Pharmaceutical composition for treating osteoporosis, preparation and preparation method thereof

Abstract

The invention discloses a pharmaceutical composition for treating osteoporosis, a preparation and a preparation method thereof, and belongs to the technical field of medicines. The pharmaceutical composition comprises notopterol and BG peptide, the amino acid sequence of the BG peptide is as shown in SEQ ID NO. 1. The invention also discloses a pharmaceutical preparation for treating osteoporosis and a preparation method thereof. The pharmaceutical composition can effectively solve the problems of poor curative effect, large side effect and limited ostealgia relieving effect of the existing medicines for treating osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a pharmaceutical composition, formulation, and preparation method for treating osteoporosis. Background Technology

[0002] Osteoporosis (OP) is a systemic bone disease characterized by low bone mass, damage to bone microstructure, and increased bone fragility. In its early stages, osteoporosis often presents with no obvious symptoms. As the disease progresses, patients may experience symptoms such as lower back pain, decreased height, and kyphosis. In severe cases, even minor external forces such as coughing, sneezing, or bending over can trigger fractures, significantly impacting patients' quality of life and physical health.

[0003] Currently, the treatment of osteoporosis focuses on the key aspect of inhibiting bone resorption. Bisphosphonates are commonly used first-line drugs in clinical practice. They can specifically bind to hydroxyapatite in bone, inhibiting osteoclast activity and thus reducing bone resorption and increasing bone density. Another mainstream drug, RANKL inhibitors, works by blocking the binding of RANKL to its receptor RANK, interfering with the differentiation, maturation, and activation of osteoclasts, thereby inhibiting bone resorption.

[0004] However, while these drugs have therapeutic effects, they also present several significant problems. Long-term use of bisphosphonates can lead to osteonecrosis of the mandible, a serious adverse reaction that not only causes immense pain but can also affect chewing function and facial appearance. Furthermore, the risk of atypical femoral fractures increases with prolonged use; these fractures often occur between the lesser trochanter of the femur and the knee, making treatment difficult and recovery time long. Moreover, these drugs are not ideal for relieving bone pain; many patients continue to suffer from bone pain even after medication, and their quality of life is unlikely to improve significantly.

[0005] Therefore, it is urgent to develop safer and more effective drugs for the treatment of osteoporosis. Summary of the Invention

[0006] The problem to be solved by the present invention is to provide a pharmaceutical composition, preparation and preparation method for treating osteoporosis. The pharmaceutical composition can effectively solve the problems of poor efficacy, large side effects and limited pain relief of current osteoporosis treatment drugs.

[0007] The technical solution adopted to solve the technical problem is to provide a pharmaceutical composition for treating osteoporosis, the pharmaceutical composition comprising notopterygium alcohol and BG peptide; the amino acid sequence of BG peptide is shown in SEQ ID NO.1.

[0008] Preferably, the molar ratio of notopterygium alcohol to BG peptide is (10~1000):1.

[0009] More preferably, the molar ratio of notopterygium alcohol to BG peptide is (50~200):1.

[0010] More preferably, the molar ratio of notopterygium alcohol to BG peptide is 100:1.

[0011] A pharmaceutical preparation for treating osteoporosis, comprising the above-described pharmaceutical composition and a pharmaceutically acceptable carrier or excipient.

[0012] More preferably, the pharmaceutical preparation is a liquid injection.

[0013] A method for preparing a pharmaceutical formulation for treating osteoporosis includes the following steps: Prepare the mother liquor of Qianghuo alcohol and the mother liquor of BG peptide separately. Mix the mother liquor of Qianghuo alcohol and the mother liquor of BG peptide according to the molar ratio of Qianghuo alcohol and BG peptide, and make up the volume. Filter to obtain the liquid injection.

[0014] Preferably, the mother liquor of Qianghuo alcohol is prepared by the following steps: dissolving Qianghuo alcohol in dimethyl sulfoxide and diluting it with PBS buffer to obtain the mother liquor of Qianghuo alcohol.

[0015] Preferably, the BG peptide stock solution is prepared by the following steps: dissolving BG peptide in water for injection to obtain the BG peptide stock solution.

[0016] Preferably, the volume is adjusted using PBS buffer; filtration is performed using a 0.22 μm sterile filter membrane.

[0017] More preferably, the pH of the PBS buffer is 7.4.

[0018] More preferably, a method for preparing a pharmaceutical formulation for treating osteoporosis includes the following steps: Weigh out Qianghuo alcohol and dissolve it in dimethyl sulfoxide at a ratio of 10 mg:1 mL. Then dilute it with PBS buffer at pH 7.4, controlling the final concentration of dimethyl sulfoxide to ≤1%, to obtain a Qianghuo alcohol stock solution with a mass concentration of 1 mg / mL. Weigh out BG peptide and dissolve it in water for injection at a ratio of 1 mg:1 mL, to obtain a BG peptide stock solution with a mass concentration of 1 mg / mL. Mix the Qianghuo alcohol stock solution and the BG peptide stock solution according to the molar ratio of Qianghuo alcohol and BG peptide, and make up to volume with PBS buffer at pH 7.4. Filter the solution using a 0.22 μm sterile filter membrane to obtain the liquid injection.

[0019] The present invention has the following beneficial effects: The BG peptide of this invention is a novel, artificially synthesized mouse RANK protein-targeting peptide. It specifically binds to key functional domains of the intracellular segment of the mouse RANK protein, forming a high-affinity interaction. This physically blocks RANK trimerization and subsequent signaling complex assembly, inhibits RANK autophosphorylation and downstream TRAF6 / TAK1 activation, thereby preventing nuclear translocation and phosphorylation of NF-κB transcription factor and significantly reducing TNF-α. α The peptide directly targets the RANK intracellular domain, avoiding off-target effects and providing more precise signal regulation. Meanwhile, the cysteine ​​residues in the sequence can enhance stability through disulfide bonds.

[0020] In this invention, notopterygium alcohol, as an upstream regulator, can gently promote osteogenic formation, while BG peptide, as an inhibitor of NF-κB signaling, can inhibit osteoclastosis and relieve pain. The combination of the two can synergistically act on the osteogenic-osteoclast balance and the neuro-skeletal axis, which can significantly reduce the required dose of each drug, avoid the toxic side effects and cost problems of high doses of single drugs, and achieve the dual efficacy of bone health and pain relief. Attached Figure Description

[0021] Figure 1 This is a diagram showing the binding relationship between the BG peptide of this invention and the intracellular segment of mouse RANK protein; Figure 2 This is the mass spectrum of BG peptide; Figure 3 The images show the HE staining results of the femurs of mice in different groups in the experimental case; (a) is the HE staining result of the femur of mice in the sham-operated group; (b) is the HE staining result of the femur of mice in the model group; (c) is the HE staining result of the femur of mice in the Qianghuo alcohol single-drug group; (d) is the HE staining result of the femur of mice in the BG single-drug group; (e) is the HE staining result of the femur of mice in the drug combination group; (f) is the HE staining result of the femur of mice in the positive control group; (g) is a magnified view of (a); (h) is a magnified view of (b); (i) is a magnified view of (c); (j) is a magnified view of (d); (k) is a magnified view of (e); and (l) is a magnified view of (f). Figure 4 The images show the femoral scan results of mice in different groups in the experimental case; (a) is the sham-operated group; (b) is the model group; (c) is the Qianghuo alcohol monotherapy group; (d) is the BG monotherapy group; (e) is the drug combination group; and (f) is the positive control group. Figure 5 The graphs show the bone morphometric analysis of mice in different groups in the experimental case; where (a) is the bone volume fraction graph; (b) is the trabecular bone thickness graph; and (c) is the trabecular bone number graph. Figure 6The images show the results of serum bone metabolism marker detection in mice from different groups in the experimental case; (a) shows the serum type I procollagen N-terminal propeptide level; (b) shows the osteocalcin level; (c) shows the tartrate-resistant acid phosphatase 5b level; and (d) shows the cathepsin K level. Figure 7 The graph shows the bone strength results of mice in different groups in the experimental case; Figure 8 The figures show the results of pain behavior tests in different groups of mice in the experimental case; where (a) is the mechanical pain threshold result; and (b) is the thermal pain threshold result. Figure 9 The graph shows the ALP activity detection results after different treatments in the experimental case. Figure 10 This is a graph showing the expression levels of related gene mRNAs after different treatments in the experimental cases; where (a) is... Dlx5 (a) The relative expression level of mRNA; (b) is Runx2 The relative expression level of mRNA; (c) is SP7 The relative expression level of mRNA; (d) is Nfatc1 The relative expression level of mRNA; (e) is Fos The relative expression level of mRNA; (f) Acp5 The relative expression level of mRNA; (g) is Ctsk The relative expression level of mRNA; Figure 11 This is a graph showing the release levels of inflammatory factors after different treatments in the experimental cases; where (a) is TNF-α. α (a) is a horizontal plot; (b) is an IL-6 horizontal plot. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0023] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0024] The features and performance of the present invention will be further described in detail below with reference to the embodiments. The BG peptide of the present invention was synthesized by Shanghai Botai Company.

[0025] Example 1 A pharmaceutical composition for treating osteoporosis includes notopterygium alcohol and BG peptide; the amino acid sequence of BG peptide is shown in SEQ ID NO.1; the molar ratio of notopterygium alcohol to BG peptide is 100:1.

[0026] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0027] A pharmaceutical preparation for treating osteoporosis, wherein the dosage form is a liquid injection, and the preparation method includes the following steps: (1) Weigh 10 mg of Qianghuo alcohol, add 1 mL of dimethyl sulfoxide, vortex to dissolve, and obtain a 10 mg / mL Qianghuo alcohol dimethyl sulfoxide solution; add PBS buffer with pH 7.4 to the Qianghuo alcohol dimethyl sulfoxide solution to dilute to a Qianghuo alcohol mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide ≤1% to obtain Qianghuo alcohol mother liquor. (2) Weigh 1 mg of BG peptide, add 1 mL of water for injection, and vortex to dissolve to obtain a BG peptide stock solution with a mass concentration of 1 mg / mL. (3) Mix 100 μL of the stock solution of Qianghuo alcohol and 1 μL of the stock solution of BG peptide according to the molar ratio of Qianghuo alcohol and BG peptide 100:1, and make up to 1 mL with PBS buffer at pH 7.4. Filter with a 0.22 μm sterile filter membrane to obtain the liquid injection. The molar concentration of Qianghuo alcohol in the liquid injection is 282 μM and the molar concentration of BG peptide is 2.82 μM.

[0028] Example 2 A pharmaceutical composition for treating osteoporosis includes notopterygium alcohol and BG peptide; the amino acid sequence of BG peptide is shown in SEQ ID NO.1; the molar ratio of notopterygium alcohol to BG peptide is 200:1.

[0029] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0030] A pharmaceutical preparation for treating osteoporosis, wherein the dosage form is a liquid injection, and the preparation method includes the following steps: (1) Weigh 10 mg of Qianghuo alcohol, add 1 mL of dimethyl sulfoxide, vortex to dissolve, and obtain a 10 mg / mL Qianghuo alcohol dimethyl sulfoxide solution; add PBS buffer with pH 7.4 to the Qianghuo alcohol dimethyl sulfoxide solution to dilute to a Qianghuo alcohol mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide ≤1% to obtain Qianghuo alcohol mother liquor. (2) Weigh 1 mg of BG peptide, add 1 mL of water for injection, and vortex to dissolve to obtain a BG peptide stock solution with a mass concentration of 1 mg / mL. (3) Mix 200 μL of the mother liquor of Qianghuo alcohol and 1 μL of the mother liquor of BG peptide according to the molar ratio of Qianghuo alcohol and BG peptide 200:1, and make up to 1 mL with PBS buffer at pH 7.4. Filter with a 0.22 μm sterile filter membrane to obtain the liquid injection. The molar concentration of Qianghuo alcohol in the liquid injection is 564 μM and the molar concentration of BG peptide is 2.82 μM.

[0031] Example 3 A pharmaceutical composition for treating osteoporosis includes notopterygium alcohol and BG peptide; the amino acid sequence of BG peptide is shown in SEQ ID NO.1; the molar ratio of notopterygium alcohol to BG peptide is 1000:1.

[0032] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0033] A pharmaceutical preparation for treating osteoporosis, wherein the dosage form is a liquid injection, and the preparation method includes the following steps: (1) Weigh 10 mg of Qianghuo alcohol, add 1 mL of dimethyl sulfoxide, vortex to dissolve, and obtain a 10 mg / mL Qianghuo alcohol dimethyl sulfoxide solution; add PBS buffer with pH 7.4 to the Qianghuo alcohol dimethyl sulfoxide solution to dilute to a Qianghuo alcohol mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide ≤1% to obtain Qianghuo alcohol mother liquor. (2) Weigh 1 mg of BG peptide, add 1 mL of water for injection, and vortex to dissolve to obtain a BG peptide stock solution with a mass concentration of 1 mg / mL. (3) Mix 1000 μL of the stock solution of Qianghuo alcohol and 1 μL of the stock solution of BG peptide according to the molar ratio of Qianghuo alcohol and BG peptide 1000:1, and make up to 1 mL with PBS buffer at pH 7.4. Filter with a 0.22 μm sterile filter membrane to obtain the liquid injection. The molar concentration of Qianghuo alcohol in the liquid injection is 2820 μM and the molar concentration of BG peptide is 2.82 μM.

[0034] Example 4 A pharmaceutical composition for treating osteoporosis includes notopterygium alcohol and BG peptide; the amino acid sequence of BG peptide is shown in SEQ ID NO.1; the molar ratio of notopterygium alcohol to BG peptide is 50:1.

[0035] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0036] A pharmaceutical preparation for treating osteoporosis, wherein the dosage form is a liquid injection, and the preparation method includes the following steps: (1) Weigh 10 mg of Qianghuo alcohol, add 1 mL of dimethyl sulfoxide, vortex to dissolve, and obtain a 10 mg / mL Qianghuo alcohol dimethyl sulfoxide solution; add PBS buffer with pH 7.4 to the Qianghuo alcohol dimethyl sulfoxide solution to dilute to a Qianghuo alcohol mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide ≤1% to obtain Qianghuo alcohol mother liquor. (2) Weigh 1 mg of BG peptide, add 1 mL of water for injection, and vortex to dissolve to obtain a BG peptide stock solution with a mass concentration of 1 mg / mL. (3) Mix 50 μL of the stock solution of Qianghuo alcohol and 1 μL of the stock solution of BG peptide according to the molar ratio of Qianghuo alcohol and BG peptide 50:1, and make up to 1 mL with PBS buffer at pH 7.4. Filter with a 0.22 μm sterile filter membrane to obtain the liquid injection. The molar concentration of Qianghuo alcohol in the liquid injection is 141 μM and the molar concentration of BG peptide is 2.82 μM.

[0037] Example 5 A pharmaceutical composition for treating osteoporosis includes notopterygium alcohol and BG peptide; the amino acid sequence of BG peptide is shown in SEQ ID NO.1; the molar ratio of notopterygium alcohol to BG peptide is 10:1.

[0038] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0039] A pharmaceutical preparation for treating osteoporosis, wherein the dosage form is a liquid injection, and the preparation method includes the following steps: (1) Weigh 10 mg of Qianghuo alcohol, add 1 mL of dimethyl sulfoxide, vortex to dissolve, and obtain a 10 mg / mL Qianghuo alcohol dimethyl sulfoxide solution; add PBS buffer with pH 7.4 to the Qianghuo alcohol dimethyl sulfoxide solution to dilute to a Qianghuo alcohol mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide ≤1% to obtain Qianghuo alcohol mother liquor. (2) Weigh 1 mg of BG peptide, add 1 mL of water for injection, and vortex to dissolve to obtain a BG peptide stock solution with a mass concentration of 1 mg / mL. (3) Mix 10 μL of the stock solution of Qianghuo alcohol and 1 μL of the stock solution of BG peptide according to the molar ratio of Qianghuo alcohol and BG peptide 10:1, and make up to 1 mL with PBS buffer at pH 7.4. Filter with a 0.22 μm sterile filter membrane to obtain the liquid injection. The molar concentration of Qianghuo alcohol in the liquid injection is 28.2 μM and the molar concentration of BG peptide is 2.82 μM.

[0040] Test case 1. BG peptide structure and binding relationship BG peptide is a novel synthetic mouse RANK protein targeting peptide synthesized by Shanghai Botai Company. Its amino acid sequence SEQ ID NO.1 is: KLVALKTNGDGNCIMHVA CQFMWGVQDTD. Its structure prediction and mass spectrometry are as follows: Figures 1-2 As shown. Figure 1 The Alphafold3 structure prediction shows that the BG peptide (red) may bind to the intracellular domain of mouse RANK protein (green). It specifically binds to key functional domains of the mouse RANK protein intracellular domain (such as the TRAF6 binding site), forming a high-affinity interaction. This physically blocks RANK trimerization and subsequent signaling complex assembly, inhibits RANK autophosphorylation and downstream TRAF6 / TAK1 activation, thereby preventing nuclear translocation and phosphorylation of NF-κB transcription factor and significantly reducing TNF-α. α The peptide directly targets the RANK intracellular domain, avoiding off-target effects and providing more precise signal regulation. Simultaneously, the cysteine ​​(C) residues in the sequence enhance stability through disulfide bonds. Figure 2 The mass spectrometry results show that the molecular weight of the synthesized BG peptide is in line with expectations.

[0041] 2. Validation of the therapeutic efficacy of a drug composition for treating osteoporosis. Sixty female C57 mice were randomly divided into six groups (n=10): sham operation group (Sham), model group (OVX), notopterygium wilfordii monotherapy group (Not), BG monotherapy group (BG), drug combination group (Not+BG), and positive control group (E2). In the sham operation group, only the periovarian adipose tissue was removed by opening the abdominal cavity, but the ovary was not removed; the other five groups of mice were used to establish an OVX (oophorectomy)-induced osteoporotic female C57 mouse model.

[0042] The high-concentration stock solution of Qianghuo alcohol was first prepared with dimethyl sulfoxide (10 mg / mL), and then diluted with PBS buffer to 1 mg / mL; BG peptide was dissolved in water for injection to a final concentration of 0.01 mg / mL.

[0043] Mice in each group were administered the drugs. The sham-operated group and model group received PBS buffer after each administration. The Qianghuo alcohol monotherapy group received 20 mg / kg of Qianghuo alcohol per administration; the BG peptide monotherapy group received 0.2 mg / kg of BG peptide per administration; the drug combination group received 5 mg / kg of Qianghuo alcohol and 0.05 mg / kg of BG peptide per administration; the positive control group received 0.1 mg / kg of estradiol per administration. Administration was intraperitoneal injection. Administered every 3 days at 18:00 for 12 weeks, for a total of 28 administrations. All data from the following validation experiments are expressed as mean ± standard deviation. Two-way ANOVA was used to compare the interaction between the drug combination group and the monotherapy group. If an interaction was found... P A value < 0.05 indicates a statistically significant synergistic effect, rather than a simple additive effect.

[0044] 2.1 HE staining of the femur After euthanizing the mice in each group, fixation and decalcification were performed, and sections were prepared in 5 μm thick longitudinal sections. The sections were then spread and dried at 60°C overnight. After dewaxing and hydration (using xylene I / II for 10 min each, followed by treatment with 100% ethanol, 95% ethanol, and 70% ethanol for 5 min each, and finally soaking in distilled water for 3-5 min), hematoxylin staining of the nuclei was performed for 5 min, followed by bluing with running water for 15 min, 1% eosin staining of the cytoplasm for 30 s, graded ethanol dehydration, xylene clearing, and mounting with neutral resin. The staining results were then observed under an optical microscope. Figure 3 As shown.

[0045] from Figure 3 As can be seen, the OVX group showed sparse and broken trabeculae, enlarged medullary cavity, and fat cell infiltration, indicating severe osteoporosis; the trabecular thickness of the Qianghuo alcohol monotherapy group and the BG monotherapy group increased in some areas, but there were still absorption depressions; the trabecular density of the drug combination group (Qianghuo alcohol + BG) was significantly restored, forming a dense network structure, and the area of ​​newly formed bone matrix (powdered area) was close to that of the positive drug E2 group.

[0046] 2.2 Micro-CT Bone Morphometric Analysis Mouse femurs were harvested and scanned using a Micro-CT scanner. Specific parameters measured included bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N). The statistical process involved manually drawing Regions of Interest (ROIs) using software (CTAn), ensuring the complete trabecular network was included and excluding cortical bone boundaries. Threshold segmentation was performed, using a fixed threshold (typically 160-170 HU) or the Otsu algorithm to distinguish bone tissue from non-bone tissue, and binarized images were extracted. The Feldkamp algorithm was used to reconstruct a 3D model, and parameters were analyzed. Results are as follows: Figure 4 As shown.

[0047] BV / TV is used to directly reflect the proportion of bone mass and is calculated according to the following formula (1); Tb.Th is calculated based on the maximum sphere fitting algorithm or the three-dimensional direct distance measurement method to calculate the average thickness of trabeculae, in μm; Tb.N is calculated using a model-independent method to calculate the number of trabeculae per unit length, in mm. -1 The result is obtained by calculation using the following formula (2). Figure 5 As shown.

[0048] BV / TV = Bone volume / Total tissue volume (1) Tb.N = (bone volume / total tissue volume) / Tb.Th (2) from Figure 4 As can be seen, the bone microstructure in the model group was more sparse and fragmented than that in the sham-operated group, with larger and irregularly distributed pores, indicating that the OVX-induced osteoporosis model in female C57 mice was fully established. Comparison of bone microstructure in the drug intervention groups revealed that the structural improvement effect of the drug combination group was significantly better than that of the single-drug group, showing that the drug combination has a synergistic effect and can more effectively improve tissue microstructure. Furthermore, from... Figure 5 The results show that the drug combination group exhibited more significant improvements in bone volume fraction, trabecular thickness, and trabecular number compared to the single-drug groups (Qianghuo alcohol single-drug group and BG peptide single-drug group) and the model group, and was even superior to the positive control group estradiol. Most indicators showed highly significant differences compared to the model group, indicating that the combined use of Qianghuo alcohol and BG peptide has a synergistic effect and can more effectively improve bone microstructure. The positive control group performed similarly to the sham-operated group in the three indicators and was significantly better than the model group. This is consistent with the expected bone-protective effect of estradiol and further verifies the effectiveness of the experimental model and detection method.

[0049] 2.3 Detection of serum bone metabolism markers Blood was collected from the abdominal aorta of mice 24 hours after the last administration. Serum was collected by centrifugation, and serum bone metabolism markers PINP, OCN, Tracp-5b, and Cath-K were detected using an ELISA kit. PINP (serum type I procollagen N-terminal propeptide) is a product of type I collagen synthesis; elevated levels indicate increased bone formation activity and are a marker reflecting bone formation status. OCN (osteocalcin) is also a marker of bone formation, synthesized and secreted by osteoblasts; its serum level is closely related to osteoblast activity and can be used to assess the state of bone formation. Tracp-5b (tartrate-resistant acid phosphatase 5b) represents a marker of bone resorption; it is mainly secreted by osteoclasts, and elevated levels indicate enhanced osteoclast activity and active bone resorption. Cath-K (cathepsin K) is a marker enzyme of osteoclasts; during bone resorption, it effectively breaks down matrix components such as bone collagen, releasing minerals such as calcium and phosphorus into the bloodstream. Changes in its activity directly affect bone metabolic balance. Results are as follows: Figure 6 As shown.

[0050] from Figure 6 As can be seen, the model group mice exhibited typical characteristics of bone metabolism imbalance, with significantly decreased levels of bone formation markers PINP and OCN, and a significantly increased level of bone resorption marker Tracp-5b. This indicates that OVX successfully induced the pathological state of decreased bone formation and increased bone resorption. Although the Qianghuochun monotherapy group and the BG monotherapy group improved the bone metabolism imbalance to some extent, as evidenced by increased levels of bone formation markers and decreased levels of bone resorption markers, the improvement was relatively limited. Building upon this, the combined use of Qianghuo alcohol and BG peptide demonstrated a more significant effect on bone metabolism regulation. Compared with the single-drug group and the model group, the levels of bone formation markers PINP and OCN were significantly increased in the drug combination group, suggesting that it can more effectively promote bone formation. Simultaneously, the level of the bone resorption marker Tracp-5b decreased more significantly, indicating a more prominent inhibitory effect on bone resorption. Furthermore, the Cath-K level increased in the OVX group, suggesting enhanced osteoclast function. The Cath-K levels in all treatment groups were lower than in the OVX group, indicating that the drugs inhibited the activity of osteoclast-related enzymes, with the combined use of Qianghuo alcohol and BG peptide showing better efficacy. The combined use of Qianghuo alcohol and BG peptide can more effectively regulate bone metabolism bidirectionally, more strongly promoting bone formation and inhibiting bone resorption, providing strong experimental evidence for developing novel combination drug strategies for treating bone metabolism-related diseases.

[0051] 2.4 Bone biomechanical testing A bone sample from the left femur was taken for a three-point bending test. The maximum load, i.e., bone strength, was measured using a bone biomechanical testing instrument. The results are as follows: Figure 7 As shown.

[0052] from Figure 7 The results show that the drug combination group can more effectively improve the biomechanical properties of bone and enhance the bone's resistance to external forces. Its bone strength is significantly higher than that of the single drug group and the model group, and there is no significant difference compared with the E2 positive control group, indicating that the generated bone quality is better.

[0053] 2.5 Pain Behavioral Testing A mouse model of neuropathic pain was established using the spinal nerve ligation (SNL) method. Mechanical pain threshold (von Frey ciliary test) and thermal pain threshold (hot plate test) were measured using a behavioral pain testing device. The results are as follows: Figure 8 As shown.

[0054] from Figure 8As can be seen, compared with the model group, the force and time values ​​of the drug composition group were significantly increased in both mechanical and thermal pain threshold tests, indicating that the drug composition can effectively improve the pain threshold of mice to mechanical and thermal stimuli and alleviate pain sensitivity. Compared with the single-drug group, the pain threshold improvement effect of the drug composition group is better, indicating that the combined use of Qianghuo alcohol and BG peptide may have a synergistic effect in regulating the pain threshold and can more effectively improve the pain sensitivity of mice. Compared with the sham-operated group, the pain threshold of the drug composition group is close, indicating that the combined use of Qianghuo alcohol and BG peptide can restore the pain threshold of mice to a near-normal level and has a synergistic analgesic effect.

[0055] 2.6 ALP Activity Assay Osteoblasts derived from MC3T3-E1 cells were established and treated with different drugs. Specifically, mouse MC3T3-E1 cells were seeded at a density of 2 × 10⁻⁶ cells / cells. 5 Patients were placed in 6-well plates using α-MEM complete medium (containing 90% α-MEM basal medium, 10% fetal bovine serum, and 1% penicillin-streptomycin mixture). 16 μL / mL of mouse osteoblast-inducing factor (batch number: AI1001-004) was added, and the plates were incubated in a 5% CO2, 37℃ incubator. The medium was replaced with fresh medium containing the inducing factor every 3 days. 2.00 mg of ≥98% purity of *Notopterygium incisum* alcohol was accurately weighed and dissolved in 100 μL of dimethyl sulfoxide to prepare a 56.54 mmol / L stock solution, which was then diluted with medium to a working concentration of 20 μmol / L (DMSO final concentration ≤0.1%). BG peptide was dissolved in water for injection to prepare a 1 mg / mL stock solution, resulting in a working concentration of 60 μg / mL. At the start of osteogenic induction, the working concentrations of *Notopterygium incisum* alcohol and / or BG peptide were added simultaneously, with the medium changed and drugs replenished every 3 days. The Model group underwent only osteogenic induction without drug administration.

[0056] ALP activity assays were performed 5 days after osteogenic induction. The culture medium was discarded, and cells were washed twice with PBS. 1 mL of ALP lysis buffer (containing protease inhibitors) was added to each well. After lysis for 10 min, cells were collected using a cell scraper. The cells were centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA method. The assay reagent was added at a loading rate of 20 μg protein, and the cells were incubated at 37°C in the dark for 10 min. The absorbance was measured at 450 nm. ALP activity was expressed as the amount of p-nitrophenol produced per unit time (nmol / min / mg protein). Results are as follows: Figure 9 As shown.

[0057] from Figure 9It can be seen that both Qianghuo alcohol and BG peptide, when used alone, can improve ALP activity to a certain extent and can partially promote osteogenic differentiation. The ALP activity of the combined use of Qianghuo alcohol and BG peptide is significantly higher than that of the single drug group and the model group, and the difference is statistically significant. This indicates that the combined use of the two drugs has a synergistic effect in improving ALP activity, can more effectively regulate ALP-related physiological processes, and promote bone differentiation more significantly.

[0058] 2.7 RT-qPCR detection of related gene mRNA expression Osteogenesis-related gene expression was detected using MC3T3-E1 cells. Osteogenesis-related genes include... Dlx5, Runx2 SP7 RAW264.7 cells were used to detect osteoclast-related genes, including: Fos , Acp5 , Ctsk , Nfatc1 The specific steps are as follows: MC3T3-E1 and RAW264.7 cells are mixed at a ratio of 2 × 10⁻⁶. 6 Cells were seeded per well in six-well plates, with 3 mL of α-MEM complete medium added to each well. After 24 h of incubation in a 37°C CO2 incubator containing 5% CO2, MC3T3-E1 cells were divided into control group (osteogenic induction), group receiving 20 μmol / L of notopterygium oleoresin (Qianghuo ol) + osteogenic induction, group receiving 60 μg / mL of BG + osteogenic induction, and group receiving 20 μmol / L of notopterygium oleoresin (Qianghuo ol) + 60 μg / mL of BG + osteogenic induction. RAW264.7 cells were divided into control group (osteoclastosis induction: 50 ng / mL RANKL inducing factor), group receiving 20 μmol / L of notopterygium oleoresin (Qianghuo ol) + 50 ng / mL RANKL, group receiving 60 μg / mL of BG + 50 ng / mL RANKL, and group receiving notopterygium oleoresin (Qianghuo ol) + 60 μg / mL of BG + 50 ng / mL RANKL. All cells were then cultured for another 48 h. After h, the culture medium in the six-well plate was discarded, and the plate was washed twice with PBS. 1 mL of Trizol reagent was added to each well for lysis. The lysed liquid was aspirated into EP tubes and incubated for 5 min. RNA was extracted according to the total RNA extraction kit and reverse transcribed into cDNA using a gradient PCR instrument. The expression levels of target genes in each group were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). 2 －ΔΔCt Method calculation Dlx5 , Runx2 , SP7 , Nfatc1 , Fos , Acp5 , Ctsk The relative expression levels of mRNA were determined, and the specific primer sequences are shown in Table 1 below. The results are as follows: Figure 10 As shown.

[0059] Table 1. List of specific primer sequences

[0060] Figure 10 qPCR results showed that the drug-treated group Runx2, SP7 and Dlx5 mRNA expression in all groups was significantly higher than in the control group, while the drug combination group was significantly higher than in the groups treated with Qianghuo alcohol and BG peptide alone; Nfatc1, Fos, Acp5, Ctsk mRNA expression in both groups was significantly lower than in the control group, while the expression in the drug combination group was significantly lower than in the groups treated with Qianghuo alcohol and BG peptide alone.

[0061] 2.8 Assay of the release level of inflammatory factors downstream of NF-κB under immune cell inflammatory stimulation RAW264.7 cells were cultured with LPS (200 ng / mL). Treatment with notopterygium nitrate and BG peptide was the same as described in section 2.6. TNF-α levels in the culture medium were measured after 4 hours. α And IL-6 levels, the results are as follows Figure 11 As shown.

[0062] In the complex physiological processes of bone metabolism, a dynamic balance is maintained between osteoblast-dominated bone formation and osteoclast-dominated bone resorption, with inflammatory factors playing a crucial regulatory role. When this balance is disrupted, such as in estrogen deficiency caused by ovariectomy, bone resorption often exceeds bone formation, leading to a series of problems including bone loss and bone microstructure damage. Figure 11 The results show that both Qianghuo alcohol and BG peptide, when used alone, can improve the release of inflammatory factors, but the level of inflammatory factors is lowest in the drug combination group, indicating that the synergistic effect of Qianghuo alcohol and BG peptide has a better effect.

[0063] In summary, when low-dose Notopterygium wilfordii and BG peptide are used in combination, they achieve a synergistic effect. Notopterygium wilfordii promotes osteogenic growth and increases bone formation, while BG peptide inhibits osteoclasts and inflammatory factors, reducing bone resorption and inhibiting osteogenic processes. This synergistic effect allows for a more effective correction of the imbalance between bone formation and resorption. Notopterygium wilfordii promotes osteoblast synthesis of more bone matrix, while BG peptide, by inhibiting osteoclast activity, prevents excessive resorption of newly synthesized bone matrix. Simultaneously, its anti-inflammatory effect provides a more favorable microenvironment for osteoblasts, further promoting bone formation. The two complement each other, working together to regulate bone metabolism and providing a potentially effective treatment strategy for improving diseases related to bone metabolic imbalances caused by various factors.

[0064] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of the present invention.

Claims

1. A pharmaceutical composition for treating osteoporosis, characterized in that, The pharmaceutical composition comprises notopterygium alcohol and BG peptide; the amino acid sequence of the BG peptide is shown in SEQ ID NO.

1.

2. The pharmaceutical composition for treating osteoporosis as described in claim 1, characterized in that, The molar ratio of the indigoferol and BG peptide is (10~1000):

1.

3. The pharmaceutical composition for treating osteoporosis as described in claim 2, characterized in that, The molar ratio of the formosan virginol to BG peptide is (50~200):

1.

4. The pharmaceutical composition for treating osteoporosis as described in claim 3, characterized in that, The molar ratio of the formosan virginol to BG peptide is 100:

1.

5. A pharmaceutical preparation for treating osteoporosis, characterized in that, Includes the pharmaceutical composition according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical preparation for treating osteoporosis as described in claim 5, characterized in that, The drug preparation is a liquid injection.

7. The method for preparing the pharmaceutical preparation for treating osteoporosis according to claim 6, characterized in that, Includes the following steps: Prepare the mother liquor of Qianghuo alcohol and the mother liquor of BG peptide separately. Mix the mother liquor of Qianghuo alcohol and the mother liquor of BG peptide according to the molar ratio of Qianghuo alcohol and BG peptide, and make up the volume. Filter to obtain the liquid injection.

8. The method for preparing the pharmaceutical formulation for treating osteoporosis as described in claim 7, characterized in that, The mother liquor of Qianghuo alcohol is prepared by the following steps: Qianghuo alcohol is dissolved in dimethyl sulfoxide and diluted with PBS buffer to obtain the mother liquor of Qianghuo alcohol.

9. The method for preparing the pharmaceutical formulation for treating osteoporosis as described in claim 7, characterized in that, The BG peptide stock solution is prepared by the following steps: dissolving BG peptide in water for injection to obtain the BG peptide stock solution.

10. The method for preparing the pharmaceutical preparation for treating osteoporosis as described in claim 7, characterized in that, The volume adjustment was performed using PBS buffer; the filtration was performed using a 0.22 μm sterile filter membrane.

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