Application of rubusoside in the preparation of medicine for preventing and treating osteoporosis

Abstract

The present invention discloses the application of rubusoside in the preparation of a drug for preventing and treating osteoporosis. The present invention finds that rubusoside as the only active ingredient can promote the osteogenic differentiation of MC3T3-E1 cells, promote the expression of ALP, BMP-2, COL, and RUNX2 mRNA, and promote the expression of ALP, BMP-2, COL, and RUNX2 proteins in MC3T3-E1 cells; under H2O2 damage conditions, rubusoside pretreatment can protect MC3T3-E1 cells from the decrease in activity and osteogenic ability caused by H2O2 damage; animal experiments show that rubusoside can improve femoral bone density in ovariectomized rats and increase the number and thickness of trabeculae. Therefore, rubusoside can be used to prevent and treat osteoporosis.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to the application of rubusoside in preparing medicine for preventing and treating osteoporosis. Background Art

[0002] Osteoporosis (OP) is a progressive bone disease characterized by decreased bone mass and destruction of bone microstructure, which leads to increased bone fragility. The clinical manifestations of the disease are pain and spinal deformity. In severe cases, fractures occur in the hip, spine and distal radius, which seriously affect the patient's quality of life. The etiology of osteoporosis is very complex. The disease is closely related not only to factors such as aging, endocrine disorders, poor calcium absorption, and limb disuse, but also to factors such as immunity, nutrition, and genetics. Osteoporosis is divided into primary osteoporosis and secondary osteoporosis. Primary osteoporosis is related to aging and is divided into postmenopausal osteoporosis (type I) and senile osteoporosis (type II). Modern Chinese medicine classifies osteoporosis into the categories of "bone arthritis", "bone atrophy", "low back pain", and "bone atrophy" based on the clinical manifestations of OP. According to TCM theory, the pathogenesis of osteoporosis is due to external pathogens invading the human body, or internal injuries and diseases that damage the spleen, liver, and kidneys, resulting in imbalance of yin and yang, qi and blood in the human body, and gradual loss of bone nutrition, which leads to the occurrence of OP.

[0003] In the treatment of osteoporosis, bisphosphonates, calcium, calcitonin, fluoride, sex hormones and other drugs are generally used clinically. Although the clinical efficacy is significant, it faces many problems such as high cost, long administration time, poor patient tolerance, large side effects and unsatisfactory long-term efficacy. Therefore, it is necessary to explore anti-osteoporosis drugs with considerable efficacy, affecting the bone remodeling process to stimulate new bone formation, or inhibiting bone resorption. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide the use of rubusoside in the preparation of a drug for preventing and treating osteoporosis. This use further increases the medicinal value of rubusoside.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The application of rubusoside in the preparation of drugs for preventing and treating osteoporosis is based on the discovery that rubusoside can promote the osteogenic differentiation of MC3T3-E1 cells, promote the expression of ALP, BMP-2, COL, and RUNX2 mRNA, and promote the expression of ALP, BMP-2, COL, and RUNX2 proteins in MC3T3-E1 cells; under H2O2 damage conditions, rubusoside pretreatment can protect MC3T3-E1 cells from the decreased activity and osteogenic ability caused by H2O2 damage; animal experiments show that rubusoside can improve the femoral bone density of ovariectomized rats and increase the number and thickness of trabeculae.

[0007] Rubusoside, also known as Rubusoside, is a highly sweet diterpene glycoside purified from Rubus serrata. Its molecular formula is C 32 H 50 O 13 , molecular weight is 642.73, CAS number is 64849-39-4.

[0008] The concentration of rubusoside in the drug for preventing and treating osteoporosis is 1-100 μmol / L, preferably 1-10 μmol / L.

[0009] The medicine for preventing and treating osteoporosis has the effect of increasing bone density, the number and thickness of trabeculae.

[0010] The medicine for preventing and treating osteoporosis also contains pharmaceutical excipients.

[0011] The dosage forms of the medicine for preventing and treating osteoporosis include decoction, tablet, injection and capsule.

[0012] Compared with the prior art, the present invention has the following advantages and effects:

[0013] (1) The present invention reports for the first time that rubusoside has the effect of promoting bone formation, thereby enriching the efficacy of rubusoside, a food-drug homologous to medicine, and providing more options for natural active ingredients in the prevention and treatment of bone metabolic diseases.

[0014] (2) Compared with stevioside (rebaudioside B, molecular formula C 38 H 60 O 18 , molecular weight 804.87, CAS number 58543-17-2), rubusoside has lower cytotoxicity and better ability to promote cell osteogenic differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the test result diagram of cell activity in different experimental groups; among them, relative to the control group, a represents P <0.05; relative to the rubusoside group, bexpress P <0.05.

[0016] Figure 2 The results of ALP activity test in different experimental groups are shown in Figure 1. Among them, ** indicates that relative to the control group, P <0.05.

[0017] Figure 3 This is a diagram showing the results of mineralized nodule formation in different experimental groups.

[0018] Figure 4 The expression results of bone-related mRNA in different experimental groups are shown in Figure 2. Compared with the control group, * indicates P <0.05, **P<0.01.

[0019] Figure 5 It is the expression result diagram of bone-related proteins in different experimental compositions; among them, the bar graph is the statistical result diagram of the hybridization graph.

[0020] Figure 6 This is the expression result of bone-related mRNA composed of rubusoside and rebaudioside B; among them, relative to the control group, a means P <0.05; relative to the rubusoside group, b means P <0.05.

[0021] Figure 7 is the test result diagram of cell activity in different experimental groups; among them, relative to the control group, a represents P <0.05; relative to H2O2 group, b means P <0.05.

[0022] Figure 8 are two-dimensional microscopic cross-sections of the femoral metaphysis of rats in each group.

[0023] Fig. 9 is the femoral bone parameter test result diagram; relative to the blank group, a represents P <0.05; relative to the model group, b means P <0.05.

[0024] Fig.10 This is the observation result of the distal femur under HE staining microscope (×40). DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0026] The detection objects used in the present invention include rubusoside (structure shown in I) and rebaudioside B. The molecular formula of rebaudioside B is C 38 H 60 O18 , molecular weight is 804.87, CAS number is 58543-17-2. Rebaudioside B is one of the sweet components of stevioside, so it is also called stevioside B (structure as shown in II). Its structural chemical structure is similar to that of rubusoside, and the difference lies in the sugar group at position 13. Rubusoside is a monosaccharide, and rebaudioside B is a disaccharide.

[0027] I II.

[0028] Example 1

[0029] (1) Detection of cytotoxicity

[0030] MC3T3-E1 (mouse calvaria preosteoblast subclone 14) cells were purchased from Wuhan Pronocell Life Science Co., Ltd. and cultured at 5×10 3 Cells / well were inoculated in a 96-well cell culture plate, and 6 replicate wells were set. After the cells adhered to the wall, the original cell culture medium was discarded and different drugs were given for intervention. The intervention method was to dissolve rubusoside and rebaudioside B in DMSO solution to prepare a mother solution with a concentration of 10mmol / L, and dilute them with culture medium to 1μmol / L, 5μmol / L, 10μmol / L, 20μmol / L, 40μmol / L, 60μmol / L, 80μmol / L, and 100μmol / L drug-containing culture medium. 200μL of blank or drug-containing culture medium was added to each well. The cells were placed in a constant temperature cell culture incubator at 37℃ and 5% CO2 for 72h. At the same time, a control group was set up, which was cultured with a culture medium without the test drug. Then 10μL of CCK-8 solution was added to each well, and the culture was continued for 1-1.5h, and the OD value at the detection wavelength of 450nm was measured with an enzyme marker. The composition of the culture medium is as follows: MEMα + 10% FBS + 1% P / S (penicillin / streptomycin).

[0031] The results are as follows Figure 1 As shown, Figure 1 It showed that compared with the blank control group, rubusoside at a concentration of 1-100 μmol / L had no significant effect on cell viability ( P >0.05), while rebaudioside B showed a decrease in cell activity at a concentration of 40-100 μmol / L; at a concentration of 40-100 μmol / L, rubusoside was superior to rebaudioside B in maintaining cell activity ( P >0.05).

[0032] (2) Detection of alkaline phosphatase (ALP) activity of MC3T3-E1 cells by rubusoside

[0033] MC3T3-E1 cells were cultured at 2×10 5 Cells / well were inoculated in a 12-well cell culture plate. After the cells adhered to the wall, the original cell culture medium was discarded, and low, medium, and high concentrations of rubusoside (1μmol / L, 5μmol / L, 10μmol / L) of drug-containing culture medium were added, 1mL per well; a control group was set up at the same time, and the control group was cultured with culture medium without rubusoside; after 72 hours of culture, the ALP content of the cell supernatant was determined according to the instructions of the ALP kit.

[0034] The results are as follows Figure 2 shown. Figure 2 The results showed that the activity of ALP in the supernatant of MC3T3-E1 cells could be significantly increased after 72 hours of intervention with low, medium and high concentrations (1μmol / L, 5μmol / L and 10μmol / L) of rubusoside-containing culture medium.

[0035] (3) Alizarin red staining to observe the effect of rubusoside on the formation of mineralized nodules in MC3T3-E1 cells

[0036] MC3T3-E1 cells were inoculated into a 12-well cell culture plate, with 1 mL of 2×10 cells per well. 5 Cell suspension of 10 cells / mL. After the cells adhered to the wall and the cell density was 90%-100%, the original culture medium was discarded, and low, medium, and high concentrations of rubusoside-containing culture medium (1μmol / L, 5μmol / L, 10μmol / L) were added to intervene respectively, 1mL per well. After 72h of culture, the original culture medium was discarded, and conditioned culture medium (α-MEM cell culture medium containing 50μg / ml vitamin C and 10mM β-glycerophosphate sodium) was added, 1mL per well. At the same time, a control group was set up. The control group was cultured with a culture medium without rubusoside, and then cultured with conditioned culture medium. Continue to culture for 21 days, during which the medium was changed every 3 days. Then Alizarin Red S staining was performed, and the formation of mineralized nodules was observed under a microscope and photographed.

[0037] The results are as follows Figure 3 shown. Figure 3 The results showed that the formation of mineralized nodules in MC3T3-E1 cells treated with low, medium and high concentrations (1μmol / L, 5μmol / L and 10μmol / L) of rubusoside was significantly increased compared with the simple induction group.

[0038] (4) Real-time PCR detection of the effect of rubusoside on the expression of ALP, RUNX2, BMP-2, and COL1 mRNA in MC3T3-E1 cells

[0039] MC3T3-E1 cells were inoculated into 6-well cell culture plates, with 2 mL of 2×10 cells per well. 5Cell suspension of 10 cells / mL. When the cells grow to about 80-90% confluence, discard the original culture medium and add low, medium and high concentrations of rubusoside containing medium (1μmol / L, 5μmol / L, 10μmol / L), 2mL per well. At the same time, set up a control group, which was cultured in a medium without rubusoside. After 48 hours of culture, extract total RNA, detect the expression of ALP, RUNX2, BMP-2, COL1 mRNA, and use GAPDH as an internal reference control.

[0040] The results are as follows Figure 4 As shown in the figure, compared with the blank group, low, medium and high concentrations of rubusoside (1μmol / L, 5μmol / L, 10μmol / L) can promote the upregulation of ALP, RUNX2, BMP-2 and COL 1 mRNA in MC3T3-E1 cells ( P <0.05).

[0041] (5) WB detection of the effects of rubusoside on the expression of ALP, BMP-2, and COL1 proteins in MC3T3-E1 cells

[0042] MC3T3-E1 cells were inoculated into 6-well cell culture plates, with 2 mL of 2×10 cells per well. 5 Cell suspension of 10 cells / mL. When the cells grow to about 80-90% confluence, discard the original culture medium and add different concentrations of rubusoside-containing culture medium (1μmol / L, 5μmol / L, 10μmol / L), 2mL per well. At the same time, a control group was set up, which was cultured in a culture medium without rubusoside. After 72 hours of culture, the whole protein was extracted, and the effect of rubusoside on the ALP, BMP-2, and COL1 protein levels of MC3T3-E1 cells was detected by Western blot.

[0043] The results are as follows Figure 5 As shown in the figure, compared with the blank group, intervention with low, medium and high concentrations of rubusoside (1μmol / L, 5μmol / L, 10μmol / L) can promote the upregulation of ALP, RUNX2, BMP-2 and COL1 protein expressions in MC3T3-E1 cells ( P <0.05).

[0044] (6) Comparison of the effects of rubusoside and rebaudioside B on the expression of ALP, RUNX2, BMP-2, and COL1 mRNA in MC3T3-E1 osteoblasts

[0045] MC3T3-E1 cells were inoculated into 6-well cell culture plates, with 2 mL of 2×10 cells per well. 5Cell suspension of 10 cells / mL. When the cells grow to about 80-90% confluence, discard the original culture medium and replace with ordinary culture medium or drug-containing culture medium. Set up a control group, a 10μmol / L rubusoside group, and a 10μmol / L rebaudioside B (Reb D) group, with 2mL per well. After 48h of culture, extract total RNA, detect the expression of ALP, RUNX2, BMP-2, and COL1 mRNA, and use GAPDH as an internal reference control.

[0046] The results are as follows Figure 6 As shown in the figure, compared with the blank group, rubusoside and rebaudioside B intervention can promote the upregulation of ALP, RUNX2, BMP-2, and COL mRNA in MC3T3-E1 cells ( P <0.05); Compared with the rebaudioside B group, the rubusoside group promoted the expression levels of ALP, BMP-2, and COL1 mRNA in MC3T3-E1 cells to a higher level, and the difference was statistically significant ( P <0.05).

[0047] (7) Protective effect of rubusoside pretreatment on MC3T3-E1 cell activity damaged by H2O2

[0048] MC3T3-E1 cells were cultured at 5 × 10 3 / mL cells were inoculated in a 96-well cell culture plate, and a blank group, a model group (H2O2 injury group), and a low, medium, and high dose intervention group of rubusoside were set up, with 6 replicates in each group. After the cells adhered to the wall, the original cell culture medium was discarded, and low, medium, and high concentrations of rubusoside (1μmol / L, 5μmol / L, and 10μmol / L) were added first, 200μL per well. The cells were then cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 for 24h, and then the culture medium of the model group and rubusoside intervention group was discarded, and a culture medium containing 400μmol / L H2O2 was added, 200μL per well, and the blank group was replaced with a normal culture medium. After 4 hours of injury, the CCK8 method was used to detect the changes in cell viability in each group.

[0049] Figure 7 The results showed that MC3T3-E1 cells pretreated with rubusoside (1μmol / L, 5μmol / L, 10μmol / L) had higher cell viability than those in the direct H2O2 injury group ( P <0.05).

[0050] (8) Rubusoside improves bone metabolism in ovariectomized rats.

[0051] Eighteen 12-week-old SPF female SD rats (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., animal license number: SCXK (Guangdong) 2022-0063) weighing 220±20g were used. During the feeding period, the rats were fed freely, with 12h light-dark rotation lighting, good ventilation, and litter changed 2-3 times a week. After one week of adaptive feeding, the rats were randomly divided into a sham operation group, an ovariectomy model group (model group), and an ovariectomy + rubusoside group (rubusoside group) by random number table method. After anesthesia, bilateral ovariectomy was performed in the model group and rubusoside group, and only the fat of the same size around the ovaries was removed in the sham operation group, while the bilateral ovaries were retained.

[0052] Modeling method: ① Preoperative anesthesia: fasting and water deprivation for 12 hours before surgery, 2% sodium pentobarbital was injected intraperitoneally at 0.2 mL / 100g body weight; ② Skin preparation: fixed in supine position on the operating board, shaved hair in the midline surgical laparotomy area of ​​the lower abdomen; ③ Disinfection: disinfection with iodine tincture and alcohol deiodination in sequence; ④ Modeling of the model group: a 2-3 cm 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 the ovaries 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 of the modeling was the same as the model group; ⑥ Prevention of infection: 75% medical alcohol was used to disinfect the surgical area and 1 mL / 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.

[0053] One week after modeling, different drug interventions were performed. Rubusoside was administered by gavage at 20 mg / kg / d using sterile distilled water as solvent, and the sham operation group and model group were gavaged with the same amount of distilled water and fed for 12 weeks.

[0054] The femurs of rats in each group were fixed with 4% paraformaldehyde, and the distal femur samples were subjected to bone microstructure detection and analysis using Micro-CT. Scanning conditions: 80 kV, 100 μA, single exposure time 50 ms, scanning resolution 25 μm, scanning angle interval 0.5 degrees. The target cancellous bone region of interest (ROI) was subjected to three-dimensional reconstruction and quantitative bone parameter analysis to observe the distal femur tissue. The results are shown in Figure 2. Figure 8 As shown in the figure, compared with the sham operation group, the cancellous bone structure of the distal femur in the model group was destroyed, with sparse, short and broken trabeculae, a large number of local voids in the bone marrow cavity, and a significant decrease in the number of trabeculae; compared with the model group, the sparse trabeculae in the rubusoside group were improved to a certain extent, and the number of trabeculae increased. The results of bone parameter analysis are shown in Fig. 9As shown, it can be seen that: bone parameter analysis showed that after intervention with rubusoside, the femoral bone volume fraction and the number of trabeculae in ovariectomized rats could be significantly increased, and the trabecular spacing could be significantly reduced. The above results were all statistically significant.

[0055] HE staining was used to observe the microstructure of the femur and lumbar vertebrae of rats in each group. The 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: Stain the slices with hematoxylin for 3-5min, rinse with running water for 1min; reverse blue with bluing solution, rinse with distilled water for 1min; dehydrate with 85% (5min) and 95% (5min) alcohol in turn, stain with eosin for 5min, and rinse 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: Sealing with neutral gum. The results are shown in Fig.10 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 thickness of the trabeculae was relatively uniform, and abundant bone marrow cells were visible in the bone marrow cavity; the trabeculae in the model group were obviously sparse, and compared with the sham operation group, the trabeculae spacing in this group was significantly increased and the free ends increased, and an orderly reticular structure could not be formed, and the number of vacuolar fat cells in the bone marrow was significantly increased; the number of trabeculae in the rubusoside 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 decreased.

[0056] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The use of rubusoside as the sole active ingredient in the preparation of drugs for preventing and treating osteoporosis.

2. The use of rubusoside according to claim 1 as the sole active ingredient in the preparation of a drug for preventing and treating osteoporosis, characterized in that: The medicine for preventing and treating osteoporosis is a medicine having the function of increasing bone density, the number and thickness of trabeculae.

3. The use of rubusoside according to claim 1 as the sole active ingredient in the preparation of a drug for preventing and treating osteoporosis, characterized in that: The concentration of the rubusoside in the medicine for preventing and treating osteoporosis is 1-100 μmol / L.

4. The use of rubusoside according to claim 1 as the sole active ingredient in the preparation of a drug for preventing and treating osteoporosis, characterized in that: The medicine for preventing and treating osteoporosis also contains pharmaceutical excipients.

5. The use of rubusoside according to claim 1 as the sole active ingredient in the preparation of a drug for preventing and treating osteoporosis, characterized in that: The dosage forms of the medicine for preventing and treating osteoporosis include decoction, tablet, injection and capsule.

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