A pharmaceutical composition for treating osteoporosis, a preparation and a preparation method thereof

Abstract

The application discloses a kind of pharmaceutical composition for treating osteoporosis, preparation and preparation method thereof, belong to medical technology field.The pharmaceutical composition includes notopterygium alcohol and BG peptide;The amino acid sequence of BG peptide is as shown in SEQ ID NO.1.The application further discloses a kind of pharmaceutical preparation for treating osteoporosis and preparation method thereof.The pharmaceutical composition can effectively solve the problems of poor curative effect, large side effect and limited bone pain relief effect of the current osteoporosis treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a kind of drug composition for treating osteoporosis, preparation and preparation method thereof. BACKGROUND

[0002] Osteoporosis (OP) is a kind of systemic bone disease with low bone mass and bone microstructure damage leading to increased bone fragility. Osteoporosis often has no obvious symptoms in the early stage, and as the disease progresses, patients may have symptoms such as back pain, height reduction and humpback. In severe cases, even slight external force such as coughing, sneezing and bending may cause bone fracture, which seriously affects the quality of life and health of patients.

[0003] At present, the treatment of osteoporosis focuses on inhibiting bone resorption. Bisphosphonate drugs are commonly used first-line drugs in clinical practice, which can specifically bind to hydroxyapatite in the bone, inhibit the activity of osteoclasts, reduce bone resorption and increase bone density. Another mainstream drug RANKL inhibitor can interfere with the differentiation, maturation and activation of osteoclasts by blocking the binding of RANKL to its receptor RANK, so as to achieve the purpose of inhibiting bone resorption.

[0004] However, while these drugs exert therapeutic effects, they also have many problems that cannot be ignored. Long-term use of bisphosphonate drugs may cause mandibular osteonecrosis, a serious adverse reaction that not only causes great pain to patients, but also affects their masticatory function and facial appearance. In addition, the risk of atypical femoral fractures increases with the length of medication, which often occurs between the lower part of the femoral trochanter and the knee, and is difficult to treat and takes a long time to recover. Moreover, these drugs have unsatisfactory effects on relieving bone pain, and many patients still suffer from bone pain after taking the drugs, making it difficult to significantly improve their quality of life.

[0005] Therefore, it is urgent to develop safer and more effective drugs for treating osteoporosis. SUMMARY

[0006] The present application solves the problem of providing a drug composition for treating osteoporosis, preparation and preparation method thereof, which can effectively solve the problems of poor efficacy, large side effects and limited bone pain relief effect of current drugs for treating osteoporosis.

[0007] To solve the technical problem, a drug composition for treating osteoporosis is provided, which comprises notopterol and BG peptide; the amino acid sequence of BG peptide is shown in SEQ ID NO. 1.

[0008] Preferably, the molar ratio of cnidain and BG peptide is (10-1000):1.

[0009] More preferably, the molar ratio of cnidain and BG peptide is (50-200):1.

[0010] More preferably, the molar ratio of cnidain and BG peptide is 100:1.

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

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

[0013] A preparation method of the pharmaceutical preparation for treating osteoporosis, comprising the following steps:

[0014] Cnidain mother liquor and BG peptide mother liquor are prepared respectively, and the cnidain mother liquor and the BG peptide mother liquor are mixed and made constant volume according to the molar ratio of cnidain and BG peptide, and a liquid injection is prepared by filtration.

[0015] Preferably, the cnidain mother liquor is prepared by the following steps: dissolving cnidain in dimethyl sulfoxide and diluting with PBS buffer, thereby obtaining the cnidain mother liquor.

[0016] Preferably, the BG peptide mother liquor is prepared by the following steps: dissolving BG peptide in water for injection, thereby obtaining the BG peptide mother liquor.

[0017] Preferably, the constant volume is made by using PBS buffer; and the filtration is made by using a 0.22 μm sterile filter membrane.

[0018] More preferably, the pH of the PBS buffer is 7.4.

[0019] More preferably, the preparation method of the pharmaceutical preparation for treating osteoporosis comprises the following steps:

[0020] Cnidain is weighed and dissolved in dimethyl sulfoxide according to a solid-liquid ratio of 10 mg:1 mL, and then diluted by adding PBS buffer with a pH of 7.4, so as to control the final concentration of dimethyl sulfoxide to be ≤1%, thereby obtaining cnidain mother liquor with a mass concentration of 1 mg / mL; BG peptide is weighed and dissolved in water for injection according to a solid-liquid ratio of 1 mg:1 mL, thereby obtaining BG peptide mother liquor with a mass concentration of 1 mg / mL; cnidain mother liquor and BG peptide mother liquor are mixed according to the molar ratio of cnidain and BG peptide, and made constant volume by using PBS buffer with a pH of 7.4, and then filtered by using a 0.22 μm sterile filter membrane, thereby obtaining a liquid injection.

[0021] The present application has the following beneficial effects:

[0022] The BG peptide of the present application is a novel mouse RANK protein targeting peptide artificially synthesized, which specifically binds to the key functional domain of the intracellular segment of mouse RANK protein to form a high-affinity interaction, thereby physically blocking RANK trimerization and subsequent signal complex assembly, inhibiting the autophosphorylation of RANK and the activation of downstream TRAF6 / TAK1, and further preventing the nuclear translocation and phosphorylation of NF-κB transcription factor, significantly reducing the expression level of inflammatory factors such as TNF- α , IL-6, etc.; the peptide segment directly targets the intracellular domain of RANK, avoids off-target effects and provides more precise signal regulation, and the cysteine residues in the sequence can enhance stability through disulfide bonds.

[0023] In the present application, the n-olivetol can mildly promote osteogenesis as an upstream regulator, and the BG peptide can inhibit osteoclasts and relieve pain as an inhibitor of NF-κB signal; the combination of the two can synergistically act on the osteogenesis-osteoclast balance and the nerve-skeletal axis, can greatly reduce the required dose of each, avoid the toxic side effects and cost problems of high-dose single drugs, and achieve the dual effects of bone health and pain relief. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a binding relationship diagram of the BG peptide segment and the intracellular segment of mouse RANK protein in the present application;

[0025] Figure 2 is a mass spectrum of the BG peptide;

[0026] Figure 3 is a femur HE staining result diagram of mice in different groups in the test example; wherein, (a) is a femur HE staining result diagram of mice in the sham operation group; (b) is a femur HE staining result diagram of mice in the model group; (c) is a femur HE staining result diagram of mice in the n-olivetol single drug group; (d) is a femur HE staining result diagram of mice in the BG single drug group; (e) is a femur HE staining result diagram of mice in the drug composition group; (f) is a femur HE staining result diagram of mice in the positive control group; (g) is a local enlarged view of the (a) diagram; (h) is a local enlarged view of the (b) diagram; (i) is a local enlarged view of the (c) diagram; (j) is a local enlarged view of the (d) diagram; (k) is a local enlarged view of the (e) diagram; (l) is a local enlarged view of the (f) diagram;

[0027] Figure 4 is a femur scanning result diagram of mice in different groups in the test example; wherein, (a) is the sham operation group; (b) is the model group; (c) is the n-olivetol single drug group; (d) is the BG single drug group; (e) is the drug composition group; (f) is the positive control group;

[0028] Figure 5 is a bone morphometry analysis diagram of mice in different groups in the test example; wherein, (a) is a bone volume fraction diagram; (b) is a bone trabecula thickness diagram; (c) is a bone trabecula number diagram;

[0029] Figure 6 The 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.

[0030] Figure 7 The graph shows the bone strength results of mice in different groups in the experimental case;

[0031] 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.

[0032] Figure 9 The graph shows the ALP activity detection results after different treatments in the experimental case.

[0033] 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;

[0034] 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

[0035] 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.

[0036] 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.

[0037] The features and performances of the present application are further described in detail below in combination with examples. The BG peptide of the present application is synthesized by Shanghai Bote Company.

[0038] Example 1

[0039] A pharmaceutical composition for treating osteoporosis, comprising cnidimide and BG peptide; the amino acid sequence of the BG peptide is shown as SEQ ID NO. 1; the molar ratio of cnidimide to BG peptide is 100:1.

[0040] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0041] A pharmaceutical preparation for treating osteoporosis, in the form of a liquid injection, the preparation method comprising the following steps:

[0042] (1) Take 10 mg of cnidimide, add 1 mL of dimethyl sulfoxide, and vortex to dissolve, to obtain a 10 mg / mL cnidimide dimethyl sulfoxide solution; dilute the cnidimide dimethyl sulfoxide solution with PBS buffer with a pH of 7.4 to a cnidimide mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide to be ≤1%, to obtain a cnidimide stock solution;

[0043] (2) Take 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;

[0044] (3) According to the molar ratio of 100:1 of cnidimide to BG peptide, take 100 μL of the cnidimide stock solution and 1 μL of the BG peptide stock solution, mix, and use PBS buffer with a pH of 7.4 to make up to 1 mL, and filter with a 0.22 μm sterile filter membrane, to obtain the liquid injection, the molar concentration of cnidimide in the liquid injection is 282 μM, and the molar concentration of BG peptide is 2.82 μM.

[0045] Example 2

[0046] A pharmaceutical composition for treating osteoporosis, comprising cnidimide and BG peptide; the amino acid sequence of the BG peptide is shown as SEQ ID NO. 1; the molar ratio of cnidimide to BG peptide is 200:1.

[0047] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0048] A pharmaceutical preparation for treating osteoporosis, in the form of a liquid injection, the preparation method comprising the following steps:

[0049] (1) Take 10 mg of Rhizoma Notopterygii alcohol, add 1 mL of dimethyl sulfoxide, vortex to dissolve, and obtain a Rhizoma Notopterygii alcohol dimethyl sulfoxide solution of 10 mg / mL; dilute the Rhizoma Notopterygii alcohol dimethyl sulfoxide solution with PBS buffer of pH 7.4 to a Rhizoma Notopterygii alcohol mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide to be ≤1%, to obtain a Rhizoma Notopterygii alcohol mother liquor;

[0050] (2) Take 1 mg of BG peptide, add 1 mL of water for injection, and vortex to dissolve, to obtain a BG peptide mother liquor of 1 mg / mL;

[0051] (3) According to a molar ratio of 200:1 of Rhizoma Notopterygii alcohol to BG peptide, take 200 μL of the Rhizoma Notopterygii alcohol mother liquor and 1 μL of the BG peptide mother liquor, mix, and use PBS buffer of pH 7.4 to make up to 1 mL, and filter using a 0.22 μm sterile filter membrane, to obtain a liquid injection, in which the molar concentration of Rhizoma Notopterygii alcohol is 564 μM and the molar concentration of BG peptide is 2.82 μM.

[0052] Example 3

[0053] A pharmaceutical composition for treating osteoporosis, comprising Rhizoma Notopterygii alcohol and BG peptide; the amino acid sequence of the BG peptide is shown as SEQ ID NO. 1; and the molar ratio of Rhizoma Notopterygii alcohol to BG peptide is 1000:1.

[0054] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0055] A pharmaceutical preparation for treating osteoporosis, in the form of a liquid injection, and a preparation method thereof, comprising the following steps:

[0056] (1) Take 10 mg of Rhizoma Notopterygii alcohol, add 1 mL of dimethyl sulfoxide, vortex to dissolve, and obtain a Rhizoma Notopterygii alcohol dimethyl sulfoxide solution of 10 mg / mL; dilute the Rhizoma Notopterygii alcohol dimethyl sulfoxide solution with PBS buffer of pH 7.4 to a Rhizoma Notopterygii alcohol mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide to be ≤1%, to obtain a Rhizoma Notopterygii alcohol mother liquor;

[0057] (2) Take 1 mg of BG peptide, add 1 mL of water for injection, and vortex to dissolve, to obtain a BG peptide mother liquor of 1 mg / mL;

[0058] (3) According to a molar ratio of 1000:1 of Rhizoma Notopterygii alcohol to BG peptide, take 1000 μL of the Rhizoma Notopterygii alcohol mother liquor and 1 μL of the BG peptide mother liquor, mix, and use PBS buffer of pH 7.4 to make up to 1 mL, and filter using a 0.22 μm sterile filter membrane, to obtain a liquid injection, in which the molar concentration of Rhizoma Notopterygii alcohol is 2820 μM and the molar concentration of BG peptide is 2.82 μM.

[0059] Example 4

[0060] A pharmaceutical composition for treating osteoporosis, comprising n- butylphthalide and BG peptide; the amino acid sequence of the BG peptide is shown as SEQ ID NO. 1; the molar ratio of n-butylphthalide to BG peptide is 50:1.

[0061] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0062] A pharmaceutical preparation for treating osteoporosis, in the form of a liquid injection, the preparation method comprising the following steps:

[0063] (1) Take 10 mg of n-butylphthalide, add 1 mL of dimethyl sulfoxide, and vortex to dissolve, to obtain a 10 mg / mL n-butylphthalide dimethyl sulfoxide solution; dilute the n-butylphthalide dimethyl sulfoxide solution with PBS buffer with a pH of 7.4 to a n-butylphthalide mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide to be ≤1%, to obtain a n-butylphthalide mother liquor;

[0064] (2) Take 1 mg of BG peptide, add 1 mL of water for injection, and vortex to dissolve, to obtain a BG peptide mother liquor with a mass concentration of 1 mg / mL;

[0065] (3) According to the molar ratio of n-butylphthalide to BG peptide of 50:1, take 50 μL of the n-butylphthalide mother liquor and 1 μL of the BG peptide mother liquor, mix, and use PBS buffer with a pH of 7.4 to make up to 1 mL, and filter using a 0.22 μm sterile filter membrane, to obtain a liquid injection, the molar concentration of n-butylphthalide in the liquid injection is 141 μM, and the molar concentration of BG peptide is 2.82 μM.

[0066] Example 5

[0067] A pharmaceutical composition for treating osteoporosis, comprising n- butylphthalide and BG peptide; the amino acid sequence of the BG peptide is shown as SEQ ID NO. 1; the molar ratio of n-butylphthalide to BG peptide is 10:1.

[0068] SEQ ID NO. 1: KLVALKTNGDGNCIMHVA CQFMWGVQDTD.

[0069] A pharmaceutical preparation for treating osteoporosis, in the form of a liquid injection, the preparation method comprising the following steps:

[0070] (1) Take 10 mg of Rhizoma Notopterygii alcohol, add 1 mL of dimethyl sulfoxide, vortex to dissolve, and obtain a 10 mg / mL Rhizoma Notopterygii alcohol dimethyl sulfoxide solution; add a PBS buffer with a pH of 7.4 to the Rhizoma Notopterygii alcohol dimethyl sulfoxide solution to dilute it to a Rhizoma Notopterygii alcohol mass concentration of 1 mg / mL, and control the final concentration of dimethyl sulfoxide to be ≤1%, to obtain a Rhizoma Notopterygii alcohol mother liquor;

[0071] (2) Take 1 mg of BG peptide, add 1 mL of water for injection, and vortex to dissolve, to obtain a BG peptide mother liquor with a mass concentration of 1 mg / mL;

[0072] (3) According to a molar ratio of 10:1 of Rhizoma Notopterygii alcohol to BG peptide, take 10 μL of the Rhizoma Notopterygii alcohol mother liquor and 1 μL of the BG peptide mother liquor, mix them, and use a PBS buffer with a pH of 7.4 to dilute them to 1 mL. Filter them using a 0.22 μm sterile filter membrane, to obtain a liquid injection. The molar concentration of Rhizoma Notopterygii alcohol in the liquid injection is 28.2 μM, and the molar concentration of BG peptide is 2.82 μM.

[0073] Test Example

[0074] 1. BG Peptide Structure and Binding Relationship

[0075] The BG peptide is a new type of mouse RANK protein targeting peptide synthesized by Shanghai Bote Company. The amino acid sequence SEQ ID NO. 1 is: KLVALKTNGDGNCIMHVA CQFMWGVQDTD. The structure prediction and mass spectrum are shown in Figures 1-2 . Figure 1 The structure prediction is shown in FIG. 1. The BG peptide segment (red) has a possible binding relationship with the intracellular segment of the mouse RANK protein (green). It specifically binds to the key functional domain (such as the TRAF6 binding site) of the intracellular segment of the mouse RANK protein, forms a high-affinity interaction, physically blocks RANK trimerization and subsequent signal complex assembly, inhibits the autophosphorylation of RANK and the activation of downstream TRAF6 / TAK1, and further prevents the nuclear translocation and phosphorylation of the NF-κB transcription factor, significantly reducing the expression level of inflammatory factors such as TNF- α , IL-6, etc. The peptide segment directly targets the intracellular domain of RANK, avoiding off-target effects and providing more precise signal regulation, while the cysteine (C) residue in the sequence can enhance stability through disulfide bonds. Figure 2 As can be seen from the mass spectrum, the molecular weight of the synthesized BG peptide meets the expectation.

[0076] 2. Verification of the Therapeutic Effect of the Osteoporosis Treatment Pharmaceutical Composition on Osteoporosis

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 2.1 HE staining of the femur

[0081] 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.

[0082] from Figure 3As 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.

[0083] 2.2 Micro-CT Bone Morphometric Analysis

[0084] 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.

[0085] 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.

[0086] BV / TV = Bone volume / Total tissue volume (1)

[0087] Tb.N = (bone volume / total tissue volume) / Tb.Th (2)

[0088] 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 5It can be seen that the drug composition group showed more significant improvement in bone volume fraction, trabecular bone thickness and trabecular bone number compared with the single drug group (Notochidanol single drug group and BG single drug group) and the model group, even better than the positive drug estradiol, and most of the indicators were extremely significantly different from the model group, indicating that the combination of notochidanol and BG peptide has a synergistic effect and can more effectively improve the bone microstructure. The positive control group showed similar performance to the sham operation group in the three indicators and was significantly better than the model group, which is consistent with the expected protective effect of estradiol on the skeleton, and further verifies the effectiveness of the experimental model and detection method.

[0089] 2.3 Serum bone metabolism marker detection

[0090] The mice in each group were taken blood from the abdominal aorta 24h after the last administration, centrifuged to obtain serum, and the serum bone metabolism markers PINP, OCN, Tracp-5b and Cath-K were detected by ELISA kit. PINP (serum type I collagen N-terminal propeptide) is a product in the synthesis process of type I collagen, and its level increase indicates that bone formation activity increases, which is a marker reflecting bone formation; OCN (osteocalcin) is also a marker of bone formation, which is synthesized and secreted by osteoblasts, and its serum content is closely related to the activity of osteoblasts and can be used to evaluate the state of bone formation; Tracp-5b (tartrate-resistant acid phosphatase 5b) represents a marker of bone resorption, which is mainly secreted by osteoclasts, and its level increase indicates that osteoclast activity is enhanced and the bone resorption process is active; Cath-K (cathepsin K) is a marker enzyme of osteoclasts, which can effectively decompose matrix components such as collagen in the bone resorption process, and release calcium, phosphorus and other minerals into the blood, and its activity change directly affects the balance of bone metabolism. The results are shown in Figure 6

[0091] 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.

[0092] 2.4 Bone biomechanical testing

[0093] 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.

[0094] 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.

[0095] 2.5 Pain Behavioral Testing

[0096] 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.

[0097] from Figure 8As can be seen from the table, compared with the model group, the force value and the time value of the drug composition group in the mechanical pain threshold and the thermal pain threshold test are significantly increased, indicating that the drug composition can effectively increase the pain threshold of mice to mechanical and thermal stimulation and relieve pain sensitivity; compared with the single drug group, the pain threshold of the drug composition group is more improved, indicating that the combination of n-menthofuran and BG peptide may have a synergistic effect on regulating the pain threshold and can more effectively improve the pain sensitivity of mice. Compared with the sham operation group, the pain threshold of the drug composition group is close, indicating that the combination of n-menthofuran and BG peptide can restore the pain threshold of mice to a normal level and has a synergistic analgesic effect.

[0098] 2.6 ALP activity detection

[0099] The MC3T3-E1-derived osteoblasts were established and intervened with different drugs. Specifically, the mouse MC3T3-E1 cells were inoculated at a density of 2x10 5 6 / well (6-well plate) using α-MEM complete culture medium (containing 90% α-MEM basic medium, 10% fetal bovine serum, 1% penicillin-streptomycin mixture), 16 μL / mL mouse osteoblast inducer (batch number: AI1001-004) was added, and the cells were cultured in a 5% CO2, 37°C constant temperature incubator. Fresh medium containing the inducer was replaced every 3 days. 2.00 mg of n-menthofuran with a purity of ≥98% was dissolved in 100 μL of dimethyl sulfoxide to prepare a 56.54 mmol / L stock solution, which was then diluted with the culture medium to a working concentration of 20 μmol / L (the final concentration of DMSO was ≤0.1%); BG peptide was dissolved in water for injection to prepare a 1 mg / mL stock solution, and the working concentration was 60 μg / mL. The working concentrations of n-menthofuran and / or BG peptide were added synchronously at the beginning of osteogenic induction, and the drugs were supplemented every 3 days when the medium was replaced. The Model group was only subjected to osteogenic induction without the addition of drugs.

[0100] ALP activity detection was performed after 5 days of osteogenic induction. The culture medium was aspirated and the cells were washed twice with PBS. 1 mL of ALP kit lysis solution containing protease inhibitors was added to each well. After lysis for 10 min, the cells were collected with a cell scraper and centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was collected, the protein concentration was determined by BCA method, 20 μg of protein was loaded, the detection reagent was added, and the absorbance was measured at 450 nm wavelength. The ALP activity was expressed as the amount of p-nitrophenol generated per unit time (nmol / min / mg protein), and the results are shown in Figure 9 .

[0101] From Figure 9As can be seen from the above table, the use of notopterol and BG peptide alone can increase the ALP activity to some extent and promote osteogenic differentiation; the ALP activity of the combination of notopterol and BG peptide is significantly higher than that of the single-drug group and the model group, and the difference is statistically significant, indicating that the combination of the two drugs has a synergistic effect on improving the ALP activity and can more effectively regulate the physiological process related to ALP and promote osteogenic differentiation.

[0102] 2.7 RT-qPCR detection of the expression of related gene mRNA

[0103] The MC3T3-E1 cells were used for the detection of the expression of osteogenic related genes, including Dlx5, Runx2, Sp7 The RAW264.7 cells were used for the detection of osteoclastic related genes, including Fos , Acp5 , Ctsk , Nfatc1 . The specific steps are as follows: the MC3T3-E1 and RAW264.7 cells were inoculated in a six-well plate at 2×10 6 cells per well, 3 mL of α-MEM complete culture medium was added to each well, and the cells were cultured in a 37°C carbon dioxide incubator containing 5% CO2 for 24 h. Then, the MC3T3-E1 cells were set as a control group (osteogenic induction), a notopterol (20 μmol / L) + osteogenic induction group, a BG (60 μg / mL) + osteogenic induction group, and a notopterol (20 μmol / L) + BG (60 μg / mL) + osteogenic induction group. The RAW264.7 cells were set as a control group (osteoclastic induction: 50 ng / mL RANKL inducer was added), a notopterol (20 μmol / L) + 50 ng / mL RANKL group, a BG (60 μg / mL) + 50 ng / mL RANKL group, and a notopterol + BG (60 μg / mL) + 50 ng / mL RANKL group. After being cultured in the incubator for 48 h, the culture medium in the six-well plate was discarded, the cells were washed twice with PBS, 1 mL of Trizol reagent was added to each well to lyse the cells, the lysed liquid was absorbed into an EP tube and stood for 5 min, and the total RNA was extracted according to the total RNA extraction kit. The cDNA was reverse transcribed into cDNA using a gradient PCR instrument. The expression levels of the target genes in each group were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) amplification. The relative expression levels of the target genes were calculated using the 2 －ΔΔCt method Dlx5 , Runx2 , Sp7 , Nfatc1 , Fos .Acp5 , Ctsk The relative expression of mRNA of the above-mentioned genes was detected by qPCR, and the specific primer sequences are shown in Table 1 below, and the results are shown in Figure 10 .

[0104] Table 1 Specific primer sequence table

[0105]

[0106] Figure 10 The qPCR results show that the mRNA expression of Runx2, Sp7 and Dlx5 in the administration group is significantly higher than that in the control group, and the drug composition group is significantly higher than the administration of Notopterygium alcohol and BG peptide alone; the mRNA expression of Nfatc1, Fos, Acp5, Ctsk in the administration group is significantly lower than that in the control group, and the drug composition group is significantly lower than the administration of Notopterygium alcohol and BG peptide alone.

[0107] 2.8 Test of the release level of inflammatory factors downstream of NF-κB under the stimulation of immune cells

[0108] RAW264.7 cells were stimulated by LPS (200 ng / mL), and Notopterygium alcohol and BG peptide were treated as described above in 2.6. After 4 hours, the levels of TNF- α and IL-6 in the culture medium were detected, and the results are shown in Figure 11 .

[0109] In the complex physiological process of bone metabolism, a dynamic balance is maintained between bone formation dominated by osteoblasts and bone resorption dominated by osteoclasts, and inflammatory factors also play an important regulatory role therein. When this balance is broken, for example, in the estrogen deficiency state caused by ovariectomy, bone resorption often exceeds bone formation, thereby triggering a series of problems such as bone mass loss and bone microstructure destruction. As can be seen from Figure 11 , Notopterygium alcohol and BG peptide alone can improve the release of inflammatory factors, but the drug composition group has the lowest level of inflammatory factors, indicating that the synergistic effect of Notopterygium alcohol and BG peptide has better effect.

[0110] In summary, when low doses of Notopterygium alcohol and BG peptide are used in combination, they achieve a synergistic effect; Notopterygium alcohol promotes osteogenesis and increases bone formation; BG peptide inhibits osteoclasts and inflammatory factors, reduces bone resorption and inhibits factors that inhibit osteogenesis. This synergistic effect makes the imbalance between bone formation and bone resorption more effectively corrected. Notopterygium alcohol can promote osteoblasts to synthesize more bone matrix, while BG peptide inhibits osteoclast activity to prevent the excessive absorption of newly synthesized bone matrix, and its anti-inflammatory effect provides a more favorable microenvironment for osteoblasts, further promoting bone formation. The two complement each other and jointly play a regulatory role in bone metabolism, providing a potential effective treatment strategy for improving bone metabolism imbalance-related diseases caused by various reasons.

[0111] The application is described in the above embodiments, it should be understood that the above embodiments do not limit the application in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of the application.

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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