Application of complanatus glycoside in the preparation of drugs for preventing and treating osteoporosis

Abstract

The present invention belongs to the field of pharmaceutical technology and specifically relates to the use of complanatus glycoside in the preparation of a drug for the prevention and treatment of osteoporosis. Complanatus glycoside can promote the osteogenic differentiation of MC3T3-E1 cells and has a protective effect on the osteogenic capacity of MC3T3-E1 cells damaged by H2O2. The present invention shows that complanatus glycoside can promote the expression of osteogenic-related proteins and the formation of mineralized nodules in MC3T3-E1 cells, showing the prospect of preparing an anti-osteoporosis drug.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to an application of complanatin 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 disruption of bone microarchitecture. The resulting brittle fractures are a major cause of disability and loss of function in the affected population. OP has become a significant health concern for people aged 50 and older in my country, particularly postmenopausal women. Driven by the continued aging of the population, OP has become a significant public health issue with its high morbidity, increased mortality, and significantly decreased quality of life. Elucidating its pathogenesis and identifying effective preventive and therapeutic strategies are crucial.

[0003] In the prevention and treatment of osteoporosis (OP), Traditional Chinese Medicine (TCM) offers significant advantages in efficacy, low toxicity, and overall regulation. Basic TCM theory holds that the kidneys are the foundation of one's constitution, responsible for bone and marrow production, and regulating growth and development, as well as all vital activities. TCM has developed a unique "kidney-dominated bone" theory, which not only clarifies the core pathogenesis of osteoporosis caused by kidney deficiency but also establishes a core framework for the prevention and treatment of OP based on a kidney deficiency-based approach to tonifying the kidney. Therefore, the use of kidney-tonifying TCM herbs is particularly important in the prevention and treatment of OP. Kidney-tonifying TCM herbs and their active ingredients also play a significant role in regulating bone metabolism. For example, icariin, total glycosides from Cistanche deserticola, polysaccharides from Cistanche deserticola, icariin, and dipsaponins have all been reported to promote bone formation or inhibit bone resorption.

[0004] Complanatoside is a flavonoid compound found in Astragalus complanatus R.Br., a plant of the Leguminosae family. It is sweet in taste, warm in nature, and enters the liver and kidney meridians. It has the effects of tonifying the kidney and promoting yang, consolidating essence and reducing urination, nourishing the liver, and improving eyesight. 28 H 32 O 16 , with a molecular weight of 624.54. Currently, there are no reports on the effect of Astragalus complanatus glycosides on promoting bone formation in bone metabolism.

[0005] Comparisonoside B is one of the main flavonoid components in Astragalus complanatus, and has a similar structure to Comparisonoside. The chemical formula of Comparisonoside 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 complanatus. Summary of the Invention

[0006] To enrich the selection of anti-osteoporosis therapeutic drugs and explore more medicinal values ​​of Compasoside, the present invention mainly provides a use of Compasoside in the preparation of a drug for promoting osteoblast precursor cell osteogenic differentiation and preventing and treating osteoporosis:

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

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

[0009] Furthermore, the cell activity of osteoblast precursor cells in the presence of complanatus glycoside within a concentration range of 1 to 100 μmol / L is greater than 70%; the cell activity of osteoblast precursor cells in the presence of complanatus glycoside at a concentration 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 fluid contains 5-10 μmol / L of complanoside.

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

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

[0014] The invention relates to an application of complanatus glycoside in the preparation of 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. Compasoside can promote the osteogenic differentiation of MC3T3-E1 cells, promote the expression of ALP, BMP-2, COL1, and RUNX2 mRNA, and promote the expression of ALP, BMP-2, and COL1 proteins in MC3T3-E1 cells. When damaged by H2O2, compasoside pretreatment can protect MC3T3-E1 cells from the decreased osteogenic capacity caused by H2O2 damage. Therefore, compasoside can be used to prepare a culture medium for promoting the osteogenic differentiation of MC3T3-E1 cells in vitro and has the potential to be developed into a drug for ameliorating osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the effects of complanatus glycosides on ALP activity. * 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 figure compares the effects of complanatus glycosides on the expression of ALP, BMP-2, and COL1 proteins in MC3T3-E1 cells. * indicates P < 0.05 relative to the blank group.

[0022] Figure 4 This is a comparison chart of the effects of complanatus glycosides on H2O2 damage in MC3T3-E1 cells.

[0023] Figure 5 Figure 2 is a graph showing the femoral bone density test results of each group; relative to the blank group, a represents P < 0.05; relative to the model group, b represents P < 0.05.

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

[0025] Figure 7 The figure shows the comparison of the cytotoxicity of complanatin and complanatin B on MC3T3-E1 cells. Among them, a indicates that the cytotoxicity of complanatin compared with the blank group is P < 0.05; b indicates that the cytotoxicity of complanatin B compared with the blank group is P < 0.05; c indicates that the cytotoxicity of complanatin B compared with the complanatin group is P < 0.05.

[0026] Figure 8 This figure compares the effects of complanoside and complanoside B on the expression of osteogenic-related mRNA in MC3T3-E1 cells. * indicates P < 0.05 relative to the blank group. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0028] Effect of Complanatus scutellariae on the activity of alkaline phosphatase (ALP) in MC3T3-E1 cells: MC3T3-E1 cells were cultured at 2×10 5 Cells were seeded per well in a 12-well cell culture plate and divided into a blank group and a group treated with different concentrations of complanatin. 1 mL of cell suspension was used per well. After cell attachment, different interventions were administered. Complanatin was dissolved in DMSO to prepare a 10 mmol / L stock solution, which was then diluted into medium containing different concentrations of complanatin (1 μmol / L, 5 μmol / L, and 10 μmol / L). 1 mL of the suspension was used per well. After 72 hours of culture, the ALP content in the cell supernatant was measured 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 1 μmol / L, 5 μmol / L, and 10 μmol / L of complanatin.

[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 seeded 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 medium, the osteogenic induction group was α-MEM cell culture medium containing 50 μg / ml vitamin C and 10 mM sodium β-glycerophosphate, and the intervention group was an osteogenic induction medium containing different concentrations of chayuanzi glycoside 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 in the results, compared with the osteogenic induction group, complanatin at concentrations of 1 μmol / L, 5 μmol / L, and 10 μmol / L all promoted the formation of mineralized nodules in 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 6-well cell culture plates. When the cells adhered to the wall and the density was 80-90%, they were divided into a blank group and a complanatus glycoside (1 μmol / L, 5 μmol / L, 10 μmol / L) intervention group. After treatment for 72 h, the total protein was extracted, and the effect of complanatus glycoside on the ALP, BMP-2 and COL1 protein levels of MC3T3-E1 cells was detected by Western blot.

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

[0034] Effect of scutellaria baicalensis glycosides on alkaline phosphatase expression in MC3T3-E1 cells damaged by H2O2: cells were plated at 1x10 4 Cells were seeded at a density of 100 cells / well in 12-well plates. Groups were arranged as before and cultured in a thermostat at 37°C and 5% CO₂. When cells reached a density of approximately 80%, they were treated with low, medium, or high concentrations of complanatin for 24 hours, after which the culture medium was discarded. The model and complanatin-treated groups were injured with 400 μmol / L H₂O₂ for 4 hours. After injury, the 12-well plates were washed three times with PBS, and the liquid was aspirated. After fixation with 4% paraformaldehyde for 15 minutes, the plates were washed three times with PBS. Alkaline phosphatase staining solution was prepared by adding the reagents in the following proportions, with 500 mL of solution added to each well to cover the cells. After incubation at room temperature in the dark for 30 minutes, the cells were observed for staining, washed three times with PBS, and the waste solution discarded. Cells were observed under a light microscope; positive cells appeared purple, and the plate was imaged within the visible range.

[0035]

[0036] The results are as follows Figure 4 The results showed that compared with the blank group, the ALP-positive area of ​​MC3T3-E1 cells in the model group after H2O2 damage was significantly reduced, and the ALP expression levels in the groups pretreated with various concentrations of complanatin were significantly higher than that in the model group, indicating that complanatin can reduce the damage of H2O2 to alkaline phosphatase in MC3T3-E1 cells.

[0037] Effects of Compasoside on Bone Mineral Density and Bone Microarchitecture in Ovariectomized Rats: Thirty three-month-old SPF female SD rats (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., animal license number: SCXK (Yue) 2022-0063) weighing 220±20g were randomly divided into a sham operation group, an ovariectomy model group (model group), and an ovariectomy + Compasoside group (Compasoside group) after one week of adaptive feeding. After anesthesia, the rats in the model group and Compasoside group underwent bilateral ovariectomy. In the sham operation group, only the fat of the same size around the ovaries was removed, while both ovaries were retained.

[0038] Modeling methods: ① Preoperative anesthesia: 12 hours before surgery, fasting and dehydration, followed by intraperitoneal injection of 2% sodium pentobarbital at 0.2 mL / 100 g body weight. ② Skin preparation: The rats were fixed in the supine position on an operating board, and the hair in the midline laparotomy area of ​​the lower abdomen was shaved. ③ Disinfection: Disinfection was performed with iodine followed by alcohol deiodination. ④ Modeling in the model group: A 2-3 cm longitudinal incision was made along the linea alba of the abdomen. The abdominal cavity was opened, and the tissues were separated layer by layer, exposing both ovaries. The ovaries were ligated at their junctions with the fallopian tubes with surgical sutures. After ovariectomy, the abdomen was closed by suturing the peritoneum, muscularis, and cortex layer by layer. ⑤ Sham-operated group modeling: Following the same laparotomy procedure as above, a small amount of periocular fat was removed, and the rest of the modeling was performed as in the model group. ⑥ Infection prevention: The surgical area was disinfected with 75% medical alcohol, and 1 mL of penicillin was injected intraperitoneally per rat postoperatively. ⑦ Postoperative observation: The rats were placed in a cage and covered with a towel to prevent hypothermia. The rats were observed for 7 days. One week after modeling, different drug interventions were administered. Comparison was administered via gavage at 15 mg / kg / d in sterile distilled water. The sham-operated and model groups received the same amount of distilled water via gavage and were maintained for 12 weeks. During this period, the rats were fed ad libitum and maintained on a 12-h light-dark cycle with good ventilation. Bedding was changed 2-3 times per 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. 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, and the difference was statistically significant (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 sequence; 2) Hydration: the dewaxed slices were immersed in anhydrous ethanol I (5min) and II (5min) in sequence, and then treated with 95% (1min), 80% (1min), and 70% (5min) ethanol in sequence, and washed with distilled water for 1min; 3) Staining: the slices were stained with hematoxylin for 3-5min, rinsed with running water for 1min; reversed with bluing solution, rinsed with distilled water for 1min; dehydrated with 85% (5min) and 95% (5min) alcohol in sequence, eosin stained 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) Transparent: the femoral slices were immersed in xylene I (5min) and xylene II (5min) in sequence; 6) Sealing: the slices were sealed with neutral gum. The results are as follows Figure 6 As shown, it can be seen that: the morphological structure of the trabeculae in the sham operation group is complete and arranged neatly and compactly, the thickness of the trabeculae is uniform, and abundant bone marrow cells can be seen in the bone marrow cavity; the cancellous bone structure in the model group is destroyed, the trabeculae are sparse, and compared with the sham operation group, the spacing between the trabeculae in this group is significantly increased and the free ends are increased, a large number of local gaps are formed in the bone marrow cavity, and the number of vacuolar fat cells in the bone marrow is significantly increased; the number of trabeculae in the complanatus glycoside group is increased compared with the model group, the reticular structure is improved to a certain extent, and the fat cells in the bone marrow cavity are relatively small.

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

[0042] Cytotoxicity test of MC3T3-E1 cells by complanatin and comparison with complanatin B: MC3T3-E1 cells were cultured at 5×10 3 Cells were seeded at 100 μg / mL in 96-well cell culture plates, and five replicate wells were set up. After cell attachment, the cells were divided into a blank group and an intervention group. The blank group was treated with standard α-MEM medium, while the intervention group was treated with different concentrations of complanoside or complanoside B (1 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, and 100 μmol / L), with 200 μl of culture medium per well. The cells were then cultured in a 37°C, 5% CO2 incubator for 72 hours. Then, 10 μl of CCK-8 solution was added to each well and cultured for 1-1.5 hours. OD values ​​at a detection wavelength of 450 nm were measured using a microplate reader to measure cell viability in different groups.

[0043] Figure 7Comparing the blank control group with the 0.1-100 μmol / L concentration of comfrey glycoside and comfrey glycoside B, the inhibitory effect of comfrey glycoside B on MC3T3-E1 cell viability was significantly greater than that of comfrey glycoside (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 complanoside and complanoside B on the expression of BMP-2 and COL1 mRNA in MC3T3-E1 osteoblasts: MC3T3-E1 cells were seeded in a 6-well cell culture plate. When the cell density was 80-90%, a blank group, a complanoside 10 μmol / L group, and a complanoside 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 results, compared with the blank group, 10 μmol / L complanoside and complanoside B could promote the upregulation of BMP-2 and COL1 mRNA in MC3T3-E1 cells (P < 0.05). The expression of mRNA in the complanoside group was upregulated by about one-fold, while the ability of the complanoside B group to promote the expression of BMP-2 and COL1 mRNA was significantly weaker than that of the complanoside group, and the ability to promote osteogenesis of MC3T3-E1 cells was worse.

[0046] Those skilled in the art can make various other corresponding changes and deformations based on the technical solutions and concepts described above, and all of these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A use of complanatus glycoside in preparing a culture material for promoting osteogenic differentiation of osteoblast precursor cells in vitro, characterized in that: The culture material is a culture medium.

2. The use of complanatus glycoside according to claim 1 in preparing a culture material for promoting osteogenic differentiation of osteoblast precursor cells in vitro, characterized in that: The culture material is culture solution.

3. The use of complanatus glycoside according to claim 1 in preparing a culture material for promoting osteogenic differentiation of osteoblast precursor cells in vitro, characterized in that: The culture medium contains 5-10 μmol / L of complanatin.

4. The use of complanatus glycoside according to claim 1 in preparing a culture material for promoting osteogenic differentiation of osteoblast precursor cells in vitro, characterized in that: The culture medium includes vitamin C and 10 mM sodium β-glycerophosphate.

5. An application of complanatus glycoside as the sole active ingredient in the preparation of a drug for preventing and treating osteoporosis.

6. The use of complanatin as the sole active ingredient in the preparation of a drug for preventing and treating osteoporosis according to claim 5, wherein: The medicine for preventing and treating osteoporosis has the effects of increasing bone density, the number and thickness of trabeculae.

7. The use of complanatus glycoside as the sole active ingredient in the preparation of a drug for preventing and treating osteoporosis according to claim 5, wherein: 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.

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