New application of hair-blacking alcohol Uvaol in preparation of products for orthopedic diseases

Abstract

The invention relates to novel application of hair blacking alcohol Uvaol in preparation of products for orthopedic diseases, and belongs to the field of medicines. The hair blacking alcohol is extracted and separated from branches, leaves and flowers of Rhododendron delavayi Franch. The compound inhibits osteoclast differentiation, maintains bone metabolism balance and prevents excessive bone resorption or bone loss in a dose-dependent manner. The compound disclosed by the invention can obviously inhibit osteoclast differentiation, and the inhibition effect is concentration-dependent, so that bone metabolism balance is well maintained, and related orthopedic diseases of bone loss caused by excessive bone resorption are prevented, and therefore, the hair-blacking alcohol has a good prospect of developing orthopedic health products or medicines for preventing bone loss and the like, and has a wide application prospect. And a solid foundation is laid for preparing anti-osteoporosis orthopedic health products or medicines.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to the novel application of urovarol in the preparation of products for orthopedic diseases. Background Technology

[0002] Bone loss is the most representative and prevalent pathogenic factor in orthopedics, manifesting significantly in orthopedic diseases such as osteoporosis and osteomyelitis. Osteoclasts are the main functional cells responsible for bone resorption, playing a crucial role in bone development, growth, repair, and remodeling. Abnormal osteoclast function leads to abnormal bone resorption. Hyperfunction can cause degenerative bone diseases such as osteoporosis, bone metastases from cancer, and arthritis; while dysfunction or decline can lead to osteosclerosis, osteogenesis imperfecta, and massive osteolysis. Therefore, the normality of osteoclast function is a major factor affecting bone loss. Screening for functional molecules that inhibit osteoclast differentiation, function, and apoptosis is an important pathway for finding drugs to treat bone-related diseases. In my country, the prevalence of osteoporosis is 3.2% in the 40-49 age group, 19.2% in the 50+ age group, and 32.0% in the 65+ age group. With the increasing incidence of osteoporosis, patients' quality of life and socioeconomic development are severely impacted. Therefore, the search for health products or drugs to prevent and treat osteoporosis, osteomyelitis, and other conditions has significant clinical importance. (Rhododendron simsii) Rhododendron delavayi Franch. belongs to the genus Rhododendron in the family Ericaceae. Rhododendron A common evergreen shrub or small tree. Its flowers and leaves have heat-clearing, detoxifying, hemostatic, and menstrual-regulating effects, and it is mainly used to treat osteomyelitis and other orthopedic diseases. In traditional folk medicine, fresh Rhododendron simsii flowers and leaves are pounded and applied externally or decocted for oral administration to prevent and treat diseases like osteomyelitis and joint pain. Phytochemical studies have found that Rhododendron simsii mainly contains a diverse range of structures including triterpenes, lignans, flavonoids, and phenolic derivatives. Modern pharmacological studies have found that extracts from this plant possess anti-inflammatory, antibacterial, and antiviral pharmacological activities. Uvaol, a characteristic triterpenoid component of Rhododendron simsii, has various biological activities, such as cardiotonic, antiarrhythmic, antibacterial, anti-inflammatory, anticancer, and cadmium poisoning-reducing effects. As a commonly used traditional medicine for orthopedic diseases, existing literature has not reported any research on the prevention and treatment of orthopedic diseases using Rhododendron simsii, and there is a lack of scientific basis and technical support for developing it as a drug for the prevention and treatment of osteoporosis, osteomyelitis, and other orthopedic diseases. Summary of the Invention

[0003] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a new application of urokinase in the preparation of products for orthopedic diseases.

[0004] The technical solution adopted to achieve the above objectives is as follows: Uvaol is being used in the preparation of products for orthopedic diseases, specifically in the preparation of drugs or health products for the treatment, prevention, reduction or relief of osteoporosis, osteomyelitis, and bone loss.

[0005] The compound inhibits osteoclast differentiation, maintains bone metabolic balance, and prevents excessive bone resorption or bone loss in a dose-dependent manner.

[0006] The compound inhibits osteoclast differentiation by blocking RANKL-induced phosphorylation of MAPKs and NF-κB nuclear translocation, and activating the expression of the osteoclast transcription factor NFATc1.

[0007] The compound improves osteoporosis by regulating MAPK phosphorylation, inhibiting NF-κB nuclear translocation and NFATc1 expression.

[0008] The compound Uvaol has the structural formula (1):

[0009] The compound umavaol was extracted from Rhododendron simsii. Fifty parts of Rhododendron simsii branches, leaves, and flowers were pulverized and extracted three times with 200 parts of 95% industrial ethanol under reflux for two hours each time. The filtrates were combined, and the ethanol was recovered and concentrated to obtain five parts of extract. The crude extract was subjected to D101 macroporous resin column chromatography, using a gradient elution of ethanol to water at a volume ratio of 1:9 to 9:1, with 100 parts of each gradient. The elution fraction with an ethanol to water volume ratio of 8:1 was collected, and the solvent was recovered to obtain the umavaol crude extract. The umavaol crude extract was then subjected to C-18 reversed-phase silica gel column chromatography, using a gradient elution of methanol to water at a volume ratio of 1:9 to 9:1, with 100 parts of each gradient collected. The elution fraction containing methanol and water in a 9:1 volume ratio was used to recover the solvent and obtain crude gentamicin extract. Further high-speed countercurrent chromatography (HSCLC) separation of the crude gentamicin extract was performed using a HSCLC solvent system composed of n-hexane-ethyl acetate-methanol-water in a 3:2:3:2 volume ratio. After thorough mixing and settling, the system was separated into upper and lower phases. The upper phase was used as the stationary phase, and the lower phase as the mobile phase. The stationary phase filled the multilayer coil separation column of the HSCLC. The HSCLC was set to 500–1000 rpm, and the mobile phase was injected at a flow rate of 0.5–5 mL / min. Detection was performed using a UV detector with a wavelength of 190–380 nm. When significant mobile phase elution was observed, the crude gentamicin extract was collected, dissolved in a 1:1 volume ratio of upper and lower phases, and injected. The elution containing gentamicin was collected based on the peak shape of the UV detector spectrum, concentrated, and dried to obtain the final product.

[0010] When used as a medicine, the compound Uvaol can be used directly or in the form of a pharmaceutical composition containing 0.1–99% Uvaol, with the remainder being a pharmaceutical carrier or excipient.

[0011] A medicine for treating, preventing, reducing or alleviating osteoporosis, osteomyelitis, or bone loss, said medicine comprising urofuranol, optionally comprising a pharmaceutically acceptable carrier.

[0012] The dosage form of the drug is any one of tablets, capsules, or granules. Attached Figure Description

[0013] Figure 1 The proton NMR spectrum of the compound ursolic acid. Figure 2 The carbon NMR spectrum of the compound ursolicol Figure 3 For MTT assay of cytotoxicity of urohydrin Figure 4 TRAP staining results after RANKL-induced osteoclast formation in RAW264.7 cells treated with urobilinogen. Figure 5 To investigate the F-Actin staining of RAW264.7 cells induced by RANKL after intervention with urobilinogen, and to detect the expression of Atp6v0d2 protein in RAW264.7 cells induced by RANKL after intervention with urobilinogen, using quantitative q-RT-PCR. Figure 6 Western blot analysis was conducted to detect the phosphorylation of MAPKs and NF-κB nuclear translocation in RAW264.7 cells induced by alpha-2-hydroxyl (AHA) intervention with RANKL. Figure 7 Micro-computed tomography and quantitative analysis of BV / TV and Tb.N in the aldosterone intervention group Figure 8 Trap staining of femoral bone samples from the aldosterone intervention group Figure 9 Phosphorylation of MAPKs and expression of NF-κB nuclear translocation-related proteins in femoral samples from the aldosterone intervention group. Detailed Implementation

[0014] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0015] Example 1: Preparation of the compound Uvaol

[0016] The preparation method of Uvaol includes the following steps: (1) Take 50 parts of Rhododendron simsii branches, leaves and flowers, crush them, use 200 parts of 95% industrial ethanol, and reflux extract three times for 2 hours each time. Combine the filtrates, recover the alcohol and concentrate to obtain 5 parts of extract. (2) The crude extract was subjected to D101 macroporous resin column chromatography, and eluted with an ethanol to water volume ratio of 1:9 to 9:1 in a gradient. 100 portions of each gradient were collected, and the eluted portion with an ethanol to water volume ratio of 8:1 was collected. The solvent was recovered to obtain the crude extract of gentamicin.

[0017] (3) The crude extract of gentamicin in step (2) was subjected to C-18 reversed-phase silica gel column chromatography. The eluent was eluted in a gradient of methanol to water with a volume ratio of 1:9 to 9:1. 100 portions of each gradient were collected. The eluted portion with a volume ratio of methanol to water of 9:1 was collected and the solvent was recovered to obtain the crude extract of gentamicin.

[0018] (4) The crude extract of gentamicin in step (3) is separated by high-speed countercurrent chromatography. A high-speed countercurrent solvent system is composed of n-hexane-ethyl acetate-methanol-water with a volume ratio of 3:2:3:2. After the high-speed countercurrent solvent system is thoroughly mixed, it is allowed to stand and separated into upper and lower phases. The upper phase is taken as the stationary phase and the lower phase is taken as the mobile phase. The stationary phase is filled into the multilayer coil separation column of the high-speed countercurrent chromatograph. The high-speed countercurrent chromatograph is set to a rotation speed of 500-1000 r / min and the mobile phase is injected at a flow rate of 0.5-5 mL / min. The ultraviolet detector with a wavelength of 190-380 nm is used for detection. When there is obvious effluent of the mobile phase, the crude extract of gentamicin is taken, dissolved in a mixed solvent with a volume ratio of 1:1 for the upper and lower phases, and injected. The effluent containing gentamicin is collected according to the peak shape of the ultraviolet detector spectrum, concentrated and dried to obtain the product.

[0019] Example 2: Structural identification of the compound ursolicol

[0020] Compounds were analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. 1 H NMR, 13 The structural formula of urohydrin was determined by comprehensive analysis of data such as C NMR, as shown below:

[0021] Uvaol: White amorphous powder. ESI-MS m / z: 445 [M + Na] + The molecular formula is C 30 H 50 O2. 1 H-NMR (600 MHz, CDCl3) δ : 5.13 (1H, t, J = 3.6 Hz, H-12), 3.21 (1H, dd,J = 11.3, 4.9 Hz, H-3), 3.52 (1H, d, J = 11.0 Hz, H-28a), 3.23 (1H, d, J = 6.5 Hz, H-28b), 0.98 (3H, d, J = 4.8 Hz, H-30), 0.80 (3H, d, J = 5.8 Hz, H-29), 1.09,0.94, 0.93, 0.92 and 0.78 (each 3H, s); 13 C-NMR (150 MHz, CDCl3) δ : 38.9 (C-1), 27.4 (C-2), 79.1 (C-3), 38.9 (C-4), 55.3 (C-5), 18.5 (C-6), 32.9 (C-7), 40.1(C-8), 47.8 (C-9), 37.0 (C-10), 23.4 (C-11), 125.1 (C-12), 138.8 (C-13), 42.2(C-14), 26.1 (C-15), 23.4 (C-16), 38.1 (C-17), 54.2 (C-18), 39.5 (C-19), 39.5(C-20), 30.7 (C-21), 35.3 (C-22), 28.3 (C-23), 15.8 (C-24), 15.8 (C-25), 16.9(C-26), 23.4 (C-27), 70.0 (C-28), 17.5 (C-29), 21.5 (C-30).

[0022] Example 3: Verification of Anti-Osteoporosis Activity To further verify the beneficial effects of the compound described in this invention, the osteoclast differentiation inhibitory activity of the compound urokinase was studied. The specific experiments are as follows: (1) MTT assay for the toxicity of urobilinogen to RAW264.7 cells: RAW264.7 cells in logarithmic growth phase were seeded into 96-well plates (8 × 10³ cells per well), cultured overnight at 37 ℃ in a 5% CO2 cell incubator, and the absorbance was measured at 540 nm to calculate the safe concentration of the drug.

[0023] (2) Utagen inhibits RANKL-induced osteoclast differentiation activity: RAW264.7 cells in logarithmic growth phase were seeded in 24-well plates (1×10⁶ cells per well). 5DMEM containing 5% FBS and 1% PS was added to cells and cultured in a 37 ℃, 5% CO2 cell culture incubator for 24 h. After cell adhesion, the DMEM was removed and replaced with αMEM containing 5% FBS and 1% PS. At the same time, drugs were added to induce culture. On day 2, Western blot was performed to detect MAPK phosphorylation and NF-κB-related protein. On day 5, tartrate-resistant acid phosphatase (TRAP), F-actin ring staining, and qRT-PCR were performed to observe osteoclast differentiation.

[0024] (3) Osteoporosis animal model construction and drug action: Sixty male C57BL / 6J mice were randomly divided into six groups: normal group, model group, high-, medium-, and low-dose ursolic acid groups, and positive control (calcitriol) group, with 10 mice in each group. Except for the normal group, all groups of mice were given retinoic acid (90 mg / kg-1) by gavage for 3 weeks to establish a mouse osteoporosis model. The positive control group was given calcitriol 0.06 μg / (kg·d) by gavage, while the high-, medium-, and low-dose ursolic acid groups were given ursolic acid solution 8, 4, and 2 mg / kg·d by gavage, respectively. The normal group and the model group were given an equal volume of physiological saline by gavage. The administration was carried out continuously for 4 weeks. After the last administration, whole blood was collected from each group of mice, and the serum was collected after centrifugation and frozen at -80℃. The femurs of the mice were completely removed, the left femur was frozen at -80℃, and the right femur was fixed in 4% paraformaldehyde for later use. Subsequent micro-computed tomography and quantitative analysis (micro-CT), tartrate-resistant acid phosphatase (TRAP) staining, and Western blot analysis further verified that urophyll has an anti-osteoporosis effect.

[0025] (3) Experimental results A. MTT assay showed that the concentration of urohydrin was between 2.5% and 40%. μ M showed no cytotoxicity to RAW264.7 cells. Figure 3 ).

[0026] B. After intervention with RANKL-induced osteoclasts, TRAP staining showed that the drug had a significant inhibitory effect on osteoclast differentiation function in a dose-dependent manner. Figure 4 F-Actin rings are cytoskeletal structures that maintain osteoclast motility and the environment for bone resorption. F-Actin staining showed that gentamicin significantly inhibited F-Actin ring formation, thereby inhibiting osteoclast differentiation. Figure 5 Bone resorption depends on the remodeling of the actin cytoskeleton into a structure rich in F-actin, and Atp6v0d2 protein plays a major role in F-actin formation. Atp6v0d2 expression was quantitatively measured using q-RT-PCR with GAPDH, and the results showed that ursodeoxycholic acid effectively inhibited the quantitative expression of GAPDH. Figure 5Western blot analysis showed that urobilinogen effectively prevented RANKL-induced MAPK phosphorylation and NF-κB nuclear translocation, and activated the expression of osteoclast transcription factor NFATc1, thereby inhibiting osteoclast differentiation. Figure 6 ).

[0027] C. Micro-computed tomography (micro-CT) analysis of femoral samples from animal model groups and groups treated with different doses of aldosterone showed core focused bone volume fraction (BV / TV) and trabecular bone number (Tb.N). Figure 7 The BV / TV and Tb.N values ​​in the medium- and high-dose urobilinogen intervention groups were significantly higher than those in the model group, indicating that the drug can effectively inhibit bone loss, promote bone tissue repair and reconstruction, increase the effective volume ratio of bone tissue, make the trabecular bone network structure denser and branched, and significantly improve the supporting function and structural stability of bone tissue.

[0028] D. Femoral samples from the animal model group and groups treated with different doses of gentamicin were subjected to TRAP staining. The number of positive cells, staining intensity, and percentage of positive area were significantly reduced in the groups treated with different doses of gentamicin (P<0.05), indicating that gentamicin has an inhibitory effect on osteoclast activity. Figure 8 ).

[0029] E. Western blot analysis of femoral samples from the animal model group and the intervention groups with different doses of gentamicin showed that, compared with the model group, the gentamicin intervention group had a significant effect on the phosphorylation of MAPKs and NF-κB nuclear translocation, particularly on upstream and downstream proteins related to NFATc1. This indicates that the drug can improve osteoporosis by regulating MAPK phosphorylation, inhibiting NF-κB nuclear translocation and NFATc1 expression. Figure 9 ).

[0030] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A novel application of umavaol in the preparation of products for orthopedic diseases, characterized by: It is used in the preparation of drugs or health products for the treatment, prevention, reduction or relief of osteoporosis, osteomyelitis, and bone loss.

2. The novel application of umavaol according to claim 1 in the preparation of products for orthopedic diseases, characterized in that: The compound inhibits osteoclast differentiation, maintains bone metabolic balance, and prevents excessive bone resorption or bone loss in a dose-dependent manner.

3. The novel application of urokinase (Uvaol) according to claim 1 in the preparation of products for orthopedic diseases, characterized in that: The compound inhibits osteoclast differentiation by blocking RANKL-induced phosphorylation of MAPKs and NF-κB nuclear translocation, and activating the expression of the osteoclast transcription factor NFATc1.

4. The novel application of urokinase (Uvaol) according to claim 1 in the preparation of products for orthopedic diseases, characterized in that: The compound improves osteoporosis by regulating MAPK phosphorylation, inhibiting NF-κB nuclear translocation and NFATc1 expression.

5. The novel application of urokinase (Uvaol) according to claim 1 in the preparation of products for orthopedic diseases, characterized in that: The compound Uvaol has the structural formula (1):

6. The novel application of umavaol according to claim 1 in the preparation of products for orthopedic diseases, characterized in that: The compound umavaol was extracted from Rhododendron simsii. Fifty parts of Rhododendron simsii branches, leaves, and flowers were pulverized and extracted three times with 200 parts of 95% industrial ethanol under reflux for two hours each time. The filtrates were combined, and the ethanol was recovered and concentrated to obtain five parts of extract. The crude extract was subjected to D101 macroporous resin column chromatography, using a gradient elution of ethanol to water at a volume ratio of 1:9 to 9:1, with 100 parts of each gradient. The elution fraction with an ethanol to water volume ratio of 8:1 was collected, and the solvent was recovered to obtain the umavaol crude extract. The umavaol crude extract was then subjected to C-18 reversed-phase silica gel column chromatography, using a gradient elution of methanol to water at a volume ratio of 1:9 to 9:1, with 100 parts of each gradient collected. The elution fraction containing methanol and water in a 9:1 volume ratio was used to recover the solvent and obtain crude gentamicin extract. Further high-speed countercurrent chromatography (HSCLC) separation of the crude gentamicin extract was performed using a HSCLC solvent system composed of n-hexane-ethyl acetate-methanol-water in a 3:2:3:2 volume ratio. After thorough mixing and settling, the system was separated into upper and lower phases. The upper phase was used as the stationary phase, and the lower phase as the mobile phase. The stationary phase filled the multilayer coil separation column of the HSCLC. The HSCLC was set to 500–1000 rpm, and the mobile phase was injected at a flow rate of 0.5–5 mL / min. Detection was performed using a UV detector with a wavelength of 190–380 nm. When significant mobile phase elution was observed, the crude gentamicin extract was collected, dissolved in a 1:1 volume ratio of upper and lower phases, and injected. The elution containing gentamicin was collected based on the peak shape of the UV detector spectrum, concentrated, and dried to obtain the final product.

7. The novel application of umavaol according to claim 1 in the preparation of products for orthopedic diseases, characterized in that: When used as a medicine, the compound Uvaol can be used directly or in the form of a pharmaceutical composition containing 0.1–99% Uvaol, with the remainder being a pharmaceutical carrier or excipient.

8. A drug for treating, preventing, reducing, or alleviating osteoporosis, osteomyelitis, and bone loss diseases, characterized in that, The drug includes uromenthol (Uvaol), optionally containing a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The dosage form of the drug is any one of tablets, capsules, or granules.

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