Use of Sangonin in preparation of product for treating osteoarthritis

Abstract

The present application relates to the technical field of medical food and health products, and particularly relates to application of morindin in preparation of products for treating osteoarthritis. The present application screens and evaluates morindin through a hydrogen peroxide-induced chondrocyte in-vitro model and an osteoarthritis animal model of subcutaneous injection of D-galactose, and finds that morindin has obvious effects of reducing joint cartilage damage, enhancing joint cartilage toughness, delaying the course of osteoarthritis, and the like, and has a broad application prospect in preparation of products for treating osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of mulberry extract in the preparation of products for treating osteoarthritis. Background Technology

[0002] Cartilage is an important component of human joints, mainly composed of chondrocytes and a large amount of extracellular matrix (ECM), providing cushioning and lubrication for joint movement. Osteoarthritis (OA) and other degenerative joint diseases impair the structure and function of cartilage, leading to joint pain, stiffness, and limited mobility, and in severe cases, disability.

[0003] Numerous studies have revealed that aging is an important pathogenic factor in osteoarthritis. Senescent cells are present in multiple tissues of OA (including cartilage, subchondral bone, synovium, and infrapatellar fat pad), exhibiting common aging-related features such as telomere shortening, increased expression of cyclin-dependent kinase inhibitors p21, p16, and p53, mitochondrial dysfunction, and increased production of reactive oxygen species (ROS). Meanwhile, various methods of inducing aging have been used to study the mechanisms of osteoarthritis and evaluate preventive drugs. For example, subcutaneous injection of D-galactose has been used to establish an animal model of osteoarthritis under aging factors (PLCγ1 deficiency inchondrocytes accelerates the age-related changes in articular cartilage and subchondral bone. Journal of Cellular and Molecular Medicine: 2024), (Effects of Fuyuan Capsules on the expression of uPA, uPAR, PAI and NF-κB in articular cartilage of rabbits with osteoarthritis; Journal of the Third Military Medical University, 2011); and hydrogen peroxide (H2O2) has been used to establish an in vitro model of osteoarthritis under aging factors (Anti-Apoptosis and Autophagy Effects of Melatonin Protect Rat Chondrocytes against Oxidative Stress via Regulation of AMPK / Foxo3 Pathways. CARTILAGE: 2021), (S-allyl cysteinereduces osteoarthritis pathology in the tert-butyl hydroperoxide-treated chondrocytes and the destabilization of the medial meniscus model mice via the Nrf2 signaling pathway. Aging-US, 2020).A growing body of research shows that drug development for intervening in the senescence of various cell types, including cartilage, subchondral bone, synovium, and infrapatellar fat pad, is highly promising (Targeting senescence to combat osteoarthritis. Science: 2017) (Cross-talk of inflammation and cell senescence: a new insight into the occurrence and progression of osteoarthritis, Bone Research, 2024).

[0004] Currently, the treatment of osteoarthritis mainly involves nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, hyaluronic acid injections, and chondroitin sulfate (such as glucosamine and chondroitin). These drugs play a role in relieving joint pain, improving inflammation, and slowing cartilage degeneration, but they also have significant limitations. While NSAIDs and glucocorticoids can quickly relieve symptoms, long-term use can cause gastrointestinal adverse reactions, kidney damage, and osteoporosis, and they cannot reverse cartilage degeneration. Hyaluronic acid injections, although helpful in improving the viscoelasticity of synovial fluid, often have short-lived effects, and repeated injections may lead to risks such as infection. Furthermore, the efficacy of chondroitin sulfate in some patients is unclear, and its bioavailability and mechanism of action are controversial, making it difficult to meet the long-term treatment needs of degenerative cartilage diseases. Therefore, safer and more effective drugs are highly anticipated. Based on the rich evidence-based medicine of traditional Chinese medicine, research into new functional factors that can intervene in the aging of articular chondrocytes and enhance the performance of articular cartilage is a highly promising direction for the prevention and control of osteoarthritis.

[0005] Maclurin is a natural flavonoid compound mainly found in plants of the Moraceae family. Its molecule contains multiple functional groups, including hydroxyl groups, which endow it with diverse biological activities such as antibacterial, antitumor, and antioxidant effects. Furthermore, studies have found that malurin may also play a role in regulating blood sugar and protecting the liver. Although the research paper "Maclurin Regulates miR-203a-3p / Smad1 to Promote Chondrocyte Differentiation of BMSCs" (Guangzhou University of Chinese Medicine, 2019) reported that "25 μg / mL of malurin has a synergistic effect on TGF-β3-induced chondrocyte differentiation of BMSCs (P<0.05)," this study faces the limitation that "chondrocyte transplantation for OA treatment faces inherent limitations such as difficulty in obtaining chondrocytes and the inability to produce cartilage-specific matrix in vitro, restricting the clinical application of this strategy." The aim was to establish an in vitro method for promoting the differentiation of mesenchymal stem cells into chondrocytes, but it lacked experimental content related to chondrocyte damage and osteoarthritis. Therefore, given the complex etiology and pathogenesis of osteoarthritis, the exact value of mulberry extract in treating osteoarthritis is unclear. Summary of the Invention

[0006] In view of the technical problems that existing osteoarthritis treatment drugs can only temporarily relieve pain and are difficult to effectively stop the progression of the disease, and that long-term use brings many side effects, this invention provides an application of mulberry extract in the preparation of products for treating osteoarthritis.

[0007] Furthermore, the osteoarthritis mentioned is aging-induced osteoarthritis and / or cartilage degenerative disease.

[0008] Furthermore, the treatment of osteoarthritis aims to enhance the toughness of articular cartilage, reduce articular cartilage damage, and delay the progression of osteoarthritis.

[0009] Furthermore, the product enhances the toughness of articular cartilage and reduces articular cartilage damage by increasing chondrocyte metabolic activity, promoting chondrocyte proliferation and extracellular matrix secretion.

[0010] Furthermore, the method of delaying the progression of osteoarthritis involves improving the characteristic pathological changes in chondrocytes caused by osteoarthritis, inhibiting the increase in cartilage water content, and inhibiting the increase in the level of inflammatory factors in articular cartilage.

[0011] Furthermore, the product contains mulberry extract or a pharmaceutically acceptable salt thereof.

[0012] Furthermore, the product is a medicine.

[0013] Furthermore, the drug consists of morin and a pharmaceutically acceptable carrier.

[0014] Furthermore, the drug also contains one or more of the following: disintegrants, humectants, binders, fillers, absorption enhancers, solvents, lubricants, buffers, surfactants, flavorings, sweeteners, antioxidants, preservatives, and colorings.

[0015] Furthermore, the drug includes tablets, capsules, injections, granules, and suspensions.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention screens and evaluates mulberry extract using an in vitro model of chondrocytes induced by hydrogen peroxide and an animal model of osteoarthritis induced by subcutaneous injection of D-galactose. It was found that mulberry extract has significant effects such as reducing articular cartilage damage, enhancing articular cartilage toughness, and delaying the course of osteoarthritis. It has broad application prospects in the preparation of products for the treatment of osteoarthritis.

[0017] 2. The application of mulberry mansoni provided in this invention in the preparation of products for treating osteoarthritis: As a natural product, mulberry mansoni not only has significant effects on improving the cartilage microenvironment and delaying cartilage degeneration, but also has low toxicity and high biocompatibility, making it more suitable as a long-term treatment option. Furthermore, mulberry mansoni has achieved intensive production, with less stringent requirements for storage and transportation conditions, greatly reducing the cost of mulberry mansoni as a drug. This invention not only provides new possibilities for treating osteoarthritis and degenerative cartilage diseases under aging conditions, but also opens up important directions for related drug development. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 These are bright-field images and statistical results of chondrocyte nodule formation in the in vitro injury model of Example 1.

[0020] Figure 2 This is a graph showing the measurement results of the anti-apoptotic ability of chondrocytes in the in vitro injury model in Example 1.

[0021] Figure 3 This is a diagram showing the results of observing chondrocytes in the in vitro injury model before and after toluidine blue staining in Example 2.

[0022] Figure 4 This is a graph showing the experimental results of the improvement effect of different doses of mulberry extract on the pathological changes of articular cartilage caused by osteoarthritis in Example 5. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0024] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0025] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in case of any conflict, the specification containing the definitions shall prevail.

[0026] The materials, reagents, and experimental equipment used in this invention are sourced from the following sources: The cell culture medium was DMEM / F12 medium supplemented with 10% FBS and 1% penicillin antibiotics, purchased from Thermo Fisher Scientific. CCK-8 solution was purchased from Wuhan Solarbio Biotechnology Co., Ltd. Sangchengsu was purchased from Qingdao Dingkang Yipin Life Technology Co., Ltd. Toluidine blue staining solution was purchased from Wuhan Solarbio Biotechnology Co., Ltd. The tissue fixative was purchased from White Shark Biotechnology Co., Ltd. EDTA decalcification solution was purchased from Senbeijia Biotechnology Co., Ltd. The fluorescence microscope was purchased from Nikon Precision Machinery (Shanghai) Co., Ltd., model ECLIPSE Ts2.

[0027] Example 1: The effect of mulberry extract on improving the reduced proliferation activity of chondrocytes in an in vitro injury model. A cell model of aging-induced osteoarthritis was established by treating chondrocytes with hydrogen peroxide solution, in order to evaluate the therapeutic effect and mechanism of action of mulberry orange extract on osteoarthritis.

[0028] (1) Accurately weigh an appropriate amount of mulberry orange powder, dissolve it in double-distilled water to prepare a 250μM stock solution, and store it at -20℃ for later use.

[0029] (2) Primary chondrocytes were cultured in 96-well plates, with blank control group, model group, low-dose group, medium-dose group, and high-dose group set up, and each group was repeated 4 times. The low-dose group, medium-dose group, and high-dose group were treated with 1 μM, 2 μM, and 4 μM mulberry extract solutions, respectively. After 24 hours of culture, except for the blank control group, all groups were treated with 350 μM hydrogen peroxide solution for 4 hours. Then, each group was treated with 10% CCK8 solution, and the OD value in the 96-well plates was measured using a microplate reader at a wavelength of 450 nm. The results are shown in Table 1. Table 1 shows that treatment with different doses of mulberry extract (low, medium, and high) can alleviate the damage of hydrogen peroxide to chondrocytes and significantly increase the proliferation activity of chondrocytes.

[0030] Table 1. Absorbance of chondrocytes in each group (Mean±Std, n=4)

[0031] Note: ## indicates comparison with the blank control group, P<0.01; **** indicates comparison with the model group, P<0.0001.

[0032] (3) Primary chondrocytes were cultured in 6-well plates, with blank control group, model group, low-dose group, medium-dose group, and high-dose group, each group was repeated 3 times. The low-dose group, medium-dose group, and high-dose group were treated with 1 μM, 2 μM, and 4 μM mulberry extract solutions, respectively. After 24 hours of culture, except for the blank control group, all groups were treated with 350 μM hydrogen peroxide solution for 4 hours. The areas of vigorous chondrocyte proliferation in each group (hereinafter referred to as cartilage nodules) were photographed and counted under bright field. The results are as follows: Figure 1 As shown. By Figure 1 It is known that medium and high doses of mulberry extract treatment can significantly increase the number of cartilage nodules.

[0033] (4) Primary chondrocytes were cultured in 6-well plates, with blank control group, low-dose group, medium-dose group, and high-dose group set up. The low-dose group, medium-dose group, and high-dose group were treated with 1 μM, 2 μM, and 4 μM mulberry extract solutions, respectively. After 24 hours of culture, except for the blank control group, all groups were treated with 350 μM hydrogen peroxide solution for 4 hours. Cell suspension was obtained, centrifuged, and the supernatant was discarded. 1 mL of 70% ethanol (pre-cooled on ice) was added, and the cells were gently mixed by pipetting. The cells were fixed at 4°C for 12 hours. After centrifugation and washing, 0.5 mL of propidium iodide staining solution was added, and the cell pellet was slowly and thoroughly resuspended. The cells were incubated at 37°C in the dark for 30 minutes. Flow cytometry was used to detect red fluorescence at an excitation wavelength of 488 nm. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that low, medium and high doses of mulberry extract can increase the proportion of cells in the G2 / M phase, and there is a clear dose-response relationship.

[0034] Based on the experimental results of (2), (3), and (4), it can be seen that the treatment with mulberry orange extract can reduce the damage of hydrogen peroxide to chondrocytes, significantly increase the proliferation activity of chondrocytes, and promote the division and proliferation of chondrocytes under osteoarthritis conditions.

[0035] Example 2: The promoting effect of mulberry mangiferin on the secretion of extracellular matrix proteoglycans in chondrocytes of an in vitro injury model. (1) Accurately weigh an appropriate amount of mulberry orange powder, dissolve it in double-distilled water to prepare a 250μM stock solution, and store it at -20℃ for later use.

[0036] (2) Primary chondrocytes were cultured in 96-well plates, and blank control group, model group, low-dose group, medium-dose group and high-dose group were set up. The low-dose group, medium-dose group and high-dose group were added with 1 μM, 2 μM and 4 μM mulberry cinnamic acid solution respectively. After culturing for 48 hours, except for the blank control group, all groups were treated with 350 μM hydrogen peroxide solution for 4 hours.

[0037] (3) Discard the supernatant of each group of culture medium and fix them with 4% paraformaldehyde. First, photograph each group of chondrocytes under bright field to observe the cell state, and then use toluidine blue staining to determine the secretion of proteoglycans in each group.

[0038] See results Figure 3 As shown, compared with the model group, the toluidine blue stained areas in each dose of mulberry mangiferin treatment group were significantly increased, demonstrating increased proteoglycan deposition in the extracellular matrix. Therefore, it can be inferred that mulberry mangiferin can enhance extracellular matrix secretory activity, promote extracellular matrix deposition, and help enhance the mechanical properties of articular cartilage under osteoarthritis conditions, thereby reducing articular cartilage damage.

[0039] Example 3: The effect of mulberry extract on improving the decreased glucose uptake capacity of hydrogen peroxide-induced chondrocytes (1) Accurately weigh an appropriate amount of mulberry orange powder, dissolve it in double-distilled water to prepare a 250μM stock solution, and store it at -20℃ for later use.

[0040] (2) Primary chondrocytes were cultured in 96-well plates, with blank control group, model group, low-dose group, medium-dose group and high-dose group set up, and each group was repeated 3 times. The low-dose group, medium-dose group and high-dose group were added with 1 μM, 2 μM and 4 μM mulberry cinnamic acid solution respectively. After culturing for 24 hours, except for the blank control group, all groups were treated with 350 μM hydrogen peroxide solution for 4 hours.

[0041] (3) The cell suspension was obtained by treating with trypsin and centrifuged at 1000 rpm for 10 minutes. After centrifugation, the supernatant and cell pellet were collected separately. The cell pellet was lysed for 30 minutes using 2% Triton X-100.

[0042] (4) Prepare the working solution with the following components: phosphate buffer 100 mmol / L, DHBS 2.0 mmol / L, 4-aminoantipyrine 1.0 mmol / L, glucose oxidase 10 kU / L, magnesium oxide 3.5 mmol / L, and peroxidase 8 kU / L.

[0043] (5) Take a 96-well plate and add 2.5 μL of supernatant or cell pellet sample, 2.5 μL of distilled water, and 2.5 μL of glucose standard to each well, and add 250 μL of working solution to each well. Incubate at 37°C for 10 minutes. Measure the absorbance of each well using a microplate reader at a wavelength of 505 nm. Calculate the glucose concentration using the following formula:

[0044] (C 标准 (Standard concentration, 5.55 mmol / L).

[0045] The results are shown in Table 2. Based on the trends in intracellular and extracellular glucose concentrations, it can be seen that mulberry extract can promote the uptake of glucose from the extracellular culture medium into chondrocytes under osteoarthritis conditions. Therefore, it can be inferred that mulberry extract can enhance the glucose uptake capacity of chondrocytes under osteoarthritis conditions, thereby promoting cellular energy metabolism and improving chondrocyte metabolic activity.

[0046] Table 2. Intracellular and extracellular glucose concentrations of chondrocytes in each group (Mean±Std, n=3)

[0047] Note: #### indicates comparison with the blank control group, P<0.0001; ** indicates comparison with the model group, P<0.01; **** indicates comparison with the hydrogen peroxide damage group, P<0.0001.

[0048] Example 4: Mulberry extract improves hydrogen peroxide-induced decline in mitochondrial function in chondrocytes (1) Accurately weigh an appropriate amount of mulberry orange powder, dissolve it in double-distilled water to prepare a 250μM stock solution, and store it at -20℃ for later use.

[0049] (2) Primary chondrocytes were cultured in 96-well plates, with blank control group, model group, low-dose group, medium-dose group and high-dose group set up, and each group was repeated 3 times. The low-dose group, medium-dose group and high-dose group were added with 1 μM, 2 μM and 4 μM mulberry cinnamic acid solution respectively. After culturing for 48 hours, except for the blank control group, all groups were treated with 350 μM hydrogen peroxide solution for 4 hours.

[0050] (3) Remove the cell culture medium, add the prepared Mito-Tracker Green staining working solution pre-incubated at 37°C, and incubate with the cells at 37°C for 30 minutes. Remove the Mito-Tracker Green staining working solution, add fresh cell culture medium pre-incubated at 37°C, and observe using a fluorescence microscope. The results were quantitatively analyzed using ImageJ software, and the results are shown in Table 3. As shown in Table 3, low, medium, and high doses of mulberry mangiferin treatment can improve the overall mitochondrial function of chondrocytes, which is consistent with the results of the sugar uptake capacity experiment and is consistent with the inference that mulberry mangiferin improves the energy metabolism effect of chondrocytes under osteoarthritis conditions.

[0051] Table 3. Overall mitochondrial function detection in chondrocytes of each group (Mean±Std, n=3)

[0052] Note: * indicates P<0.05 compared with the hydrogen peroxide damage group; ** indicates P<0.01 compared with the model group.

[0053] Example 5: Mulberry extract improves characteristic pathological changes in articular cartilage caused by osteoarthritis: Aging is the most important pathogenic factor in osteoarthritis. An animal model of aging-induced osteoarthritis was established by subcutaneous injection of D-galactose to evaluate the efficacy of mulberry extract in the treatment of osteoarthritis.

[0054] (1) Accurately weigh an appropriate amount of mulberry orange powder, dissolve it in double-distilled water to prepare a 500μM stock solution, and store it at -20℃ for later use.

[0055] (2) Twenty 6-8 week old SD rats were randomly divided into 5 groups of 4 rats each: blank control group, model group, low-dose group, medium-dose group, and high-dose group. The treatment methods for each group were as follows, and the experimental period was 14 days: ① Blank control group: No intervention was performed; ② Model group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 3 mL of double-distilled water was administered by gavage daily; ③ Low-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 1.5 μM / g mulberry orange extract was administered by gavage daily according to body weight; ④ Medium-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 3 μM / g mulberry orange extract was administered by gavage daily according to body weight; ⑤ High-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 6 μM / g mulberry orange extract was administered by gavage daily according to body weight.

[0056] (3) After the experiment, the rats were sacrificed, and the cartilage tissue was removed. After fixation with acetone, it was dehydrated, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (HE) for pathological examination. The results are as follows: Figure 4 As shown.

[0057] Depend on Figure 4 It was found that in the blank control group, the hypertrophic cells were uniformly distributed, with regular cell morphology and a clear and uniform extracellular matrix structure, consistent with the typical physiological characteristics of normal cartilage histology. In the model group using D-galactose to induce osteoarthritis, the hypertrophic cells exhibited nuclear pyknosis, and the cell distribution was significantly abnormal, with damage to the extracellular matrix structure and a large number of inflammatory cells aggregated. The low-dose group showed some improvement in the morphology and distribution of hypertrophic cells compared to the model group, but inflammatory cells were still present in the extracellular matrix. The medium-dose group had a hypertrophic cell morphology similar to the blank group, and the extracellular matrix structure was close to normal, but a small number of inflammatory cells were visible. The high-dose group had a hypertrophic cell morphology similar to the blank group, and the extracellular matrix structure was close to normal. In conclusion, mulberry extract can improve the pathological changes in articular cartilage caused by osteoarthritis.

[0058] Example 6: The effect of different doses of mulberry extract on improving the increased cartilage water content caused by osteoarthritis. (1) Accurately weigh an appropriate amount of mulberry orange powder, dissolve it in double-distilled water to prepare a 500μM stock solution, and store it at -20℃ for later use.

[0059] (2) Twenty 6-8 week old SD rats were randomly divided into 5 groups of 4 rats each: blank control group, model group, low-dose group, medium-dose group, and high-dose group. The treatment methods for each group were as follows, and the experimental period was 14 days: ① Blank control group: No intervention was performed; ② Model group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 3 mL of double-distilled water was administered by gavage daily; ③ Low-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 1.5 μM / g mulberry orange extract was administered by gavage daily according to body weight; ④ Medium-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 3 μM / g mulberry orange extract was administered by gavage daily according to body weight; ⑤ High-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 6 μM / g mulberry orange extract was administered by gavage daily according to body weight.

[0060] (3) After the experiment, the rats were euthanized, and the hip joint cartilage was immediately removed. The surface moisture was carefully absorbed using filter paper, and the cartilage weight was accurately measured. Subsequently, the cartilage tissue was placed in acetone for dehydration treatment for 24 hours. After dehydration, the cartilage was continuously vacuum dried at room temperature until the weight was constant, at which point the dry weight of the cartilage was accurately measured. Finally, the moisture content was calculated according to the formula: Moisture content = (Wet weight - Dry weight) ÷ Wet weight × 100%, and the results are shown in Table 4.

[0061] Table 4. Measurement results of articular cartilage water content in each group of rats

[0062] Note: #### indicates a significant difference compared to the blank control group (p<0.0001); **** indicates a relatively significant difference compared to the model group (p<0.0001).

[0063] Table 4 shows that, compared with the blank control group, the water content of articular cartilage in the model group rats was significantly increased, consistent with the clinical pathological manifestation of increased articular cartilage water content in age-related osteoarthritis, thus verifying the effectiveness of the modeling method. Meanwhile, the articular cartilage water content in the low-dose, medium-dose, and high-dose groups showed a significant dose-dependent decreasing trend. This indicates that low, medium, and high doses of mulberry extract can effectively improve the increased cartilage water content caused by osteoarthritis and delay the progression of osteoarthritis.

[0064] Example 7: Mulberry extract improves local inflammatory response caused by osteoarthritis (1) Accurately weigh an appropriate amount of mulberry orange powder, dissolve it in double-distilled water to prepare a 500μM stock solution, and store it at -20℃ for later use.

[0065] (2) Twenty 6-8 week old SD rats were randomly divided into 5 groups of 4 rats each: blank control group, model group, low-dose group, medium-dose group, and high-dose group. The treatment methods for each group were as follows, and the experimental period was 14 days: ① Blank control group: No intervention was performed; ② Model group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 3 mL of double-distilled water was administered by gavage daily; ③ Low-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 1.5 μM / g mulberry orange extract was administered by gavage daily according to body weight; ④ Medium-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 3 μM / g mulberry orange extract was administered by gavage daily according to body weight; ⑤ High-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and 6 μM / g mulberry orange extract was administered by gavage daily according to body weight.

[0066] (3) After the experiment, the rats were euthanized, and the hip joint cartilage was removed. The levels of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) in the joint cartilage were measured by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 5.

[0067] Table 5. Levels of inflammatory factors in the articular cartilage of rats in each group

[0068] Note: #### indicates a significant difference compared to the blank control group (p<0.0001); **** indicates a relatively significant difference compared to the model group (p<0.0001).

[0069] As shown in Table 5, mulberry extract can effectively inhibit the increase in the level of inflammatory factors in articular cartilage caused by osteoarthritis, and this effect is dose-dependent.

Claims

1. The application of mulberry extract in the preparation of products for treating osteoarthritis, characterized in that, The product is a drug, the drug is administered orally, the osteoarthritis is osteoarthritis and / or degenerative changes in cartilage induced by aging factors, and the treatment of osteoarthritis is to reduce damage to articular cartilage and delay the progression of osteoarthritis.

2. The application as described in claim 1, characterized in that, The method of delaying the progression of osteoarthritis is to improve the characteristic pathological changes of chondrocytes caused by osteoarthritis, inhibit the increase of cartilage water content, and inhibit the increase of inflammatory factor levels in articular cartilage.

3. The application as described in claim 1, characterized in that, The drug contains morin or a pharmaceutically acceptable salt thereof.

4. The application as described in claim 1, characterized in that, The drug consists of morin and a pharmaceutically acceptable carrier.

5. The application as described in claim 1, characterized in that, The drug also contains one or more of the following: disintegrants, humectants, binders, fillers, absorption enhancers, solvents, lubricants, buffers, surfactants, flavorings, sweeteners, antioxidants, preservatives, and colorants.

6. The application as described in claim 1, characterized in that, The dosage form of the drug is tablets, capsules, granules, or suspension.

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