Application of aporphine alkaloid Tinopaine A in hemsleya amabilis ox gall in preparation of medicine for treating bone destruction related diseases

Abstract

The invention discloses application of aporphine alkaloid Tinopaine A in hemsleya amabilis ox gall in preparation of a medicine for treating bone destruction related diseases, and belongs to the technical field of biological medicine. Experiments prove that the Tinpaine A monomer has an inhibition effect on osteoclast differentiation formation in vivo and in vitro; by regulating and controlling key factors for osteoclast formation, the compound has an inhibiting effect on in-vitro osteoclast formation under the concentration of 1-10 mu M and on in-vivo osteoclast formation of mice under the dosage of 2-20 mg / kg, can effectively reduce the number of osteoclasts and protect bone trabecula structures, and has an excellent treatment effect on bone destruction related diseases. The invention relates to a medicine for inhibiting osteoclast differentiation in vivo and in vitro and treating diseases related to bone destruction, which takes Tinopaine A as an active ingredient and can relieve bone destruction by regulating and controlling bone metabolism balance so as to achieve the purpose of treating the diseases related to bone destruction.

Description

Technical Field

[0001] This invention relates to the field of biomedicine technology, and in particular to the application of Tinopaine A, an apophrine-like alkaloid from bovine bile, in the preparation of drugs for treating bone destruction-related diseases. Background Technology

[0002] Osteoporosis, rheumatoid arthritis, and other bone-related diseases caused by bone metabolism imbalances have become significant health problems affecting the population. Inhibiting excessive activation of osteoclasts is a major strategy for treating these diseases. With the rise of natural drug research, active ingredients derived from traditional Chinese medicinal herbs have gradually become a research focus in the pharmaceutical field due to their high safety and diverse target mechanisms. *Achyranthes bidentata*, a plant in the Menispermaceae family, is a commonly used medicinal herb in traditional Chinese and Dai medicine. Its modern pharmacological value in areas such as hypoglycemia and antioxidation has been gradually discovered. Apophene alkaloids, as rigid planar active molecules abundant in this type of plant, have potential anti-inflammatory and cellular function-regulating effects that urgently need further exploration, providing new research directions for the treatment of bone-related diseases.

[0003] Osteoclasts, as the only cells in the body responsible for bone resorption, rely on a dynamic balance between bone formation and bone resorption for differentiation and activation. This process is dominated by the RANKL / RANK signaling pathway, which recruits TRAF6 to form a complex with c-Src, activating signaling pathways such as NF-κB and MAPK, and ultimately regulating the expression of downstream factors such as c-Fos and NFATc1. Apophene alkaloids are widely found in plants of the Magnoliaceae and Menispermaceae families, and their unique tetracyclic or pentacyclic structures endow them with rich pharmacological activities. *Achyranthes bidentata*, a representative medicinal herb of the Menispermaceae family, has traditionally been used for dispelling wind and unblocking meridians, clearing heat and detoxifying. Modern research has confirmed its multifaceted medicinal potential, but specific research on apophene alkaloids in its extracts is still in its early stages.

[0004] Although the medicinal value of *Boletus edulis* has been partially recognized, and the pharmacological activity of apophrine alkaloids has attracted much attention, there are currently no reports on the activity of Tinopaine A, an apophrine alkaloid extracted from *Boletus edulis*. Especially in the areas of inflammation regulation and bone metabolism balance, whether it can inhibit inflammation-mediated bone destruction by intervening in osteoclast activation signaling pathways has not been specifically studied. This has led to the underutilization of the medicinal value of this potential active ingredient and left a gap in the development of natural drugs for bone-related diseases. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides the application of Tinopaine A, an apophene alkaloid from bovine bile, in the preparation of osteoclast differentiation inhibitors, nuclear factor expression inhibitors, and therapeutic agents for bone destruction-related diseases. By regulating bone metabolism balance, it alleviates bone destruction, thereby achieving the goal of treating bone destruction-related diseases.

[0006] To achieve the above objectives, the present invention provides the following solution: In a first aspect, the present invention provides an apocynine alkaloid, Tinopaine A, from bovine bile, the chemical structural formula of which is shown below: .

[0007] Secondly, the present invention provides an application of Tinopaine A, an apophrine-like alkaloid from the above-mentioned *Ilex chinensis* bovine bile, for the preparation of an osteoclast differentiation inhibitor.

[0008] Thirdly, the present invention provides an application of the apophrine alkaloid Tinopaine A from the above-mentioned *Ilex chinensis* bile in the preparation of nuclear factor expression inhibitors, wherein the nuclear factor is selected from one or more of NF-κB p65, c-Src, c-Fos, NFATc1, DC-STAMP, CTSK, and JNK.

[0009] Fourthly, the present invention provides an application of Tinopaine A, an apophrine alkaloid from the above-mentioned *Cynanchum paniculatum* ox bile, wherein Tinopaine A is used to prepare a drug that inhibits osteoclast differentiation in vivo.

[0010] Fifthly, the present invention provides the application of Tinopaine A, an apophene alkaloid from bovine bile, in the preparation of a therapeutic agent for bone destruction-related diseases, wherein the bone destruction-related diseases are one or more of osteoporosis, Paget's disease, rheumatoid arthritis, periodontitis, bone metastasis of tumors, and multiple myeloma.

[0011] In a sixth aspect, the present invention provides a drug for inhibiting osteoclast differentiation, the drug comprising the apophene alkaloid Tinopaine A from the above-mentioned *Cynanchum paniculatum* bile, and the drug, in addition to being in tablet, suspension, pill, or capsule form, also includes pharmaceutically acceptable excipients.

[0012] Preferably, the concentration of Tinopaine A is not less than 1 μM, and the drug is an apophine alkaloid with an effective concentration of 1 μM to 10 μM.

[0013] Preferably, the dosage of the drug in animals is 2 mg / kg to 20 mg / kg.

[0014] Preferably, the dosage form of the drug includes gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0015] Preferably, the dosage form of the drug includes conventional dosage forms, sustained-release and controlled-release dosage forms, targeted dosage forms, and immediate-release dosage forms.

[0016] According to specific embodiments provided by the present invention, the present invention discloses the following technical effects: This invention provides the application of Tinopaine A, an apocynine-like alkaloid from bovine bile, in the preparation of osteoclast differentiation inhibitors, nuclear factor expression inhibitors, and therapeutic agents for bone destruction-related diseases. Experimental studies have verified that the Tinopaine A monomer significantly inhibits osteoclast differentiation in vitro at a concentration of 1 μM, exhibits no cytotoxicity at concentrations up to 10 μM, and can target and inhibit the expression of key nuclear factors such as NF-κB p65, c-Src, JNK, c-Fos, and NFATc1. In vivo, at doses of 2 mg / kg-20 mg / kg, it effectively reduces the number of osteoclasts and protects bone trabecular structure, demonstrating excellent therapeutic effects on osteoporosis, rheumatoid arthritis, osteoarthritis, and other bone destruction-related diseases.

[0017] A drug for inhibiting osteoclast differentiation in vitro and in vivo and for treating bone destruction-related diseases. The drug uses Tinopaine A as its active ingredient and is available in various dosage forms, including gastrointestinal, non-gastrointestinal, conventional, sustained-release, and targeted formulations. It can alleviate bone destruction by regulating bone metabolism balance, thereby achieving the goal of treating bone destruction-related diseases. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Effects of different concentrations of Tinopaine A (TA) on the viability of mouse osteoclast precursor cells (BMMs) after 96 hours of treatment; Figure 2 To investigate the effect of different concentrations of Tinopaine A (TA) on osteoclast formation; among which, Figure 2 A in the image represents a representative micrograph of osteoclasts stained with TRAP under different concentrations of drug. Figure 2 B in the figure represents a quantitative statistical bar chart of the number of TRAP-positive multinucleated osteoclasts; Figure 3 To investigate the effects of different concentrations of Tinopaine A (TA) on the expression of NFATc1, a key transcription factor in RANKL-induced osteoclast differentiation; among which, Figure 3 In the figure, A represents the representative bands of NFATc1 protein, internal reference protein PCNA and GAPDH expressed under different concentrations of TA and RANKL, as detected by Western blotting. Figure 3 B in the figure represents a quantitative statistical bar chart of the relative expression levels of the NFATc1 protein band; Figure 4 To investigate the effects of different concentrations of Tinopaine A (TA) on the expression of phosphorylated Src protein, a key signaling molecule in RANKL-induced osteoclast differentiation; among which, Figure 4 In the diagram, A represents the representative bands of phosphorylated Src protein, total Src protein, and internal reference protein GAPDH expressed under different concentrations of TA and RANKL, as detected by Western blotting. Figure 4 B in the figure represents a quantitative statistical bar chart of the relative expression levels of phosphorylated Src protein bands; Figure 5 To investigate the effects of different concentrations of Tinopaine A (TA) on the expression of p65, a key signaling protein in RANKL-induced osteoclast differentiation; among which, Figure 5 In the diagram, A represents the representative bands of p65 protein, internal reference protein PCNA, and GAPDH expressed under different concentrations of TA and RANKL, as detected by Western blotting. Figure 5 B in the figure represents a quantitative statistical bar chart of the relative expression levels of the p65 protein band; Figure 6 To investigate the effects of different concentrations of Tinopaine A (TA) on the phosphorylation levels of key proteins in the RANKL-induced osteoclast differentiation-related MAPK signaling pathway; among which, Figure 6 In the figure, A represents the representative bands of phosphorylated JNK, total JNK, phosphorylated ERK, total ERK, phosphorylated p38, total p38, and internal reference protein GAPDH under different concentrations of TA and RANKL, as detected by Western blotting. Figure 6 B in the figure is a quantitative statistical bar chart of the relative phosphorylation levels of p-JNK, p-ERK, and p-p38 protein bands; Figure 7 To investigate the effects of different concentrations of Tinopaine A (TA) on the expression of c-Fos protein, a key transcription factor in RANKL-induced osteoclast differentiation; among which, Figure 7In the figure, A represents the representative bands of c-Fos protein and internal reference protein GAPDH in total protein under different concentrations of TA and RANKL, as well as their corresponding quantitative statistical bar charts, as detected by Western blotting. Figure 7 In the figure, B represents the representative bands and corresponding quantitative statistical bar charts of c-Fos protein, internal reference protein PCNA and GAPDH in nuclear proteins under different concentrations of TA and RANKL, as detected by Western blotting. Figure 8 To investigate the effects of different concentrations of Tinopaine A on the expression of DC-STAMP and CTSK proteins, key factors in RANKL-induced osteoclast differentiation; among which, Figure 8 In the figure, A represents the representative bands of DC-STAMP protein and internal reference protein GAPDH under different concentrations of TA and RANKL, as well as their corresponding quantitative statistical bar charts, as detected by Western blotting. Figure 8 In the figure, B represents the representative bands of CTSK protein and internal reference protein GAPDH under different concentrations of TA and RANKL, as detected by Western blotting, and their corresponding quantitative statistical bar charts. Figure 9 To investigate the effects of different doses of Tinopaine A (TA) administered via gavage on LPS-induced pathological changes and osteoclast formation in mouse femoral tissue; among which, Figure 9 A in the image shows representative images of the femoral trabecular structure observed by hematoxylin-eosin (HE) staining in the control group (CTL), LPS model group (LPS), LPS combined with low-dose TA treatment group (LPS+TA (2mg / kg)), and LPS combined with high-dose TA treatment group (LPS+TA (20mg / kg)). Figure 9 B in the image represents a representative image of osteoclasts in the femur observed by tartrate-resistant acid phosphatase (TRAP) staining in the corresponding experimental group. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] This invention provides an apocynine alkaloid, Tinopaine A, from the bile of *Ilex chinensis*, the chemical structural formula of which is shown below: .

[0023] Specifically, the apophene alkaloid Tinopaine A in the above-mentioned *Botrytis cinerea* bile has a significant inhibitory effect on osteoclast differentiation. It can significantly inhibit osteoclast differentiation at a concentration of 1 μM, and therefore the concentration of Tinopaine A is not less than 1 μM.

[0024] Based on the apophrine alkaloid Tinopaine A from *Botrytis cinerea* bile, it can be used to prepare osteoclast differentiation inhibitors. It can also be used in the preparation of nuclear factor expression inhibitors, wherein the nuclear factor is selected from one or more of NF-κB p65, c-Src, c-Fos, NFATc1, DC-STAMP, CTSK, and JNK.

[0025] Specifically, when RANKL induces osteoclast differentiation, it first binds to the adaptor protein TRAF6 to form a trimer complex, which, in conjunction with c-Src, further activates the downstream NF-κB and MAPK pathways, thereby promoting the activation of the key osteoclast differentiation factor NFATc1. Phosphorylation of JNK (c-Jun N-terminal kinase) in the MAPK pathway activates downstream c-Fos. Once activated, c-Fos translocates into the nucleus, promoting the expression of NFATc1 and the transcription and translation of osteoclast-related genes: CathepsinK and DC-STAMP.

[0026] NFATc1 is a key transcription factor downstream of the RANK / RANKL signaling pathway, and its activation is a hallmark event of osteoclast formation. Its expression is regulated at multiple levels. In addition to NF-κB and c-Fos directly activating NFATc1, NFATc1 also synergistically binds to c-Fos in its own promoter region for self-amplification.

[0027] Furthermore, Tinopaine A can also be used to prepare drugs that inhibit osteoclast differentiation in vivo. It can also be used in the preparation of therapeutic agents for bone destruction-related diseases, specifically osteoporosis, Paget's disease, rheumatoid arthritis, periodontitis, bone metastases from tumors, and multiple myeloma. Osteoporosis is currently the most common metabolic bone disease, particularly affecting postmenopausal women and the elderly. Inflammatory factors activate osteoclasts, leading to joint bone erosion and destruction. Over-activation of osteoclasts increases bone resorption, resulting in osteoporosis and related inflammatory bone diseases. In mice, Tinopaine A can be used as a therapeutic agent for osteoarthritis, reducing bone damage caused by LPS-induced osteoclast over-activation. Therefore, the drug in this embodiment effectively inhibits osteoclast generation and differentiation in mice, and the inhibitory effect is achieved at doses of 2 mg / kg to 20 mg / kg in animals.

[0028] This invention also provides a drug for inhibiting osteoclast differentiation, comprising the apophene alkaloid Tinopaine A from *Brachys pubescens* bile, and in addition to tablets, suspensions, pills, and capsules, the drug also includes pharmaceutically acceptable excipients. Furthermore, the drug is an apophene alkaloid, and experimental verification has shown that Tinopaine A at a concentration of 1 μM can effectively inhibit osteoclast differentiation. Increasing the drug concentration to 10 μM has little impact on cell survival, thus determining the effective drug concentration to be 1 μM to 10 μM.

[0029] Furthermore, the dosage forms of the aforementioned drugs include gastrointestinal and non-gastrointestinal dosage forms, specifically including conventional dosage forms, sustained-release and controlled-release dosage forms, targeted dosage forms, and immediate-release dosage forms. Gastrointestinal dosage forms include tablets (conventional tablets, coated tablets, chewable tablets, dispersible tablets, sustained-release tablets, etc.), capsules (hard capsules, soft capsules, enteric-coated capsules), granules, pills, and liquid preparations such as oral liquids, syrups, and emulsions. Non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, transdermal dosage forms, and mucosal dosage forms.

[0030] The above content will be further explained below through specific implementation methods. The provided embodiments are only some embodiments of the present invention.

[0031] Example 1 This embodiment aims to detect the cytotoxicity of Tinopaine A using the MTT assay, specifically examining its effect on the survival of osteoclast precursor cells (BMMs). BMMs cells at 2×10 4Cells were seeded at a density of 100 μL / well of α-MEM full culture in 96-well plates, with an appropriate amount of M-CSF added to achieve a final concentration of 50 ng / mL to promote cell proliferation and differentiation. The plates were then incubated in a CO2 incubator for 2 days until cell adhesion and proliferation were observed. The blank control group (CTL) received only 200 μL of full culture. The DMSO group received the same treatment as the blank control group, with an added amount of DMSO to achieve a final concentration ≤0.1%. The drug group was further divided into three treatment groups with different working concentrations of TA: 1 μM, 5 μM, and 10 μM, with three replicates in each group. The wells were replenished with M-CSF as described above. After 4 days, the supernatant of each well was discarded, and 100 μL of 0.5 mg / mL MTT solution diluted with sterile PBS was added to each well. The wells were then incubated at 37°C for 4 hours. After that, the supernatant was discarded, and 150 μL of DMSO was added to react. The crystals were thoroughly dissolved by high-speed vortex mixing for 15 minutes using a digital display vortex mixer. Finally, the 96-well plate was placed in a multi-functional microplate reader, and the density value of each well was measured at a wavelength of 570 nm. The cell viability was calculated as the percentage of each group relative to the blank control group.

[0032] Based on the above, the results are as follows: Figure 1 As shown, after treating BMMs cells with Tinopaine A at concentrations of 1 μM, 5 μM, and 10 μM for 96 h, cell survival was not significantly affected, indicating that Tinopaine A at concentrations ≤10 μM is non-toxic to osteoclast precursor cells (BMMs) in vitro.

[0033] Example 2 Tinopaine A significantly inhibits RANKL-induced osteoclastogenesis. Therefore, this embodiment uses bone marrow-derived macrophages (BMMs) as the experimental subject to detect the effect of Tinopaine A on cell differentiation into osteoclasts, specifically including: Bone marrow cells were collected from female C57BL / 6 mice aged 6-12 weeks. After centrifugation to remove the supernatant, the cells were resuspended in α-MEM and cultured in α-MEM medium for one day. The supernatant was then collected by centrifugation, and the cells were evenly seeded at a rate of 20,000 cells / well in 96-well plates. Five groups were set up with three replicates per group. After the cells adhered for 24 hours, drugs with final concentrations of 1 μM, 5 μM, and 10 μM, MCSF with a final concentration of 50 ng / ml, and RANKL with a final concentration of 100 ng / ml were added. The cells were cultured for another 4-5 days until osteoclasts were clearly visible in the RANKL group, at which point TRAP staining of the osteoclasts was performed. Before staining, aspirate the cell supernatant, wash once with PBS, add 100 μL of 4% tissue fixative to each well, and fix at 4°C for 20 min. Simultaneously prepare the staining solution. After fixation, wash again with PBS, add the staining solution, and incubate at 37°C for 30 min. After staining, observe the staining under a microscope, rinse with sterile water, air dry, and photograph using an automated cell imaging system. The results are as follows: Figure 2 As shown, where Figure 2 In the image, A represents a representative micrograph of osteoclasts stained with TRAP after treatment with different concentrations of the drug. Figure 2 B in the figure represents a quantitative statistical bar chart of the number of TRAP-positive multinucleated osteoclasts; from Figure 2 Results A and B show that Tinopaine A can inhibit RANKL-induced osteoclast differentiation at a concentration of 1 μM, and the inhibitory effect on osteoclast differentiation is more obvious at a concentration of 10 μM.

[0034] Example 3 This embodiment uses mouse peritoneal macrophages RAW264.7 as the experimental subject to detect the effect of Tinopaine A on key osteoclast formation factors, specifically including: Western blotting experiments were performed using the RAW264.7 cell line. After culturing for 2-3 generations, cells were seeded evenly in 6-well plates at a density of 800,000 cells / well. After 24 hours of cell adhesion, a drug was added for 4 hours of treatment, followed by targeted stimulation with RANKL (30 minutes for phosphorylated proteins, and 48 hours for downstream proteins such as NFATC1 and DC-STAMP). After stimulation, the culture medium was discarded, the cells were washed twice with PBS, and lysis buffer was added. The cells were incubated for 10 minutes, and then scraped off and collected into 1.5 ml centrifuge tubes, removing air bubbles. The tubes were placed on ice and vortexed every 5 minutes. After 30 minutes, the supernatant was collected by centrifugation and 5× Loading agent was added. The buffer was denatured by boiling in a metal bath at 100°C for 10 min; then, 20 μL of protein sample was loaded into each well for electrophoresis. The electrophoresis was initially run at a constant voltage of 80V until the orange marker appeared, then adjusted to 120V. Once the indicator buffer reached the bottom, the electrophoresis was switched to a constant current of 300mA for 90 min. After electrophoresis, the membrane was incubated with primary antibody overnight, and the bands were washed three times with TBST for 5 min each time; then incubated with secondary antibody for 1 h, washed with TBST, and developed using a near-infrared laser imaging system. Based on... Figure 3 From A and B in the data, it can be concluded that Tinopaine A can inhibit the expression of NFATc1 in the nucleus; based on Figure 4 From A and B in the data, it can be concluded that Tinopaine A has an inhibitory effect on the expression of the upstream signaling molecule p-Src; based on Figure 5 From A and B in the data, it can be concluded that Tinopaine A can inhibit the expression of NF-κB p65 nuclear protein; simultaneously, Tinopaine A can act on the MAPK pathway, based on... Figure 6 From A and B in the data, it can be concluded that it can affect the phosphorylation of JNK, a key factor in osteoclast apoptosis, formation, and differentiation; based on Figure 7 From A and B in the equation, it can be concluded that it further affects the expression of the downstream transcription factor c-Fos; based on Figure 8 From A and B in the data, it can be concluded that it also has a regulatory effect on the expression of key osteoclast differentiation factors DC-STAMP and CTSK.

[0035] Example 4 This embodiment uses C57BL / 6 female mice as an animal experimental model to investigate the effect of Tinopaine A on LPS-induced inflammatory bone destruction, specifically including: Eight-week-old female C57BL / 6 mice were selected and housed in an environment with relative humidity controlled at 40-70% and temperature maintained at 22-24℃. Mice were randomly divided into a control group (CTL), a model group (LPS), a low-dose group (LPS+TA (2 mg / kg)), and a high-dose group (LPS+TA (20 mg / kg)). On days 1 and 4 of the experiment, except for the control group (CTL) which received PBS, all other groups received intraperitoneal injections of 5 mg / kg LPS to induce inflammation. Mice were weighed daily during the experiment. The low-dose group (LPS+TA (2 mg / kg)) received 2 mg / kg Tinopaine A via gavage daily, and the high-dose group (LPS+TA (20 mg / kg)) received 20 mg / kg Tinopaine A via gavage daily. Seven days later, mouse leg bones were harvested, excess muscle tissue was removed, and the bones were fixed with 4% tissue fixative. The right leg bones of all mice underwent decalcification for one month to prepare sections, while the left leg bones were subjected to CT scans. After the tissue sections were prepared, HE staining and TRAP staining were performed respectively. For HE staining, the decalcified leg bone was first placed in an embedding cassette and incubated overnight in running water. The next day, it was dehydrated stepwise with 50%, 60%, 70%, 80%, 90%, 95%, and 100% ethanol (30 min per step), followed by dehydration with xylene for 20 min. Then, it was immersed in paraffin at 62℃ for 6 h. After embedding, it was cut into 7 μm thick sections using a microtome. The paraffin around the bone tissue was melted on a 62℃ baking machine, and then baked at 37℃ overnight. After dewaxing and rehydrating the sections stepwise with xylene, 100%, 90%, 80%, 70%, 60%, and 50% ethanol (10 min per step), it was stained with hematoxylin for 3 min, differentiated with 1% hydrochloric acid differentiation solution, and stained with eosin for 2 min. Finally, it was immersed in 95% ethanol, 100% ethanol, and xylene in that order. After mounting with neutral resin and drying, it was placed in an automated cell imaging system for photography and observation. The steps for decalcification, embedding, dewaxing, and rehydration of bone tissue for TRAP staining are the same as those for HE staining. After dewaxing and rehydration, TRAP staining solution is prepared. The stained area of ​​bone tissue is delineated with an immunohistochemical pen and staining solution is added. The tissue is placed in a light-proof box at 37°C for 30-45 minutes for staining. Nuclear staining solution is added and stained for 1 minute. The tissue is then immersed in water to wash away the staining solution. The tissue is then soaked in 95% ethanol, 100% ethanol, and xylene in that order. The tissue is then mounted with neutral resin and air-dried.

[0036] Experimental results are as follows Figure 9 As shown, based on Figure 9 As can be seen from A, after LPS inflammatory stimulation, the number of trabeculae in the femur of mice in the model group (LPS) was significantly reduced, while the number of trabeculae in both the low-dose group (LPS+TA (2mg / kg)) and the high-dose group (LPS+TA (20mg / kg)) was significantly increased after gavage treatment with Tinopaine A (TA). Figure 9As can be seen from B, compared with the model group (LPS), the number of osteoclasts in the femur of mice in the low-dose group (LPS+TA (2mg / kg)) and the high-dose group (LPS+TA (20mg / kg)) was significantly reduced. Therefore, Tinopaine A can be used as a treatment agent for osteoarthritis and can effectively reduce bone damage caused by excessive activation of osteoclasts induced by LPS.

[0037] Based on Examples 1 to 4, it was found that Tinopaine A, an apocynine alkaloid from *Botrytis cinerea* bile, exhibited excellent bioactivity and safety in in vitro experiments. It showed no cytotoxicity at concentrations of 1 μM, 5 μM, and 10 μM, and significantly inhibited osteoclast differentiation, with even a 1 μM concentration achieving effective inhibition. Mechanistically, Tinopaine A precisely regulates the signaling pathways of osteoclast formation, downregulating the phosphorylation of upstream signaling factors NF-κB p65, JNK, and c-Src, and reducing the expression levels of downstream key factors c-Fos, NFATc1, DC-STAMP, and CTSK. Through synergistic regulation of upstream and downstream signaling pathways, it achieves highly efficient inhibition of osteoclast differentiation.

[0038] In vivo experiments further validated the therapeutic value of Tinopaine A, which can effectively inhibit the formation of osteoclasts in the femur of mice and significantly reduce the degree of trabecular bone loss. In particular, at doses of 2 mg / kg-20 mg / kg, it can effectively improve LPS-induced inflammatory bone destruction, fully demonstrating its effectiveness and applicability in the in vivo environment.

[0039] Based on consistent results from in vitro and in vivo experiments, Tinopaine A, an apophene alkaloid from bovine bile, has shown excellent efficacy in treating bone destruction-related diseases such as rheumatoid arthritis and osteoporosis. This not only provides a new treatment direction for these diseases but also has great potential to replace bisphosphonates and dennosumab as targeted drugs for bone-related diseases. Furthermore, it has clarified its broad application prospects in the preparation of osteoclast differentiation inhibitors and therapeutic agents for bone destruction-related diseases.

[0040] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0041] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. An aporphine alkaloid Tinopaine A in Gjelderia insignis, characterized by, The chemical structural formula of the Tinopaine A is as shown in the following: 。 2. Use of the aporphine alkaloid Tinopaine A from Gastrodia elata Blume. according to claim 1, characterized in that, The Tinopaine A is used for preparing an inhibitor of osteoclast differentiation.

3. Use of the aporphine alkaloid Tinopaine A from G. tinctoria according to claim 1 for the preparation of an inhibitor of the expression of nuclear factors, characterized in that, The nuclear factor is selected from one or more of NF-κB p65, c-Src, c-Fos, NFATc1, DC-STAMP, CTSK, JNK.

4. The use of aporphine alkaloid Tinopaine A in G. tinus according to claim 1, characterized by, The Tinopaine A is used for preparing a medicine for inhibiting osteoclast differentiation in vivo.

5. Use of the aporphine alkaloid Tinopaine A in G. tinus for the preparation of a medicament for the treatment of bone destruction-related diseases, according to claim 1, characterized in that, The kind of the bone destruction related disease is one or more of osteoporosis, Paget's disease of bone, rheumatoid arthritis, periodontitis, tumor bone metastasis, multiple myeloma.

6. A medicament for inhibiting differentiation of osteoclasts, characterized by, The medicine comprises the aporphine alkaloid Tinopaine A in Gastrodia elata Blume as claimed in claim 1, and the medicine further comprises a pharmaceutically acceptable auxiliary agent in addition to tablets, suspensions, pills, and capsules.

7. The medicament for inhibiting differentiation of osteoclast according to claim 6, wherein The concentration of the Tinopaine A is not less than 1 μM, and the medicine is an aporphine alkaloid, and the medicine action concentration is 1 μM-10 μM.

8. The medicament for inhibiting differentiation of osteoclast according to claim 6, wherein The dose of the medicine in an animal body is 2 mg / kg-20 mg / kg.

9. The medicament for inhibiting differentiation of osteoclast according to claim 6, wherein The dosage form of the medicine comprises a gastrointestinal administration dosage form and a non-gastrointestinal administration dosage form.

10. The medicament for inhibiting differentiation of osteoclast according to claim 9, wherein The dosage form of the medicine comprises a conventional dosage form, a sustained and controlled release dosage form, a targeted dosage form, and an immediate release dosage form.

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