A flavonoid compound in eaglewood and a preparation method and application thereof

Abstract

The application discloses a flavonoid compound in eaglewood as well as a preparation method and application of the flavonoid compound, and relates to the technical field of medicines. The application extracts and separates a new compound from natural eaglewood, and the new compound is used in the preparation of anti-osteoporosis drugs. The compound is detected in vitro to obtain an induction effect of the compound on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts, so as to affect the formation of osteoclasts. Meanwhile, the compound can promote the bone development of zebra fish through RT-qPCR detection, which indicates that the compound has anti-osteoporosis activity. The separation method of the compound is simple and easy to operate.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel flavonoid compound extracted and isolated from the natural plant Rosewood Heartwood and its application as an anti-osteoporosis drug. Background Technology

[0002] Osteoporosis is a common metabolic bone disease characterized by decreased bone mass and deterioration of bone microstructure, leading to increased bone fragility and a higher risk of fractures. This condition primarily results from an imbalance in bone metabolism, specifically a disruption of the dynamic balance between bone formation and bone resorption. Normally, osteoblasts are responsible for bone formation, while osteoclasts are responsible for bone resorption; the two maintain a dynamic balance, ensuring continuous bone tissue renewal and repair after injury. However, in osteoporosis, bone resorption exceeds bone formation, leading to bone loss.

[0003] According to the diagnostic criteria developed by the WHO using the peak bone mass-related BMD standard deviation score in healthy young women, osteoporosis is defined as a bone mineral density (BMD) T score of -2.5 or lower. A BMD T score between -1 and -2.5 is considered to indicate osteopenia and low bone mass. However, BMD is only one of the risk factors for fractures; most fragility fractures occur in individuals with BMD T scores higher than -2.5, indicating that BMD is a limited indicator of osteoporosis clinically. It is well known that under normal circumstances, bone resorption lasts approximately 4 to 6 weeks, while bone formation lasts approximately 4 to 6 months. The balance between bone resorption and bone formation, and the regulation of these processes, are essential for maintaining bone mineral density and homeostasis under healthy conditions. Osteoporosis results from an imbalance in the remodeling process, where bone resorption exceeds bone formation. Several conditions can lead to an imbalance in the remodeling process, thus resulting in osteoporosis. While postmenopause and old age are the leading causes of osteoporosis, other risk factors, including medications, endocrine disorders, immobilization, inflammatory joint disease, hematopoietic dysfunction, and malnutrition, may also contribute. Given the increasing global life expectancy, osteoporosis will impact individual quality of life and impose an economic burden in most countries. Therefore, osteoporosis should be properly managed using effective methods, which can be achieved by understanding the pathogenesis of this disease. The three main mechanisms leading to osteoporosis are: insufficient peak bone mass, excessive bone resorption, and insufficient new bone formation during remodeling; the interaction of these three mechanisms ultimately results in fragile bone tissue. To date, bisphosphonates, which inhibit bone resorption, are among the most commonly used drugs for treating osteoporosis. However, due to the side effects and limited efficacy of currently used drugs, there is an urgent need to develop new drugs that are highly effective and have no toxic side effects.

[0004] The root cause of osteoporosis is that osteoclasts resorb bone more than osteoblasts form bone. However, bone marrow mesenchymal stem cells (BMSCs), located in the bone marrow, are pluripotent adult stem cells that can differentiate into both osteoblasts and adipocytes. Studies have found that in childhood and adolescence, BMSCs differentiate more into osteoblasts and less into adipocytes; however, in old age, the opposite is true, with BMSCs differentiating more into adipocytes and less into osteoblasts. These studies indicate that the differentiation direction of BMSCs is closely related to the occurrence of osteoporosis. Therefore, effectively promoting osteogenic differentiation of BMSCs or inhibiting adipogenic differentiation of BMSCs can effectively promote bone formation, thereby achieving a therapeutic effect on osteoporosis. Numerous studies have found that drugs can promote osteoblast bone formation by directionally inducing the osteogenic differentiation process of BMSCs, thereby alleviating osteoporosis.

[0005] Multidimensional explorations in modern pharmacological experiments have provided solid evidence for the contributions of traditional Chinese medicine (TCM) in the prevention and treatment of osteoporosis. These experiments reveal that the effectiveness of TCM in increasing bone mass and optimizing bone quality does not solely rely on the direct calcium-supplementing effect of increasing mineral salt content. Rather, it achieves a deeper level of comprehensive regulation of the complex bone metabolism network by acting on multiple systems, organs, and tissues of the body. This regulation involves multiple links, factors, and multi-level mechanisms of action, collectively promoting increased bone density, improved bone microstructure, and enhanced bone mechanical strength, thereby significantly reducing the risk of fractures. Against this backdrop, exploring and developing highly effective, safe, and non-toxic new drugs from the treasure trove of TCM to address this global health challenge of osteoporosis has become a hot topic. TCM treatment of osteoporosis holds broad research potential. Summary of the Invention

[0006] This invention provides a substance with anti-osteoporosis activity, providing a material basis for the development and research of anti-osteoporosis drugs. Specifically, six components were separated from agarwood extract by medium- and low-pressure liquid chromatography. The most bioactive component, CX-3, was screened. CX-3 was further purified by semi-preparative liquid chromatography to obtain 19 components. After natural solvent evaporation, crystals were obtained. This compound, CX-3-3, ranked third among the 19 components and is a previously unreported novel compound.

[0007] One of the objectives of this invention is to provide a flavonoid compound from agarwood.

[0008] The second objective of this invention is to provide a method for preparing flavonoids from agarwood.

[0009] A third objective of this invention is to provide the application of flavonoids in agarwood in the preparation of a drug for the prevention and treatment of osteoporosis.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0011] In a first aspect, the present invention provides a flavonoid compound from agarwood, the Chinese name of which is: 2-[(1 E )-2-(4-hydroxyphenyl)vinyl]-6,7-dimethoxy-4 H -Cryrophenone-4-one, English name: (E)-2-(4-hydroxystyryl)-6,7-dimethoxychroman-4-one, molecular formula: C 19 H 18 O5 has the structure shown in equation (I):

[0012] Formula (I)

[0013] Agarwood, belonging to the Aquilaria sinensis plant of the Thymelaeaceae family, is primarily used medicinally for its purplish-brown woody parts. It is distributed in Guangdong, Hainan, Guangxi Zhuang Autonomous Region, Fujian, and other regions. One reason for its medicinal value is its sedative effect, reducing excessive excitation of the respiratory center and thus alleviating asthma. Secondly, as a traditional Chinese medicine, agarwood can relieve pain by promoting blood circulation, commonly used to treat symptoms such as chest and abdominal distension and pain, achieving a good ventilatory effect. Additionally, it has kidney-tonifying effects. Thirdly, agarwood possesses a certain degree of dampness-drying properties, eliminating dampness from the body and relieving itching symptoms, including those caused by skin problems such as eczema and urticaria. Furthermore, while agarwood has a pungent and bitter taste, its warm nature effectively alleviates symptoms such as abdominal distension and loss of appetite caused by food stagnation.

[0014] This invention discloses a new compound, which is a novel substance extracted and isolated from the natural plant agarwood.

[0015] Secondly, the present invention provides a method for preparing the flavonoids in agarwood, comprising the following steps:

[0016] The stem bark of natural agarwood is crushed and placed in a round-bottom flask. Then anhydrous ethanol (99.5% ethanol solution) is added and refluxed at 50-80℃ for 4-5 hours. The mixture is filtered while hot, the filtrate is collected, the solvent is distilled under reduced pressure, and the mixture is dried under vacuum to obtain agarwood extract.

[0017] Agarwood extract was dissolved in anhydrous methanol (99.5% methanol solution) as the mother liquor, and separated and identified by liquid chromatography to obtain 6 components. Each component was tested for alkaline phosphatase (ALP) in bone marrow mesenchymal stem cells, and the component with the highest bioactivity was screened and denoted as CX-3.

[0018] Component CX-3 was dissolved in anhydrous methanol as CX-3 mother liquor. After separation and purification by semi-preparative liquid chromatography, 19 components were obtained. Component 3 was obtained by natural evaporation of solvent to crystallize and then recrystallize (hereinafter referred to as CX-3-3). Alkaline phosphatase (ALP) of bone marrow mesenchymal stem cells was detected. Among all components, CX-3-3 had the highest biological activity.

[0019] The ethanol extract of agarwood was separated into 6 components by AKTA primeplus liquid chromatography with a Spherical C18 column of 20-35 μm and 100 Å. CX-3 was separated and purified into 19 components by Waters 2545 semi-preparative liquid chromatography with Positisil ODS-P column of 5 μm and 100 Å. The third component was naturally crystallized, and the precipitated part was recrystallized.

[0020] Comprehensive and in-depth structural analysis using mass spectrometry, nuclear magnetic resonance (NMR), and X-ray diffraction (XRD) techniques confirmed that CX-3-3 is a novel compound obtained in this invention, intended for use in treating osteoporosis. This invention demonstrates a good therapeutic effect on osteoporosis.

[0021] Thirdly, the present invention provides the application of the flavonoids in the above-mentioned agarwood in the preparation of a medicine for the prevention and treatment of osteoporosis.

[0022] The compound was used to detect its induction effect on bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts in vitro. The experiment showed that it can affect osteoclast formation, demonstrating that it has anti-osteoporosis activity and can be used to prepare drugs for treating osteoporosis.

[0023] Beneficial effects:

[0024] This invention extracts and isolates a novel flavonoid compound from the natural plant agarwood. Its induction effect on osteoblast differentiation of bone marrow mesenchymal stem cells (BMSCs) was investigated in vitro, and its influence on osteoblast differentiation of rat bone marrow mesenchymal stem cells was studied to explore its application value in the treatment and prevention of osteoporosis. The efficacy of this natural plant extract in treating osteoporosis was preliminarily determined. The anti-osteoporosis activity of the compound was detected in vitro using BMSCs, establishing the material basis for the treatment of osteoporosis by this natural plant extract, providing a new approach for the prevention and treatment of osteoporosis.

[0025] The present invention has been described in detail above; however, the above embodiments are merely illustrative in nature and are not intended to limit the invention. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following embodiments. Attached Figure Description

[0026] Figure 1 Chromatograms of the six components separated from agarwood by medium- and low-pressure liquid chromatography;

[0027] Figure 2 The chromatograms are of the 19 components of component CX-3 separated by semi-preparative liquid chromatography.

[0028] Figure 3 The H-NMR spectrum of CX-3-3;

[0029] Figure 4 The mass spectrum of compound CX-3-3 is shown below.

[0030] Figure 5 The XRD results for compound CX-3-3 are shown below.

[0031] Figure 6 Image showing the morphological results of rat bone marrow mesenchymal stem cells (BMSCs);

[0032] Figure 7 ALP staining pattern for identifying osteogenic differentiation of BMSCs;

[0033] Figure 8 Alizarin red staining image for identifying osteogenic differentiation of BMSCs;

[0034] Figure 9 The results show the mRNA expression of osteogenic-related genes after 24 h, 48 h and 96 h of osteogenic induction culture of BMSCs by compound CX-3-3.

[0035] Figure 10 The results show the expression of osteogenic-related proteins in BMSCs after 48 h and 96 h of osteogenic induction culture by compound CX-3-3.

[0036] Figure 11 Results of gene expression levels of alp, bglap, ocn, runx2a, and sp7 in zebrafish sample CX-3-3 exposed from 3 dpf to 9 dpf. Detailed Implementation

[0037] The present invention will be further described below with reference to the embodiments. It should be noted that the following embodiments are provided for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention.

[0038] Unless otherwise specified, the raw materials, reagents, and methods used in the embodiments are all conventional raw materials, reagents, and methods in the art.

[0039] Example 1

[0040] 100 g of agarwood powder (from Hainan, purchased from Bozhou Guisikang Pharmaceutical Co., Ltd.) was accurately weighed and placed in a 500 mL round-bottom flask. 300 mL of 99.5% anhydrous ethanol was then added, and the mixture was refluxed at 70 °C for 4 h. The mixture was filtered while hot, and the filtrate was collected. The solvent was distilled under reduced pressure, and the mixture was dried under vacuum to obtain 5.76 g of agarwood extract.

[0041] Dissolve 5.76g of agarwood extract in 100mL of 99.5% anhydrous methanol to obtain the mother liquor;

[0042] The mother liquor was separated and identified by medium-low pressure liquid chromatography (GE AKTA primeplus). The chromatographic conditions were as follows: column: Spherical C18 (4.6 mm × 250.0 mm, 20–35 µm, 100 Å); mobile phase: methanol (A)-water (B); gradient elution: 0–200 min, 40%–100% methanol; 200.1–340 min, 100% methanol; flow rate: 3 mL / min. -1 Column temperature: 25℃; UV detection: 200–360 nm; injection volume: 2 mL. Chromatogram as shown. Figure 1 As shown, there are 6 characteristic peaks, namely component CX-1 (60-80 min), component CX-2 (205-215 min), component CX-3 (225-240 min), component CX-4 (242-260 min), component CX-5 (260-267 min), and component CX-6 (267-275 min). The samples of the above components were collected, concentrated and dried, and the 6 components were successfully separated.

[0043] The obtained components CX-1, CX-2, CX-3, CX-4, CX-5, and CX-6 were used to detect alkaline phosphatase (ALP) activity in bone marrow mesenchymal stem cells (BMSCs). The specific detection method is as follows: BMSCs were seeded in 12-well plates and cultured until 90% confluence. Osteogenic induction was then performed according to the experimental groups. The activity of ALP, an early bone formation marker, typically reaches its peak after 9 days. After drug treatment, ALP activity was measured according to the operating instructions of the Nanjing Jiancheng alkaline phosphatase (ALP) test kit.

[0044] (1) Set up blank tubes, standard tubes, and test tubes, and add triple-distilled water, 0.1 mg / mL phenol standard solution, and the osteogenic induction culture medium to be tested (containing 10 mg / mL phenol) to each tube. -7mol / L dexamethasone, 10 -2 Samples from various concentration groups after treatment with L-DMEM medium containing mol / L β-glycerophosphate, 50 μg / ml vitamin C, and 10% fetal bovine serum;

[0045] (2) Add buffer solution and matrix solution to each tube, mix thoroughly, and then incubate in a water bath at 37°C for 15 min;

[0046] (3) After incubation, add the colorimetric reagent, mix immediately, and transfer 200 μl into a 96-well plate;

[0047] (4) Detect the absorbance value at 520 nm using an ELISA reader;

[0048] (5) Calculate ALP activity according to the formula:

[0049]

[0050] C 标准 Phenol standard solution concentration: 0.1 mg / mL;

[0051] Cpr: Sample protein concentration, gprot / mL (prot refers to protein).

[0052] The results are shown in Table 1:

[0053] Table 1

[0054]

[0055] It is evident that component CX-3 exhibits the highest activity, and component CX-3 was selected for further separation and purification.

[0056] Example 2

[0057] 1 g of component CX-3 was dissolved in 10 mL of anhydrous methanol to obtain the CX-3 mother liquor. The CX-3 mother liquor was purified by semi-preparative liquid chromatography (Waters 2545) under the following chromatographic conditions: Positisil ODS-P (20 mm × 250 mm, 5 µm) column; mobile phase: methanol (A) - aqueous solution (B); elution program: 0–6 min, 10% A; 7–30 min, 10%–50% A; 31–45 min, 50%–95% A; flow rate: 5 mL / min. -1 The column temperature was 30℃; the detection wavelength was 254 nm, and 19 components were finally obtained, such as... Figure 2 As shown, in order are (Named CX-3-1 to CX-3-19).

[0058] By collecting and accumulating multiple batches of the same component, samples of components CX-3-1 to CX-3-19 can be prepared.

[0059] Nine fractions (CX-3-2 to CX-3-10) with relatively high content were isolated and subjected to alkaline phosphatase (ALP) activity assay in bone marrow mesenchymal stem cells (as described above). The results are shown in Table 2.

[0060] Table 2

[0061]

[0062] CX-3-3 showed the highest activity.

[0063] Example 3 CX-3-3 Structure Identification

[0064] A comprehensive and in-depth structural analysis was conducted using mass spectrometry, nuclear magnetic resonance (NMR), and X-ray diffraction (XRD) techniques. Figure 2 , Figure 3 ):

[0065] Compound CX-3-3, (E)-2-(4-hydroxystyryl)-6,7-dimethoxychroman-4-one, is a yellow crystal. Its molecular formula was derived as C1 by TOF-ESI-MS (m / z 347.0899 [mH]). 19 H 18 O5 has an unsaturation degree of 9. 1 H-NMR showed: 1 ¹H NMR (600 MHz, MeOD) δ 7.58 (s, 1H), 7.55 (s, 1H), 7.33 (s, 1H), 6.89 (s, 1H), 6.88 (s, 1H), 6.79 (s, 1H), 6.41 (s, 1H), 6.38 (s, 1H), 6.35 (d, J = 12.4 Hz, 1H), 3.99 (d, J = 11.7 Hz, 6H). XRD single-crystal diffraction data indicate that the CX-3-3 compound is a chiral flavonoid.

[0066] CX-3-3 is a novel compound obtained in this invention. The above compounds include those with different stereoconfigurations. The structure of the new compound is shown below. Figure 5 .

[0067] Experimental Example: In vivo bioactivity experiment of compound CX-3-3

[0068] 1. Identification of rat bone marrow mesenchymal stem cells (BMSCs)

[0069] (1) Cell morphology observation

[0070] Experimental methods: The growth of primary and passaged cells was observed and photographed under an inverted biological microscope, recording the morphology, adhesion, density, and confluence. Results are as follows: Figure 6 As shown, the left image is 48 hours after primary inoculation, and the right image is 72 hours after primary inoculation.

[0071] (2) Detection of CD molecules, surface markers of BMSCs

[0072] Experimental methods: BMSCs were extracted, isolated, and cultured using the whole bone marrow adherent culture method. Cell morphology was observed, and cells in good growth condition (90% confluence) at passage 4 were prepared into single-cell suspensions using the passage method. Cells were then cultured at 3 × 10⁶ cells per tube. 5 Cells were aliquoted into 6 flow cytometry tubes, centrifuged, and the supernatant was discarded. The cells were rinsed twice and resuspended in 100 μL buffer. Then, 2 μL of primary antibodies against CD90, CD44, CD73, CD45, CD34, and CD11b / c were added, and the mixture was thoroughly mixed. The cells were incubated at 4°C in the dark for 30 min. After centrifugation, the supernatant was discarded, and the cells were rinsed twice. The cells were resuspended in 100 μL buffer, and 2 μL of FITC-labeled fluorescent secondary antibodies were added. The mixture was thoroughly mixed and incubated at 4°C in the dark for 30 min. After centrifugation, the supernatant was discarded, and the cells were rinsed twice. The cells were resuspended in 400 μL buffer and then analyzed by flow cytometry.

[0073] Experimental results: Flow cytometry was used to detect CD molecules on the surface of fourth-generation cells. The results showed high expression of mesenchymal stem cell surface markers CD90, CD44, and CD73, and low expression of hematopoietic stem cell surface markers CD45, CD34, and CD11b / c. The cells also showed good adipogenic and osteogenic differentiation potential, indicating that the cultured cells were BMSCs.

[0074] (3) Identification of osteogenic differentiation of BMSCs

[0075] Experimental methods: Primary cells were selected for osteogenic differentiation induction. After the primary cells reached about 90% confluence, they were added to classic osteogenic induction medium. The medium was changed every 3 days. On the 9th day of osteogenic induction of BMSCs, ALP staining was performed to observe the ALP staining status. On the 12th day of osteogenic induction of BMSCs, Alizarin Red staining was performed to observe the formation of mineralized nodules.

[0076] Experimental results: ALP staining, observed under an inverted microscope, revealed that the blue-stained cells gradually formed concentric nodules of ALP-positive cells, as shown in the image. Figure 7 As shown; on day 12 of osteogenic induction, primary BMSCs were observed under an inverted microscope, where mineral salt particles accumulated to form visible white patches (×20). Figure 8After staining with alizarin red, red calcified nodule deposits were observed under a microscope (×4, Figure 8 ).

[0077] 2. Screening of the optimal concentration of agarwood-induced osteogenic BMSCs

[0078] Experimental Methods: BMSCs were seeded in 12-well plates and cultured to 90% confluence. Osteogenic induction was then performed according to experimental groups. After drug treatment, ALP activity was measured according to the Nanjing Jiancheng ALP assay kit instructions. Different concentrations of the agarwood compound CX-3-3 (3×10⁻⁶) were compared. -2 mol / L ~ 3×10 -8 The effect of agarwood (mg / mL) on ALP activity after 9 days of osteogenic induction culture of BMSCs was investigated to determine the optimal osteogenic induction concentration for subsequent experiments.

[0079] Experimental results: show that 3×10 -6 The agarwood compound CX-3-3 at a concentration of mg / mL exhibited the best activity in promoting osteogenic differentiation of BMSCs. The results are shown in Table 1 above.

[0080] 3. RT-qPCR detection of the effect of agarwood compound CX-3-3 on osteogenic-related gene expression in BMSCs

[0081] Experimental Methods: After 90% cell fusion of BMSCs, osteogenic induction was induced by culturing them at the optimal concentration of the agarwood compound CX-3-3 selected above. Treatment times were set at 24h, 48h, and 96h, with a blank control group. After the agarwood intervention, total RNA was extracted using the TRIzol one-step method. The reverse transcription system, RT-qPCR amplification system, and reaction conditions were all set according to the manufacturer's instructions. The effects on the mRNA expression of osteogenic-related genes (Collagen1, RUNX2, BMP-2) were detected.

[0082] Experimental results: After CX-3-3 treatment of BMSCs for 24h, 48h, and 96h, the mRNA expression levels of Collagen1, RUNX2, and BMP-2 in the agarwood group were significantly higher than those in the control group. The results are as follows: Figure 9 As shown (CON refers to the blank group without agarwood).

[0083] 4. Western blotting to detect the effect of agarwood compound CX-3-3 on osteogenic-related protein expression in bone marrow mesenchymal stem cells (BMSCs)

[0084] Experimental Methods: After 90% confluence of BMSCs, osteogenic induction was induced by culturing them at the optimal concentration of the agarwood compound CX-3-3 selected above. Treatment times were set at 48 h and 96 h, with a blank control group. After the drug intervention, total protein was extracted, and the effects of agarwood on the expression of osteogenic-related proteins (Collagen1, RUNX2, BMP-2) were detected using the BCA protein quantification method to determine the influence of agarwood on the osteogenic differentiation of BMSCs.

[0085] Experimental results: After CX-3-3 treatment of BMSCs for 48 h and 96 h, the expression levels of Collagen1, RUNX2, and BMP-2 proteins in the agarwood group were significantly higher than those in the control group. The results are as follows: Figure 10 As shown (CON refers to the blank group without agarwood).

[0086] 5. RT-qPCR detection of the effect of agarwood compound CX-3-3 on the expression level of genes related to osteophyte development in zebrafish.

[0087] Experimental Methods: Zebrafish with uniform development at 3 days post-flop (dpf) were randomly selected and cultured in petri dishes. The control group was cultured normally in E3 culture, while the model group was exposed to 10 μM dexamethasone. The positive control group was co-treated with 20 μg / mL alendronate sodium tablets in addition to the modeling treatment. The model + sample group was co-treated with different concentrations of the agarwood compound CX-3-3 in addition to the modeling treatment. The culture medium was changed daily. Paramecium was introduced at 5 dpf, and Paramecium plus brine shrimp was introduced at 7 dpf. Treatment was completed at 9 dpf. After treatment, 30 fish from each group were randomly selected, homogenized, and RNA was extracted using a kit. cDNA was obtained by reverse transcription, and the expression levels of the alp, sp7, runx2a, bglap, and ocn genes were quantitatively detected.

[0088] Experimental Results: To investigate whether agarwood affects zebrafish bone development, a model was established in juvenile fish, and they were exposed to different concentrations of CX-3-3 at 9 dpf. The expression levels of related genes alp, sp7, runx2a, bglap, and ocn were detected by quantitative real-time qPCR. Compared with the control group, alp (see...) showed significantly higher expression levels. Figure 11 (A), bglap (see) Figure 11 (Chinese B), ocn (see) Figure 11 (in C), runx2a (see...) Figure 11 (middle D), sp7 (see) Figure 11 The expression levels of all five genes in the model group (E) showed an upregulation trend; compared with the model group, the expression levels of five genes (alp, sp7, runx2a, bglap, and ocn) in the sample group also showed an upregulation trend; one-way ANOVA analysis was used to determine the statistical differences. This means P < 0.0001.

[0089] Summary: The above experimental results indicate that BMSCs were successfully isolated and cultured, and 3×10⁶ BMSCs were screened. -6 mg / mL of agarwood compound CX-3-3 is the optimal drug concentration for inducing osteogenic differentiation of bone mesenchymal stem cells (BMSCs); CX-3-3 can promote the osteogenic activity of BMSCs; CX-3-3 can promote zebrafish bone development.

[0090] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and substance defined by the claims of the present invention; and such modifications or substitutions are still within the scope defined by the claims of the present invention.

Claims

1. The use of flavonoids in the preparation of a drug for preventing and treating osteoporosis by inducing the osteogenic differentiation of BMSCs. The Chinese name of the flavonoid compound is: 2-[(1 E )-2-(4-hydroxyphenyl)vinyl]-6,7-dimethoxy-4 H -chromen-4-one, having the structure shown in formula (I): Formula (I); The concentration of the flavonoid compound is 3 x 10 -6 mg / mL.

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