Application of fermented gastrodia elata water extract in preparation of products for preventing and treating androgenic alopecia

Abstract

The application discloses application of a fermented Gastrodia elata water extract in preparation of a product for preventing and treating androgen alopecia, and belongs to the technical field of probiotic fermentation and application, wherein the fermented Gastrodia elata water extract is prepared from fermented Gastrodia elata by Lactobacillus salivarius AACE1. The fermentation process realizes biological transformation of Gastrodia elata raw medicinal materials, reduces potential irritation of the Gastrodia elata, and makes the product more suitable for long-term scalp care. Animal model efficacy evaluation of the water extract proves that the water extract can significantly improve androgen alopecia symptoms and has a remarkable effect on preventing and treating androgen alopecia.

Description

Technical Field

[0001] This invention relates to the field of probiotic fermentation and application technology, specifically to the application of fermented Gastrodia elata water extract in the preparation of products for preventing and treating androgenetic alopecia. Background Technology

[0002] Androgenetic alopecia (AGA), also known as seborrheic alopecia, is the most common type of hair loss in clinical practice. It is characterized by the selective attack of hair follicles in specific scalp areas (such as the forehead and crown) by androgens (primarily dihydrotestosterone, DHT) against a genetically predisposed individual. This leads to follicle miniaturization, with terminal hairs gradually transforming into vellus hairs and eventually falling out. While not life-threatening, this condition significantly impacts patients' mental health and quality of life, thus creating a substantial demand for treatment.

[0003] Currently, clinical treatment options for androgenetic alopecia are limited and have significant limitations. Finasteride, a systemic 5α-reductase inhibitor, can effectively reduce serum and scalp DHT levels, but it may cause side effects such as male sexual dysfunction, and its efficacy is difficult to maintain after discontinuation, leading to poor patient compliance. Minoxidil, a topical vasodilator, has not fully elucidated its exact mechanism of action in promoting hair growth and may cause adverse reactions such as contact dermatitis and hirsutism; its efficacy also varies among individuals. Furthermore, while hair transplantation surgery provides obvious results, it is expensive and cannot stop the continued atrophy of hair follicles in non-transplanted areas, requiring continuous drug treatment. Therefore, developing a prevention and treatment strategy that is derived from natural sources, has diverse mechanisms of action, is safe, and has few side effects has become a critical issue that urgently needs to be addressed in this field. Summary of the Invention

[0004] In order to solve the above-mentioned technical problems, the purpose of this invention is to provide an application of fermented Gastrodia elata water extract in the preparation of products for preventing and treating androgenetic alopecia. This water extract can significantly improve the symptoms of androgenetic alopecia and has a significant effect in preventing and treating androgenetic alopecia.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: providing the application of fermented Gastrodia elata water extract in the preparation of products for preventing and treating androgenetic alopecia, wherein the fermented Gastrodia elata water extract is obtained by fermenting Gastrodia elata with Lactobacillus salivarius AACE1.

[0006] Furthermore, Lactobacillus salivarius AACE1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20700.

[0007] Furthermore, the fermented gastrodia elata water extract is obtained through the following steps:

[0008] After steaming the Gastrodia elata until it is just cooked, chop it and mix it with water. Then, break the cell wall to obtain a Gastrodia elata homogenate. Inoculate the Gastrodia elata homogenate with a bacterial solution containing Lactobacillus salivarius AACE1 for anaerobic fermentation. When the pH of the fermentation broth stabilizes at 4.0-4.2, stop the fermentation, centrifuge and collect the supernatant. Adjust the pH to 5.5-6.0 to obtain the fermented Gastrodia elata water extract.

[0009] Furthermore, steam the gastrodia elata at 94℃ for 4-8 minutes to allow it to fully mature.

[0010] Furthermore, the mass ratio of Gastrodia elata to water is 1:7-9.

[0011] Furthermore, the inoculum size was 1-5 vt; the viable count of *Lactobacillus salivarius* AACE1 in the inoculum was 1.0-4.8 × 10⁻⁶. 9 CFU / mL.

[0012] Further, anaerobic fermentation at 37℃ for 96-120 hours.

[0013] Furthermore, fermentation was terminated by heating the fermentation broth to 72°C and holding it at that temperature for 30 minutes to kill the bacteria.

[0014] Furthermore, the products include pharmaceuticals and cosmetics.

[0015] Specifically, it can be a product in various forms such as lotion, serum, and shampoo.

[0016] The present invention has the following beneficial effects:

[0017] The fermented Gastrodia elata water extract prepared by this invention can significantly improve the symptoms of androgenetic alopecia, specifically by increasing the hair coverage density in the bald area, promoting significant hair growth, increasing the total weight of hair per unit area, and significantly increasing the density of active hair follicles. It has the effect of preventing androgenetic alopecia and has significant effects. Attached Figure Description

[0018] Figure 1 The effect of AACE1 fermented Gastrodia elata aqueous extract on promoting hair growth in mice;

[0019] Figure 2 Figure 1 shows the hair length measurement results of mice with androgenetic alopecia induced by AACE1 fermented Gastrodia elata aqueous extract.

[0020] Figure 3 The image shows the hair weight results of mice with androgenetic alopecia induced by AACE1 fermented Gastrodia elata aqueous extract.

[0021] Figure 4 Comparison of hair follicle environment in mice to show the effect of AACE1 fermented Gastrodia elata water extract on promoting hair growth;

[0022] Figure 5 H&E staining results of skin tissue in mice with androgenetic alopecia treated by AACE1-fermented Gastrodia elata aqueous extract;

[0023] Figure 6 Masson staining results of skin tissue from mice with androgenetic alopecia treated by AACE1-fermented Gastrodia elata aqueous extract;

[0024] Figure 7 The image shows the hair follicle density results of mice with androgenetic alopecia treated by AACE1-fermented Gastrodia elata aqueous extract. Detailed Implementation

[0025] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] The symptoms of androgenetic alopecia differ between men and women. The pattern of androgenetic alopecia in men follows the Hamilton-Norwood classification: a receding hairline at the frontal hairline is usually an early sign, forming a typical "M" shaped bald patch; thinning hair on the top of the head, with the hair gradually thinning and falling out in the crown area, forming an "O" shaped bald patch; as the condition progresses, the "M" shaped area at the frontal hairline and the "O" shaped area on the top of the head will merge together, eventually leaving only a ring of hair on the sides and back of the head, the so-called "horseshoe" bald patch pattern. Female androgenetic alopecia follows the Ludwig classification: diffuse thinning at the crown; unlike men, women typically do not experience a significant receding hairline, but rather a diffuse thinning of hair starting from the midline of the crown, increasing scalp visibility; it mainly presents a "Christmas tree" pattern, and in severe cases, the thinning area from the forehead hairline to the crown gradually widens, resembling the outline of a Christmas tree; and the forehead hairline is preserved, which is one of the most crucial differences from male androgenetic alopecia (AGA), as women's forehead hairline is usually preserved intact.

[0027] The overall concept of this invention is as follows:

[0028] Lactobacillus salivarius is a microorganism belonging to the family Lactobacillus and the genus Lactobacillus. It is widely colonized in the intestinal system of humans and animals and has been proven to have significant probiotic potential.

[0029] Gastrodia elata Bl., a perennial herb belonging to the genus Gastrodia in the Orchidaceae family, is a traditional and valuable Chinese medicine. Its main active components, gastrodin and 4-hydroxybenzyl alcohol, exhibit significant anti-inflammatory, antioxidant, and neuroprotective effects. Given the oxidative stress and microenvironmental inflammation involved in hair follicle miniaturization, Gastrodia elata extract theoretically possesses the potential to intervene in AGA (anti-inflammatory glycosuria), but its direct application faces limitations such as low bioavailability and unclear mechanisms of action. Moderate moist heat treatment (steaming until partially cooked) can moderately soften the cell walls of Gastrodia elata, gelatinize starch, and disrupt some structures, without completely dissolving and eroding it. This provides a more easily decomposed substrate for subsequent lactic acid bacteria fermentation, improving the efficiency of biotransformation.

[0030] This invention relates to fermented Gastrodia elata water extract, which refers to the metabolites obtained by biotransformation of Gastrodia elata homogenate with Lactobacillus salivarius AACE1 under suitable conditions. Specifically, the process involves co-culturing the active ingredients of Gastrodia elata with Lactobacillus salivarius to induce directional fermentation and transformation, ultimately yielding a complex system containing bacterial metabolites and transformed active components, collectively referred to as fermented Gastrodia elata water extract. To date, no systematic reports have been found in domestic and international literature and patents regarding the directional fermentation of Gastrodia elata using Lactobacillus salivarius to prepare products with this specific application, nor are there corresponding standardized preparation processes. This invention, against this technological gap, through systematic research and process development, achieves the construction and application of this fermentation system for the first time.

[0031] Example 1

[0032] A fermented gastrodia elata water extract is prepared through the following steps:

[0033] Gastrodia elata was steamed at 94℃ for 8 minutes to soften it, then chopped and mixed with water at a mass ratio of 1:8. The mixture was then subjected to cell wall disruption at 12000 rpm for 8 minutes to obtain a Gastrodia elata homogenate. A bacterial culture containing Lactobacillus salivarius AACE1 was inoculated into the Gastrodia elata homogenate and anaerobic fermented at 37℃ for 120 hours. The inoculum size was 4 vt%, and the viable cell count in the culture was 4.8 × 10⁻⁶. 9 CFU / mL, when the pH of the fermentation broth stabilizes at 4.0, heat treatment at 72℃ for 30 min is used to terminate the fermentation. The supernatant is collected by centrifugation at 8000 rpm and 4℃ for 15 min, and the pH is adjusted to 5.8 to obtain the fermented Gastrodia elata water extract.

[0034] Subsequently, samples were aliquoted into pretreated 0.2 mL EP tubes using sterilized pipettes and rapidly immersed in liquid nitrogen for instantaneous quenching to quickly fix the biochemical state of the samples and avoid changes in physicochemical properties caused by time delays in subsequent operations. After quenching, all samples were immediately transferred to an ultra-low temperature freezer at -80°C for long-term storage for subsequent analysis or application.

[0035] Experimental Example 1

[0036] 1. Establishment of a mouse model of androgenetic alopecia

[0037] Laboratory animals: SPF grade C57BL / 6J male mice, 5 weeks old, weighing 18-22 g, provided by Spiford (Beijing) Biotechnology Co., Ltd.

[0038] Mice were anesthetized via intraperitoneal injection using tribromoethanol (Avorin) anesthetic solution at a dose of 20 μL / g. After the mice were in a stable anesthetized state, the hair on their backs was completely shaved using an electric shaver, and depilatory cream was evenly applied to thoroughly remove any remaining short hairs. Subsequently, the skin was repeatedly washed with sterile saline-soaked cotton balls until all depilatory cream residue was removed. The cleaned skin was dried with sterile gauze and then thoroughly disinfected with 75% ethanol cotton pads. Before the procedure, the skin on the backs of the mice was visually and tactilely examined to ensure that the skin in the area to be modeled was intact and free of any visible trauma, damage, or scratches, meeting the basic skin conditions required for experimental modeling.

[0039] Androgenetic alopecia model was established by subcutaneous injection of testosterone propionate solution at multiple points in the hairless area on the back of mice. On day 14 of modeling, mice were sacrificed and complete skin tissue samples were obtained from the back. Each skin specimen was immediately fixed in 4% paraformaldehyde, then dehydrated, cleared, and embedded in paraffin to prepare 5 μm thick serial sections for hematoxylin-eosin (H&E) staining.

[0040] After staining, the tissue morphology was systematically observed under an optical microscope. To quantitatively assess hair follicle density, three non-overlapping typical regions were randomly selected at 200x magnification (corresponding to an actual field diameter of 0.5 mm for the section), and the total number of hair follicles with complete hair bulb structures in each field of view was counted. The arithmetic mean of the number of hair follicles in the three fields of view was calculated as the hair follicle density per unit area of ​​the sample. Finally, one-way ANOVA was used to statistically compare the hair follicle density data between the experimental group and the control group. Successful model establishment criteria: ① Decreased hair growth density on the back of mice, dull coat color, prolonged telogen phase, and visible sebum discharge on the back; ② HE staining showed that the hair follicle pigment was lighter, the diameter was smaller, the number of hair follicles was reduced, and the hair follicles were miniaturized.

[0041] 2. Grouping, treatment, and administration of experimental animals

[0042] Experimental grouping: The experiment was randomly divided into 6 groups, including blank group, model group, positive control group (5% minoxidil), fermented Gastrodia elata water extract group with Lactobacillus salivarius AACE1, Lactobacillus salivarius AACE1 group, and Gastrodia elata group.

[0043] The treatment methods for each group of mice are shown in Table 1.

[0044] Table 1. Drug administration in mice

[0045] Group drug Dosage Injection status Blank group none 0.2 mL / animal No injection Model group PBS phosphate buffer 0.2 mL / animal Subcutaneous injection of testosterone propionate Positive drug group 5% Minoxidil 0.2 mL / animal Subcutaneous injection of testosterone propionate AACE1 Fermented Gastrodia elata Water Extract AACE1 Fermented Gastrodia elata Water Extract 0.2 mL / animal Subcutaneous injection of testosterone propionate AACE bacterial culture group AACE bacterial liquid 0.2 mL / animal Subcutaneous injection of testosterone propionate Gastrodia elata group Gastrodia elata homogenate 0.2 mL / animal Subcutaneous injection of testosterone propionate

[0046] (1) Blank group: Hair was removed from the back of mice (area 2 cm × 3 cm), and no other experimental treatment was performed;

[0047] (2) Model group: The back of the mice was shaved (area of ​​2 cm × 3 cm). After the mice were injected subcutaneously into the back of the mice at multiple points according to the dosage to model the condition, 0.2 mL of PBS solution was evenly applied to the back of the mice every day.

[0048] (3) Positive control group: The back of mice was shaved (area 2 cm × 3 cm). After the mice were modeled by subcutaneous injection of testosterone propionate at multiple points on the back, 0.2 mL of 5% minoxidil was evenly applied to the back of the mice at regular intervals every day. The medication was administered only once a day.

[0049] (4) Lactobacillus salivarius AACE1 fermented Gastrodia elata water extract group: The back of mice was dehaired (area 2 cm × 3 cm). After the mice were modeled by subcutaneous injection of testosterone propionate at multiple points on the back of the mice according to the dosage, 0.2 mL of Lactobacillus salivarius AACE1 fermented Gastrodia elata water extract prepared in Example 1 was evenly applied to the back of the mice once a day.

[0050] (5) Lactobacillus salivarius AACE1 group: The back of mice was shaved (area 2 cm × 3 cm), and after the mice were modeled by subcutaneous injection of testosterone propionate at multiple points on the back of the mice according to the dosage, 0.2 mL of Lactobacillus salivarius AACE1 bacterial solution in Example 1 was evenly applied to the back of the mice once a day at regular intervals.

[0051] (6) Gastrodia elata group: The back of mice was shaved (area 2 cm × 3 cm). After the mice were modeled by subcutaneous injection of testosterone propionate at multiple points on the back, 0.2 mL of Gastrodia elata homogenate from Example 1 was applied evenly to the back of the mice once a day.

[0052] 3. The therapeutic effect and evaluation of gastrodia elata water extract fermented by Lactobacillus salivarius AACE1 on male pattern baldness.

[0053] (1) Comparison of the effects of Lactobacillus salivarius AACE1 fermented Gastrodia elata water extract on promoting hair regeneration in male pattern mice.

[0054] Strict drug administration procedures were followed during the experiment. Changes in new hair growth on the backs of mice were closely observed, and photographs were taken and recorded weekly. Results are as follows: Figure 1 As shown.

[0055] Day 7 of drug administration during modeling:

[0056] from Figure 1 As can be seen, on the 7th day of modeling and drug administration, the skin of mice in all six groups showed melanin deposition and darkening of different sizes in the drug administration area. Among them, the skin darkening area of ​​mice in the AACE1 bacterial culture fermentation Gastrodia elata water extract group and the 5% minoxidil group was slightly smaller than that of the blank group.

[0057] Day 14 of drug administration during modeling:

[0058] Model group: Large areas of smooth, bare skin on the back, with only a very small number of colorless, transparent, fine, soft hairs visible, and weak signs of new hair follicle activation, confirming that the androgenic alopecia model has been successfully established and maintained.

[0059] In the 5% minoxidil group: newly grown hair entered an active growth phase, with clearly distributed black stubble covering approximately 30-50% of the skin area. The hair began to show terminal hair characteristics, but there was still considerable room for growth in density and length, indicating that the drug intervention had initiated an effective hair follicle regeneration process.

[0060] The AACE1 bacterial broth fermented Gastrodia elata water extract group showed the most significant hair growth, with newly grown black stubble densely covering more than 60% of the skin area. The hair density was significantly higher than that of the positive control group, with good growth synchronization, and some hairs had clearly protruded from the skin surface, demonstrating excellent hair follicle activation and growth promotion capabilities.

[0061] AACE1 bacterial solution group: Localized patchy new hair growth was observed, covering an area of ​​approximately 15-25%. The hair was generally fine and soft, with lighter pigmentation and a significantly slower growth rate, indicating that the bacterial solution alone had a limited activating effect on hair follicles.

[0062] In the Gastrodia elata group, the newly grown hair appeared as scattered, light-colored stubble, with skin coverage of approximately 10-20%. Insufficient hair growth vitality confirmed that traditional water extracts had a weak effect in promoting hair follicle regeneration at this stage.

[0063] Day 21 of drug administration during modeling:

[0064] Model group: The skin on the back of the mice was still largely bare, and the new hair growth was extremely sparse, with only a small number of colorless or light-colored fine and soft hairs visible. The activity of hair follicles was continuously inhibited, confirming that the hair loss model was stable.

[0065] 5% Minoxidil group: New hair growth entered a rapid growth phase, covering most of the back area. Hair density increased significantly, with most hairs transforming into thick terminal hairs, and obvious melanin deposition was visible, indicating that hair follicle function was effectively restored.

[0066] The AACE1 bacterial culture fermented Gastrodia elata water extract group showed the most significant increase in hair growth rate and maturity. New terminal hairs had almost completely covered the skin, forming a dense and uniform felt. The hair density, length, and pigmentation level all reached or exceeded the levels of the positive drug group at the same time, demonstrating excellent ability to promote hair follicle regeneration and maturation.

[0067] AACE1 bacterial solution group: Patchy growth of new hair was observed, but overall coverage was insufficient. The hair was generally fine and soft, short in length, and lightly pigmented, indicating that although the hair follicles were activated, the degree of miniaturization reversal was limited, and the efficacy of using it alone was moderate.

[0068] In the Gastrodia elata group, the growth of new hair was characterized by localized and sparse hair coverage, with most hairs being fine and soft intermediate hairs and a low proportion of terminal hairs, confirming the insufficient activity of traditional water extracts.

[0069] Day 28 of drug administration during modeling:

[0070] Model group: The back skin continued to be largely exposed, the new hair growth was extremely sparse, with only a small number of colorless or light-colored fine vellus hairs visible, and the hair follicle miniaturization characteristics were significant, confirming that the androgenic alopecia model was stably maintained during the experimental period.

[0071] In the 5% minoxidil group: high-quality hair regeneration was achieved on the back skin, with dense new terminal hair covering most areas. The hair was thick and robust, with significant melanin deposition and a marked recovery in hair shaft diameter, approaching normal physiological condition, confirming the effectiveness of the positive drug intervention.

[0072] The AACE1-fermented Gastrodia elata water extract group exhibited optimal hair growth efficacy. New terminal hair completely covered the skin, forming a dense felt. Hair density, thickness, and pigmentation all reached or exceeded the levels of the positive control group, indicating that this fermentation product has a very strong ability to promote hair follicle regeneration and maturation.

[0073] AACE1 bacterial solution group: Moderate hair regeneration was observed, but the density and quality of the new hair were significantly insufficient. The hair was mostly fine, soft, and short, covering patchy areas with light pigmentation, indicating that the bacterial solution alone has a certain physiological regulatory effect, but the therapeutic effect is limited.

[0074] In the Gastrodia elata group, the growth of new hair was characterized by localized, sparse, fine and soft hair coverage, which failed to effectively promote the formation of thick terminal hair, confirming that although traditional water extracts have activity, their bioavailability or efficacy is insufficient.

[0075] A comprehensive comparison of macroscopic mouse skin data revealed that the AACE1-fermented Gastrodia elata aqueous extract group and the positive control group had the largest black stubble coverage area on day 14. The model group, the Gastrodia elata group, and the AACE1-fermented group showed localized, sparse, light-colored stubble coverage. The regrowth of new hair in the AACE1-fermented Gastrodia elata aqueous extract group was superior to that in the positive control group.

[0076] (2) Effects of AACE1 fermented gastrodia elata water extract on hair length and weight in androgenic alopecia model mice.

[0077] On days 14, 21, and 28 after drug administration, 10 hairs were randomly plucked from each mouse in each group, and the average value was taken as the length of newly grown hair for each mouse. The mean hair length for each group was calculated. One-way ANOVA was used for analysis using SPSS 21.0 software. Quantitative data are expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant. Results are as follows: Figure 2 As shown.

[0078] The results showed that, compared with the model group, the AACE1 bacterial culture fermentation of Gastrodia elata water extract group significantly increased hair length (p<0.001); compared with the minoxidil positive group, there was no statistically significant difference.

[0079] After the hair length measurement was completed on day 28, the hair in the modeling area was carefully shaved off using an animal shaver. The weighing paper was collected and stored, and then weighed using a 1 / 100,000 balance. The gross weight value was recorded. The results are as follows: Figure 3 As shown in the figure. The results showed that, compared with the model control group, the AACE1 bacterial culture fermentation of Gastrodia elata water extract group significantly increased hair weight (p < 0.001).

[0080] The above results indicate that the AACE1 bacterial culture fermentation of Gastrodia elata water extract can promote hair growth in AGA mice, and has the same effect as minoxidil positive drug in increasing hair length and weight.

[0081] (3) Effects of Lactobacillus salivarius AACE1 fermented Gastrodia elata water extract on hair follicle development and collagen fiber pathological changes in androgenetic alopecia in mice.

[0082] To dynamically and quantitatively evaluate the changes in hair growth in each group of subjects, follicular endoscopy was used to periodically photograph and record the skin on the backs of mice in each group. This method aims to overcome the limitations of traditional endpoint sampling and observation, and to achieve precise quantitative analysis of key indicators in the hair regeneration process (including but not limited to hair follicle activation time, hair density, hair diameter, coverage area, and terminal hair / vellus hair conversion rate) by obtaining continuous and intuitive macroscopic imaging evidence.

[0083] Before and after photos of hair follicles Figure 4 As shown, the results indicate that:

[0084] On day 9, the skin surface of the model group (M) was smooth and there were no signs of hair follicle activation; in the minoxidil group (D), tiny black dots (stubble) began to appear at the openings of some hair follicles, indicating that the hair follicles had been activated and entered the early growth phase; the number of black stubble in the AACE1 bacterial solution fermented Gastrodia elata water extract group (GL) was significantly greater than that in the positive drug group, and the distribution was dense and uniform, indicating that its hair follicle activation time was earlier and more synchronous; only sporadic and sparse light-colored stubble was visible in the AACE1 bacterial solution group (L) and the Gastrodia elata group (G). On day 14, the condition of the model group (M) remained unchanged with no significant improvement; the black stubble in the minoxidil group (D) increased significantly and became longer, covering about 40-60% of the observation area, and the hair began to break through the skin surface; the hair growth advantage of the AACE1 bacterial solution fermented Gastrodia elata water extract group (GL) further expanded, forming a continuous and dense black felt-like coverage with an area coverage rate of over 70%, and the hair length was generally better than that of the minoxidil group; the stubble in the AACE1 bacterial solution group (L) and the Gastrodia elata group (G) water extract group increased, but the growth was slow, the distribution was uneven, and it was patchy, with a coverage rate of about 20-40%. On day 21, the model group (M) showed sparse hair; the minoxidil group (D) showed further hair growth, covering most of the area, with thicker and darker hair; the AACE1 bacterial solution fermented Gastrodia elata water extract group (GL) showed that the back skin was basically completely covered by thick, dense, and pigmented terminal hairs, with the hair shaft diameter significantly larger than other experimental groups, approaching or reaching the standard of normal terminal hairs; the AACE1 bacterial solution group (L) and the Gastrodia elata group (G) showed continued hair growth, but the density and thickness were significantly insufficient, the hairs were still fine and soft, and some skin was visible.

[0085] To investigate the effects of Lactobacillus salivarius AACE1 bacterial culture fermentation of Gastrodia elata aqueous extract on the skin histopathology and hair follicle development of androgenetic alopecia mice, tissue sections were prepared from the backs of the mice in the above groups and subjected to H&E staining and Masson staining.

[0086] H&E staining results are as follows Figure 5 As shown, the results indicate that:

[0087] On day 28, skin sections from mice in the model group (M) showed epidermal atrophy and significantly thinner dermis. The number of skin appendages was sparse, with numerous resting hair follicles, characterized by a reduced or absent hair bulb and missing or only remnant hair shafts. The remaining hair follicles were significantly miniaturized, with both diameter and depth significantly smaller than in the normal group. No obvious signs of new hair follicle growth or activation were observed in the dermis, confirming the successful establishment of the androgenetic alopecia model and the continuous inhibition of hair follicle growth. In the minoxidil group (D), compared to the model group, the skin tissue structure of the positive control group was significantly improved. Dermal thickness increased, and a significant number of anagen-phase hair follicles were visible. These follicles were deep, extending into the subcutaneous fat layer, with full and intact hair bulbs, clearly visible outer and inner root sheaths, and accompanied by normally sized dermal papillae. The dense stromal cells surrounding the hair follicles indicate that the follicles are in an active regeneration phase. The AACE1-fermented Gastrodia elata aqueous extract group (GL) exhibited the best histological structure. It had the highest follicle density in the dermis, with numerous robust follicles in the mid-to-late anagen phase, distributed in clusters. The follicle structure was intact, the hair bulb was enlarged, and the dermal papilla was significantly larger and cell-rich, suggesting strong hair regeneration capacity. The rich vascular distribution in the dermis provided a favorable microenvironment for follicle growth. In the Lactobacillus salivarius AACE1-fermented group (LS), the number of follicles showed some recovery, but most follicles were still in the early anagen phase or transitioning from the telogen phase to the anagen phase. The overall follicle depth was shallower, the diameter was smaller, and some follicles still exhibited miniaturization characteristics. Its effect was significantly better than the model group, but it was inferior to the positive drug group and the fermentation group of this invention in terms of hair follicle density, maturity, and structural integrity. The Gastrodia elata group (G) showed similar tissue morphology to the AACE1 bacterial solution group, exhibiting mild hair follicle regeneration. The number of hair follicles was sparse, with most being small follicles, and only a few showing signs of entering the growth phase. The results indicate that the unfermented Gastrodia elata aqueous extract has limited ability to activate and promote the maturation of hair follicles.

[0088] Masson staining results are as follows: Figure 6 As shown, the results indicate that:

[0089] On day 28, in the model group (M), the collagen fibers in the dermis were sparse and disordered, with a lighter blue staining, and the overall collagen density was significantly reduced. Around the hair follicles, especially around the atrophied miniaturized follicles, there was a lack of dense collagen fiber encapsulation and support, resulting in a loose structure. Collagen fiber breakage was observed in some areas, indicating that the supporting structure of the hair follicles also deteriorated after atrophy, and the hair follicle microenvironment worsened. In the minoxidil group (D), the dermal collagen fiber deposition was significantly improved. The collagen fiber density increased significantly, the blue staining deepened, and a relatively dense and orderly network structure was formed. Especially around newly formed hair follicles, clear and regularly arranged collagen fiber sheaths were visible, providing good structural support for the hair follicles and reflecting effective repair of the hair follicle microenvironment. In the *Gastrodia elata* group (G), the collagen fiber condition was similar to the bacterial solution group, showing a slight increase in collagen deposition, but the overall collagen network structure remained relatively loose, providing weak structural support for the hair follicles. In the *Lactobacillus salivarius* AACE1 bacterial solution group (LS), the dermal collagen density was increased compared to the model group, but the collagen fibers were still slightly thin and their arrangement was generally not very orderly. Although collagen sheaths were visible around the hair follicles, their thickness and density were limited, indicating that they had some improvement effect on the dermal matrix, but the strength was insufficient. The *Lactobacillus salivarius* AACE1 fermented *Gastrodia elata* water extract group (GL) showed the most obvious collagen remodeling effect. Its dermal layer exhibited dense and uniform blue staining, and the collagen fiber density reached or exceeded the level of the positive drug group. The results show that the group fermented with Lactobacillus salivarius AACE1 and Gastrodia elata water extract is far superior to other groups in terms of skin collagen fiber recovery, and even slightly superior to the positive drug group.

[0090] To observe the total number of hair follicles, HE-stained sections of skin from the experimental area on the back of mice were analyzed. Cross-sections of hair follicles were photographed under a microscope (×200), and five fields of view were randomly selected under a microscope (×100). The total number of hair follicles was counted using ImageJ software, and statistical analysis of the number of hair follicles in different groups was performed using one-way ANOVA with SPSS 21.0 software. The results are as follows: Figure 7 As shown, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0091] The results showed that, compared with the model control group, the minoxidil-positive group (p < 0.001), the Lactobacillus salivarius AACE1-fermented Gastrodia elata aqueous extract group (p < 0.001), the Lactobacillus salivarius AACE1 group (p < 0.001), and the Gastrodia elata group (p < 0.001) all significantly increased the total number of hair follicles. Furthermore, compared with the minoxidil-positive group, there was no significant difference in the total number of hair follicles in the Lactobacillus salivarius AACE1-fermented Gastrodia elata aqueous extract group.

[0092] The above results indicate that the Lactobacillus salivarius AACE1-fermented Gastrodia elata water extract group, the Lactobacillus salivarius AACE1 group, and the Gastrodia elata group can all increase the total number of hair follicles in AGA mice. The Lactobacillus salivarius AACE1-fermented Gastrodia elata water extract group has the same effect as minoxidil positive drug.

[0093] The above embodiments fully verify the outstanding effect of AACE1-fermented Gastrodia elata aqueous extract in preventing and treating androgenetic alopecia. Experimental data show that the fermentation composition and its application in the preparation of corresponding products possess novelty, inventiveness, and practicality. It is particularly noteworthy that the effect of the fermentation product is significantly superior to the sum of the effects of the Gastrodia elata aqueous extract and AACE1 bacterial solution used alone; this synergistic effect is not revealed in the prior art. Therefore, this invention claims protection for the application of AACE1-fermented Gastrodia elata aqueous extract in the preparation of products for preventing and treating androgenetic alopecia.

Claims

1. The application of fermented Gastrodia elata aqueous extract in the preparation of products for preventing and treating androgenetic alopecia, characterized in that, The fermented Gastrodia elata water extract was obtained by fermenting Gastrodia elata with Lactobacillus salivarius AACE1.

2. The application as described in claim 1, characterized in that, The Lactobacillus salivarius AACE1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 20700.

3. The application as described in claim 1, characterized in that, The fermented Gastrodia elata water extract was obtained through the following steps: After steaming the Gastrodia elata until it is just cooked, chop it and mix it with water. Then, perform cell wall breaking treatment to obtain a Gastrodia elata homogenate. Inoculate the Gastrodia elata homogenate with the bacterial solution containing the Lactobacillus salivarius AACE1 and carry out anaerobic fermentation. When the pH value of the fermentation broth stabilizes at 4.0-4.2, stop the fermentation, centrifuge and collect the supernatant. Adjust the pH to 5.5-6.0 to obtain the fermented Gastrodia elata water extract.

4. The application as described in claim 3, characterized in that, The mass ratio of Gastrodia elata to water is 1:7-9.

5. The application as described in claim 3, characterized in that, The inoculum size of the bacterial solution is 1-5 vt; the viable count of Lactobacillus salivarius AACE1 in the bacterial solution is 1.0-4.8 × 10⁻⁶. 9 CFU / mL.

6. The application as described in claim 3, characterized in that, Anaerobic fermentation at 37℃ for 96-120 h.

7. The application as described in claim 1, characterized in that, Fermentation was terminated by heating the fermentation broth to 72°C and holding it at that temperature for 30 minutes to kill the bacteria.

8. The application as described in claim 1, characterized in that, The products include pharmaceuticals and cosmetics.

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