Use of armillaria mellea in preparation of drugs for preventing and treating bone metabolism or osteoporosis and armillaria mellea preparation

Abstract

The application discloses an application of Armillaria mellea in preparation of medicines for preventing and treating bone metabolism or osteoporosis and an Armillaria mellea preparation, wherein the medicine is Armillaria mellea powder or an ethanol extract of the Armillaria mellea powder, and the Armillaria mellea powder is obtained by drying, crushing and sieving a wet mycelium of Armillaria mellea strain after fermentation culture. The Armillaria mellea powder and the ethanol extract of the Armillaria mellea powder are used for treating osteoporosis, and a remarkable curative effect is achieved without side effects. A bottleneck that medicines of single Chinese medicine such as Gastrodia and Chinese cress seed for treating osteoporosis are not remarkable is broken through. Meanwhile, a range of medicines prepared from the Armillaria mellea for mainly treating central nervous system diseases in the current market is expanded.

Description

(I) Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to new applications of Armillaria mellea, particularly the application of Armillaria mellea in the preparation of drugs for the prevention and treatment of bone metabolism or osteoporosis and Armillaria mellea preparations. (II) Background Technology

[0002] Osteoporosis (OP) is a metabolic disease of the skeletal system characterized by progressive bone loss, decreased bone density, and degenerative changes in bone microstructure, leading to increased bone fragility and a higher risk of fractures (i.e., fragility fractures and low-traumatic fractures). Postmenopausal osteoporosis is the most common type of OP, with 158 million women over 50 years of age worldwide at high risk of osteoporotic fractures, and this number is projected to double by 2040. The imbalance between bone formation and bone resorption caused by decreased estrogen levels after menopause is the main mechanism of bone loss in postmenopausal women. Treatment for postmenopausal osteoporosis is mostly etiological, involving estrogen, calcium, and vitamin supplementation. However, long-term intake of endogenous estrogen and calcium supplements carries the risk of inducing breast cancer, hypercalcemia, and endometrial cancer.

[0003] In Traditional Chinese Medicine (TCM), osteoporosis (OP) is often categorized under "bone atrophy," with treatment primarily focused on tonifying the kidneys, supplemented by regulating the liver and spleen, and promoting blood circulation and removing blood stasis. TCM offers advantages in preventing and treating OP through holistic approaches, high safety profiles, and the presence of some medicinal and edible ingredients. The development of estrogen-like herbal preparations has become a research hotspot in recent years; however, the content of estrogen-like components in TCM herbs is currently low, extraction and separation costs are high, and the efficacy of single-herb anti-osteoporosis preparations remains insignificant.

[0004] Armillaria mellea, a symbiotic fungus of Gastrodia elata, is a well-known edible and medicinal fungus with similar pharmacological effects to Gastrodia elata, including antioxidant, anti-aging, blood pressure lowering, blood sugar lowering, hypnotic, antibacterial, anticancer, antitumor, and immunomodulatory effects. The main chemical components of Armillaria mellea are terpenes, sterols, polysaccharides, adenosine, and amino acids. However, current research does not document any therapeutic activity of Armillaria mellea for osteoporosis.

[0005] Therefore, in response to the side effects of long-term use of endogenous estrogen in the treatment of postmenopausal osteoporosis and the high cost and insignificant efficacy of single-herb Chinese medicine preparations, there is a need to provide new anti-osteoporosis preparations. (III) Summary of the Invention

[0006] The purpose of this invention is to provide an application of *Armillaria mellea* in the preparation of drugs for the prevention and treatment of bone metabolism or osteoporosis, particularly drugs for postmenopausal osteoporosis. This invention utilizes animal experiments, employing an ovariectomized (OVX) rat model to construct a postmenopausal osteoporosis model, to investigate the effects of *Armillaria mellea* powder and its ethanol extract on bone mineral density, bone biomechanics, and bone microstructure in these rats. The results showed good anti-osteoporosis efficacy with no side effects. This invention solves the side effects problem associated with long-term use of endogenous estrogen in existing technologies and significantly improves efficacy compared to existing single-herb Chinese herbal preparations.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides the application of Armillaria mellea in the preparation of drugs for the prevention and treatment of bone metabolism or osteoporosis.

[0009] Furthermore, the drug is a drug for the prevention or treatment of postmenopausal osteoporosis or senile osteoporosis in women.

[0010] Furthermore, the drug is Armillaria mellea powder or an ethanol extract of Armillaria mellea powder, wherein the Armillaria mellea powder is obtained by drying, pulverizing, and sieving the wet mycelium of Armillaria mellea strain after fermentation culture.

[0011] Furthermore, the *Armillaria mellea* powder is prepared by the following method: *Armillaria mellea* inoculum is inoculated onto potato dextrose agar (PDA) slant, statically cultured at 30°C for activation, and once the mycelial cords have fully grown in the test tube, the *Armillaria mellea* mycelial cords are inoculated into PDA liquid medium and statically cultured at 30°C for 22 days to obtain the seed culture, which is then stored at 4°C for later use; the seed culture is inoculated into a comprehensive PDA liquid medium at a volume concentration of 5%, statically cultured at 30°C for 25 days, filtered through gauze, and rinsed with distilled water to obtain wet mycelium, which is then dried at 60°C and pulverized to obtain *Armillaria mellea* powder. The PDA culture medium consists of: 200g potato, 20g glucose, 20g agar powder, 1000mL distilled water, natural pH; the PDA liquid culture medium consists of: 200g potato, 20g glucose, 1000mL distilled water, natural pH; the comprehensive PDA liquid culture medium consists of: 200g potato, 20g maltodextrin, 2g peptone, 1g yeast extract, 0.5g magnesium sulfate, 0.5g potassium dihydrogen phosphate, 1g dipotassium hydrogen phosphate, 10mg vitamin B1, 1000ml water, natural pH.

[0012] Furthermore, the preferred Armillaria mellea strain is Armillaria mellea ACCC 50801, purchased from the Shaanxi Institute of Microbiology's Culture Collection Center.

[0013] Furthermore, the pulverization refers to passing the material through a 2-100 mesh sieve.

[0014] Furthermore, the ethanol extract of the *Armillaria mellea* powder is prepared by the following method: *Armillaria mellea* powder is added to an aqueous ethanol solution with a volume concentration of 20-70%, soaked at 18-25°C for 2-7 hours, then extracted with ultrasonic assistance for 0.5-4 hours, and then refluxed for 0.5-4 hours. The mixture is filtered, and the filtrate is concentrated to a relative density of 5-30 mg / mL to obtain a concentrated solution. The concentrated solution is loaded onto a macroporous resin chromatography column, and eluted sequentially with water, an aqueous ethanol solution with a volume concentration of 30%, and an aqueous ethanol solution with a volume concentration of 60%. The 60% aqueous ethanol solution eluent is collected and concentrated to a relative density of 10-50 mg / mL to obtain the *Armillaria mellea* ethanol extract.

[0015] Furthermore, the volume of the 20-70% ethanol aqueous solution used is 20-50 mL / g based on the total weight of the Armillaria mellea powder, preferably 20 mL / g.

[0016] Furthermore, the ultrasonic-assisted extraction conditions are 20–45 kHz.

[0017] Furthermore, the volumes of water, 30% ethanol aqueous solution, and 60% ethanol aqueous solution are all 3 to 6 times the volume of the macroporous resin packed in the column, and the elution rate is 10 mL / min for all of them. The preferred type of macroporous resin is D101.

[0018] Furthermore, the ethanol extract of the *Armillaria mellea* powder is prepared by the following method: *Armillaria mellea* powder is added to a 20-70% (v / v) ethanol aqueous solution for a first extraction, soaked at 18-25°C for 2-7 hours, then extracted with ultrasonic assistance at 20-45 kHz for 0.5-4 hours, followed by reflux extraction for 0.5-4 hours, and filtered to obtain a primary filtrate and a primary residue; the primary residue is then added to a 20-70% (v / v) ethanol aqueous solution for a second extraction, followed by ultrasonic extraction at 20-45 kHz for 0.5-4 hours, followed by reflux extraction for 0.5-4 hours, and filtered to obtain a secondary filtrate and a secondary residue. The two filtrates are combined and concentrated to a relative density of 5-30. mg / mL was used to obtain a concentrated solution; the concentrated solution was then adsorbed onto a D101 macroporous resin column and loaded onto a chromatography column at a volume of 10-30% of the column volume. Gradient elution was performed sequentially with 3-6 times the volume of macroporous resin water, 30% ethanol aqueous solution, and 60% ethanol aqueous solution at a rate of 10 mL / min. The eluent from the 60% ethanol aqueous solution was collected, concentrated to a relative density of 10-50 mg / mL, and dried at 40-80℃ to obtain the *Armillaria mellea* ethanol extract. The volume of 20-70% ethanol aqueous solution used in both the primary and secondary extractions was 20-50 mL / g of *Armillaria mellea* powder, preferably 20 mL / g.

[0019] Furthermore, the drug also includes medically acceptable adjuvants, including preservatives, flavoring agents, antioxidants, colorants, surfactants, emulsifiers, stabilizers, binders, suspending agents, disintegrants, etc., as specified in the pharmacopoeia.

[0020] Furthermore, the drug dosage form of the present invention includes any one of tablets, pills, granules, capsules, injections, drop pills, or topical preparations.

[0021] Furthermore, the drug described in this invention is a drug that increases bone alkaline phosphatase levels and decreases tartrate-resistant acid phosphatase 5b levels.

[0022] Furthermore, the drug described in this invention is a drug that increases serum estrogen levels.

[0023] Furthermore, the drug is a drug that inhibits the level of tumor necrosis factor α.

[0024] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0025] (1) This invention has developed new functions of Armillaria mellea: Armillaria mellea powder and Armillaria mellea powder ethanol extract prepared by this invention as a symbiotic fungus of the traditional Chinese medicine Gastrodia elata are used to treat osteoporosis and have achieved significant therapeutic effects. This has broken through the bottleneck that single Chinese medicines such as Gastrodia elata and leek seed, which are mainly used to treat "lower back and knee pain", are not effective in treating osteoporosis. At the same time, it has expanded the scope of drugs prepared by Armillaria mellea on the market that mainly treat central nervous system diseases.

[0026] (2) The use of Armillaria mellea in the preparation of drugs for treating osteoporosis has no toxic side effects, which solves the problem that long-term use of existing estrogen drugs for treating osteoporosis may cause side effects such as endometrial hyperplasia.

[0027] (3) The present invention has achieved significant progress: the *Armillaria mellea* powder and its ethanol extract can significantly increase bone density in rats by 42% and 85%, respectively; in terms of improving bone biomechanical properties, the *Armillaria mellea* powder and its ethanol extract can increase the maximum load on rat femurs by 18% and 33%, respectively, and increase the maximum strain on rat femurs by 28% and 55%, respectively; in terms of protecting the microstructure of cancellous bone, the *Armillaria mellea* powder and its ethanol extract can increase the bone volume of rat femurs by 11% and 18%, respectively, increase the bone surface area of ​​rat femurs by 10% and 18%, respectively, and reduce the trabecular bone size of rat femurs. The separation rates were 13% and 21%; in regulating bone metabolism indicators, *Armillaria mellea* powder and its ethanol extract increased bone alkaline phosphatase by 13% and 26%, respectively, and decreased tartrate-resistant acid phosphatase 5b by 9% and 18%, respectively; in regulating rat serum estrogen levels, *Armillaria mellea* powder and its ethanol extract increased rat serum estrogen levels by 9% and 22%, respectively; in regulating rat serum tumor necrosis factor α levels, *Armillaria mellea* powder and its ethanol extract decreased rat serum tumor necrosis factor α levels by 12% and 18%, respectively.

[0028] (4) The *Armillaria mellea* powder and *Armillaria mellea* ethanol extract have significant therapeutic effects in preventing and treating osteoporosis, and have the effects of promoting bone formation, inhibiting bone resorption, promoting estrogen secretion, and inhibiting the expression of inflammatory factors. This is reflected in the following: Both *Armillaria mellea* powder and *Armillaria mellea* ethanol extract can increase the level of bone alkaline phosphatase, a marker of bone formation, and decrease the level of tartrate-resistant acid phosphatase 5b, a marker of bone resorption, in osteoporosis model rats, indicating that *Armillaria mellea* powder and *Armillaria mellea* ethanol extract can promote bone formation and inhibit bone resorption; *Armillaria mellea* powder and *Armillaria mellea* ethanol extract can also increase the level of estrogen in the serum of osteoporosis model rats, indicating that *Armillaria mellea* powder and *Armillaria mellea* ethanol extract can correct the hormone secretion disorder in ovariectomized rats, and that *Armillaria mellea* powder and *Armillaria mellea* ethanol extract have estrogen-like effects; *Armillaria mellea* powder and *Armillaria mellea* ethanol extract can also inhibit the level of tumor necrosis factor α in the serum of osteoporosis model rats, indicating that *Armillaria mellea* powder and *Armillaria mellea* ethanol extract can inhibit the inflammatory state in ovariectomized rats. (iv) Description of the attached drawings

[0029] Figure 1 This is a representative image of the rat uterus from Example 3; a represents the sham-operated group; b represents the model control group; c represents the estradiol group; d represents the Gastrodia elata ethanol extract group; e represents the Allium tuberosum seed ethanol extract group; f represents the Armillaria mellea powder group; g represents the Armillaria mellea ethanol extract group; h represents the Armillaria mellea water extract group.

[0030] Figure 2This is a representative diagram of the microstructure of the distal femur cancellous bone in rats in Example 3; a represents the sham-operated group; b represents the model control group; c represents the estradiol group; d represents the Gastrodia elata ethanol extract group; e represents the Allium tuberosum seed ethanol extract group; f represents the Armillaria mellea powder group; g represents the Armillaria mellea ethanol extract group; h represents the Armillaria mellea water extract group. (V) Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0032] The raw materials of Gastrodia elata and Allium tuberosum seeds used in this invention were collected and prepared in accordance with the 2020 edition of the Chinese Pharmacopoeia. The Armillaria mellea used was purchased from the China Agricultural Microbial Culture Collection Center, with the number ACCC 50801. Unless otherwise specified, all percentage concentrations mentioned in this invention are volume concentrations. The room temperature mentioned in this invention refers to 25-30℃.

[0033] Example 1: Method for preparing Armillaria mellea powder

[0034] Armillaria mellea ACCC 50801 strain was inoculated onto potato dextrose agar (PDA) slant and activated by static incubation at 30°C. Once the mycelial cords had fully colonized the test tubes, the mycelial cords were inoculated into PDA liquid medium and incubated statically at 30°C for 22 days to obtain the seed culture, which was then stored at 4°C for later use. At a volume concentration of 5%, the seed culture was inoculated into 10 bottles of 800 mL / bottle of comprehensive PDA liquid medium. After static incubation at 30°C for 25 days, the culture was filtered through gauze and rinsed with distilled water to obtain wet mycelium. This wet mycelium was dried at 60°C, pulverized, and passed through a 50-mesh sieve to obtain 148 g of Armillaria mellea powder.

[0035] Example 2: Preparation method of Armillaria mellea ethanol extract

[0036] Take 50g of Armillaria mellea powder prepared by the method in Example 1, add 1000mL of 60% ethanol aqueous solution for a first extraction, soak at 20℃ for 2h, then extract with ultrasonic assistance at 35kHz for 1h, and then heat and reflux for 1h. Filter to obtain a first filtrate and a first residue. Add 1000mL of 60% ethanol aqueous solution to the first residue for a second extraction, then extract with ultrasonic assistance at 35kHz for 1h, and then heat and reflux for 1h. Filter to obtain a second filtrate and a second residue. Combine the first and second filtrates and concentrate to a relative density of 30mg / m³. L, obtain the concentrated solution; after activating the D101 macroporous resin, pack it into a chromatography column (a glass column with a height of 1m and a diameter of 6.5cm, and a column height of 80cm), load the concentrated solution, the loading volume is 20% (16cm) of the column resin volume, and perform gradient elution sequentially with 3 times the column macroporous resin volume of water, 30% ethanol aqueous solution and 60% ethanol aqueous solution, the elution rate of each is 10mL / min, collect the eluent of 60% ethanol aqueous solution, concentrate to a relative density of 40mg / mL, and obtain 50mL of Armillaria mellea ethanol extract.

[0037] Comparative Example 1: Armillaria mellea water extract

[0038] Take 50g of Armillaria mellea powder prepared by the method in Example 1, add 1000mL of distilled water for a first extraction, soak at 20℃ for 2h, then extract with ultrasonic assistance at 35kHz for 1h, and then heat under reflux for 1h. Filter to obtain a first filtrate and a first residue. Add 1000mL of distilled water to the first residue for a second extraction, then extract with ultrasonic assistance at 35kHz for 1h, and then heat under reflux for 1h. Filter to obtain a second filtrate and a second residue. Combine the first and second filtrates and concentrate to a relative density of 30mg / mL to obtain... The concentrated solution was obtained; after activating the D101 macroporous resin, it was packed into a chromatography column (a glass column with a height of 1m and a diameter of 6.5cm, and a column height of 80cm). The concentrated solution was loaded onto the column, and the loading volume was 20% (16cm) of the resin volume. Gradient elution was performed sequentially with 3 times the volume of macroporous resin water, 30% ethanol aqueous solution, and 60% ethanol aqueous solution, with an elution rate of 10mL / min for each. The eluent of 60% ethanol aqueous solution was collected and concentrated to a relative density of 40mg / mL to obtain 50mL of Armillaria mellea aqueous extract.

[0039] Comparative Example 2: Gastrodia elata ethanol extract

[0040] Take 50g of Gastrodia elata, pulverize it through a 50-mesh sieve, add 1000mL of 60% ethanol aqueous solution for a first extraction, soak at 20℃ for 2 hours, then extract with ultrasonic assistance at 35 kHz for 1 hour, and then heat and reflux for 1 hour. Filter to obtain a first filtrate and a first residue. Add 1000mL of 60% ethanol aqueous solution to the first residue for a second extraction, then extract with ultrasonic assistance at 35 kHz for 1 hour, and then heat and reflux for 1 hour. Filter to obtain a second filtrate and a second residue. Combine the first and second filtrates and concentrate to a relative density of 30mg. / mL, to obtain the concentrated solution; after activating the D101 macroporous resin, pack it into a chromatography column (a glass column with a height of 1m and a diameter of 6.5cm, and a column height of 80cm), load the concentrated solution, the loading volume is 20% (16cm) of the column resin volume, and perform gradient elution sequentially with 3 times the volume of macroporous resin water, 30% ethanol aqueous solution and 60% ethanol aqueous solution, the elution rate is 10mL / min, collect the eluent of 60% ethanol aqueous solution, concentrate to a relative density of 40mg / mL, to obtain 30mL of Gastrodia elata ethanol extract.

[0041] Comparative Example 3: Leek Seed Ethanol Extract

[0042] Take 50g of leek seeds, crush them through a 50-mesh sieve, add 1000mL of 60% ethanol aqueous solution for a first extraction, soak at 20℃ for 2 hours, then extract with ultrasonic assistance at 35 kHz for 1 hour, followed by reflux extraction for 1 hour, filter, and obtain a first filtrate and a first residue. Add 1000mL of 60% ethanol aqueous solution to the first residue for a second extraction, then extract with ultrasonic assistance at 35 kHz for 1 hour, followed by reflux extraction for 1 hour, filter, and obtain a second filtrate and a second residue. Combine the first and second filtrates and concentrate to a relative density of 30mg. / mL, to obtain the concentrated solution; after activating the D101 macroporous resin, pack it into a chromatography column (a glass column with a height of 1m and a diameter of 6.5cm, and a column height of 80cm), load the concentrated solution, the loading volume is 20% (16cm) of the column resin volume, and perform gradient elution sequentially with 3 times the volume of macroporous resin water, 30% ethanol aqueous solution and 60% ethanol aqueous solution, the elution rate is 10mL / min, collect the eluent of 60% ethanol aqueous solution, concentrate to a relative density of 40mg / mL, to obtain 25mL of leek seed ethanol extract.

[0043] Example 3: The efficacy of Armillaria mellea powder and Armillaria mellea ethanol extract on osteoporosis.

[0044] To verify the significant therapeutic effects of the *Armillaria mellea* powder and *Armillaria mellea* ethanol extract of the present invention on osteoporosis, animal experiments were conducted on the *Armillaria mellea* powder prepared according to the method of Example 1, the *Armillaria mellea* ethanol extract prepared according to the method of Example 2, the *Armillaria mellea* aqueous extract prepared according to the method of Comparative Example 1, the *Gastrodia elata* ethanol extract prepared according to the method of Comparative Example 2, and the *Allium tuberosum* seed ethanol extract prepared according to the method of Comparative Example 3.

[0045] 1. Experimental Materials

[0046] 1.1 Experimental animals: 64 SPF-grade female SD rats, 7-8 weeks old, weighing (250±15)g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., license number SCXK(Sichuan)2020-030; and were housed in the SPF-grade animal housing room (SYXK(Shaanxi)2022-008) of the Shaanxi Provincial and Ministerial Collaborative Innovation Center for the Industrialization of Traditional Chinese Medicine Resources.

[0047] 1.2 Experimental Reagents: The *Armillaria mellea* powder prepared according to the method of Example 1 was diluted with distilled water to a solution with a concentration of 8 g / 100 mL. The *Armillaria mellea* ethanol extract prepared according to the method of Example 2 was diluted with distilled water to a solution with a concentration of 8 g / 100 mL. The *Armillaria mellea* aqueous extract prepared according to the method of Comparative Example 1 was diluted with distilled water to a solution with a concentration of 8 g / 100 mL. The *Gastrodia elata* ethanol extract prepared according to the method of Comparative Example 2 was diluted with distilled water to a solution with a concentration of 8 g / 100 mL. The *Allium tuberosum* seed ethanol extract prepared according to the method of Comparative Example 3 was diluted with distilled water to a solution with a concentration of 8 g / 100 mL. Sodium penicillin for injection (800,000 units, Harbin Pharmaceutical Group Pharmaceutical Factory), and 3% sodium pentobarbital solution were also used.

[0048] 2 Experimental Methods

[0049] 2.1 Animal grouping: Sixty-four SPF-grade female SD rats were acclimatized for one week and then randomly divided into eight groups using a random number table: Group 1 was the sham-operated group, Group 2 was the model control group, Group 3 was the estradiol group, Group 4 was the Gastrodia elata ethanol extract group, Group 5 was the Allium tuberosum seed ethanol extract group, Group 6 was the Armillaria mellea powder group, Group 7 was the Armillaria mellea ethanol extract group, and Group 8 was the Armillaria mellea water extract group, with eight rats in each group.

[0050] 2.2 Animal Model Establishment: Sixty-four rats were anesthetized via intraperitoneal injection of 3% sodium pentobarbital solution. Groups 2-8 were the surgical group, where bilateral ovariectomy was performed after skin preparation, fixation, and disinfection. Group 1 underwent only a small amount of adipose tissue around the ovary removed, without ligation of the fallopian tubes; the rest was the same as the surgical group. Postoperatively, rats were injected with penicillin for three consecutive days, and the abdominal incision was observed for bleeding, infection, and their overall condition. Vaginal cytology smears were performed for three consecutive days starting on the second postoperative day, confirming complete ovariectomy and successful establishment of the ovariectomy rat model.

[0051] 2.3 Animal drug administration: Seven days after successful model establishment, animals in each group were administered the drug via gavage. The dosage was based on the conversion factor between the standard human and experimental rat equivalent doses. The gavage volume for each group of rats was 10 mL / kg.

[0052] Rats in Group 1 (sham surgery) and Group 2 (model control group) were administered distilled water by gavage. Rats in Group 3 (estradiol group) were administered estradiol valerate aqueous solution at a concentration of 90 μg / kg / day. Rats in Group 4 (Gastrodia elata ethanol extract group) were administered Gastrodia elata raw material at a dose of 0.80 g / kg / day. Rats in Group 5 (Allium tuberosum seed ethanol extract group) were administered Allium tuberosum seed raw material at a dose of 0.80 g / kg / day. Rats in Group 6 (Armillaria mellea powder group) were administered Armillaria mellea powder at a dose of 0.80 g / kg / day. Rats in Group 7 (Armillaria mellea ethanol extract group) were administered Armillaria mellea powder at a dose of 0.80 g / kg / day. Rats in Group 8 (Armillaria mellea aqueous extract group) were administered Armillaria mellea powder at a dose of 0.80 g / kg / day. Each group of rats was administered the corresponding drug by gavage once daily for 3 consecutive months, with normal feeding and ample water intake, and body weight measured weekly. After 3 months of administration, rat organs were observed, and bone mineral density, bone microstructure, bone biomechanical properties, serum bone metabolism, inflammation, and estrogen-related indicators were measured.

[0053] 3-indicator detection

[0054] 3.1 After dissection, observe whether there are any pathological changes in the heart, liver, spleen, lung, kidney, thymus, brain, and uterus tissues of rats in each group.

[0055] 3.2 Bone mineral density and bone microstructure determination: Bone microstructure was scanned using Micro-CT. Analysis 12 software was used to measure and analyze the bone mineral density, trabecular bone volume, bone surface area, and trabecular separation of the right proximal femur in rats. Scanning parameters: planar image resolution was 1024×1024, pixel size was 20×20μm, and slice interval was 20μm. Calculations of trabecular-related parameters included bone volume, bone surface area, and trabecular separation.

[0056] 3.3 Biomechanical Performance Analysis of Rat Femur: The biomechanical properties of the left femur were measured using a CMT4304 electronic universal testing machine. The sample was placed on a support frame with the physiological curvature of the femur facing upwards. The midpoint of the femur was used as the compression point, with a compressor diameter of 6 mm and a support span of 20 mm. Compression was applied to the mid-fracture of the femur at a rate of 0.5 mm / min until fracture. The load-displacement curves were recorded by computer, and the maximum load and maximum strain were measured.

[0057] 3.4 Determination of serum bone metabolism, inflammation, and estrogen-related indicators in rats: The levels of bone alkaline phosphatase, tartrate-resistant acid phosphatase 5b, estrogen, and tumor necrosis factor α in rat serum were measured strictly according to the ELISA kit procedure.

[0058] 3.5 Statistical Methods: Experimental data are expressed as mean ± standard deviation (mean ± SD). Covariance analysis was used to analyze the effect of the drug on BMD and biomechanical performance indicators, eliminating the influence of body weight on these parameters. One-way analysis of variance (ANOVA) was used to test the variances among the groups for other indicators. The least significant difference (LSD) test was used when variances were homogeneous, and Tamhane's T² test was used when variances were non-homogeneous. All analyses were performed using SPSS 16.0 statistical software, and P < 0.05 was considered statistically significant.

[0059] 4. Experimental Results

[0060] 4.1 Representative images of the uterus of each group of rats are shown below. Figure 1 Compared with the sham-operated group, the model control group rats showed uterine atrophy, the estradiol group rats showed endometrial hyperplasia leading to thicker uterine walls, the uterine morphology of rats in the *Armillaria mellea* powder group and *Armillaria mellea* ethanol extract group tended to be normal, while the uterus of rats in the *Gastrodia elata* ethanol extract group, *Allium tuberosum* seed ethanol extract group, and *Armillaria mellea* water extract group remained atrophic. No pathological changes were observed in the heart, liver, spleen, lungs, kidneys, thymus, and brain tissues of rats in any group. Preliminary results indicate that *Armillaria mellea* powder and *Armillaria mellea* ethanol extract do not have toxic side effects such as endometrial hyperplasia.

[0061] 4.2 The bone mineral density (BMD) values ​​of the right femur in each group of rats are shown in Table 1. Compared with the sham-operated group, the femoral BMD value in the model control group decreased significantly, and the difference was statistically significant (p<0.05). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group increased the femoral BMD of rats by 64%, 42%, and 85%, respectively, and the differences were statistically significant (p<0.01 or p<0.05). The difference between the Armillaria mellea ethanol extract group and the Armillaria mellea powder group was statistically significant (p<0.01). Compared with the model control group, there were no significant differences in the Gastrodia elata ethanol extract group, the Allium tuberosum seed ethanol extract group, and the Armillaria mellea water extract group (p>0.05).

[0062] Table 1. Comparison of bone mineral density in the left femur of rats

[0063]

[0064]

[0065] Note: Mean ± Standard Deviation, n = 8 # p<0.05, ## p<0.01 vs. sham surgery group; *p<0.05, **p<0.01 vs. model control group; &&p<0.01 vs. Armillaria mellea powder group.

[0066] Representative microstructures of the distal femur cancellous bone in each group of rats are shown in the figure. Figure 2 Compared with the sham-operated group, rats in the model control group showed a significant reduction in cancellous bone after ovariectomy. The estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group significantly reversed the reduction in trabecular bone, while the Gastrodia elata ethanol extract group, Allium tuberosum ethanol extract group, and Armillaria mellea water extract group did not show significant reversal of the reduction in trabecular bone.

[0067] Further analysis of the microstructural parameters of the distal femur cancellous bone in each group of rats is shown in Table 2. Compared with the sham-operated group, the bone volume and surface area of ​​the right femur in the model control group were significantly decreased (p<0.01), while the trabecular separation was significantly increased (p<0.01). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group increased the femoral bone volume by 29%, 11%, and 18%, respectively, with statistically significant differences (p<0.05). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group increased the femoral bone surface area by 23%, 10%, and 18%, respectively, with statistically significant differences (p<0.01 or p<0.05). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group decreased the trabecular separation of the femur by 18%, 13%, and 21%, respectively, with statistically significant differences (p<0.01 or p<0.05). The differences between the Armillaria mellea ethanol extract group and the Armillaria mellea powder group were statistically significant (p<0.01 or p<0.05). Compared with the model control group, there were no significant differences between the Gastrodia elata ethanol extract group, the Allium tuberosum seed ethanol extract group, and the Armillaria mellea water extract group (p>0.05).

[0068] Table 2. Comparison of microstructure of cancellous bone in the distal femur of rats

[0069]

[0070] Note: Mean ± Standard Deviation, n = 8 # p<0.05, ## p<0.01 vs. sham surgery group; *p<0.05, **p<0.01 vs. model control group; & p<0.05, && p<0.01 vs. Armillaria mellea powder group.

[0071] 4.3 Comparison of biomechanical properties of the left femur in each group of rats is shown in Table 3. Compared with the sham-operated group, the maximum load and maximum strain of the femur in the model control group were significantly decreased, with statistically significant differences (p<0.01). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group increased the maximum load of the rat femur by 35%, 18%, and 33%, respectively, with statistically significant differences (p<0.01 or p<0.05). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group increased the maximum strain of the rat femur by 91%, 28%, and 55%, respectively, with statistically significant differences (p<0.01 or p<0.05). There was a statistically significant difference between the Armillaria mellea ethanol extract group and the Armillaria mellea powder group (p<0.01). Compared with the model control group, there were no significant differences in the maximum load and maximum strain in the Gastrodia elata ethanol extract group, Allium tuberosum seed ethanol extract group, and Armillaria mellea water extract group (p>0.05).

[0072] Table 3. Comparison of biomechanical properties of the left femur in rats

[0073]

[0074] 4.4 Comparison of serum bone metabolism indicators among different groups of rats is shown in Table 4. Compared with the sham-operated group, the serum bone formation indicator, bone alkaline phosphatase, was significantly decreased in the model control group (p<0.01), while the bone resorption indicator, tartrate-resistant acid phosphatase 5b, was significantly increased (p<0.01). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group increased serum bone alkaline phosphatase by 21%, 13%, and 26%, respectively, with statistically significant differences (p<0.01 or p<0.05). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group decreased serum tartrate-resistant acid phosphatase 5b by 18%, 9%, and 18%, respectively, with statistically significant differences (p<0.01). The difference between the Armillaria mellea ethanol extract group and the Armillaria mellea powder group was statistically significant (p<0.01 or p<0.05). Compared with the model control group, there were no significant differences in the Gastrodia elata ethanol extract group, Allium tuberosum ethanol extract group, and Armillaria mellea water extract group (p>0.05).

[0075] Table 4. Comparison of serum bone metabolism indicators in rats

[0076]

[0077]

[0078] Note: Mean ± Standard Deviation, n = 8 # p<0.05, ## p<0.01 vs. sham surgery group; *p<0.05, **p<0.01 vs. model control group;& p<0.05, && p<0.01 vs. Armillaria mellea powder group.

[0079] 4.5 Comparison of serum estrogen levels in rats across different groups is shown in Table 5. Compared with the sham-operated group, serum estrogen levels in the model control group were significantly decreased (p<0.01). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group increased serum estrogen levels in rats by 48%, 9%, and 22%, respectively, with statistically significant differences (p<0.01 or p<0.05). The difference between the Armillaria mellea ethanol extract group and the Armillaria mellea powder group was statistically significant (p<0.01). Compared with the model control group, there were no significant differences in the Gastrodia elata ethanol extract group, Allium tuberosum seed ethanol extract group, and Armillaria mellea water extract group (p>0.05).

[0080] Table 5. Comparison of serum estrogen levels in rats

[0081]

[0082] Note: Mean ± Standard Deviation, n = 8 # p<0.05, ## p<0.01 vs. sham surgery group; *p<0.05, **p<0.01 vs. model control group; && p<0.01 vs. Armillaria mellea powder group.

[0083] 4.6 Comparison of serum tumor necrosis factor α levels in different groups of rats is shown in Table 6. Compared with the sham-operated group, the serum tumor necrosis factor α level in the model control group was significantly increased (p<0.01). Compared with the model control group, the estradiol group, Armillaria mellea powder group, and Armillaria mellea ethanol extract group reduced the serum tumor necrosis factor α level in rats by 10%, 12%, and 18%, respectively, with statistically significant differences (p<0.01 or p<0.05). The difference between the Armillaria mellea ethanol extract group and the Armillaria mellea powder group was statistically significant (p<0.05). Compared with the model control group, there were no significant differences in the Gastrodia elata ethanol extract group, Allium tuberosum seed ethanol extract group, and Armillaria mellea water extract group (p>0.05).

[0084] Table 6. Comparison of serum tumor necrosis factor α levels in rats

[0085]

[0086]

[0087] Note: Mean ± Standard Deviation, n = 8 # p<0.05, ## p<0.01 vs. sham surgery group; *p<0.05, **p<0.01 vs. model control group;& p<0.05 vs. Armillaria mellea powder group.

[0088] 5. Experimental Conclusions

[0089] Experimental results showed that ovariectomy in rats significantly reduced bone mineral density, and biomechanical parameters such as maximum load and maximum strain were significantly decreased. Bone microstructure parameters, including bone volume and bone surface area, were significantly reduced, while trabecular separation was significantly increased, indicating successful establishment of the osteoporosis model. Gavage administration of *Armillaria mellea* powder and its ethanol extract significantly increased bone mineral density, improved biomechanical properties, and protected bone microstructure in rats. The difference between the ethanol extract group and the powder group was statistically significant. These findings suggest that both *Armillaria mellea* powder and its ethanol extract can prevent and treat osteoporosis, with the ethanol extract showing a more significant effect than the powder.

[0090] Further analysis of serum biochemical parameters in rats revealed that ovariectomized rats exhibited decreased levels of bone alkaline phosphatase (BAP), a marker of bone formation, increased levels of tartrate-resistant acid phosphatase 5b (TA-AP), decreased serum estrogen levels, and increased levels of tumor necrosis factor-α (TNF-α). Both *Armillaria mellea* powder and ethanol extract increased serum BAP levels and decreased tartrate-resistant acid phosphatase 5b levels in osteoporosis model rats, indicating that they promote bone formation and inhibit bone resorption. Furthermore, both powder and extract increased serum estrogen levels in osteoporosis model rats, suggesting they can correct hormonal imbalances in ovariectomized rats and possess estrogen-like effects. Finally, they inhibited TNF-α levels in ovariectomized rats, indicating they can suppress inflammatory states in ovariectomized rats.

[0091] In summary, the Armillaria mellea powder and Armillaria mellea ethanol extract prepared according to this process have significant therapeutic effects in preventing and treating osteoporosis, and also have the effects of promoting bone formation, inhibiting bone resorption, promoting estrogen secretion, and inhibiting the expression of inflammatory factors.

Claims

1. The application of *Armillaria mellea* in the preparation of drugs for the prevention and treatment of postmenopausal osteoporosis or senile osteoporosis, characterized in that... The Armillaria mellea is Armillaria mellea ( Armillaria mellea According to ACCC 50801, the drug is Armillaria mellea powder or an ethanol extract of Armillaria mellea powder, wherein the Armillaria mellea powder is obtained by drying, pulverizing, and sieving the wet mycelium of Armillaria mellea strain after fermentation culture.

2. The application as described in claim 1, characterized in that, The *Armillaria mellea* powder was prepared as follows: *Armillaria mellea* inoculum was inoculated onto a PDA medium slant and activated by static incubation at 30°C. Once the mycelial cords had fully colonized the test tube, the mycelial cords were inoculated into PDA liquid medium and incubated at 30°C for 22 days to obtain a seed culture, which was then stored at 4°C for later use. The seed culture was then inoculated into a comprehensive PDA liquid medium at a volume concentration of 5% and incubated at 30°C for 25 days. The culture was then filtered through gauze and rinsed with distilled water to obtain wet mycelium, which was dried at 60°C and pulverized to obtain *Armillaria mellea* powder. Culture medium A consists of: 200g potato, 20g glucose, 20g agar powder, 1000mL distilled water, natural pH; the PDA liquid culture medium consists of: 200g potato, 20g glucose, 1000mL distilled water, natural pH; the comprehensive PDA liquid culture medium consists of: 200g potato, 20g maltodextrin, 2g peptone, 1g yeast extract, 0.5g magnesium sulfate, 0.5g potassium dihydrogen phosphate, 1g dipotassium hydrogen phosphate, 10mg vitamin B1, 1000ml water, natural pH.

3. The application as described in claim 1, characterized in that, The ethanol extract of Armillaria mellea powder is prepared by the following method: Armillaria mellea powder is added to an aqueous ethanol solution with a volume concentration of 20-70%, soaked at 18-25℃ for 2-7 hours, then extracted with ultrasonic assistance for 0.5-4 hours, and then heated under reflux for 0.5-4 hours. The mixture is filtered, and the filtrate is concentrated to a relative density of 5-30 mg / mL to obtain a concentrated solution. The concentrated solution is loaded onto a macroporous resin chromatography column, and eluted sequentially with water, an aqueous ethanol solution with a volume concentration of 30%, and an aqueous ethanol solution with a volume concentration of 60%. The 60% aqueous ethanol solution eluent is collected and concentrated to a relative density of 10-50 mg / mL to obtain the Armillaria mellea ethanol extract.

4. The application as described in claim 3, characterized in that, The volume of the 20-70% ethanol aqueous solution used is 20-50 mL / g based on the total weight of the Armillaria mellea powder.

5. The application as described in claim 1, characterized in that, The drug is used to increase bone alkaline phosphatase levels and decrease tartrate-resistant acid phosphatase 5b levels.

6. The application as described in claim 1, characterized in that, The drug is one that increases serum estrogen levels and inhibits tumor necrosis factor α levels.

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