Use of neoruscogenin in the preparation of drugs for treating osteoporosis

Abstract

This invention provides the application of Neoruscogenin in the preparation of anti-osteoporosis drugs, belonging to the field of pharmaceutical biotechnology. It was found that Neoruscogenin can significantly increase bone volume fraction, bone surface area to tissue volume ratio, bone mineral density, trabecular bone number, and trabecular bone thickness in osteoporotic mice, while significantly reducing trabecular bone separation; that is, Neoruscogenin can alleviate low bone mass in postmenopausal osteoporotic mice. Furthermore, Neoruscogenin treatment for 8 weeks did not significantly affect the major organs of mice, suggesting the biocompatibility of Neoruscogenin.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, and in particular relates to the application of Neoruscogenin in the preparation of drugs for treating osteoporosis. Background Technology

[0002] Osteoporosis, a systemic skeletal disease characterized by decreased bone density and destruction of bone microstructure, is currently showing a significant upward trend globally, leading to fragility fractures and placing a heavy burden on healthcare systems.

[0003] Currently, medications for treating osteoporosis include various categories such as hormones, estrogen receptor modulators, and bone resorption inhibitors. While these conventional drug treatments are effective, they are expensive and have significant side effects. Traditional Chinese medicine (TCM) active substances have gained widespread attention in the treatment of osteoporosis due to their milder effects and fewer side effects. In particular, flavonoids, polyphenols, saponins, polysaccharides, coumarins, and alkaloids show broad application prospects in osteoporosis treatment, providing new treatment strategies for clinical prevention and treatment. However, existing drugs suffer from unsatisfactory clinical efficacy, numerous side effects, and high prices, necessitating the discovery of safe, effective, and controllable alternative drugs.

[0004] Neoruscogenin is a saponin-based active substance in traditional Chinese medicine; however, it is currently unclear whether Neoruscogenin can alleviate osteoporosis. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide the use of Neoruscogenin in the preparation of medicaments for treating osteoporosis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of Neoruscogenin in the preparation of drugs for treating osteoporosis, wherein the structure of Neoruscogenin is shown in Formula I:

[0007] Formula I.

[0008] Preferably, the osteoporosis includes decreased bone density and / or destruction of bone microstructure.

[0009] Preferably, the anti-osteoporosis includes increasing bone volume fraction, bone surface area to tissue volume ratio, bone density, number of trabeculae and / or trabeculae thickness; The anti-osteoporosis measures also include reducing trabecular separation.

[0010] Preferably, the concentration of Neoruscogenin used is 0.01~1 mg / ml.

[0011] Preferably, the dosage form of the drug includes an oral dosage form.

[0012] Preferably, the drug includes pharmaceutically acceptable excipients.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of Neoruscogenin in the preparation of anti-osteoporosis drugs. It was found that Neoruscogenin can significantly increase bone volume fraction, bone surface area to tissue volume ratio, bone mineral density, trabecular bone number, and trabecular bone thickness in osteoporotic mice, while significantly reducing trabecular separation. In other words, Neoruscogenin can alleviate low bone mass in postmenopausal osteoporotic mice. Furthermore, Neoruscogenin treatment for 8 weeks did not significantly affect the major organs of mice, suggesting the biocompatibility of Neoruscogenin. Attached Figure Description

[0014] Figure 1 Micro-CT analysis of cancellous bone mass in mouse femurs; Figure 2 Image of HE staining of mouse femur; Figure 3 Image showing HE staining of mouse heart, liver, spleen, lungs, and kidneys. Detailed Implementation

[0015] This invention provides the use of Neoruscogenin in the preparation of drugs for treating osteoporosis. The molecular formula of Neoruscogenin is C 27 H 40 O4, molecular weight 428.60, CAS No. 17676-33-4. Its specific structural formula is shown in Formula I:

[0016] Formula I.

[0017] In this invention, the osteoporosis preferably includes decreased bone density and / or destruction of bone microstructure.

[0018] In this invention, the anti-osteoporosis measures preferably include increasing bone volume fraction, bone surface area to tissue volume ratio, bone density, number of trabeculae and / or trabeculae thickness; the anti-osteoporosis measures also preferably include reducing trabeculae separation.

[0019] In this invention, the concentration of Neoruscogenin used is preferably 0.01~1 mg / ml, more preferably 0.1~0.5 mg / ml, and even more preferably 0.3 mg / ml.

[0020] In this invention, the dosage form of the drug preferably includes an oral dosage form.

[0021] In this invention, the drug preferably includes pharmaceutically acceptable excipients.

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Source of materials

[0024] Neoruscogenin was purchased from MCE Ltd., product number: HY-N2253; Eight-week-old female C57BL / 6J mice were purchased from Vitonil, catalog number: 219; DMSO was purchased from Sigma, part number: D8418; Labrafil® M 1944 CS was purchased from Jaffa Lion (Shanghai) Trading Co., Ltd., item number: 198381; Paraformaldehyde was purchased from Beyotime, product number: P0099.

[0025] Example 1

[0026] Add 1% DMSO + 99% Labrafil® M 1944 CS in the following order. Dissolve Neoruscogenin in the above solvents and shake continuously until the solution is clear to prepare a 0.3 mg / ml Neoruscogenin solution.

[0027] A bilateral ovariectomy was used to establish an osteoporosis model, which is currently the most internationally recognized and commonly used method for simulating postmenopausal osteoporosis in women. The specific implementation method is as follows: Twenty mice were acclimatized in an animal facility and then anesthetized with isoflurane gas and fixed in a prone position. The fur on the back of the mice was shaved and disinfected with iodine. A longitudinal incision (0.5 cm in length) was made on each side of the spine, 0.5 cm away. The muscle layer was bluntly dissected to locate the fat pad surrounding the ovary. The fallopian tube-uterus junction was ligated with 8-0 surgical sutures, and the ovary was completely removed. The muscle and skin were sutured layer by layer with 7-0 surgical sutures. Amoxicillin capsules (0.25 mg) were evenly applied topically to the wound to prevent infection. The sham surgery group (n=10) underwent the same surgical procedure as the above modeling except that the ovary was not removed.

[0028] Grouping and administration: Experimental group (OVX+NRS group): 0.3 mg / ml Neoruscogenin solution was administered by gavage; Sham group: 1% DMSO + 99% Labrafil® M 1944 CS solvent was administered by gavage.

[0029] Osteoporosis model group (OVX group): 1% DMSO + 99% Labrafil® M 1944 CS solvent was administered by gavage.

[0030] Ten animals were in each group. Starting from the second week after surgery, the animals were administered the drug by gavage at a dose of 3 mg / kg once a day for 8 consecutive weeks.

[0031] Eight weeks after administration, mice were euthanized, and their femurs, hearts, livers, spleens, lungs, and kidneys were fixed in 4% paraformaldehyde to obtain the test samples.

[0032] Example 2

[0033] To clarify the therapeutic effect of Neoruscogenin, micro-CT was used to perform bone phenotypic analysis on the distal femur.

[0034] The sample to be tested prepared in Example 1 (femur used here, vertebral bone (L5) can also be used) was scanned using a Micro-CT scanner, and parameter analysis and three-dimensional reconstruction were performed. The mouse femur or vertebra was placed clockwise on a self-made circular foam carrier, then placed in a sample tube, and loaded into the sample stage of the machine for scanning. Scanning parameters: scanning voltage 60kV, scanning current 134μA, resolution 22μm.

[0035] Analysis of femoral cancellous bone: A 200-layer scan was performed starting from the lowest point of the growth plate closure point, and the entire area was reconstructed in three dimensions, with analysis of trabecular bone morphology parameters. The analytical indicators were bone volume fraction (BV / TV), bone surface area to tissue volume ratio (BS / TV), bone mineral density (BMD), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone separation (Tb.Sp). The experimental results of the OVX+NRS group, the sham group, and the OVX group were compared. Figure 1 As shown: The results showed that compared with the sham group, the OVX group had significantly lower bone volume fraction (BV / TV), bone surface area to tissue volume ratio (BS / TV), bone mineral density (BMD), number of trabeculae (Tb.N), and trabecular thickness (Tb.Th), while its trabecular separation (Tb.Sp) was significantly increased. Compared with the OVX group, the OVX+NRS group had significantly higher bone volume fraction (BV / TV), bone surface area to tissue volume ratio (BS / TV), bone mineral density (BMD), number of trabeculae (Tb.N), and trabecular thickness (Tb.Th), while its trabecular separation (Tb.Sp) was significantly decreased.

[0036] Example 3

[0037] H&E staining

[0038] (3.1) Tissue embedding and sectioning

[0039] (3.1.1) Decalcification: The test sample (prepared in Example 1) that has been fixed with paraformaldehyde was placed in EDTA decalcification solution for about 3 weeks to decalcify; (3.1.2) Dehydration and embedding: The decalcified bone tissue, heart, liver, spleen, lungs, and kidneys were placed in an embedding cassette and rinsed with tap water for 8 hours. The following steps were performed for gradient ethanol dehydration and paraffin embedding, as shown in Table 1: Table 1. Ethanol gradient dehydration and paraffin embedding

[0040] (3.2) H&E staining

[0041] (3.2.1) Dewaxing: Immerse the slices in xylene for 8 minutes.

[0042] (3.2.2) Hydration: The tissue sections were sequentially placed in 100%, 95%, 85%, 75% ethanol and pure water for 2 min each for gradient hydration.

[0043] (3.2.3) Hematoxylin staining: Add hematoxylin solution to the tissue section, incubate at room temperature for 2 min, and then rinse the section with tap water.

[0044] (3.2.4) Differentiation and blueing: Immerse the tissue sections in 1% hydrochloric acid alcohol differentiation solution for 3 seconds, and then blue them in tap water for 30 minutes.

[0045] (3.2.5) Eosin staining: Immerse the tissue sections in eosin solution for 1 min and rinse with tap water.

[0046] (3.2.6) Dehydration: Place the tissue sections in 95% ethanol for 2 min to dehydrate, and then place them in 100% ethanol until the red color of the bone tissue completely fades and only the cartilage remains red.

[0047] (3.2.7) Transparent: Place in xylene for 3 minutes to become transparent.

[0048] (3.2.8) Mounting: Apply neutral resin to the tissue and mount it. Observe under a microscope. The results are as follows: Figure 2 As shown.

[0049] The results showed that H&E staining analysis revealed that the bone mass of the cancellous bone in the OVX group was significantly lower than that in the Sham group, while the bone mass in the NRS+OVX group was significantly higher than that in the OVX group, indicating that gavage administration of Neoruscogenin can alleviate low bone mass in postmenopausal osteoporotic mice.

[0050] To further clarify whether oral administration of Neoruscogenin has toxicity to the major organs (heart, liver, spleen, lung, and kidney) of mice, the H&E results showed that the morphology of the heart, liver, spleen, lung, and kidney tissues of mice treated for 8 weeks did not show significant changes compared with the OVX group. Figure 3 This indicates that administration of Neoruscogenin via gavage for 8 weeks did not produce any toxicity to the major organs of mice.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Neoruscogenin in the preparation of drugs for treating osteoporosis, characterized in that, The structure of Neoruscogenin is shown in Equation I: Formula I.

2. The application according to claim 1, characterized in that, The osteoporosis includes decreased bone density and / or destruction of bone microstructure.

3. Use according to claim 2, characterized in that, The anti-osteoporosis measures include increasing bone volume fraction, bone surface area to tissue volume ratio, bone density, number of trabeculae and / or trabeculae thickness; The anti-osteoporosis measures also include reducing trabecular separation.

4. Use according to claim 3, characterized in that, The concentration of Neoruscogenin used is 0.01~1 mg / ml.

5. Use according to claim 4, characterized in that, The dosage form of the drug includes oral dosage forms.

6. Use according to claim 5, characterized in that, The drug includes pharmaceutically acceptable excipients.

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