Promoter with bone growth effect and application thereof

Abstract

The invention belongs to the field of medicines, foods and health-care products, and particularly relates to an accelerant with a bone growth effect and application of the accelerant in the fields of medicines, health-care foods and functional foods. According to the application disclosed by the invention, the inhibiting effect of the morurin on osteogenesis promoting and osteoporosis resisting marker post target osteosclerosis protein is found through high-throughput virtual screening for the first time and is verified through experiments; meanwhile, in-vitro and animal experiments prove the osteogenic differentiation promoting effect of the morusin for the first time. According to the present invention, the morusin and the pharmaceutically acceptable salt thereof can be adopted as the bone growth promoter, can be singly or jointly used, can be used for promoting bone growth and resisting osteoporosis, and can be used in the forms of drugs, health-care foods, functional foods, food additives and the like.

Description

Technical Field

[0001] The invention belongs to the field of medicine, food and health care products, and specifically relates to a promoter of bone growth, mulberry orange, and its application in the fields of medicine, health care food and functional food. Background Art

[0002] The high incidence of fracture injuries and osteoporosis has become a major threat to human health. Fractures lead to the destruction of bone structure and dysfunction, which not only affects the bones themselves, but also causes damage to surrounding tissues, nerves, blood vessels and organs throughout the body. Osteoporosis is characterized by decreased bone mass and increased bone brittleness. The decreased motor system capacity and high risk of fractures caused by osteoporosis have brought serious social problems and economic burdens. Therefore, the need for the prevention, diagnosis and treatment of fracture injuries and osteoporosis is becoming increasingly urgent.

[0003] At present, there are many types of drugs that promote bone growth and fight osteoporosis, and their targets and mechanisms of action are also different (Chinese Pharmaceutical Association Hospital Pharmacy Committee, "Expert Consensus on the Rational Use of Osteoporosis Treatment Drugs (2023)" Editorial Group, Zhang Yu. Expert Consensus on the Rational Use of Osteoporosis Treatment Drugs (2023) [J]. Chinese Journal of Hospital Pharmacy, 2024, 44(9): 985-1006.), mainly in two aspects: First, bone formation promoters. The most representative ones are sclerostin inhibitors (Marini F, Giusti F, Palmini G, et al. Role of Wnt signaling and sclerostinin bone and as therapeutic targets in skeletal disorders[J]. Osteoporosis International, 2022, 34(2):213-238. DOI:10.1007 / s00198-022-06523-7.). In 2019, the US FDA approved the new sclerostin inhibitor drug Evenity for marketing, which also became the first new anti-osteoporosis drug with the dual effects of increasing bone formation and reducing bone resorption.

[0004] Sclerostin is a glycoprotein encoded by the sost gene and secreted by osteogenic cells that inhibits bone formation. On the one hand, sclerostin inhibits the Wnt signaling pathway by competitively inhibiting the co-receptor LRP-5 / 6, thereby inhibiting the signaling pathway and inhibiting bone formation. On the other hand, after being embedded in matrix proteins, osteocytes begin to secrete sclerostin, which is then transported to osteoblasts on the bone surface through the bone tissue network system, thereby inhibiting osteoblast-mediated bone formation. Due to the inhibitory effect of sclerostin on bone formation, inhibiting its expression or activity can significantly increase bone mass and bone strength without obvious adverse reactions outside the bone, making it an effective therapeutic target for osteoporosis. Animal models and clinical studies have shown that sclerostin inhibitors can significantly increase bone density and bone strength, and compared with traditional drugs such as alendronate and teriparatide, sclerostin inhibitors are more effective in increasing bone density.

[0005] Second, bone resorption inhibitors. These include bisphosphonates and calcitonins. Bisphosphonates mainly act on osteoclasts, inhibiting the dissolution of bones by osteoclasts, reducing bone loss, and thus increasing bone density. Such drugs, such as alendronate sodium and zoledronic acid, are suitable for patients at risk of fracture or who have already had a fracture. Calcitonins target osteoclasts, and rapidly inhibit osteoclast activity to reduce bone dissolution. They also have analgesic effects and are suitable for acute pain relief and prevention of complications, such as eel calcitonin analogs and salmon calcitonin.

[0006] Currently, anti-osteoporosis sclerostin protein inhibitors have shortcomings such as high cost, poor injection compliance, and potential side effects. Therefore, there is an urgent need for anti-osteoporosis products with clear molecular mechanisms, easy use, good safety, and low price.

[0007] Drug screening and in vitro and animal study evaluation are feasible methods for discovering sclerostin inhibitors. Virtual screening technology, also known as virtual screening in computer screening or computer-aided drug design, is a method that uses computer technology and professional application software to select potentially effective drug candidate molecules from a large number of compounds. It can significantly reduce the time and cost of drug development and improve the efficiency of new drug discovery (Zeng J, Han J, Liu Z, et al. Pentagalloylglucose disrupts the PALB2-BRCA2 interaction and potentiatestumor sensitivity to PARP inhibitor and radiotherapy [J]. Cancer letters, 2022, 546: 215851. DOI: 10.1016 / j.canlet.2022.215851.). The results of virtual screening need to be evaluated by in vitro cell experiments and animal experiments. In in vitro cell research and evaluation, the MC3T3-E1 cell model is a commonly used cell model for studying bone diseases and mechanisms (Zheng W, Luo Q, Shi D, et al. FDPS-lnfluenced Transcriptome Alternations in MC3T3-E1 Cells are Associated with the Pathogenesis of Osteoporosis[J]. Science of advanced materials, 2023(8):15.), and is used in osteogenic differentiation, osteoporosis (Liu Ning. Study on the role and mechanism of miR-148a-3p in osteogenic differentiation of MC3T3-E1 cells and osteoporotic bone remodeling[D]. China Medical University, 2023. DOI:10.27652 / d.cnki.gzyku.2023.001911.) and other studies.

[0008] Thermal drift experiments are an effective means to study the binding of compounds to target proteins in recent years. They are used to detect the interaction between drugs and proteins in vitro and to screen specific protein candidate drugs in vitro. When cells are treated with drugs, the drug binds to the target protein, enhancing the thermal stability of the target protein and increasing the melting temperature (Wang Li, Liu Jingfang, Li Weilin, et al. Standard operating procedures for identifying drug targets in living cells based on cell-based thermal drift assay (CETSA) technology [J]. Acta Microbiologica Sinica, 2023, 63(6): 2488-2501.).

[0009] In the animal research and evaluation of sclerostin protein inhibitors, the in situ osteogenesis model can be used to evaluate the effects of sclerostin protein inhibitors on bone tissue and provide a strong experimental basis. The in situ osteogenesis model refers to inducing or promoting the formation and regeneration of bone tissue in a specific part of an animal through specific experimental methods. When establishing the model, osteoblasts can be induced to differentiate in vitro, injected or transplanted into the subcutaneous tissue of mice or other animals, and the regeneration of bone tissue can be evaluated by imaging or histological examination (Ma D, Zhong C, Yao H, et.al. Engineering injectable bone using bone marrowstromal cell aggregates. Stem Cells Dev. 2011 Jun;20(6):989-99.). In addition to ALP, Runx2 indicators also play a vital role in the bone formation process. Their changes directly reflect the state of bone metabolism and the activity of bone formation.

[0010] Traditional Chinese medicine has a valuable foundation of thousands of years of evidence-based medicine. It is a feasible strategy to explore new methods to fight osteoporosis from a large number of records and reports of effective applications of bone and joint diseases. Mulberry, as a traditional Chinese medicine with a long history, has a wide range of application value. "Compendium of Materia Medica" and "Shennong's Herbal Classic" record that mulberry can "benefit the joints of the five internal organs", "Newly Revised Materia Medica" in the Tang Dynasty mentioned that mulberry can treat "weak waist and knees", and "Sui Xi Ju Diet" mentioned that mulberry "nourishes the liver and kidneys, and strengthens walking". However, it is still unknown which active ingredients in mulberry play the role of "benefiting the joints of the five internal organs" and "nourishing the liver and kidneys, and strengthening walking". Therefore, using modern biotechnology to study the active ingredients in mulberry that are closely related to bone growth is a very valuable research work for the prevention and treatment of bone diseases. Summary of the invention

[0011] In view of the problems existing in the prior art, the present invention provides a promoter of moringin that can inhibit sclerostin protein and has a bone growth effect. It is used to promote bone growth, resist osteoporosis and other common bone problems, and clarifies its application value in the fields of medicine, health food, functional food, etc.

[0012] The technical contents of the present invention are as follows: The invention provides a promoter with bone growth effect, the effective component of which is mulberry orange or a pharmaceutically acceptable salt thereof.

[0013] The accelerator has the following functions: (1) Inhibit the activity of sclerostin protein; (2) Promote osteogenic differentiation of osteoblast precursor cells; a. Enhance the transcriptional activity of TCF / LEF1 in MC3T3-E1 cells and activate the Wnt / β-catenin signaling pathway; b. Enhanced the alkaline phosphatase activity in MC3T3-E1 cells to induce osteoblast differentiation; c. Promote the formation of calcified nodules in MC3T3-E1 cells; (3) Promote bone formation in mice; a. Promote the expression of ALP in mouse bone tissue; b. Promote the expression of Runx2 in mouse bone tissue.

[0014] The present invention also provides the use of the promoter in the preparation of a product for promoting bone growth. Specifically, the promoter can promote osteoblast differentiation and mineralization, thereby increasing bone density and bone toughness.

[0015] Furthermore, the promoter can be used to prepare products for promoting fracture healing and / or resisting osteoporosis.

[0016] The accelerators are used alone or in combination to prepare medicines, health foods, functional foods and food additives.

[0017] The medicine, health food, functional food and food additive contain moringin or a pharmaceutically acceptable salt thereof.

[0018] The medicine, health food or functional food is composed of mulberry orange extract and a pharmaceutically acceptable carrier or a food acceptable carrier; or, the medicine, health food or functional food also contains one or more of a disintegrant, a wetting agent, a binder, a filler, an absorption promoter, a solvent, a lubricant, a surfactant, a flavoring agent, a sweetener, an antioxidant, a preservative and a pigment, an ointment base, and a transdermal enhancer.

[0019] The medicines include tablets, capsules, injections, granules and suspensions; the health foods include tablets, hard capsules, soft capsules, oral liquids, milk powder, biscuits, candies, beverages and wine; the functional foods include functional dairy products, bread and beverages.

[0020] Compared with the prior art, the present invention has the following beneficial values: 1. The present invention discovered for the first time through virtual screening the inhibitory effect of mulberry orange pigment on the osteogenesis benchmark target sclerostin protein, and confirmed it through in vitro experiments.

[0021] 2. The present invention, through the MC3T3-E1 cell model experiment, confirms for the first time the effect of mulberry orange peel on promoting osteogenic differentiation of mouse osteoblast precursor cells MC3T3-E1. Specifically, mulberry orange peel can enhance the TCF / LEF1 transcription activity in MC3T3-E1 cells, activate the Wnt / β-catenin signaling pathway, enhance the alkaline phosphatase activity that induces osteoblast differentiation in MC3T3-E1 cells, and promote the formation of calcified nodules in MC3T3-E1 cells.

[0022] 3. The present invention, by constructing an in situ osteogenesis model, confirmed for the first time the osteogenesis-promoting effect of mulberry orange on mice. The changes in bone tissue size before and after the action of mulberry orange and the differences in the expression of ALP and Runx2 in bone tissue confirmed that mulberry orange could promote bone formation in mice.

[0023] 4. The present invention proposes that mulberry orange peel is used as a bone growth promoter, which can be used alone or in combination to prepare products for preventing and treating fractures and osteoporosis, and the product forms include medicines, health foods and functional foods. As a natural compound, mulberry orange peel can be extracted from mulberries, does not rely on limited chemical raw materials and complex synthesis processes, and has the advantages of safety, effectiveness, and clear molecular mechanisms. Compared with currently marketed drugs, it has potential advantages in safety and compliance for long-term use. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 , molecular weight score distribution diagram of natural product compound library combined with sclerostin 2K8P structure screening results; Figure 2 , luciferase assay of candidate compounds; Figure 3 , the effective dose of mulberry orange to enhance luciferase activity; Figure 4 , Morin inhibits the activity of sclerostin protein Figure 5 , the safety of mulberry orange in MC3T3-E1 cells; Figure 6 , ALP staining of MC3T3-E1 cells after 7 days of treatment with moringin; Figure 7 , ALP activity of MC3T3-E1 cells after 7 days of treatment with moringin; Figure 8 , fluorescence intensity of the orange orange calcium ion probe; Fig. 9 , comparison of heterotopic transplanted tissue size in mouse in situ osteogenesis experiment; Fig.10 , Immunofluorescence staining of ALP in mouse bone tissue sections; Fig.11, Immunofluorescence staining of Runx2 in mouse bone tissue sections. DETAILED DESCRIPTION

[0025] To make the technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified, all raw materials used in the embodiments are commercially available.

[0026] Example 1 Virtual screening revealed the inhibitory effect of mulberry orange on the osteoblastic target sclerostin protein The virtual screening of drugs using sclerostin protein as a target includes the following steps: (1) Processing of sclerostin protein structure The SOST_HΜMAN protein structure was processed as a target protein using the Protein Preparation Wizard module in the Schrödinger software, and the prepared structure was used as a receptor file for subsequent virtual screening.

[0027] (2) Preparation of small molecule compound library The natural product compound library is processed to ensure the global conformation of small molecules during virtual screening, and conformation generation is performed on small molecules, with a maximum of 32 conformations generated for each small molecule. The prepared compound library is used as a ligand file for subsequent virtual screening.

[0028] (3) Molecular docking The prepared receptor files and ligand files are screened using the SP (standard precision mode) precision screening mode, with docking parameters set to default values, using the small molecule flexible docking method, and performing energy optimization after docking. Based on the docking results, the small molecules to be screened in the next step, i.e., the candidate drugs, are screened in the next step of in vitro experiments.

[0029] (4) Evaluation of docking results Affinity scoring: Schrödinger software was used to perform molecular docking on each compound in the compound library and calculate the affinity score between the compound and the 2K8P structure of SOST_HΜMAN. The data generated by the docking process of each compound library is as follows Figure 1 As shown, there are 279 compounds in the compound library that bind to the 2K8P structure, and their affinities range from -11.8030 kcal / mol to -5.2366 kcal / mol (the lower the score, the better the evaluation result); Structural diversity analysis: Structural diversity analysis was performed on 279 compounds in the compound library combined with the 2K8P structure. Structure-based clustering was performed using MOE software. The fingerprint clustering algorithm was the Jarvis-Patrick clustering method. The structural similarity was set to 70%. The 279 molecules in the compound library and the 2K8P structure were divided into 165 categories. Visual binding mode analysis: The binding conformations of molecules and proteins were visually checked for 279 molecules, and unreasonable structures were removed. A total of 74 candidate drugs, including mulberry orange, were retained for the next step of in vitro experimental screening.

[0030] Example 2 TCF / LEF1-Luc-Puro Reporter Lentivirus was used to construct TCF / LEF1-LucMC3T3-E1 cell line in vitro, and the activation effect of 10 μM moringin on wnt signaling in MC3T3-E1 cells was verified The 74 candidate drugs obtained by virtual screening in Example 1 were screened in vitro. The compounds used for screening were derived from the natural product compound library of Taoshu Biological Co., Ltd. and dissolved in DMSO to be stored at 10 mM. The specific operation of the in vitro screening is as follows: (1) Model construction First, extract the TCF / LEF1-Luc-Puro Reporter plasmid, then perform TCF / LEF1 lentivirus packaging and titer determination, extract the lentivirus vector and its auxiliary packaging original vector plasmid with high purity and no endotoxin, use HGTransgeneTM Reagent to co-transfect the constructed lentivirus vector and its auxiliary packaging original vector plasmid into HEK-293T cells, add Enhancing buffer 10-12 hours after transfection, replace fresh culture medium 8 hours later, continue to culture for 48 hours, collect the cell supernatant rich in lentivirus particles, concentrate it to obtain a high-titer lentivirus concentrate, and determine and calibrate the virus titer in HEK-293T cells. Then, infect MC3T3-E1 cells with the packaged TCF / LEF1-Luc lentivirus to obtain the TCF / LEF1-Luc MC3T3-E1 stable strain. For cell samples of MC3T3-E1 cells infected with TCF / LEF1-Luc lentivirus, Real Time PCR was used to verify the overexpression effects of luciferase and puro.

[0031] Wnt proteins are secreted lipid-modified glycoproteins that activate different intracellular signal transduction pathways. They regulate cell growth, function, differentiation, and cell death and are essential for normal embryonic development. Wnt proteins activate Frz receptors, causing β-catenin proteins to translocate into the nucleus, bind to TCF / LEF transcription factor binding sites, activate downstream pathways, and thus activate the expression of luciferase. Luciferase readouts represent the activation effect of the signaling pathway and can therefore be used to evaluate the in vitro effects of Wnt pathway-related drugs. Using a Wnt-β-specific TCF / LEF-β-driven reporter system optimized for Wnt 3a response, a natural product library was screened in vitro, and a cell-based screening assay identified a variety of sclerostin protein inhibitors with the ability to enhance Wnt / β-catenin signaling.

[0032] (2) Compound treatment Remove the cells from the incubator, digest and centrifuge to collect the cell pellet, resuspend and count with complete medium. Then adjust the cell concentration to 1.5×10 5 cells / mL. Use a dispenser to add 100 μL of cells / well to the middle well. Add 100 μL of PBS to the surrounding wells. Cover the plate and incubate in a 37°C cell culture incubator. After 24 hours, discard the complete medium, add 0.02 μg / mL of Wnt3a protein and 10 μM of drug to each well, and incubate in a 37°C cell culture incubator for 24 hours.

[0033] (3) Fluorescence detection The 96-well plate was equilibrated at room temperature for 5 minutes, and then 100 μL of luciferase detection reagent was added to each well to lyse the cells. The cells were blown up and down 5 times with a pipette tip to make the cells lyse more fully. Wait for 5 minutes to allow the cells to be fully lysed. 100 μL of the mixture (detection reagent + cell culture medium) was pipetted into each well on the white detection plate. The fluorescence value was detected at 570 nm using a Molecular Devices Spectra Max L microplate reader.

[0034] The results are as follows Figure 2 Among the candidate compounds, the luciferase value of mulberry orange peel was significantly higher than that of the blank group, indicating that mulberry orange peel can bind to the TCF / LEF transcription factor binding site and activate the classic Wnt signaling pathway. The Wnt signaling pathway can promote osteoblast differentiation and help delay osteoporosis. Figure 3 We studied the effective dose of mulberry orange peel, and the results showed that 5, 10, and 20 μM mulberry orange peel could enhance luciferase activity in a dose-dependent manner.

[0035] Example 3 In vitro experiments demonstrate the inhibitory effect of moringin on sclerostin protein To verify that the activation of Wnt signaling by morin is due to binding to sclerostin protein and inhibiting its activity, a thermal shift experiment was performed, including the following steps: (1) Cell stimulation and treatment MC3T3-E1 cells were stimulated with 10 μM moringin for 24 h. After washing the cells with PBS buffer, the cells were digested with trypsin and processed into a suspension with PBS solution containing protease inhibitors.

[0036] (2) Thermal drift experiment The cell suspension was heated at 37, 42, 47, 52, and 57° C. The binding ability of moringin to sclerostin protein was detected by Western blotting.

[0037] (3) Analysis of binding ability and inhibition The grayscale value of the protein bands was quantified using ImageJ software, and the binding of moringin to sclerostin protein (inhibiting the activity of sclerostin protein) was quantified and analyzed by Graphpad prism9.0.

[0038] The results are as follows Figure 4 Thermal drift experiments confirmed that 10 μM mulberry orange can bind to sclerostin protein and reduce its activity. Mulberry orange has the function of sclerostin inhibitor.

[0039] Example 4 Cytotoxicity experiments prove that mulberry orange extract has no cytotoxicity In order to study the toxicity of mulberry orange in MC3T3-E1 cells, CCK8 experiment was performed. The specific operation is as follows: Cell Counting Kit-8 (Mei Lun Biotech) was used to detect cell viability according to the instructions. 100 μL of MC3T3-E1 cell suspension was inoculated into a 96-well plate at a density of 5 × 10 3 cells and cultured at 37°C for 24h. Then, moringin was added to the culture medium in sequence, and at the specified time point (48h), 100μL of 10% CCK-8 solution was added to each well. After incubation at 37°C for 1h, the absorbance value was measured at a wavelength of 450nm on a microplate reader, and the cell viability (%) was calculated using the untreated control cells. Each experiment was repeated three times.

[0040] The results are as follows Figure 5 Compared with the control group, after MC3T3-E1 cells were treated with 5, 10, and 20 μM moringin for 48 h, the cell viability of the moringin group was greater than 90%, indicating that moringin had no cytotoxicity at this dose and could be used to evaluate osteogenic differentiation experiments.

[0041] Example 5 ALP staining was used to verify the effect of mulberry orange on alkaline phosphatase activity in MC3T3-E1 cells, thereby proving that mulberry orange promotes osteogenic differentiation of MC3T3-E1 cells ALP staining and enzyme activity determination, the specific operations are as follows: (1) MC3T3-E1 cell suspension was prepared at 1×10 4 Cells were seeded at a density of 100 cells / well in a 12-well plate and incubated at 37°C. After 24 hours, the complete medium was replaced with osteogenic differentiation medium, and then orange peel was added in sequence for 7 days. ALP staining and ALP activity were detected using an ALP kit (Beyotime) and an ALP activity kit (Beyotime). The cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 30 minutes, and then BCIP / NBT working solution was added. Incubate at room temperature in the dark for 5-30 minutes, wash 3 times with PBST, 10 minutes each time, and observe the pictures under a light microscope. The results are as follows Figure 6 .

[0042] (2) For ALP activity determination, cells were collected with cell lysis buffer. The lysed cells were mixed with pNPP (5 mM pNPP, 1 mM MgCl2, 1 mM ZnCl2) substrate solution (0.1 Mglycine buffer, pH = 10.4) at room temperature for 30 min, and 4 M NaOH solution was added to stop the enzymatic reaction. The absorbance value was measured at 405 nm, and the ALP activity was calculated based on the p-nitrophenol (pNP) standard curve and calibrated with the total protein concentration determined by the BCA method. The results are shown in Figure 7 .

[0043] Osteoblasts (OB) are the main functional cells of bone formation and are responsible for new bone formation. During the osteogenic differentiation process, its basic biological characteristics are bone matrix synthesis, secretion, mineralization and maturation. Osteoblasts first synthesize extracellular matrices such as type I collagen (collagen-I, COL-I), osteocalcin (osteocalcin, OC), and osteopontin (osteopontin, OPN), and release calcium ions and enzymes such as alkaline phosphatase (alkaline phosphatase, ALP) through matrix vesicles. Calcium ions are precipitated on collagen fibers under the action of alkaline phosphatase, completing the matrix mineralization process and finally forming bone tissue. Type I collagen begins to be expressed from the proliferation stage and reaches its peak in the matrix synthesis stage; alkaline phosphatase is an early marker of osteoblast differentiation, which begins to appear in the bone matrix synthesis stage and reaches its peak in the mineralization stage.

[0044] We treated MC3T3-E1 cells with 5, 10, and 20 μM moringin for 7 days and then performed ALP staining. Figure 6 and Figure 7, compared with the control group, blue precipitates appeared in the cytoplasm of the three doses of moringin groups, indicating that osteoblast differentiation was promoted.

[0045] Example 6 Alizarin red staining was used to demonstrate that moringin promoted osteoblast matrix deposition and mineralization.

[0046] After culturing the cell samples for 21 days, the culture medium was discarded, the cells were washed with PBS 1-2 times, fixed with 4% paraformaldehyde for 10 min, washed with PBS again 1-2 times, and stained with 0.1% Alizarin Red-S (Beyotime) solution for 10 min. Finally, the cells were rinsed with PBS and observed under an inverted optical microscope. The OD value was measured at 562 nm using 10% hexadecylpyridine (Aladdin).

[0047] Osteocalcin is a marker of osteoblast differentiation and maturation. It is generally expressed in the early stage of mineralization and reaches its peak after the mineralized nodules mature. Mineralized nodules are a sign of osteoblast differentiation and maturation, and are also the main morphological characteristics of osteoblasts in performing their osteogenic function. Observing the mineralized nodules of osteoblasts is one of the commonly used technical methods to study osteoblast differentiation. In addition, the current pathophysiological research on osteoporosis relies heavily on 2D in vitro cell culture. However, the gene expression of human osteoblasts cultured by this 2D method is limited and does not represent the complexity of biophysical clues in vivo. The osteocyte-selective gene expression (E11 / GP 38) of differentiated human osteoblasts cultured in 3D (with biphasic calcium phosphate particles) is comparable to that of mature human cortical bone cells. Therefore, we used calcium ion probes on a 3D organoid model to further evaluate the ability of morugin to promote osteoblast mineralization.

[0048] The results are as follows Figure 8 Compared with the blank group, the fluorescence intensity of the calcium ion probe of 10μM mulberry orange pigment was significantly enhanced. And on the 2D model, the mineralized nodules of mulberry orange pigment were significantly stronger than those of the blank group, indicating that mulberry orange pigment promoted the mineralization of osteoblast matrix, thereby promoting bone formation.

[0049] Example 7 Construction of an in situ osteogenesis model in mice to confirm that mulberry orange extract promotes bone formation in mice Three-week-old NOD / SCID mice were used for in situ osteogenesis experiments. MC3T3 cells were pretreated one week before ectopic transplantation of osteoblasts: each group was treated with osteogenic differentiation induction culture, and the mulberry orange group was treated with osteogenic differentiation induction medium combined with mulberry orange 10μM, and the medium was changed every 3 days. At the same time, 1 / 1000 DMSO was added as the negative control group of this experiment. The back skin of the mouse was selected as the injection site, and 2×10 6cells / 200μL. The longest diameter (L), the longest horizontal diameter perpendicular to the longest diameter (W), and the height (H) of the transplanted tissue were photographed and recorded at 1 week and 2 weeks, and the tissue volume was calculated according to V=π / 6×L×W×H. After tissue sampling, paraffin sections were obtained after fixation, dehydration, and embedding, and immunofluorescence was used to detect changes in ALP and Runx2 proteins.

[0050] The results are as follows Fig. 9 After measuring the volume of tissue 14 days after transplantation, it was found that the in situ tissue of mice in the mulberry orange extract-treated group was significantly larger than that in the blank group, confirming that mulberry orange extract can not only promote the proliferation of osteoblasts, but also protect the activity of osteoblasts.

[0051] ALP is produced by osteoblasts and plays an important role in the growth and development of bones. Its main physiological function is to hydrolyze phosphates during osteogenesis, providing the necessary phosphate for the deposition of hydroxyapatite. It also hydrolyzes pyrophosphates to relieve their inhibitory effect on bone salt formation, which is beneficial to osteogenesis. The enhanced expression activity of ALP is an obvious feature of osteoblast differentiation. Fig.10 The results of paraffin section staining showed that the expression of ALP was significantly enhanced in the in situ bone tissue of mice in the mulberry orange group, confirming that mulberry orange has the ability to promote the mineralization of mouse bone tissue.

[0052] Runx2 is a specific transcriptional regulator required for osteogenic differentiation. Gene mutation and deletion can lead to bone development disorders and affect intramembranous ossification and cartilage ossification. Runx2 can upregulate the synthesis of extracellular matrix of osteoblasts, accelerate cell proliferation and differentiation, and promote anabolism and osteogenic response. During osteoblast differentiation, Runx2 is upregulated in preosteoblasts, reaches the highest level in immature osteoblasts, and is downregulated in mature osteoblasts. Fig.11 The results of paraffin section staining showed that the expression of Runx2 was significantly enhanced in the in situ bone tissue of mice in the moringin group, indicating that the level of osteogenic differentiation in the tissue was enhanced under the induction of moringin.

Claims

1. A bone growth promoter, characterized in that: The active ingredient is mulberry orange or its pharmaceutically acceptable salt.

2. The bone growth promoter according to claim 1, characterized in that: (1) Inhibit the activity of sclerostin protein; (2) Promote osteogenic differentiation of osteoblast precursor cells; (3) Promote bone formation in mice.

3. Use of the promoter according to claim 1 in the preparation of a product for promoting bone growth.

4. The use according to claim 3, characterized in that: The promoter is used for preparing products promoting osteoblast differentiation and mineralization.

5. The use according to claim 4, characterized in that: The promoter is used for preparing products for promoting fracture healing and / or resisting osteoporosis.

6. The use according to claim 3, characterized in that: The accelerators are used alone or in combination to prepare medicines, health foods, functional foods and food additives.

7. The use according to claim 6, characterized in that: The medicine, health food, functional food and food additive contain moringin or a pharmaceutically acceptable salt thereof.

8. The use according to claim 7, characterized in that: The medicine, health food or functional food is composed of mulberry orange extract and a pharmaceutically acceptable carrier or a food acceptable carrier; or, the medicine, health food or functional food also contains one or more of a disintegrant, a wetting agent, a binder, a filler, an absorption promoter, a solvent, a lubricant, a surfactant, a flavoring agent, a sweetener, an antioxidant, a preservative and a pigment, an ointment base, and a transdermal enhancer.

9. The use according to claim 8, characterized in that: The medicines include tablets, capsules, injections, granules and suspensions; the health foods include tablets, hard capsules, soft capsules, oral liquids, milk powder, biscuits, candies, beverages and wine; the functional foods include functional dairy products, bread and beverages.

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