Promoter having bone growth effect and application thereof

Abstract

Provided is an application of a bone growth promoter using maclurin as an active ingredient in the preparation of drugs, health supplements, and functional foods. As a bone growth promoter, maclurin and a pharmaceutically acceptable salt thereof can be used alone or in combination for promoting bone growth and resisting osteoporosis. The product forms thereof comprise drugs, health supplements, functional foods, food additives, etc.

Description

Promoter of bone growth and its application TECHNICAL FIELD

[0001] The present application belongs to the field of medical food and health products, and specifically relates to a promoter of bone growth, mulberry orange flavonoid, and its application in the fields of medicine, health food and functional food. BACKGROUND

[0002] Fracture injury and osteoporosis have a high incidence, which is an important threat to human health. Fracture leads to the destruction of bone structure and dysfunction, not only affecting the bone itself, but also causing damage to the surrounding tissues, nerves, blood vessels and other systemic organs. Osteoporosis is characterized by decreased bone mass and increased bone fragility. The decline in motor system ability and high incidence of fracture risk caused by osteoporosis have brought serious social problems and economic burden. Therefore, the prevention, diagnosis and treatment of fracture injury and osteoporosis are increasingly urgent.

[0003] There are many types of drugs for promoting bone growth and anti-osteoporosis, and their target points and mechanisms are different (Zhang Y. Expert consensus on rational application of osteoporosis treatment drugs (2023) [J]. Chinese Journal of Hospital Pharmacy, 2024, 44(9):985-1006.). There are two main aspects:

[0004] First, bone formation promoters. The most representative one is sclerostin inhibitor (Marini F, Giusti F, Palmini G, et al. Role of Wnt signaling and sclerostin in 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 FDA approved the sclerostin inhibitor drug Evenity for marketing, which is also the first anti-osteoporosis drug with dual effects of increasing bone formation and reducing bone resorption.

[0005] Osteoprotegerin is a glycoprotein encoded by the gene sost, which is secreted by osteogenic cells and has the effect of inhibiting bone formation. On the one hand, osteoprotegerin inhibits the Wnt signaling pathway by competitively inhibiting the co-receptor LRP-5 / 6 in the Wnt signaling pathway, thereby inhibiting bone formation. On the other hand, osteoprotegerin is secreted by bone cells after being embedded in the matrix protein, and then transported to the osteoblasts on the bone surface through the bone tissue tube network system, thereby inhibiting the bone formation mediated by osteoblasts. Due to the inhibitory effect of osteoprotegerin on bone formation, inhibiting its expression or activity can significantly increase bone mass and bone strength without obvious adverse reactions outside the bone, so it has become an effective therapeutic target for osteoporosis. Animal models and clinical studies have shown that osteoprotegerin inhibitors can significantly increase bone density and bone strength, and compared with traditional drugs such as alendronate and teriparatide, osteoprotegerin inhibitors show more significant effects in increasing bone density.

[0006] Second, bone resorption inhibitors. Including bisphosphonate drugs and calcitonin drugs. Bisphosphonate drugs mainly act on osteoclasts, inhibit the dissolution of bone by osteoclasts, reduce bone loss, and thus increase bone density. Such drugs, such as alendronate and zoledronate, are suitable for patients at risk of bone fracture or who have already experienced bone fracture. The target of calcitonin drugs is osteoclasts, which can quickly inhibit the activity of osteoclasts to reduce bone dissolution, and also has analgesic effect, and is suitable for acute pain relief and prevention of complications, such as eel calcitonin analogs and salmon calcitonin.

[0007] At present, the osteoprotegerin inhibitor for anti-osteoporosis has the disadvantages of high cost, poor injection compliance, potential side effects, and therefore there is an urgent need for anti-osteoporosis products with clear molecular mechanism, convenient use, good safety and low price.

[0008] It is a feasible method to find sclerostin protein inhibitors through drug screening and in vitro and animal research evaluation. Virtual screening technology, also known as computer screening or virtual screening in computer-aided drug design, is a method that uses computer technology and professional application software to select potential effective drug candidates from a large number of compounds. It can significantly reduce the time and cost of drug research and development, and improve the efficiency of new drug discovery (Zeng J, Han J, Liu Z, et al. Pentagalloylglucose disrupts the PALB2-BRCA2 interaction and potentiates tumor 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 the evaluation of in vitro cell research, 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-Influenced Transcriptome Alternations in MC3T3-E1 Cells are Associated with the Pathogenesis of Osteoporosis [J]. Science of advanced materials, 2023(8):15.), which is used for osteogenic differentiation, osteoporosis (Liu N. The role and mechanism of miR-148a-3p in MC3T3-E1 cell osteogenic differentiation and osteoporosis bone remodeling [D]. China Medical University, 2023. DOI:10.27652 / d.cnki.gzyku.2023.001911.) and other researches.

[0009] Thermal shift experiment is an effective means of studying the binding of compounds to target proteins in recent years, which is used to detect the interaction between drugs and proteins in vitro to screen specific protein candidate drugs. When cells are treated with drugs, the binding of drugs to target proteins enhances the thermal stability of target proteins and increases the melting temperature value (Wang L, Liu JF, Li WL, et al. Standard operating procedure for identifying drug targets in living cells based on cellular thermal shift assay (CETSA) [J]. Acta Microbiologica Sinica, 2023, 63(6):2488-2501.).

[0010] In the animal study evaluation of sclerostin protein inhibitors, in situ bone formation model can be used to evaluate the effect of sclerostin protein inhibitors on bone tissue, and provide strong experimental evidence. The in situ bone formation model refers to inducing or promoting the formation and regeneration of bone tissue in a specific part of an animal through a specific experimental method. When establishing the model, bone cell induced differentiation can be carried out in vitro, injected or transplanted into mice or other animals subcutaneously, and the regeneration of bone tissue is evaluated by imaging or histological examination (Ma D, Zhong C, Yao H, et.al. Engineering injectable bone using bone marrow stromal cell aggregates. Stem Cells Dev. 2011 Jun;20(6):989-99.). In addition, in addition to ALP, Runx2 indicators also play a crucial role in the process of bone formation, and their changes directly reflect the state of bone metabolism and the activity of bone formation.

[0011] Traditional Chinese medicine has a valuable foundation of evidence-based medicine for thousands of years, and it is a feasible strategy to explore new methods for preventing and treating osteoporosis from a large number of effective application experience records and reports of bone and joint diseases. Mulberry is a traditional Chinese medicine with a long history and has wide application value. "Bencao Gangmu" and "Shen Nong Bencao Jing Shu" record that mulberry can "benefit five internal organs and joints", Tang Dynasty "Xinxiu Bencao" mentions that mulberry can treat "waist and knee soreness", and "Sui Xi Ju Yinshi" mentions that mulberry "nourishes liver and kidney and strengthens walking". However, it is not known which effective component in mulberry plays the role of "benefiting five internal organs and joints" and "nourishing liver and kidney and strengthening walking", therefore, it is very valuable to study the effective component in mulberry closely related to bone growth by using modern biological technology for the prevention and treatment of bone diseases. SUMMARY

[0012] In view of the problems in the prior art, the application provides a mulberroside capable of inhibiting sclerostin protein and having a bone growth promoting effect, which is used for promoting bone growth and resisting common bone problems such as osteoporosis, and the application value of the mulberroside in the fields of medicine, health food, functional food and the like is clear.

[0013] The technical content of the application is as follows:

[0014] The application provides a bone growth promoting agent, and an effective component of the bone growth promoting agent is mulberroside or a pharmaceutically acceptable salt thereof.

[0015] The bone growth promoting agent has the following functions:

[0016] (1) inhibiting the activity of sclerostin protein;

[0017] (2) promoting osteogenic differentiation of osteogenic precursor cells;

[0018] a. enhancing TCF / LEF1 transcriptional activity in MC3T3-E1 cells, activating Wnt / β-catenin signaling pathway;

[0019] b. enhancing alkaline phosphatase activity in MC3T3-E1 cells for inducing osteoblast differentiation;

[0020] c. promoting calcified nodule formation in MC3T3-E1 cells;

[0021] (3) promoting bone formation in mice;

[0022] a. promoting ALP expression in bone tissue of mice;

[0023] b. promoting Runx2 expression in bone tissue of mice.

[0024] The application also provides use of the promoter in preparation of products for promoting bone growth, in particular, the promoter can promote osteoblast differentiation and mineralization, thereby increasing bone density and bone toughness.

[0025] Further, the promoter can be used in preparation of products for promoting fracture healing and / or anti-osteoporosis.

[0026] The promoter is used alone or in combination to prepare medicines, health foods, functional foods and food additives.

[0027] The medicines, health foods, functional foods and food additives contain morin or a pharmaceutically acceptable salt thereof.

[0028] The medicines, health foods or functional foods consist of morin and a pharmaceutically acceptable carrier or a food acceptable carrier; or the medicines, health foods or functional foods further contain one or more of disintegrants, wetting agents, binders, fillers, absorption promoters, solvents, lubricants, surfactants, flavoring agents, sweeteners, antioxidants, preservatives and pigments, ointment bases, transdermal absorption promoters.

[0029] 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, wine; the functional foods include functional dairy products, bread, beverages.

[0030] Compared with the prior art, the application has the following advantages:

[0031] 1. The application first discovers the inhibitory effect of morin on sclerostin protein, an osteogenic marker target, through virtual screening, and verifies the effect through in vitro experiments.

[0032] 2. The application first confirms the promoting effect of morindon on the osteogenic differentiation of mouse osteogenic precursor cells MC3T3-E1 through MC3T3-E1 cell model experiments, specifically, morindon can enhance the TCF / LEF1 transcriptional activity in MC3T3-E1 cells, activate the Wnt / β-catenin signaling pathway, enhance the alkaline phosphatase activity in MC3T3-E1 cells for inducing osteoblast differentiation, and promote the formation of calcified nodules in MC3T3-E1 cells.

[0033] 3. The application first confirms the promoting effect of morindon on the osteogenesis of mice by constructing an in situ osteogenesis model, and confirms that morindon can promote the bone formation of mice through the size change of bone tissue and the expression difference of ALP and Runx2 in bone tissue before and after the action of morindon.

[0034] 4. The application proposes that morindon can be used alone or in combination as a bone growth promoter for preparing products for preventing and treating bone fracture and osteoporosis, and the product forms include drugs, health foods and functional foods. As a natural compound, morindon can be extracted from mulberries, does not depend on limited chemical raw materials and complex synthesis process, has advantages of safety, effectiveness and clear molecular mechanism, and has potential advantages in long-term use safety and compliance compared with currently marketed drugs. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a molecular weight scoring distribution diagram of the screening results of the binding of the natural product compound library with the sclerostin 2K8P structure;

[0036] Figure 2 is luciferase detection of the candidate compound;

[0037] Figure 3 is the effective dose of morindon for enhancing luciferase activity;

[0038] Figure 4 is the activity of morindon for inhibiting sclerostin protein;

[0039] Figure 5 is the safety of morindon in MC3T3-E1 cells;

[0040] Figure 6 is the ALP staining diagram of MC3T3-E1 cells after being acted on by morindon for 7 days;

[0041] Figure 7 is the ALP activity of MC3T3-E1 cells after being acted on by morindon for 7 days;

[0042] Figure 8 is the fluorescence intensity of the calcium ion probe of morindon;

[0043] Figure 9 is a comparison of the sizes of the ectopic transplanted tissues in the in situ osteogenesis experiment of mice;

[0044] Figure 10 is the immunofluorescence staining of ALP in the bone tissue sections of mice;

[0045] Figure 11. Immunofluorescence staining of Runx2 in mouse bone tissue sections. Embodiments of the present application

[0046] In order to make the technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the present application will be further described below in combination with specific embodiments. All raw materials used in the embodiments are commercially available, unless otherwise specified.

[0047] Example 1. Virtual screening found that Sangonin inhibited the osteogenic marker target sclerostin protein

[0048] Virtual screening of drugs was carried out with sclerostin protein as a target, including the following steps:

[0049] (1) Processing of sclerostin protein structure

[0050] The Protein Preparation Wizard module in Schrödinger software was used to process the target protein structure of SOST_HUMAN protein structure, and the prepared structure was used as a receptor file for subsequent virtual screening.

[0051] (2) Preparation of small molecule compound library

[0052] The natural product compound library was processed to ensure the global conformation of the small molecules in the virtual screening process, and the conformation of the small molecules was generated, with a maximum of 32 conformations for each small molecule. The prepared compound library was used as a ligand file for subsequent virtual screening.

[0053] (3) Molecular docking

[0054] The prepared receptor file and ligand file were screened using the SP (standard precision mode) accuracy screening mode, with the docking parameters set to the default value, and the small molecule flexible docking method was used for docking, followed by energy optimization. According to the docking results, the small molecules that entered the next step of screening, i.e. candidate drugs, were determined for the next step of in vitro experimental screening.

[0055] (4) Evaluation of docking results

[0056] Affinity scoring: Schrödinger software was used to dock each compound in the compound library to calculate the affinity score of the compound with the 2K8P structure of SOST_HUMAN. The data generated during the docking process of each compound library is shown in Figure 1. There are 279 compounds in the compound library that bind to the 2K8P structure, and their affinity is distributed between -11.8030 kcal / mol and -5.2366 kcal / mol (the lower the score, the better the evaluation result);

[0057] Structure diversity analysis: Structure diversity analysis was performed on 279 compounds of the compound library combined with the 2K8P structure. Structure-based clustering was performed by MOE software, the fingerprint clustering algorithm was Jarvis-Patrick clustering method, the structure similarity was set to 70%, and the 279 molecules of the compound library and the 2K8P structure were divided into 165 classes;

[0058] Visual binding mode analysis: 279 molecules were checked by visual inspection of the binding conformation of the molecule and the protein, and unreasonable structures were removed. A total of 74 candidate drugs including santalactone were retained for further in vitro experimental screening.

[0059] Example 2: TCF / LEF1-Luc-Puro Reporter Lentivirus was used to construct TCF / LEF1-Luc MC3T3-E1 cell strain in vitro, and the activation effect of 10 μM santalactone on MC3T3-E1 cell wnt signal was verified.

[0060] The 74 candidate drugs obtained by virtual screening in Example 1 were subjected to in vitro experimental screening. The compounds used for screening were derived from the natural product compound library of Tao Shu Biotechnology Co., Ltd. and were dissolved in DMSO to prepare 10 mM for storage. The specific operation of in vitro experimental screening is as follows:

[0061] (1) Model construction

[0062] First, the TCF / LEF1-Luc-Puro Reporter plasmid was extracted, and then TCF / LEF1 lentivirus packaging and titer determination were performed. The extracted high-purity, endotoxin-free lentivirus vector and its auxiliary packaging element vector plasmid were used to co-transfect the HEK-293T cells with the constructed lentivirus vector and its auxiliary packaging element vector plasmid. After 10~12 h of transfection, Enhancing buffer was added, and after 8 h, fresh culture medium was replaced and continued to be cultured for 48 h. The cell supernatant rich in lentivirus particles was collected, and after concentration, high-titer lentivirus concentrate was obtained. The virus titer was determined and calibrated in HEK-293T cells. Then, the packaged TCF / LEF1-Luc lentivirus was used to infect MC3T3-E1 cells to obtain TCF / LEF1-Luc MC3T3-E1 stable strain. For the cell sample of MC3T3-E1 cells infected with TCF / LEF1-Luc lentivirus, the overexpression effect of luciferase and puro was verified by Real Time PCR.

[0063] Wnt proteins are secreted lipid-modified glycoproteins that activate diverse intracellular signaling pathways. They regulate cell growth, function, differentiation, and cell death and are required for normal embryonic development. Wnt proteins activate Frz receptors, which cause the translocation of β-catenin protein into the nucleus, bind to TCF / LEF transcription factor binding sites, activate downstream pathways, and thus activate the expression of luciferase. The luciferase reading value represents the activation effect of the signaling pathway, and thus can be used for in vitro effect evaluation of Wnt pathway-related drugs. An in vitro screening of a natural product library was performed using a Wnt-β-specific TCF / LEF-β-driven reporter system optimized for Wnt 3a response, and a variety of sclerostin protein inhibitors with enhanced Wnt / β-catenin signaling ability were identified in a cell-based screening test.

[0064] (2) Compound treatment

[0065] The cells were taken out of the incubator, digested, and the cell pellets were collected by centrifugation, resuspended with complete medium and counted. The cell concentration was adjusted to 1.5 x 10 5 cells / mL with complete medium. A row gun was used to add 100 μL of cells per well to the middle wells. 100 μL of PBS was added to the surrounding wells. The plate was covered and incubated in a 37°C cell incubator. After 24 h, the complete medium was discarded, 0.02 μg / mL of Wnt3a protein and 10 μM of drug were added to each well, and incubated in a 37°C cell incubator for 24 h.

[0066] (3) Fluorescence detection

[0067] The 96-well plate was equilibrated at room temperature for 5 min, then 100 μL of luciferase detection reagent was added to each well to lyse the cells, and the cells were blown 5 times with a gun head to make the cell lysis more complete. Wait for 5 min for the cells to lyse completely. 100 μL of the mixture (detection reagent + cell culture medium) was taken from each well to a white detection plate. The fluorescence value was detected at 570 nm using a Molecular Devices Spectra Max L enzyme marker.

[0068] The results are shown in FIG. 2. Among the candidate compounds, the luciferase value of morindin was significantly higher than that of the blank group, indicating that morindin 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 is beneficial for delaying osteoporosis. As shown in FIG. 3, we studied the effective dose of morindin, and the results showed that 5, 10, and 20 μM of morindin can enhance the luciferase activity, and the effect is dose-dependent.

[0069] Example 3 In vitro experiments demonstrate the inhibitory effect of morindin on sclerostin protein

[0070] To verify that the activation of wnt signal by Sangonin is due to the binding of sclerostin protein and inhibiting its activity, a heat shift experiment was performed, including the following steps:

[0071] (1) Cell stimulation and treatment

[0072] 10 μM of Sangonin stimulated MC3T3-E1 cells for 24 h, and after washing the cells with PBS buffer, the cells were trypsinized and treated into suspension with PBS solution containing protease inhibitors.

[0073] (2) Heat shift experiment

[0074] The cell suspension was heated at 37, 42, 47, 52, and 57°C, respectively, using a PCR instrument. The binding ability of Sangonin to sclerostin protein was detected by Western blotting.

[0075] (3) Analysis of binding force and inhibition

[0076] The gray value of the protein band was quantified by ImageJ software, and the binding of Sangonin to sclerostin protein (inhibiting the activity of sclerostin protein) was quantified and analyzed by Graphpad prism 9.0.

[0077] The results are shown in Figure 4. The heat shift experiment confirmed that 10 μM of Sangonin could bind to sclerostin protein and reduce its activity. Sangonin has the function of sclerostin protein inhibitor.

[0078] Example 4: Cell toxicity experiment proves that Sangonin has no cytotoxicity

[0079] To study the toxicity of Sangonin in MC3T3-E1 cells, a CCK8 experiment was performed. The specific operation is as follows:

[0080] The cell viability was detected by Cell Counting Kit-8 kit (Meiren Biology) 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 per well, and cultured at 37°C for 24 h. Then, Sangonin was added to the culture medium, and at the designated time point (48 h), 100 μL of 10% CCK-8 solution was added to each well. After incubation at 37°C for 1 h, the absorbance value was measured on a microplate reader at a wavelength of 450 nm, and the cell viability (%) was calculated based on the untreated control cells. Each experiment was repeated three times.

[0081] Results as shown in Figure 5, compared with the control group, the cell viability of the Morin groups was greater than 90% after 48h treatment of MC3T3-E1 cells with 5, 10, 20μM Morin, indicating that Morin has no cytotoxicity at this dose and can be used to evaluate the osteogenic differentiation experiment.

[0082] Example 5 Verify the effect of Morin on the activity of alkaline phosphatase in MC3T3-E1 cells by ALP staining, and further prove that Morin promotes the osteogenic differentiation of MC3T3-E1 cells

[0083] ALP staining and enzyme activity determination, the specific operation is as follows:

[0084] (1) MC3T3-E1 cell suspension was inoculated in 12-well plates at a density of 1×10 4 cells / well, and incubated at 37℃. After 24h, the complete culture medium was replaced with osteogenic differentiation medium, and then Morin was added 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 and fixed with 4% paraformaldehyde (PFA) for 30min, then BCIP / NBT working solution was added. Incubate at room temperature for 5-30min, wash with PBST for 3 times, 10min each time, observe the pictures under the light microscope, and the results are shown in Figure 6.

[0085] (2) For ALP activity determination, the cells were collected with cell lysis solution. The lysed cells were mixed with pNPP (5mM pNPP, 1mM MgCl2, 1mM ZnCl2) substrate solution (0.1M glycine buffer, pH=10.4) at room temperature for 30min, and 4M NaOH solution was added to stop the enzymatic reaction. The absorbance value was measured at 405nm, and the ALP activity was calculated according to the standard curve of p-nitrophenol (pNP), and the total protein concentration was calibrated by BCA method, and the results are shown in Figure 7.

[0086] Osteoblasts (OB) are the main functional cells of bone formation, responsible for new bone formation. During the process of osteogenic differentiation, the basic biological characteristics are bone matrix synthesis, secretion, mineralization and maturation. Osteoblasts first synthesize extracellular matrix such as collagen-I (COL-I), osteocalcin (OC), osteopontin (OPN), and release calcium ions and enzyme substances such as alkaline phosphatase (ALP) through matrix vesicles. Calcium ions precipitate on collagen fibers under the action of alkaline phosphatase, completing the matrix mineralization process, and finally forming bone tissue. Collagen-I is expressed from the proliferation stage, and the matrix synthesis peaks at the peak; alkaline phosphatase is an early marker of osteoblast differentiation, and bone matrix synthesis begins to appear, and mineralization peaks at the peak.

[0087] We applied 5, 10, 20 μΜ morindin to MC3T3-E1 cells for 7 days, and then performed ALP staining. The results are shown in Figures 6 and 7. Compared with the control group, blue precipitates appeared in the cytoplasm of the three dose morindin groups, indicating that osteoblast differentiation was promoted.

[0088] Example 6. By alizarin red staining, it is proved that morindin promotes osteoblast matrix deposition and mineralization.

[0089] After 21 days of cell sample culture, the culture medium was discarded, washed with PBS for 1-2 times, fixed with 4% paraformaldehyde for 10 min, washed with PBS again for 1-2 times, and stained with 0.1% alizarin red-S (Beyotime) solution for 10 min. Finally, PBS was washed, and observed under an inverted optical microscope. The OD value was measured at 562 nm with 10% cetylpyridine (Aladdin).

[0090] Osteocalcin is a marker of osteoblast differentiation and maturation, generally expressed at the early stage of mineralization, and peaks at the mature stage of mineralization nodule. Mineralization nodule is a marker of osteoblast differentiation and maturation, and also a main morphological feature of osteoblasts to perform osteogenic function. Observing mineralization nodule of osteoblasts is one of the common technical methods for studying osteoblast differentiation. Moreover, the pathophysiological study of osteoporosis currently relies largely 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 in vivo biophysical cues. The bone cell-selective gene expression (E11 / GP 38) of differentiated human bone cells cultured in 3D (with biphasic calcium phosphate particles) is comparable to that of mature human cortical bone cells. Therefore, we further evaluated the ability of morindin to promote osteoblast mineralization using a calcium ion probe on a 3D organoid model.

[0091] Results as shown in Figure 8, compared with the blank group, the fluorescence intensity of calcium ion probe of 10 μM morindin was significantly enhanced. And on the 2D model, the mineralization nodule of morindin was significantly stronger than the blank group, indicating that morindin promoted the matrix mineralization of osteoblasts, thereby promoting bone formation.

[0092] Example 7: Construction of mouse in situ osteogenesis model to confirm that morindin promotes bone formation in mice

[0093] 3-week-old NOD / SCID mice were used for in situ osteogenesis experiments, and MC3T3 cells were pretreated one week before ectopic transplantation of osteoblasts: each group was treated with osteogenic differentiation induction culture, and the morindin group was treated with osteogenic differentiation induction medium combined with 10 μM morindin, and the medium was changed every 3 days. At the same time, 1 / 1000 DMSO was added as a negative control group in this experiment. The back skin of the mouse was selected as the injection site, and 2x10 6 cells / 200 μL were injected into each injection site according to the grouping. At 1 week and 2 weeks, the longest diameter (L), the longest transverse diameter perpendicular to the longest diameter (W), and the height (H) of the tumor mass of the transplanted tissue were recorded by taking pictures, and the volume of the tissue was calculated according to V=π / 6xLxWxH. After the tissue was taken, it was fixed, dehydrated, and embedded to obtain paraffin sections, and the changes in ALP and Runx2 protein were detected by immunofluorescence.

[0094] Results as shown in Figure 9, after measuring the volume of the tissue 14 days after transplantation, it was found that the in situ tissue of the mouse in the morindin treatment group was significantly larger than that of the blank group, confirming that morindin not only promotes the proliferation of osteoblasts, but also protects the activity of osteoblasts.

[0095] ALP is produced by osteoblasts and plays an important role in bone growth and development. Its main physiological function is to hydrolyze phosphate in the process of osteogenesis, providing the necessary phosphate for the deposition of hydroxyapatite, and at the same time hydrolyzing pyrophosphate to remove its inhibition of bone salt formation, which is conducive to osteogenesis. The enhancement of its expression activity is a clear feature of osteoblast differentiation. As shown in Figure 10, paraffin section staining results showed that the expression of ALP in the in situ bone tissue of the mouse in the morindin group was significantly enhanced, confirming that morindin has the ability to promote the mineralization of mouse bone tissue.

[0096] Runx2 is a specific transcriptional regulator essential for osteogenic differentiation. Mutations and deletions of the gene can lead to bone development disorders, affecting intramembranous and endochondral ossification. Runx2 can up-regulate the synthesis of extracellular matrix of osteoblasts, accelerate cell proliferation and differentiation, and promote anabolic and osteogenic responses. During osteoblast differentiation, Runx2 expression is up-regulated in pre-osteoblasts, reaches the highest level in immature osteoblasts, and is down-regulated in mature osteoblasts. As shown in FIG. 11, paraffin section staining results showed that the expression of Runx2 was significantly enhanced in the in situ bone tissue of mice in the Sangju Orange group, suggesting that the level of osteogenic differentiation in the tissue was enhanced under the induction of Sangju Orange.

Claims

1. A bone growth promoter, characterized in that: An effective component is morindon or a pharmaceutically acceptable salt thereof.

2. The agent for promoting bone growth according to claim 1, wherein: (1) the agent inhibits sclerostin protein activity; (2) the agent promotes osteogenic differentiation of osteogenic precursor cells; and (3) the agent promotes bone formation in mice.

3. Use of the agent according to claim 1 in the manufacture of a product for promoting bone growth. The agent is used in the manufacture of a product for promoting osteoblast differentiation and mineralization. The agent is used in the manufacture of a product for promoting bone fracture healing and / or anti-osteoporosis. The agent is used alone or in combination to manufacture a medicine, health food, functional food, and food additive.

4. Use according to claim 3, wherein: The medicine, health food, functional food, and food additive contain morindon or a pharmaceutically acceptable salt thereof.

5. The use according to claim 4, wherein: The medicine, health food, or functional food consists of morindon and a pharmaceutically acceptable carrier or a food acceptable carrier; or the medicine, health food, or functional food further 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 colorant, an ointment base, a transdermal enhancer.

6. The use according to claim 3, characterized in that: The medicine includes tablets, capsules, injections, granules, and suspensions; the health food includes tablets, hard capsules, soft capsules, oral liquids, milk powder, biscuits, candies, beverages, and wine; and the functional food includes functional dairy products, bread, and beverages.

7. Use according to claim 6, wherein: ​ 8. Use according to claim 7, wherein: ​ 9. Use according to claim 8, wherein: ​

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