Application of arbutin derivative in preparation of medicine for preventing or treating osteoporosis

Abstract

The invention belongs to the technical field of medicines, and discloses application of an arbutin derivative in preparation of a medicine for preventing or treating osteoporosis diseases. The structural formula of the arbutin derivative is as follows: # imgabs0 #. Experiments on the osteoblast proliferation and differentiation promoting activity and postmenopausal osteoporosis resisting effect of the arbutin derivative by using an in-vitro cell model and an ovariectomized induced osteoporosis mouse model prove that the arbutin derivative can be used for treating osteoporosis. The arbutin derivative has good activity of promoting osteoblast proliferation and differentiation and resisting postmenopausal osteoporosis, can be used as an active ingredient of a medicine for preventing or treating osteoporosis, and has wide application.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and relates to the application of an ursool derivative in preparing a medicine for preventing or treating osteoporosis. Background Art

[0002] Osteoporosis (OP) is a systemic bone disease characterized by decreased bone mass, destruction of bone microstructure, increased bone brittleness, and increased risk of fractures. Osteoporosis can be divided into primary and secondary types according to the cause, and its incidence gradually increases with age. Studies have shown that osteoporosis has become an important problem that troubles the health and quality of life of middle-aged and elderly people, especially for postmenopausal women. Postmenopausal osteoporosis (PMOP) is a type of primary osteoporosis, accounting for about 80% of osteoporosis patients. Its cause is not clear enough and is often related to estrogen deficiency. It mainly occurs in women within 5 to 10 years after menopause. At present, the commonly used drugs and therapies for the clinical treatment of PMOP include bisphosphonates, estrogen replacement therapy, selective estrogen receptor modulators, and calcium agents. However, many adverse reactions or poor efficacy have occurred after long-term clinical use. Therefore, it is of great significance to develop safe and effective drugs for the prevention and treatment of postmenopausal osteoporosis.

[0003] Pentacyclic triterpenoids are widely distributed in the plant kingdom and are mainly found in fruits, leaves, and barks of medicinal plants. These secondary plant metabolites are gaining increasing interest due to their beneficial anti-inflammatory, antidiabetic, and antimicrobial effects. Studies have also reported that pentacyclic triterpenoids have immunomodulatory, glycemic, antihypertensive, anti-skin inflammatory, and antitumor effects. Therefore, given the wide range of biological activities of triterpenes, there is growing interest in using pentacyclic triterpenoids for pharmacological studies and potential new drug development.

[0004] Uvaol is a bioactive molecule of the ursane-type pentacyclic triterpenoids, mainly distributed in olives and olive tree leaves, and can also be isolated from different parts of plants such as bayberry, camellia oil, oleander, lavender, and apple. Studies have shown that urvaol has a variety of pharmacological activities, including anti-inflammatory, cardiotonic, anti-arrhythmic and anti-tumor properties, and can promote the healing of skin wounds.

[0005] However, due to the poor solubility and low bioavailability of ursolic acid, as well as the limited number of hydrogen bond donors in the parent nucleus and the limited number of rotatable bonds in the rigid skeleton, further chemical and biological research on ursolic acid is limited. In order to improve these properties of ursolic acid, researchers mainly use chemical synthesis and biotransformation to modify the structure of ursolic acid. Conventional chemical synthesis methods are costly, and it is particularly difficult to introduce functional groups on saturated CH bonds. Microbial transformation is a simple and effective natural compound structure modification technology that can introduce functional groups regioselectively and stereoselectively. It has the advantages of high efficiency, economy, and environmental protection, which is difficult to achieve with conventional chemical modification. As an efficient modification tool, microbial transformation technology can modify the structure of natural active compounds with different skeletons and has been widely used in the structural modification of various compounds.

[0006] We have previously conducted a systematic study on the microbial transformation of ursutol, obtained a series of ursutol derivatives with novel structures, and discovered ursutol derivatives with potential anti-Alzheimer's disease. The previous research results "Antineuroinflammatory Potential of Uvaol Transformation Products by Three Fungal Species" (J. Agric. Food Chem., 2024, 72: 26762-26776), patent CN118787652A, patent CN118994295A, etc. reported the preparation method of ursutol derivatives of formula I-III of the present invention and their anti-tumor and anti-Alzheimer's disease activities. At present, there is no report on the application of ursutol derivatives of formula I-III in the treatment of osteoporosis. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide an ursolic acid derivative or a pharmaceutically acceptable salt thereof for use in preparing a drug for preventing or treating osteoporosis, and further to provide an ursolic acid triterpene derivative having a 7β,21β-dihydroxy-13,28-epoxy structure or a pharmaceutically acceptable salt thereof for use in preparing a drug for promoting osteoblast proliferation and preparing a drug for treating postmenopausal osteoporosis.

[0008] The technical solution provided by the present invention is as follows:

[0009] The present invention provides an application of an ursolic acid derivative represented by any of the following structural formulas or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating osteoporosis:

[0010]

[0011] In some embodiments of the present invention, the drug for treating osteoporosis is a drug that promotes the proliferation and differentiation of osteoblasts.

[0012] In some embodiments of the present invention, the drug for treating osteoporosis is a drug for treating postmenopausal osteoporosis.

[0013] In some embodiments of the present invention, the drug further contains pharmaceutically acceptable excipients.

[0014] In some embodiments of the present invention, the pharmaceutically acceptable excipient is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier and a lubricant.

[0015] Compared with the prior art, the present invention tests the osteoblast proliferation activity and anti-postmenopausal osteoporosis effect of ursolic acid derivatives in an in vitro osteoblast model and an ovariectomy-induced osteoporosis rat model, confirming that the ursolic acid derivatives mentioned in the present invention have good osteoblast proliferation and anti-postmenopausal osteoporosis activities, can be used as active ingredients of drugs for preventing or treating osteoporosis, and have a wide range of uses. DETAILED DESCRIPTION

[0016] To further illustrate the present invention, the use of the ursodiol derivatives or pharmaceutically acceptable salts thereof provided by the present invention for preparing a drug for preventing or treating osteoporosis is described in detail below in conjunction with examples.

[0017] In this embodiment, ursoyl alcohol derivatives of formula I to formula III were synthesized and purified according to the method disclosed in the document "Antineuroinflammatory Potential of Uvaol Transformation Products by Three Fungal Species" (J. Agric. Food Chem., 2024, 72: 26762-26776).

[0018] Example 1: Effects of arbutin derivatives of formula I to III on proliferation and differentiation of MC3T3-E1 cells

[0019] 1) Cell culture: MC3T3-E1 cells were cultured at 5×10 3 Cells / cm 2 The cells were inoculated at a density of 1.50 μg / mL in a culture flask, and 25 mL of complete culture medium containing 10% fetal bovine serum was added. When the cells were fused to 80% to 90%, the basal culture medium was replaced with osteogenic differentiation induction medium (OM, dexamethasone 0.1 μmol / L, vitamin C 50 mg / L, and β-glycerophosphate 10 mmol / L). The induction medium was replaced every 48 h.

[0020] 2) Cell grouping: Take MC3T3-E1 cells in the logarithmic growth phase and adjust the cell concentration to 1×10 5 / ml were inoculated in 96-well plates. They were divided into blank control group, H2O2 group (0.5mM), H2O2 + compound low-dose group (2.5μM), H2O2 + compound medium-dose group (5μM) and H2O2 + compound high-dose group (10μM). The culture was continued for 72h.

[0021] 3) CCK-8 cell activity assay: Take out the 96-well plate, add 10 μL of CCK-8 solution directly to each well in the dark, and place in a 37°C, 5% CO2 cell culture incubator for 1 hour. Use a multifunctional microplate reader to detect the absorbance value of each well at OD 450nm and calculate the cell survival rate.

[0022] 4) Alkaline phosphatase (ALP) activity assay: MC3T3-E1 cells were treated with 0.5mM H2O2 for 24h, and osteogenic induction solution containing different concentrations of compounds was added to induce differentiation for 7 days. After fixing the cells with 4% paraformaldehyde, alkaline phosphatase staining working solution was prepared according to the instructions for staining for 30 minutes, and 70μL of Ripa lysis solution was added to each well to lyse the cells. The supernatant was collected as a sample after centrifugation. The standard and activity assay working solution were prepared according to the instructions. After sufficient oscillation and mixing, 50μL of substrate, 30μL of buffer and 20μL of sample were added to each well of the 96-well plate, with a total volume of 100μL, and 3 replicates were set for each group. Incubate at 37℃ for 15min, and add 100μL of reaction stop solution to each well to terminate the reaction. The absorbance value at 405nm was detected by an enzyme reader; the ALP activity enzyme assay standard curve was drawn according to the absorbance value of the standard provided by the kit, and the specific content of alkaline phosphatase was obtained according to the standard curve.

[0023] The results are shown in Tables 1 and 2.

[0024] Table 1. Effects of arbutin derivatives of formula I to III on the survival rate of MC3T3-E1 cells ( n=3)

[0025]

[0026]

[0027] Compared with the control group * P<0.05; compared with H2O2 group # P<0.05

[0028] Table 2. Effects of arbutin derivatives of formula I to III on alkaline phosphatase (ALP) activity ( n=3)

[0029]

[0030] Compared with the control group * P<0.05; compared with H2O2 group # P<0.05

[0031] The above experimental results show that the ursolic alcohol derivatives of formula I to formula III of the present invention can promote the proliferation of osteoblasts, inhibit the cytotoxicity induced by H2O2, reverse the osteogenic differentiation dysfunction induced by H2O2, and significantly increase the ALP activity. This shows that the ursolic alcohol derivatives of formula I to formula III can be used as active ingredients of anti-osteoporosis drugs.

[0032] Example 2: Effects of arbutin derivatives of formula I to III on bone density in model mice

[0033] The present invention uses bilateral ovariectomized (OVX) to establish a postmenopausal osteoporosis model in mice. Eight-week-old female C57BL / 6J mice were randomly divided into a sham operation group, a model group, and a low-dose group (2 mg / kg) and a high-dose group (5 mg / kg) treated with a compound, with 6 mice in each group. Sham operation group (cut the skin and peritoneum of the mouse, expose the ovary, return to the cavity and re-suture without destroying the peritoneal structure), model group (ovariectomy), low-dose group (administered 2 mg / kg of the compound after ovariectomy) and high-dose group (administered 5 mg / kg of the compound after ovariectomy). After the modeling surgery was completed, the mice were given a one-week postoperative recovery period. After one week, the low-dose group and the high-dose group were gavaged with the compound every day, and the model group mice were gavaged with the same volume of 0.9% sodium chloride solution for 8 weeks. After the animals were anesthetized, the bone density (BMD) values ​​of the fourth lumbar vertebra and right femur of the rats were measured using a bone densitometer. SPSS 22.0 software was used for analysis of variance and LSD-t test, and the results are shown in Table 3.

[0034] Table 3. Effects of arbutin derivatives of formula I-III on bone density in OVX mice ( n=6)

[0035]

[0036] Compared with the blank group * P<0.05; compared with the model group # P<0.05

[0037] The experimental results showed that compared with the model group, the bone density values ​​of the fourth lumbar vertebra and right femur in the low-dose group and high-dose group of ursolic alcohol derivatives Formula I-Formula III were significantly increased (P < 0.05). This indicates that ursolic alcohol derivatives Formula I-Formula III can significantly increase the bone density of postmenopausal osteoporotic mice in a dose-dependent manner and can be used as active ingredients of anti-osteoporosis drugs.

Claims

1. Use of an ursolic acid derivative represented by any of the following structural formulas or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating osteoporosis, 2. The use according to claim 1, characterized in that: The drug for treating osteoporosis is a drug that promotes the proliferation and differentiation of osteoblasts.

3. The use according to claim 1, characterized in that: The medicine for treating osteoporosis is a medicine for treating postmenopausal osteoporosis.

4. The use according to claim 1, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The auxiliary material is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

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