation extract. Alternatively, the anhydrous ethanol extraction method is anhydrous ethanol Soxhlet reflux extraction; preferably, the anhydrous ethanol Soxhlet reflux extraction method includes: crushing the Penicillium cicadae-Sophora flavescens bidirectional fermentation product, passing it through a 50-mesh sieve, extracting it with anhydrous ethanol by Soxhlet reflux at a mass ratio of 1:25 for 3 hours, then extracting it with anhydrous ethanol by Soxhlet reflux at a mass ratio of 1:15 for 3 hours, combining the two filtrates, rotary evaporating at 50°C, and then freeze-drying to obtain the Soxhlet ginseng fermentation extract.

8. A fermented extract of Sophora flavescens prepared by any one of claims 1-7.

9. The use of the fermented extract of Sophora flavescens as described in claim 8 in the manufacture of anti-hair loss products.

10. A hair loss prevention product, characterized in that, It includes the fermented extract of Sophora flavescens as described in claim 8, preferably including 1 wt% of the fermented extract of Sophora flavescens; More preferably, the anti-hair loss product is an essence, formulated by weight percentage from the following raw materials: propylene glycol 3%, glycerin 3%, panthenol 1%, carbomer 940 0.6%, the fermented extract of Sophora flavescens 1%, triethanolamine 0.35%, p-hydroxyacetophenone 0.5%, 1,2-hexanediol 0.5%, and the balance being deionized water.

Images