Application of flatstem milkvetch seed glycoside in preparation of medicine for preventing and treating osteoporosis

Abstract

The invention belongs to the technical field of medicine, and particularly relates to application of flatstem milkvetch seed glycoside in preparation of medicine for preventing and treating osteoporosis, and the flatstem milkvetch seed glycoside can promote osteogenic differentiation of MC3T3-E1 cells and has a protection effect on osteogenic ability damage of H2O2 to the MC3T3-E1 cells. The invention shows that the flatstem milkvetch seed glycoside can promote the expression of osteogenesis-related protein and the formation of mineralized nodules of the MC3T3-E1 cell, and has the prospect of preparing the anti-osteoporosis medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to an application of complanatus glycoside in the preparation of a drug for preventing and treating osteoporosis. Background Art

[0002] Osteoporosis (OP) is a chronic disease characterized by decreased bone mass and density and destruction of bone microstructure. The resulting brittle fractures are the main cause of disability and disuse in the affected population. OP has become one of the important factors affecting the health of people over 50 years old in my country, especially postmenopausal women. Affected by the continuous deepening of the aging population, the high incidence, increased mortality and significantly reduced quality of life of OP have become an important public health issue. It is very important to clarify its pathogenesis and seek effective prevention and treatment methods.

[0003] In the research on the prevention and treatment of OP, traditional Chinese medicine has significant advantages in terms of efficacy, low toxicity and side effects, and overall regulation. The basic theory of traditional Chinese medicine believes that the kidney is the foundation of congenitality, responsible for bone and marrow production, and regulates the growth and development of the body and all life activities. Traditional Chinese medicine has formed a unique "kidney-dominated bone" theory, which not only clarifies the core pathogenesis of osteoporosis kidney deficiency syndrome, but also forms a core system for the prevention and treatment of OP based on kidney deficiency syndrome-kidney tonification method. Therefore, the use of kidney-tonifying Chinese medicine is particularly important in the prevention and treatment of OP. Kidney-tonifying Chinese medicine and its active ingredients also play a significant role in regulating the process of bone metabolism. For example, icariin, total glycosides of Cistanche deserticola, polysaccharides of Cistanche deserticola, icariin, and radix dipsaponins have all been reported to promote bone formation or inhibit bone resorption.

[0004] Complanatoside is a flavonoid compound found in the traditional Chinese medicine Astragalus complanatus R.Br. It has a sweet taste, warm nature, and enters the liver and kidney meridians. It has the effects of nourishing the kidney and yang, consolidating essence and reducing urine, and nourishing the liver and improving eyesight. 28 H 32 O 16 , with a molecular weight of 624.54. There is no report on the effect of Astragalus complanatus glycoside on promoting bone formation in bone metabolism.

[0005] Compasoside B is one of the main flavonoid components in Astragalus compascens, and has a similar structure to Compasoside B. The chemical formula of Compasoside B is C 33 H 40 O 20 , molecular weight 756.66, is one of the most abundant components in the total flavonoids of Astragalus membranaceus. Summary of the invention

[0006] In order to enrich the selection of anti-osteoporosis therapeutic drugs and explore more medicinal values ​​of Compasoside, the present invention mainly provides an application of Compasoside in the preparation of drugs for promoting osteogenic differentiation of osteoblast precursor cells and preventing and treating osteoporosis:

[0007] The invention discloses an application of complanatus glycoside in the preparation of a drug for promoting osteogenic differentiation of osteoblast precursor cells, thereby up-regulating the expression of osteoblast-related proteins and mRNA in osteoblast precursor cells.

[0008] Furthermore, the activity of alkaline phosphatase in osteoblast precursor cells was increased.

[0009] Furthermore, the cell activity of osteoblast precursor cells in the concentration range of 1-100 μmol / L of complanatus glycoside is greater than 70%; the cell activity of osteoblast precursor cells in the concentration of complanatus glycoside below 60 μmol / L is greater than 80%.

[0010] The invention discloses an application of complanatus glycoside in preparing a culture material for promoting osteogenic differentiation of osteoblast precursor cells in vitro, wherein the culture material is a culture medium or a culture fluid.

[0011] Furthermore, the culture medium or culture solution contains 5-10 μmol / L of complanatus glycoside.

[0012] Furthermore, the culture medium or culture solution includes vitamin C and 10 mM sodium β-glycerophosphate.

[0013] The invention discloses an application of complanatus glycoside in the preparation of a medicine for inhibiting hydrogen peroxide damage to osteoblast precursor cells, and inhibits 1-50% peroxidation damage to alkaline phosphatase.

[0014] The invention discloses an application of complanatus glycoside in preparing a medicine for preventing and treating osteoporosis.

[0015] Furthermore, the drug for preventing and treating osteoporosis has the effect of increasing bone density, the number and thickness of trabeculae.

[0016] Furthermore, the dosage form of the drug is an oral preparation or an injection; the dosage form of the oral preparation is tablets, pills, oral liquid, capsules, syrups, pellets or granules; the injection includes injectable powder or injection solution.

[0017] By adopting the above scheme, the method of the present invention has the following advantages:

[0018] 1. Astragalus complanatus glycosides can promote the osteogenic differentiation of MC3T3-E1 cells, the expression of ALP, BMP-2, COL1, and RUNX2 mRNA, and the expression of ALP, BMP-2, and COL1 proteins in MC3T3-E1 cells. 2O 2 Complanatus glycosides pretreatment protected MC3T3-E1 cells from H 2 O 2 The bone formation ability is decreased due to the injury. Therefore, complanatus glycoside can be used to prepare a culture medium for promoting the osteogenic differentiation of MC3T3-E1 cells in vitro, and has the prospect of being developed into a drug for improving osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the effect of complanatus glycoside intervention on ALP activity. Among them, * indicates P < 0.05 relative to the blank group.

[0020] Figure 2 This is a comparison chart of the effects of complanatus glycosides on the formation of mineralized nodules in MC3T3-E1 cells.

[0021] Figure 3 This is a comparison of the effects of complanatus glycosides on the expression of ALP, BMP-2, and COL1 proteins in MC3T3-E1 cells. Compared with the blank group, * indicates P < 0.05.

[0022] Figure 4 Is the effect of complanatine on MC3T3-E1 cells 2 O 2 Comparison chart of the effects of damage.

[0023] Figure 5 The figure is the femoral bone density test results of each group; wherein, relative to the blank group, a means P < 0.05; relative to the model group, b means P < 0.05.

[0024] Figure 6 This is the observation result of the distal femur under HE staining microscope (×40).

[0025] Figure 7 This is a comparison of the cytotoxicity of complanatus glycoside and complanatus glycoside B on MC3T3-E1 cells. Among them, a means that the complanatus glycoside is compared with the blank group, P < 0.05; b means that the complanatus glycoside B is compared with the blank group, P < 0.05; c means that the complanatus glycoside B is compared with the complanatus glycoside group, P < 0.05.

[0026] Figure 8 This is a comparison of the effects of complanatus glycoside and complanatus glycoside B on the expression of osteogenic-related mRNA in MC3T3-E1 cells, where * indicates P < 0.05 relative to the blank group. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Effect of Astragalus complanatus glycoside on the activity of alkaline phosphatase (ALP) in MC3T3-E1 cells: MC3T3-E1 cells were cultured at 2×10 5 Cells were inoculated in 12-well cell culture plates and divided into blank group and different concentrations of scutellaria baicalensis glycoside intervention group, with 1 mL of cell suspension in each well. After the cells adhered to the wall, different intervention measures were given. Scutellaria baicalensis glycoside was dissolved in DMSO solution to prepare a 10 mmol / L concentration mother solution, which was diluted into different concentrations of scutellaria baicalensis glycoside (1 μmol / L, 5 μmol / L, 10 μmol / L) containing medium, 1 mL per well, and after 72 hours of culture, the ALP content of the cell supernatant was determined according to the instructions of the ALP kit.

[0029] Depend on Figure 1 It can be seen that compared with the blank group, the activity of ALP in the supernatant of MC3T3-E1 cells was significantly increased after 72 hours of intervention with 1μmol / L, 5μmol / L, and 10μmol / L of complanatus glycosides.

[0030] Alizarin red staining was used to observe the effect of chayuanzi glycoside on the formation of mineralized nodules in MC3T3-E1 cells: MC3T3-E1 cells were inoculated in 12-well cell culture plates, with 2 mL of cell suspension per well. After the cells adhered to the wall and the cell density reached 90%-100%, they were divided into a blank group, an osteogenic induction group, and a low (1 μmol / L), medium (5 μmol / L), and high (10 μmol / L) dose group of chayuanzi glycoside. The blank group was α-MEM culture medium, the osteogenic induction group was α-MEM cell culture medium containing 50 μg / ml vitamin C and 10 mM β-sodium glycerophosphate, and the intervention group was a culture medium containing different concentrations of chayuanzi glycoside in the osteogenic induction medium, 1 mL per well, and cultured continuously for 18-21 days, with the medium changed every 3 days. After the mineralized nodules were formed, they were fixed and stained with Alizarin Red S, and the formation of mineralized nodules was observed under a microscope and photographed.

[0031] The results are as follows Figure 2 As shown, compared with the osteogenic induction group, 1μmol / L, 5μmol / L, and 10μmol / L concentrations of complanatus glycosides all promoted the formation of mineralized nodules of MC3T3-E1 cells, and the intervention effect was better than that of osteogenic induction.

[0032] WB detection of the effect of complanatus glycoside on the expression of ALP, BMP-2 and COL1 proteins in MC3T3-E1 cells: MC3T3-E1 cells were seeded in a 6-well cell culture plate. When the cells were attached to the wall and the density was 80-90%, they were divided into a blank group and complanatus glycoside (1μmol / L, 5μmol / L, 10μmol / L) intervention groups. After 72 hours of treatment, the total protein was extracted, and the effect of complanatus glycoside on the protein levels of ALP, BMP-2 and COL1 in MC3T3-E1 cells was detected by Western blot.

[0033] Results from Figure 3 As shown, compared with the blank group, the intervention of 1μmol / L, 5μmol / L, and 10μmol / L of complanatus glycosides could promote the upregulation of ALP, BMP-2, and COL1 protein expressions in MC3T3-E1 cells (P<0.05).

[0034] Complanatus glycosides 2 O 2 Effect of damage on alkaline phosphatase expression in MC3T3-E1 cells: 1x10 cells per well 4 The cells were seeded at a density of 100 μg / ml on a 12-well plate and grouped as before at 37°C and 5% CO. 2 When the cells grew to a density of about 80%, low, medium, and high concentrations of glycosides were added for intervention for 24 h, and then the culture medium was discarded. The model group and the glycosides intervention group were treated with 400 μmol / L H 2 O 2 Injury for 4 hours. After the injury, wash the 12-well plate with PBS solution 3 times and remove the liquid, fix it with 4% paraformaldehyde for 15 minutes, wash it with PBS 3 times, add each reagent in sequence according to the proportion in the table below to prepare the alkaline phosphatase staining working solution, add 500mL of working solution to each well to cover the cells, incubate at room temperature in the dark for 30 minutes, observe the staining effect, wash it with PBS 3 times, and then discard the waste liquid. Observe the cells under an optical microscope. Positive cells are purple. Adjust to the visible range and take pictures.

[0035]

[0036] The results are as follows Figure 4 Compared with the blank group, H 2 O 2 The ALP-positive area of ​​MC3T3-E1 cells in the model group after injury was significantly reduced, while the ALP expression levels in the groups pretreated with various concentrations of complanatus glycosides were significantly higher than those in the model group, indicating that complanatus glycosides can reduce H 2 O 2 Alkaline phosphatase damage to MC3T3-E1 cells.

[0037] Effect of Compasoside on Bone Mineral Density and Bone Microarchitecture in Ovariectomized Rats: Thirty 3-month-old SPF female SD rats (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal license number: SCXK (Guangdong) 2022-0063), weighing 220±20g, were randomly divided into sham operation group, ovariectomy model group (model group), and ovariectomy + Compasoside group (Compasoside group) by random number table method after one week of adaptive feeding. After anesthesia, the rats in the model group and Compasoside group underwent bilateral ovariectomy, and the sham operation group only removed the same size of fat around the ovaries and retained the bilateral ovaries.

[0038] Modeling method: ① Preoperative anesthesia: fasting and water deprivation for 12 hours before surgery, 2% sodium pentobarbital was injected intraperitoneally at 0.2mL / 100g body weight; ② Skin preparation: fixed on the operating board in the supine position, shaved the hair in the surgical laparotomy area in the middle of the lower abdomen; ③ Disinfection: disinfected with iodine tincture and deiodinated with alcohol in sequence; ④ Modeling of the model group: a 2-3cm longitudinal incision was made along the linea alba of the rat abdomen, the abdominal cavity was opened, the tissues were separated layer by layer, the bilateral ovaries were exposed, and the connection between them and the fallopian tubes was ligated with surgical sutures. After the ovaries were removed, the peritoneum, muscle layer, and cortex were sutured layer by layer to close the abdomen; ⑤ Modeling of the sham operation group: after the above laparotomy, a small amount of fat around the ovaries was removed, and the rest was the same as the model group; ⑥ Prevention of infection: 75% medical alcohol was used to disinfect the surgical area and 1ml / rat of penicillin was injected intraperitoneally after surgery; ⑦ Postoperative observation: the rats were placed in a mouse cage after surgery and covered with a towel to prevent hypothermia. The rats were observed for 7 days after surgery. One week after modeling, different drug interventions were performed. Comparative glycosides were administered by gavage at a dose of 15 mg / kg / d using sterile distilled water as solvent, and the sham operation group and the model group were gavaged with the same amount of distilled water for 12 weeks. During the feeding period, the rats were free to eat, the light was rotated for 12 hours, the ventilation was good, and the bedding was changed 2 to 3 times a week.

[0039] After 12 weeks, the femoral DAX bone density of rats in each group was measured. The rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital at 0.2 mL / 100 g body weight, and then fixed on the testing bed with their limbs abducted and their backs facing upwards for testing. Figure 5 As shown in the data, compared with the sham operation group, the femoral bone density of the rats in the model group was significantly reduced. After intervention with complanatus glycosides, the femoral bone density of the model group was significantly increased, with statistically significant differences (P < 0.05).

[0040] HE staining was used to observe the pathological changes of the femoral structure of rats in each group: HE staining method was as follows: 1) Dewaxing: the femoral slices were immersed in xylene I (20min) and II (20min) in turn; 2) Hydration: the dewaxed slices were immersed in anhydrous ethanol I (5min) and II (5min) in turn, and then treated with 95% (1min), 80% (1min), and 70% (5min) ethanol in turn, and washed with distilled water for 1min; 3) Staining: hematoxylin staining slices for 3-5min, rinsed with running water for 1min; blueing with bluing solution, rinsed with distilled water for 1min; dehydrated with 85% (5min) and 95% (5min) alcohol in turn, eosin staining for 5min, and rinsed with distilled water for 1min; 4) Dehydration: the femoral slices were immersed in anhydrous ethanol I, II, and III for 5min each; 5) Transparency: the femoral slices were immersed in xylene I (5min) and xylene II (5min) in turn; 6) Sealing: neutral gum sealing. The results are as follows Figure 6 As shown, it can be seen that: the trabeculae in the sham operation group were complete in morphology and structure, arranged neatly and compactly, the trabeculae were of uniform thickness, and abundant bone marrow cells were visible in the bone marrow cavity; the cancellous bone structure in the model group was destroyed, the trabeculae were sparse, and compared with the sham operation group, the trabecular spacing in this group was significantly increased and the free ends increased, a large number of local gaps were formed in the bone marrow cavity, and the vacuolar fat cells in the bone marrow increased significantly; the number of trabeculae in the complanatus glycoside group was increased compared with the model group, the reticular structure was improved to a certain extent, and the fat cells in the bone marrow cavity were relatively small.

[0041] Comparative Example: Comparative experiment was conducted using Comparative glycoside B, which has a similar structure to Comparative glycoside, and Comparative glycoside:

[0042] Detection of cytotoxicity of chayuanzi glycoside on MC3T3-E1 cells and comparison with cytotoxicity of chayuanzi glycoside B: MC3T3-E1 cells were cultured at 5×10 3 / mL cells were inoculated in a 96-well cell culture plate, and 5 replicate wells were set. After the cells adhered to the wall, they were divided into a blank group and an intervention group. The blank group was intervened with ordinary α-MEM culture medium, and the intervention group was added with different concentrations of scutellaria baicalensis glycoside or scutellaria baicalensis glycoside B, with a concentration gradient of 1μmol / L, 5μmol / L, 10μmol / L, 20μmol / L, 40μmol / L, 60μmol / L, 80μmol / L, and 100μmol / L, with 200μl of culture medium per well. The cells were then placed in a constant temperature cell culture incubator at 37°C and 5% CO2 for 72h. Then 10μl of CCK-8 solution was added to each well, and the culture was continued for 1-1.5h. The OD value at a detection wavelength of 450nm was measured with an enzyme marker to measure the cell activity in different groups.

[0043] Figure 7The results showed that the inhibitory effects of 0.1-100 μmol / L concentrations of comfrey glycoside and comfrey glycoside B on MC3T3-E1 cell viability were significantly higher than those of the blank control group (P < 0.05). The strong cytotoxicity makes comfrey glycoside B unsuitable for the preparation of anti-osteoporosis drugs, while comfrey glycoside is significantly more suitable.

[0044] Comparison of the effects of complanatus glycoside and complanatus glycoside B on the expression of BMP-2 and COL1 mRNA in osteoblasts of MC3T3-E1 cells: MC3T3-E1 cells were seeded in a 6-well cell culture plate. When the cell density was 80-90%, a blank group, a complanatus glycoside 10 μmol / L group, and a complanatus glycoside B 10 μmol / L group were set up and given different interventions. After 48 hours of culture, total RNA was extracted to detect the expression of BMP-2 and COL1 mRNA, and GAPDH was used as an internal reference control.

[0045] Figure 8 As shown in the data, compared with the blank group, 10 μmol / L complanatus glycoside and complanatus glycoside B could promote the up-regulation of BMP-2 and COL1 mRNA in MC3T3-E1 cells (P < 0.05). The expression of mRNA in the complanatus glycoside group was up-regulated by about one-fold, while the ability of the complanatus glycoside B group to promote the expression of BMP-2 and COL1 mRNA was significantly weaker than that of the complanatus glycoside group, and the ability to promote osteogenesis of MC3T3-E1 cells was even worse.

[0046] For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all of these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A use of complanatus glycoside in the preparation of a drug for promoting osteogenic differentiation of osteoblast precursor cells, characterized in that: The expression of osteoblast-related proteins and mRNA in osteoblast precursor cells is upregulated.

2. The use of complanatus glycoside according to claim 1 in the preparation of a drug for promoting osteogenic differentiation of osteoblast precursor cells, characterized in that: Increases the activity of alkaline phosphatase in osteoblast precursor cells.

3. The use of complanatus glycoside according to claim 1 in the preparation of a drug for promoting osteogenic differentiation of osteoblast precursor cells, characterized in that: The cell activity of osteoblast precursor cells in the concentration range of 1-100 μmol / L of complanatus glycoside is greater than 70%; the cell activity of osteoblast precursor cells in the concentration of complanatus glycoside below 60 μmol / L is greater than 80%.

4. A use of complanatus glycoside in the preparation of a culture material for promoting osteogenic differentiation of osteoblast precursor cells in vitro, characterized in that: The culture material is a culture medium or a culture solution.

5. The use of complanatus glycoside according to claim 4 in the preparation of a culture material for promoting osteogenic differentiation of osteoblast precursor cells in vitro, characterized in that: The culture medium or culture solution contains 5-10 μmol / L of complanatine.

6. The use of complanatus glycoside according to claim 4 in the preparation of a culture material for promoting osteogenic differentiation of osteoblast precursor cells in vitro, characterized in that: The culture medium or culture solution includes vitamin C and 10 mM sodium β-glycerophosphate.

7. Use of complanatus glycoside in the preparation of a drug for inhibiting hydrogen peroxide damage to osteoblast precursor cells, characterized in that: Inhibits 1-50% peroxidation damage to alkaline phosphatase.

8. Use of Compasaena glycoside in the preparation of drugs for preventing and treating osteoporosis.

9. The use of complanatus glycoside according to claim 8 in the preparation of a drug for preventing and treating osteoporosis, characterized in that: The medicine for preventing and treating osteoporosis has the effect of increasing bone density, the number and thickness of trabeculae.

10. The use of complanatus glycoside according to claim 8 in the preparation of a drug for preventing and treating osteoporosis, characterized in that: The dosage form of the drug is an oral preparation or an injection; the dosage form of the oral preparation is tablets, pills, oral liquid, capsules, syrups, pellets or granules; the injection includes an injection powder or injection solution.

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