CN111920800A - Application of oroxylin A in treating osteoporosis and promoting fracture healing - Google Patents

Abstract

The invention discloses an application of oroxylin A in preparation of a pharmaceutical composition for treating osteoporosis and promoting fracture healing. The effective concentration of oroxylin A is 1-400 mg/kg. The invention discovers a mechanism of oroxylin A for treating osteoporosis for the first time, and the medicine is screened based on an L-plastic target and has the functions of inhibiting osteoclast precursor cell fusion and promoting bone formation.

Description

Application of oroxylin A in treating osteoporosis and promoting fracture healing

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to application of oroxylin A in treating osteoporosis and promoting fracture healing.

Background

The bone metabolism is regulated by the bone absorption function of osteoclast and the bone formation function of osteoblast, and the metabolic balance of the bone is maintained. Over-activation of osteoclasts plays an important role in a range of bone-related diseases. Currently, there are more than 2 billion patients with osteoporosis worldwide. The osteoporotic patients lose a great deal of bone mass, which leads to a great increase in their fracture risk and mortality. The main drugs currently used in clinical practice to inhibit bone resorption are bisphosphates, which interfere with bone remodeling and inhibit osteoblast bone formation function at the same time as inhibiting bone resorption function. Meanwhile, studies have shown that long-term administration of bisphosphonates produces a series of complications, such as necrosis of the jaw, atypical fracture, etc. In recent years, the biological agents have received increasing attention from researchers for the treatment of pathological bone loss.

Various osteoporosis treatment methods have been reported in the prior art, and for example, patent document CN 110151747a describes that Ferrostatin-1 can significantly alleviate bone mass loss caused by menopause; patent document CN110840882A describes that a composition containing isoimperatorin as a main active ingredient and dihydromyricetin can inhibit osteoclast differentiation and bone resorption, promote osteoblast differentiation and bone formation, and exert an effect of treating osteoporosis by regulating the balance between osteoblasts and osteoclasts; patent document CN 111166884a describes that siFoxf1 may play an important role in the treatment of PMOP and may be used for the treatment of osteoporosis and the like.

In addition, patent document CN 110478345a describes that a novel psoralea fruit isoflavone inhibits the activation of NF- κ B pathway and MAPK pathway by inhibiting the binding between RANK and TRAF6, and inhibits the activation of AKT pathway by inhibiting the binding between RANK and C-Src, thereby finally inhibiting the expression of the osteoclast key transcription factor NFATc1, inhibiting the generation of osteoclast, and reducing the bone loss.

Patent document CN110787207A describes that a persimmon leaf flavonoid extract can reduce the level of osteoclast differentiation and maturation marker factor mRNA; can promote the expression of mitochondrial complex I so as to reduce the generation of ROS in the differentiation and maturation process of osteoclast; inhibits the differentiation of osteoclast by inhibiting the activation of PI3K-AKT-mTOR, JNK and p38 signal channels, thereby achieving the effect of preventing and treating osteoporosis.

The oroxylin A is an extract of traditional Chinese medicine scutellaria baicalensis, and researches show that oroxylin A plays an important role in resisting inflammation and tumors, but the influence of oroxylin A on bone metabolism is not clear.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of oroxylin A in treating osteoporosis and promoting fracture healing.

The purpose of the invention is realized by the following technical scheme:

the invention provides application of oroxylin A in preparation of a pharmaceutical composition for treating osteoporosis and promoting fracture healing.

Preferably, the effective concentration of the oroxylin A is 1-400mg/kg, and the effective concentration is the in vivo administration concentration.

Preferably, the effective concentration of the oroxylin A is 30-80mg/kg, and more preferably, the effective concentration is 50 mg/kg.

The invention also provides a pharmaceutical composition for treating osteoporosis and promoting fracture healing, which comprises oroxylin A and pharmaceutically acceptable auxiliary materials.

Preferably, in the pharmaceutical composition, the effective concentration of oroxylin A is 1-400 mg/kg.

Preferably, the excipients include carriers or excipients, such as lactose hydrate, microcrystalline cellulose, mannitol, sodium citrate, calcium phosphate, glycine, starch; disintegrants such as crospovidone, copovidone, sodium starch glycolate, croscarmellose sodium and certain complex silicates; binding agents such as polyvinylpyrrolidone, Hydroxypropylmethylcellulose (HPMC), Hydroxypropylcellulose (HPC), sucrose, gelatin, gum arabic and the like.

The invention also provides application of oroxylin A in preparation of a preparation for inhibiting osteoclast precursor cell fusion, wherein the effective concentration of oroxylin A is 1-400 mg/kg.

The invention also provides application of oroxylin A in preparation of a preparation for promoting H-type angiogenesis, wherein the effective concentration of oroxylin A is 1-400 mg/kg.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers a mechanism of oroxylin A for treating osteoporosis for the first time, and the medicine is screened based on an L-plastic target and has the functions of inhibiting osteoclast precursor cell fusion and promoting bone formation. This is quite different from the action mechanism of the flavonoid compound reported previously, and the theory of treating osteoporosis by using osteoclast precursor cells is proposed by the invention for the first time.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 shows TRAP staining and TRAP positive cell numbers 3 days after RANKL induction of bone marrow primary cells of example 1;

FIG. 2 shows TRAP staining results and TRAP-positive cell numbers 3 days after RANKL induction of RAW264.7 cells in example 2;

FIG. 3 is the Hoechst staining results of example 3 to count the osteoclast precursor cell membrane fusion rate at 3 days of RANKL induction;

FIG. 4 shows the results of LPL and F-actin staining and number of pseudopodia (Filopodia) 3 days after RANKL induction of bone marrow primary cells of example 4;

FIG. 5 shows TRAP staining results and osteoclast (Osteoclasts) numbers after 6 days of RANKL induction of bone marrow primary cells of example 5;

FIG. 6 is the results of TRAP staining and osteoclast (Osteoclasts) numbers 6 days after RANKL induction of RAW264.7 cells of example 6;

FIG. 7 is the bone plate bone absorption area of example 7;

FIG. 8 shows the results of the drug affinity experiments for LPL and oroxylin of example 8;

FIG. 9 shows the results of the experiment in example 9; wherein, FIG. 9A is Micro CT image of mouse femur; FIG. 9B shows the results of bone density, bone volume/tissue volume, trabecular number; FIG. 9C is the results of cortical bone thickness; FIG. 9D shows the results of H & E staining for histomorphology; FIG. 9E is the result of trabecular bone area;

FIG. 10 shows the results of the femoral angiography test of example 10 and the level of blood vessels in the femur; wherein, fig. 10A is a contrast result; FIG. 10B is a graph of blood vessel surface area and volume results;

FIG. 11 is the CD31 of example 10^hiEMCN^hiEndothelial vascular staining results and CD31^hiEMCN^hiEndothelial blood vessel percentage; wherein, FIG. 11A is CD31^hiEMCN^hiResults of endothelial vessel staining, FIG. 11B is CD31^hiEMCN^hiEndothelial blood vessel percentage;

FIG. 12 shows CD31 obtained by flow cytometry in example 10^hiEMCN^hiResults of endothelial cell number statistics;

FIG. 13 shows the results of ELISA analysis performed in example 10;

FIG. 14 is a Micro CT image of femur and a fracture healing mouse of example 11;

FIG. 15 shows the results of mechanical analysis in example 11.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1 bone marrow Primary cell osteoclast differentiation experiment

Mature osteoclasts are further formed by bone marrow mononuclear cells gradually forming osteoclast precursor cells under the induction of macrophage colony stimulating Factor (M-CSF) and Nuclear Factor kappa B Receptor Activator Ligand (RANKL). This example provides an experiment of using a nontoxic dose of oroxylin (10 μ M) to interfere with the osteoclast differentiation process, the specific procedure is as follows:

the 4-week mice were placed in a sterile operating table, dissected with scissors sterilized at high temperature and high pressure, and the mouse femurs were isolated. The isolated mouse femur was immersed in a 4% double antibody solution for 30 minutes. The mouse femurs were removed and rinsed with PBS solution. Meanwhile, both ends of the femur of the mouse were cut with scissors of a high-temperature autoclave, and the mouse bone marrow cells were flushed out by extracting the medium containing 10% serum with a 1mL syringe. Culturing the flushed cells in an incubator, taking cell culture medium supernatant after 12 hours, discarding adherent cells, placing the cell culture medium supernatant in a constant temperature centrifuge for centrifugation at 3000rpm for 5 minutes, resuspending the cell precipitate, adding a culture medium containing M-CSF (50ng/mL), and inducing the cells. After 3 days of M-CSF induction of cells, the cells were allowed to adhere to the medium, digested with trypsin, seeded at a density of 50000 cells per well in 12-well plates, and induced by addition of M-CSF (30ng/mL) and RANKL (50 ng/mL).

After 3 days, TRAP (tartrate-resistant acid phosphatase-positive) staining results are shown in FIG. 1, control (Con) is a control group, Oroxylin A (Oro) is a oroxylin A-treated group, and as a result, oroxylin was found to have no significant effect on osteoclast formation.

Example 2

The RAW264.7 cell line is a cell line of a commonly used osteoclast differentiation experiment, and is verified in the RAW264.7 cell line, and the specific operation steps are as follows:

RAW264.7 cells were seeded in a 12-well plate at a density of 50000 per well, after the cells were attached to the wall, the cell supernatant was discarded, and changed to a DMEM medium containing RANKL (50ng/mL), after 3 days of induction, after osteoclast precursor cells were formed, the medium supernatant was discarded, and fixed with 4% paraformaldehyde solution to prepare for TRAP staining.

The results are shown in figure 2, oroxylin a (10 μ M) intervention did not inhibit osteoclast precursor cell formation.

Example 3

Osteoclast precursor cell nuclei were stained with Hoechst dye, and osteoclast precursor cell membranes were stained with Dil dye. The specific operation steps are as follows:

the 4-week mice were placed in a sterile operating table, dissected with scissors sterilized at high temperature and high pressure, and the mouse femurs were isolated. The isolated mouse femur was immersed in a 4% double antibody solution for 30 minutes. The mouse femurs were removed and rinsed with PBS solution. Meanwhile, both ends of the femur of the mouse were cut with scissors of a high-temperature autoclave, and the mouse bone marrow cells were flushed out by extracting the medium containing 10% serum with a 1mL syringe. Culturing the flushed cells in an incubator, taking cell culture medium supernatant after 12 hours, discarding adherent cells, placing the cell culture medium supernatant in a constant temperature centrifuge for centrifugation at 3000rpm for 5 minutes, resuspending the cell precipitate, adding a culture medium containing M-CSF (50ng/mL), and inducing the cells. After 3 days of M-CSF induction of cells, the cells were allowed to adhere to the medium, digested with trypsin, seeded at a density of 50000 cells per well in 12-well plates, and induced by addition of M-CSF (30ng/mL) and RANKL (50 ng/mL). Meanwhile, oroxylin was added, and after 2 days, cell supernatant was aspirated off with a pipette tip, suspended cells were removed, and cells were washed with PBS. Staining the cell nuclei of a group of cells with blue dye Hoechst, incubating for 5 minutes in a constant temperature incubator at 37 ℃, sucking off the dye with a gun, adding PBS solution, washing the cells 3 times on a shaker, staining the cell membranes with red dye DIL, incubating for 5 minutes in the constant temperature incubator at 37 ℃, sucking off the dye with the gun, adding PBS solution, and washing the cells 3 times on a shaker. Two groups of cells were seeded in 12-well plates at a density of 50000 cells per well. The cells were observed for fusion at 24 hours.

The results are shown in fig. 3, where the osteoclast precursor cell membrane fusion rate was significantly decreased at 3 days of RANKL induction.

Example 4

The method is characterized in that F-actin is used for staining the pseudopodia of osteoclast precursor cells, and the specific operation steps are as follows:

the 4-week mice were placed in a sterile operating table, dissected with scissors sterilized at high temperature and high pressure, and the mouse femurs were isolated. The isolated mouse femur was immersed in a 4% double antibody solution for 30 minutes. The mouse femurs were removed and rinsed with PBS solution. Meanwhile, both ends of the femur of the mouse were cut with scissors of a high-temperature autoclave, and the mouse bone marrow cells were flushed out by extracting the medium containing 10% serum with a 1mL syringe. Culturing the flushed cells in an incubator, taking cell culture medium supernatant after 12 hours, discarding adherent cells, placing the cell culture medium supernatant in a constant temperature centrifuge for centrifugation at 3000rpm for 5 minutes, resuspending the cell precipitate, adding a culture medium containing M-CSF (50ng/mL), and inducing the cells. After 3 days of M-CSF induction of cells, after the cells were attached to the full medium, the cells were digested with pancreatin and seeded at a density of 50000 cells per well in a 12-well plate, and M-CSF (30ng/mL) and RANKL (50ng/mL) were added for induction, and LPL and F-actin staining were performed after 3 days: (1) firstly, removing the cell culture medium supernatant, and washing for 3 times by using a PBS solution; (2) adding 4% paraformaldehyde solution for fixation, and fixing for 20 minutes; (3) TRITON was transparent for 10 min; (4) sucking off the TRITON solution by using a gun head, and washing for three times by using a PBS solution; (5) preparing a phalloidin staining solution (5 mug/mL), and incubating for one hour at 37 ℃; (6) sucking with a gun head, discarding the phalloidin staining solution, and washing with PBS solution for 3 times; 3 minutes each time; (7) dripping DAPI staining solution, incubating for 5 minutes in a dark place, carrying out nuclear staining on the specimen, and washing for 3 times by using PBS solution; (8) LPL and F-actin fluorescent staining.

The results are shown in figure 4, with the intervention of oroxylin a (10 μ M), the pseudopodogenesis of osteoclast precursor cells is significantly reduced.

Example 5

We stained the formation of mature osteoclasts by TRAP staining, as follows:

the 4-week mice were placed in a sterile operating table, dissected with scissors sterilized at high temperature and high pressure, and the mouse femurs were isolated. The isolated mouse femur was immersed in a 4% double antibody solution for 30 minutes. The mouse femurs were removed and rinsed with PBS solution. Meanwhile, both ends of the femur of the mouse were cut with scissors of a high-temperature autoclave, and the mouse bone marrow cells were flushed out by extracting the medium containing 10% serum with a 1mL syringe. Culturing the flushed cells in an incubator, taking cell culture medium supernatant after 12 hours, discarding adherent cells, placing the cell culture medium supernatant in a constant temperature centrifuge for centrifugation at 3000rpm for 5 minutes, resuspending the cell precipitate, adding a culture medium containing M-CSF (50ng/mL), and inducing the cells. After 3 days of M-CSF induction of cells, the cells were allowed to adhere to the medium, digested with pancreatin, seeded at a density of 50000 cells per well in 12-well plates, and induced by addition of M-CSF (30ng/mL) and RANKL (100 ng/mL). After 6 days, osteoclasts were formed, the medium was discarded, and the fixed cells were ready for TRAP staining.

The results are shown in fig. 5, and indicate that the intervention of oroxylin a (10 μ M) can significantly inhibit the formation of mature osteoclasts.

Example 6

The RAW264.7 cells are adopted for verification, and the specific operation steps are as follows:

RAW264.7 cells were seeded in a 12-well plate at a density of 50000 per well, after the cells were attached to the wall, the cell supernatant was discarded and replaced with a DMEM medium containing RANKL (100ng/mL), after 6 days of induction, after osteoclast precursor cells were formed, the medium supernatant was discarded and fixed with 4% paraformaldehyde solution to prepare for TRAP staining.

The results are shown in fig. 6, that oroxylin a (10 μ M) intervention significantly inhibited the formation of mature osteoclasts.

Example 7

In this example, the function of oroxylin a in bone resorption by osteoclasts was explored, and the specific experimental steps were as follows:

the 4-week mice were placed in a sterile operating table, dissected with scissors sterilized at high temperature and high pressure, and the mouse femurs were isolated. The isolated mouse femur was immersed in a 4% double antibody solution for 30 minutes. The mouse femurs were removed and rinsed with PBS solution. Meanwhile, both ends of the femur of the mouse were cut with scissors of a high-temperature autoclave, and the mouse bone marrow cells were flushed out by extracting the medium containing 10% serum with a 1mL syringe. Culturing the flushed cells in an incubator, taking cell culture medium supernatant after 12 hours, discarding adherent cells, placing the cell culture medium supernatant in a constant temperature centrifuge for centrifugation at 3000rpm for 5 minutes, resuspending the cell precipitate, adding a culture medium containing M-CSF (50ng/mL), and inducing the cells. After 3 days of M-CSF induction of cells, the cells were allowed to adhere to the medium, digested with pancreatin, seeded at a density of 50000 cells per well in 12-well plates, and induced by addition of M-CSF (30ng/mL) and RANKL (100 ng/mL). After 6 days, osteoclasts were formed, the medium was discarded, mature osteoclasts were obtained by digestion with trypsin, osteoclasts were seeded on the bone plate, and after 2 days, osteoclasts on the surface of the bone plate were removed by digestion with trypsin and ultrasonic vibration. The plate was washed 3 times with PBS solution and observed under a mirror. And counted with image J software.

The results are shown in fig. 7, which shows that oroxylin a (10 μ M) significantly inhibits the bone resorption function of osteoclasts. After oroxylin drying, the absorptive area of the bone plate is significantly reduced.

Example 8

In this example, a surface plasmon resonance analysis experiment was performed, and the specific experimental steps were as follows:

surface plasmon resonance analysis: SPR analysis and data processing were performed on the Biacore T200 system. The recombinant protein was first diluted to 100. mu.g/mL in 10mM sodium acetate buffer and then immobilized on CM5 chips by EDC/NHS cross-linking reaction. PBS containing 5% DMSO was used as a flow buffer. The compound molecules were formulated into solutions ranging in concentration from 2. mu.M to 64. mu.M using running buffer and placed in the instrument for automated analysis. At the end of each analysis, the intermediate concentrations were repeatedly determined to confirm the stability of the sensor surface. Finally, the calculation of the affinity equilibrium dissociation constant (KD) was performed on the instrument using a steady-state affinity model

Drug affinity response target stability validation: extracting total cell protein and quantifying by BCA. 50 μ g of protein was selected for each sample, drug solution (usually 3 concentration gradients) was added and incubated overnight at 4 ℃ or one hour at room temperature. A control group without drug addition was required. After completion of incubation, hydrolysis was performed with the appropriate concentration of Pronase protease for 30 minutes at room temperature. A control group without adding hydrolase was set. Immediately after the hydrolysis, a protein loading buffer was added and denatured at 100 ℃ for 10 minutes. The samples were subjected to SDS-PAGE electrophoresis. And carrying out silver nitrate dyeing on the obtained glue according to the specification of a silver nitrate dyeing kit, and searching for protein difference bands of the drug adding group and the drug non-adding group under the hydrolysis condition. And excavating the bands with the difference to carry out MADI-TOF mass spectrum identification, and matching the protein from the protein library. The target point verification step mainly comprises the steps of purchasing an antibody corresponding to the protein after mass spectrum identification and matching are successfully carried out to obtain possible target protein, repeating the sample preparation step to prepare a sample, turning a membrane after SDS-PAGE electrophoresis is finished, incubating the antibody, observing the hydrolysis degree of the protein under different drug concentrations, and verifying whether the protein has an affinity effect with the drug.

As a result, as shown in FIG. 8, oroxylin A was able to bind to L-plastic. SPR (surface plasmon resonance) detection shows that the protein has very good binding capacity.

Example 9

In view of the remarkable inhibitory effect of oroxylin a on osteoclast formation and osteoclastic bone resorption function, the following in vivo experiment was designed to explore its role in postmenopausal osteoporosis and fracture healing in a C57BL/6 mouse model.

Firstly, bilateral ovariectomy operation is carried out on a mouse to simulate the condition of postmenopausal osteoporosis of women in clinic, uterine atrophy is observed after 8 weeks, and if the bone mass is reduced, the operation is considered to be successful, and modeling is completed. The mouse model oroxylin (50mg/kg) was then administered daily for treatment by intraperitoneal injection. Mice were sacrificed after 8 weeks and their femurs and serum tissues were analyzed, and the results are shown in fig. 9.

The femur of the mice was analyzed by Micro-CT and the results of fig. 9A show that the bone mass and cortical bone thickness of the mice decreased significantly at 8 weeks after ovariectomy. The bone density (BMD), bone volume/tissue volume (BV/TV), trabecular number (Tb, N), and cortical bone thickness (Ct, Th) indices of mice successfully modeled (OVX group) were significantly reduced compared to normal mice (Sham group). Whereas the relevant indices were significantly alleviated after oroxylin treatment (OVX + Oro group) (fig. 9B and 9C).

Further, by histomorphometric H & E staining (staining method specifically: drawing and fixing mouse femur, dehydrating and transparentizing, wax-dipping and embedding, slicing and pasting, dewaxing, staining, dehydrating and transparentizing, sealing), it was found that the decrease in trabecular bone area (Tb, area) in mice was significantly alleviated after oroxylin a treatment (fig. 9D and 9E).

Example 10

How much bone formation is closely related to the formation of blood vessels in bone tissue, especially CD31, an EMCN positive endothelial blood vessel, and we further performed experiments in order to study the vascularization in bone tissue.

Firstly, blood vessels in the femurs of mice are imaged, and the method comprises the following specific steps:

(1) firstly, a 3% chloral hydrate solution is prepared, and a mouse is anesthetized by intraperitoneal injection of 0.1 mL; (2) then, the chest cavity of the mouse is cut by scissors, and the heart is exposed; (3) gently cutting open the right auricle of the mouse heart with an ophthalmic scissors; (4) preparing a 20mL syringe, extracting physiological saline, puncturing a needle from the left ventricle of the heart of the mouse, injecting, and flushing blood in the body of the mouse, wherein the step is repeated twice; (5) preparing a 20mL injector, extracting 4% paraformaldehyde solution, puncturing a needle from the left ventricle of the mouse, injecting, and internally fixing the blood vessel in the femur of the mouse; (6) preparing a 5mL injector, extracting the prepared vascular dye, and puncturing a needle from the left ventricle of the mouse for injection to fill the blood vessel of the mouse with the contrast medium; (7) the mice were placed in a 4-degree thermostatted refrigerator overnight; (8) taking out the mouse from the constant temperature refrigerator, separating the femur of the mouse by using scissors, removing soft tissues attached to the femur, (9) scanning the femur by using Micro CT, detecting the angiogenesis condition of the femur, and analyzing the data of the angiogenesis condition.

The results of examination by Micro-CT are shown in fig. 10, in which the intrafemoral vascular levels were significantly inhibited after OVX surgery (OVX group) and were significantly restored after oroxylin a intervention (Oro group).

We further treated CD31 in the femur of mice^hiEMCN^hiEndothelial vessels were stained and the results are shown in FIG. 11, CD31 of mice (OVX group) after successful modeling^hiEMCN^hiEndothelial vessels were stained and, as a result, the number of CD31, EMCN-positive vessels was found to be significantly reduced compared to normal mice (Sham group) and after oroxylin-dried (OV)Group X + Oro), CD31^hiEMCN^hiThe proportion of endothelial vessels to the area of all vessels increases.

We performed flow cytometry on CD31 in mouse femurs^hiEMCN^hiThe endothelial cell number was counted, and as a result, as shown in FIG. 12, CD31 was found after the oroxylin A was dried^hiEMCN^hiEndothelial cell numbers were significantly restored.

ELISA analysis is carried out on serum in a mouse, and specific analysis is respectively carried out on an osteoclast related index CTX-1, TRAcp5b and an osteoblast related index OCN in the serum, and the result is shown in figure 13.

Example 11

This example further studies the effect of oroxylin a on healing of femoral fractures in mice. The specific operation method comprises the following steps:

first, we anesthetize mice with 5% chloral hydrate solution. The skin of the mouse femur was cut with scissors to expose the femur, and a thin needle was inserted from the mouse knee along the long axis of the femur to secure it in the femur. The muscles were separated with forceps and the mouse femur was transected from the femoral midline with scissors. The muscle is recovered and sewed on the skin. Administration was started 3 days after recovery of the mice, and oroxylin (50mg/kg) was administered by intraperitoneal injection once for 2 days for 3 weeks.

FIG. 14 is a graph of Micro CT of mouse femur and percentage of fracture healing mice (wherein the Vehicle (Vel group) is PBS-treated control group), showing that oroxylin A intervention promotes healing of femur fracture in mice.

The mechanical analysis is shown in fig. 15, and the result shows that the mechanical recovery of the fracture healing of the mice is better after the oroxylin A treatment.

The invention has many applications, and the above description is only a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made without departing from the principles of the invention and these modifications are to be considered within the scope of the invention.

Claims (7)

1. An application of oroxylin A in preparing the medicine for treating osteoporosis and promoting union of fracture is disclosed.

2. Use according to claim 1, characterized in that the effective concentration of oroxylin a is between 1 and 400 mg/kg.

3. Use according to claim 1, characterized in that the effective concentration of oroxylin A is between 30 and 80 mg/kg.

4. A pharmaceutical composition for treating osteoporosis and promoting fracture healing is characterized by comprising oroxylin A and pharmaceutically acceptable auxiliary materials.

5. The pharmaceutical composition for treating osteoporosis and promoting fracture healing according to claim 3, wherein the effective concentration of oroxylin A in the pharmaceutical composition is 1-400 mg/kg.

6. The application of oroxylin A in preparing a preparation for inhibiting osteoclast precursor cell fusion is characterized in that the effective concentration of oroxylin A is 30-80 mg/kg.

7. The application of oroxylin A in preparation of a preparation for promoting H-type angiogenesis is characterized in that the effective concentration of oroxylin A is 30-80 mg/kg.

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