Application of morusin in preparation of product for treating osteoarthritis

Abstract

The invention relates to the technical field of medicines, foods and health-care products, in particular to application of morusin in preparation of a product for treating osteoarthritis. A hydrogen peroxide induced chondrocyte in-vitro model and a D-galactose subcutaneous injection osteoarthritis animal model are used for screening and evaluating the morusin, and it is found that the morusin has the obvious effects of relieving articular cartilage injury, enhancing articular cartilage toughness, delaying the course of osteoarthritis and the like. Wide application prospects are realized in the aspect of preparing products for treating osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical, food and health products, and particularly relates to the application of maclurin in the preparation of products for treating osteoarthritis. Background Art

[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. The cartilage structure and function in degenerative joint diseases such as osteoarthritis (OA) are damaged, causing joint pain, stiffness and limited mobility, and severe cases may lead to disability.

[0003] Numerous studies have revealed that aging is an important risk factor for osteoarthritis. Senescent cells are present in multiple tissues of OA (including cartilage, subchondral bone, synovium, and infrapatellar fat pad, etc.), showing common senescence-related characteristics, 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). At the same time, various methods of inducing senescence have been used in the research on the mechanism of osteoarthritis and the evaluation of prevention and control drugs. For example, a subcutaneous injection of D-galactose was used to establish an animal model of osteoarthritis under aging factors (PLCγ1 deficiency in chondrocytes accelerates the age-related changes in articular cartilage and subchondral bone. Journal of Cellular and Molecular Medicine: 2024), (The effect of Fuyuan Capsule on the expression of uPA, uPAR, PAI, and NF-κB in articular cartilage of rabbits with osteoarthritis; Acta Academiae Medicinae Militaris Tertiae, 2011); hydrogen peroxide (H2O2) was 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 cysteine reduces 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).More and more studies have shown that the development of drugs to intervene in the aging of various types of cells, such as cartilage, subchondral bone, synovium, and infrapatellar fat pad, is very promising (Targeting senescence to combat osteoarthritis. Science: 2017), (Cross-talk of inflammation and cell μLarsenescence: a new insight into the occurrence and progression of osteoarthritis, Bone Research, 2024).

[0004] At present, osteoarthritis is mostly treated with drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, hyaluronic acid injections, and chondroitin (such as glucosamine and chondroitin). These drugs have played a certain role in relieving joint pain, improving inflammation, and delaying cartilage degeneration, but they also have significant limitations. Although nonsteroidal anti-inflammatory drugs and glucocorticoids can quickly relieve symptoms, long-term use can cause gastrointestinal adverse reactions, renal damage, osteoporosis and other side effects, and cannot reverse cartilage degeneration. Although hyaluronic acid injections help improve the viscoelasticity of synovial fluid, the effect is often short-lived, and repeated injections may lead to risks such as infection. In addition, the efficacy of chondroprotective agents in some patients is still unclear, and their bioavailability and mechanism of action are also controversial, making it difficult to meet the long-term treatment needs of cartilage degeneration diseases. Therefore, safer and more effective drugs are highly anticipated. Based on the rich evidence-based medicine of traditional Chinese medicine, the study of new functional factors that can intervene in the aging of articular chondrocytes and enhance the performance of articular cartilage is a very promising direction for the prevention and control of osteoarthritis.

[0005] Maclurin is a natural flavonoid compound mainly found in plants such as Moraceae. Its molecule contains multiple hydroxyl groups and other functional groups, and these structural features endow it with diverse biological activities such as antibacterial, anti-tumor, and antioxidant. In addition, studies have found that Maclurin may also have certain effects in regulating blood sugar and protecting the liver. Although the research paper "Maclurin Regulates miR-203a-3p / Smad1 to Promote the Chondrogenic Differentiation of BMSCs" (Guangzhou University of Chinese Medicine, 2019) reported that "25 μg / mL of Maclurin has a synergistic effect on TGF-β3-induced chondrogenic differentiation of BMSCs (P<0.05)", the problem faced by this study is that "the method of chondrocyte transplantation for the treatment of OA faces its own limitations such as difficulty in obtaining chondrocytes and no longer producing cartilage-specific matrix in vitro culture, which limits the clinical application of this strategy". The purpose is to establish a method for promoting the differentiation of mesenchymal stem cells into chondrocytes in vitro, and there is no relevant experimental content for chondrocyte injury and osteoarthritis. Therefore, in the face of the complex etiology and pathogenesis of osteoarthritis, the exact value of Maclurin in the treatment of osteoarthritis is not clear. Summary of the Invention

[0006] In view of the technical problem that existing drugs for the treatment of osteoarthritis can only relieve pain temporarily, are difficult to effectively prevent the progression of the disease, and bring many side effects when used for a long time, the present invention provides the use of Maclurin in the preparation of a product for the treatment of osteoarthritis.

[0007] Further, the osteoarthritis is osteoarthritis induced by aging factors and / or cartilage degenerative lesions.

[0008] Further, the treatment of osteoarthritis is to enhance the toughness of articular cartilage, reduce articular cartilage damage, and delay the course of osteoarthritis.

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

[0010] Further, the delay in the process of osteoarthritis is to improve the characteristic pathological changes of chondrocytes caused by osteoarthritis, inhibit the increase in cartilage water content, and inhibit the increase in the level of articular cartilage inflammatory factors.

[0011] Further, the product contains Maclurin or a pharmaceutically acceptable salt thereof.

[0012] Further, the product is a drug, health food, functional food, or food additive.

[0013] Further, the drug, health food, or functional food is composed of Maclurin and a pharmaceutically acceptable carrier or a food-acceptable carrier.

[0014] Furthermore, the drug, health food or functional food further contains one or more of a disintegrant, a wetting agent, a binder, a filler, an absorption enhancer, a solvent, a lubricant, a buffer, a surfactant, a flavoring agent, a sweetening agent, an antioxidant, a preservative, a pigment, an ointment base, and a transdermal enhancer.

[0015] Furthermore, the drug includes tablets, capsules, injections, granules, and suspensions; the health food includes tablets, hard capsules, soft capsules, oral liquids, milk powder, biscuits, candies, beverages, and wines; the functional food includes functional dairy products, bread, and beverages.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. The present invention screens and evaluates maclurin through an in vitro model of chondrocytes induced by hydrogen peroxide and an osteoarthritis animal model of subcutaneous injection of D-galactose, and finds that maclurin has obvious effects such as reducing articular cartilage damage, enhancing the toughness of articular cartilage, and delaying the course of osteoarthritis, and has broad application prospects in the preparation of products for treating osteoarthritis.

[0018] 2. The application of maclurin provided by the present invention in the preparation of products for treating osteoarthritis. As a natural product, maclurin not only has significant effects in improving the cartilage microenvironment and delaying cartilage degeneration, but also has low toxicity and side effects and high biological safety, and is more suitable as a long-term treatment plan. In addition, maclurin has been intensively produced, and the requirements for storage and transportation conditions are not high, which greatly reduces the cost of maclurin as a drug. The present invention not only provides new possibilities for the treatment of osteoarthritis and cartilage degenerative diseases under aging conditions, but also opens up an important direction for the research and development of related drugs. Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 It is a bright-field photograph and statistical result graph of chondron formation of chondrocytes in the in vitro injury model in Example 1.

[0021] Figure 2 It is a measurement result graph of the anti-apoptosis ability of chondrocytes in the in vitro injury model in Example 1.

[0022] Figure 3 It is an observation result graph of chondrocytes in the in vitro injury model before and after toluidine blue staining in Example 2.

[0023] Figure 4 It is the experimental result diagram of the improvement effect of different doses of maclurin on the pathological changes of articular cartilage caused by osteoarthritis in Example 5. Specific implementation manners

[0024] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0025] The processes, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no particularly restricted content. For the experimental methods without specific conditions noted in each embodiment, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0026] Unless otherwise specified, the meanings of all professional terms and scientific terms used in this specification are the same as those generally understood by those skilled in the technical field to which the present invention belongs. However, in case of conflict, the specification containing the definitions shall prevail.

[0027] The sources of the materials, reagents and experimental equipment used in the present invention are as follows:

[0028] The cell culture medium is DMEM / F12 medium supplemented with 10% FBS and 1% double antibody, purchased from Thermo Fisher Scientific;

[0029] The CCK-8 solution is purchased from Wuhan Solarbio Science & Technology Co., Ltd.;

[0030] Maclurin is purchased from Qingdao Dingkang Yipin Life Science Co., Ltd.;

[0031] Toluidine blue staining solution is purchased from Wuhan Solarbio Science & Technology Co., Ltd.;

[0032] The tissue fixative is purchased from Biosaitech Co., Ltd.;

[0033] EDTA decalcifying solution is purchased from Senbeijia Biological Technology Co., Ltd.;

[0034] The fluorescence microscope is purchased from Nikon Precision Instruments (Shanghai) Co., Ltd., model ECLIPSE Ts2.

[0035] Example 1 Improvement effect of maclurin on the reduction of proliferation activity of chondrocytes in an in vitro injury model

[0036] Chondrocytes were treated with hydrogen peroxide solution to establish a cell model of osteoarthritis induced by aging factors, which was used to evaluate the therapeutic effect and mechanism of action of mulberry orange extract on osteoarthritis.

[0037] (1) Accurately weigh an appropriate amount of moringin powder, dissolve it in double distilled water to prepare a 250 μM stock solution, and store it at -20°C for later use.

[0038] (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, with 4 replicates in each group. 1μM, 2μM, and 4μM mulberry orange solution were added to the low-dose group, medium-dose group, and high-dose group, respectively. After 24 hours of culture, 350μM hydrogen peroxide solution was added to all groups except the blank control group for 4 hours. Then, CCK-8 solution with a final concentration of 10% was added to each group, and the OD value in the 96-well plate was measured using an enzyme reader at a wavelength of 450nm. The results are shown in Table 1. As can be seen from Table 1, treatment with different doses of mulberry orange can reduce the damage of hydrogen peroxide to chondrocytes and significantly increase the proliferation activity of chondrocytes.

[0039] Table 1 Absorbance of chondrocytes in each group (Mean±Std, n=4)

[0040] Group Absorbance Blank control group 1.589±0.01870 Model group <![CDATA[1.508±0.02545 ## > Low-dose group <![CDATA[1.694±0.04661 **** > Medium-dose group <![CDATA[1.716±0.05693 **** > High-dose group <![CDATA[1.683±0.01638 **** >

[0041] Note: ## indicates that compared with the blank control group, P<0.01; **** indicates that compared with the model group, P<0.0001.

[0042] (3) Primary chondrocytes were cultured in 6-well plates, and blank control group, model group, low-dose group, medium-dose group, and high-dose group were set up, with 3 replicates in each group. 1μM, 2μM, and 4μM moringin solution were added to the low-dose group, medium-dose group, and high-dose group, respectively. After 24 hours of culture, 350μM hydrogen peroxide solution was added to all groups except the blank control group 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 shown in the figure. Figure 1 As shown. Figure 1 It can be seen that medium and high doses of moringin treatment can significantly increase the number of cartilage nodules.

[0043] (4) The primary chondrocytes were cultured in 6-well plates, and blank control 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 respectively added with 1 μM, 2 μM, and 4 μM maclurin solutions. After culturing for 24 hours, except for the blank control group, 350 μM hydrogen peroxide solution was added and treated for 4 hours. The cell suspension was obtained, the supernatant was discarded after centrifugation, 1 mL of ice-bath pre-cooled 70% ethanol was added, gently pipetted and mixed evenly, and fixed at 4 °C for 12 hours. After centrifugation and washing, 0.5 mL of propidium iodide staining solution was added, the cell precipitate was slowly and fully resuspended, and incubated at 37 °C in the dark for 30 minutes. The red fluorescence at the excitation wavelength of 488 nm was detected by flow cytometry, and the results were as Figure 2 shown. It can be seen from Figure 2 that the treatment with maclurin at different low, medium, and high doses can all increase the proportion of cells in the G2 / M phase, and there is an obvious dose-effect relationship.

[0044] Based on the experimental results of (2), (3), and (4) comprehensively, it can be known that the treatment with maclurin 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.

[0045] Example 2 Promoting effect of maclurin on the secretion of extracellular matrix proteoglycan by chondrocytes in an in vitro injury model

[0046] (1) An appropriate amount of maclurin powder was accurately weighed and dissolved in double-distilled water to prepare a 250 μM stock solution, which was stored at -20 °C for later use.

[0047] (2) The 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 respectively added with 1 μM, 2 μM, and 4 μM maclurin solutions. After culturing for 48 hours, except for the blank control group, 350 μM hydrogen peroxide solution was added and treated for 4 hours.

[0048] (3) The culture medium supernatant of each group was discarded and fixed with 4% paraformaldehyde. First, the chondrocytes of each group were photographed under bright field to observe the cell state, and then toluidine blue staining was used to determine the secretion of proteoglycan in each group.

[0049] The results are shown in Figure 3 shown. Compared with the model group, the toluidine blue positive staining areas in the maclurin treatment groups at each dose were significantly increased, indicating an increase in the deposition of proteoglycan in the extracellular matrix. It can be inferred from this that maclurin can improve the secretion activity of the extracellular matrix, promote the deposition of the extracellular matrix, contribute to enhancing the mechanical properties of articular cartilage under osteoarthritis conditions, and reduce the damage of articular cartilage.

[0050] Example 3 Improvement effect of maclurin on the reduction of glucose uptake ability in hydrogen peroxide-induced chondrocytes

[0051] (1) Weigh an appropriate amount of maclurin powder accurately, dissolve it with double-distilled water to prepare a 250 μM mother liquor, and store it at -20 °C for later use.

[0052] (2) Culture primary chondrocytes in a 96-well plate, set up a blank control group, a model group, a low-dose group, a medium-dose group, and a high-dose group, with 3 replicates in each group. Add 1 μM, 2 μM, and 4 μM maclurin solutions to the low-dose group, medium-dose group, and high-dose group respectively. After culturing for 24 hours, except for the blank control group, add 350 μM hydrogen peroxide solution and treat for 4 hours.

[0053] (3) Treat with trypsin to obtain a cell suspension, centrifuge at 1000 revolutions per minute for 10 minutes. After centrifugation, collect the supernatant and cell pellet respectively. Use 2% TritonX-100 to lyse the cell pellet for 30 minutes.

[0054] (4) Prepare the working solution, and the content of each component is as follows: 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, peroxidase 8 kU / L.

[0055] (5) Take a 96-well plate, add 2.5 μL of the supernatant or cell pellet sample of each group, 2.5 μL of distilled water, and 2.5 μL of glucose standard respectively, and then add 250 μL of the working solution. Incubate at 37 °C for 10 minutes. Measure the absorbance value of each well at a wavelength of 505 nm using an enzyme-linked immunosorbent assay reader. Calculate the glucose concentration using the following formula:

[0056]

[0057] (C standard: standard concentration, 5.55 mmol / L).

[0058] The results are shown in Table 2. Combining the changing trends of intracellular and extracellular glucose concentrations, it can be seen that maclurin can promote the uptake of glucose from the extracellular culture medium by chondrocytes into the cells under osteoarthritis conditions. It can be inferred that maclurin can enhance the glucose uptake ability of chondrocytes under osteoarthritis conditions, thereby promoting the energy metabolism of cells and improving the metabolic activity of chondrocytes.

[0059] Table 2 Intracellular and extracellular glucose concentrations of chondrocytes in each group (Mean±Std, n = 3)

[0060]

[0061]

[0062] Note: # indicates P < 0.0001 compared with the blank control group; ** indicates P < 0.01 compared with the model group;

[0063] **** indicates P < 0.0001 compared with the hydrogen peroxide injury group.

[0064] Example 4 Moracin improves the decline of chondrocyte mitochondrial function induced by hydrogen peroxide

[0065] (1) Accurately weigh an appropriate amount of moracin powder, dissolve it with double-distilled water to prepare a 250 μM mother liquor, and store it at -20 °C for later use.

[0066] (2) Culture primary chondrocytes in a 96-well plate, set up a blank control group, a model group, a low-dose group, a medium-dose group, and a high-dose group, with 3 replicates in each group. Add 1 μM, 2 μM, and 4 μM moracin solutions to the low-dose group, medium-dose group, and high-dose group respectively. After culturing for 48 hours, except for the blank control group, add 350 μM hydrogen peroxide solution and treat for 4 hours.

[0067] (3) Remove the cell culture medium, add the prepared Mito-Tracker Green staining working solution pre-warmed at 37 °C, and co-incubate with the cells at 37 °C for 30 minutes. Remove the Mito-Tracker Green staining working solution, add the freshly pre-warmed cell culture medium at 37 °C, and observe using a fluorescence microscope. Use ImageJ software to perform quantitative analysis on the captured results. The results are shown in Table 3. It can be seen from Table 3 that treatment with low, medium, and high doses of moracin can all improve the overall mitochondrial function of chondrocytes, which is consistent with the results of the glucose uptake ability experiment and conforms to the inference of the effect of moracin on improving the energy metabolism of chondrocytes under osteoarthritis conditions.

[0068] Table 3 Detection of the overall mitochondrial function of chondrocytes in each group (Mean ± Std, n = 3)

[0069] Group Average fluorescence intensity (AU) Blank control group 14.12±1.847 Model group 12.50±0.8453 Low-dose group <![CDATA[18.15±3.724 * > Medium-dose group <![CDATA[21.62±1.965 ** > High-dose group <![CDATA[18.42±1.910 * >

[0070] Note: * indicates P < 0.05 compared with the hydrogen peroxide injury group; ** indicates P < 0.01 compared with the model group.

[0071] Example 5 Moracin improves the characteristic pathological changes of articular cartilage caused by osteoarthritis:

[0072] Aging is the most important pathogenic factor in the development of osteoarthritis. An animal model of osteoarthritis induced by aging factors is established by subcutaneous injection of D-galactose for evaluating the effect of moracin in the treatment of osteoarthritis.

[0073] (1) Accurately weigh an appropriate amount of moracin powder, dissolve it with double-distilled water to prepare a 500 μM mother liquor, and store it at -20 °C for later use.

[0074] (2) Twenty 6 - 8 - week - old SD rats were randomly divided into 5 groups, with 4 rats in each group, namely the blank control group, the model group, the low - dose group, the medium - dose group, and the high - dose group. The treatment methods for each group were as follows, and the experimental period was 14 days in total:

[0075] ① Blank control group: Without any intervention;

[0076] ② Model group: Subcutaneously injected with D - galactose at a dose of 250 mg / kg daily, and gavaged with 3 mL of double - distilled water daily;

[0077] ③ Low - dose group: Subcutaneously injected with D - galactose at a dose of 250 mg / kg daily, and gavaged with 1.5 μM / g of morin mother liquor according to body weight daily;

[0078] ④ Medium - dose group: Subcutaneously injected with D - galactose at a dose of 250 mg / kg daily, and gavaged with 3 μM / g of morin mother liquor according to body weight daily;

[0079] ⑤ High - dose group: Subcutaneously injected with D - galactose at a dose of 250 mg / kg daily, and gavaged with 6 μM / g of morin mother liquor according to body weight daily.

[0080] (3) After the experiment, the rats were sacrificed, and the cartilage tissues were taken out. After fixation with acetone, dehydration and paraffin - embedding sectioning were carried out, and HE pathological section staining was performed. The results were as Figure 4 shown.

[0081] It can be Figure 4 seen that in the blank control group, the cells in the hypertrophic zone were evenly distributed as a whole, and the cell morphology was regular. The extracellular matrix structure was clear and uniform, which conformed to the typical physiological characteristics of normal cartilage histology. In the model group with osteoarthritis induced by H2O2, karyopyknosis occurred in the cells of the hypertrophic zone, and the cell distribution was significantly abnormal. The extracellular matrix structure was damaged, and a large number of inflammatory cells were seen to aggregate. In the low - dose group, the cell morphology and distribution in the hypertrophic zone were improved to a certain extent compared with the model group, but there were still inflammatory cells in the extracellular matrix. In the medium - dose group, the cell morphology in the hypertrophic zone was close to that of the blank group, and the extracellular matrix structure was close to normal, but a small number of inflammatory cells were visible. In the high - dose group, the cell morphology in the hypertrophic zone was close to that of the blank group, and the extracellular matrix structure was close to normal. In summary, it can be known that morin can improve the pathological changes of articular cartilage caused by osteoarthritis.

[0082] Example 6 Improvement effect of different doses of morin on the increased cartilage water content caused by osteoarthritis

[0083] (1) Weigh an appropriate amount of morin powder accurately, dissolve it with double - distilled water to prepare a 500 μM mother liquor, and store it at - 20 °C for later use.

[0084] (2) Twenty 6-8-week-old SD rats were randomly divided into 5 groups, with 4 rats in each group, namely the blank control group, the model group, the low-dose group, the medium-dose group, and the high-dose group. The treatment methods for each group are as follows, and the experimental period is 14 days in total:

[0085] ① Blank control group: Without any intervention;

[0086] ② Model group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 3 mL of double-distilled water daily;

[0087] ③ Low-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 1.5 μM / g of morin mother liquor according to body weight daily;

[0088] ④ Medium-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 3 μM / g of morin mother liquor according to body weight daily;

[0089] ⑤ High-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 6 μM / g of morin mother liquor according to body weight daily.

[0090] (3) After the experiment, the rats were sacrificed, and the hip joint cartilage was immediately taken out, and the surface moisture was carefully blotted dry with filter paper, and the weight of the cartilage was accurately weighed. Subsequently, the cartilage tissue was placed in acetone for dehydration for 24 hours. After dehydration, the cartilage was continuously vacuum-dried at room temperature until the weight was constant, and then the dry weight of the cartilage was accurately weighed. 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.

[0091] Table 4 Measurement results of the moisture content of the articular cartilage of rats in each group

[0092]

[0093]

[0094] Note: # indicates a significant difference compared with the blank control group (p < 0.0001); **** indicates a relatively significant difference compared with the model group (p < 0.0001).

[0095] As can be seen from Table 4, compared with the blank control group, the moisture content of the articular cartilage of the rats in the model group increased significantly, which was in line with the clinical pathological manifestation of the increased moisture content of the articular cartilage caused by aging, verifying the effectiveness of the modeling method. At the same time, the moisture content of the articular cartilage in the low-dose group, the medium-dose group, and the high-dose group showed an obvious decreasing trend in a dose-dependent manner. It indicates that low, medium, and high doses of morin can effectively improve the increase in the moisture content of cartilage caused by osteoarthritis and delay the course of osteoarthritis.

[0096] Example 7 Moracin improves local inflammatory response caused by osteoarthritis

[0097] (1) Weigh an appropriate amount of moracin powder accurately, dissolve it with double-distilled water to prepare a 500 μM mother liquor, and store it at -20 °C for later use.

[0098] (2) Randomly divide 20 6-8-week-old SD rats into 5 groups, with 4 rats in each group, namely the blank control group, the model group, the low-dose group, the medium-dose group, and the high-dose group. The treatment methods for each group are as follows, and the experimental period is 14 days in total:

[0099] ① Blank control group: Without any intervention;

[0100] ② Model group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and gavage 3 mL of double-distilled water daily;

[0101] ③ Low-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and gavage 1.5 μM / g of moracin mother liquor according to body weight daily;

[0102] ④ Medium-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and gavage 3 μM / g of moracin mother liquor according to body weight daily;

[0103] ⑤ High-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and gavage 6 μM / g of moracin mother liquor according to body weight daily.

[0104] (3) After the experiment, sacrifice the rats, take out the hip joint cartilage, and use the enzyme-linked immunosorbent assay to measure the levels of tumor necrosis factor (TNF-α) and interleukin 6 (IL-6) in the articular cartilage. The results are shown in Table 5.

[0105] Table 5 Levels of inflammatory factors in articular cartilage of rats in each group

[0106] Group TNF-α (ng / mL) IL-6 (ng / mL) Blank control group 11.45±1.43 7.92±0.56 Model group <![CDATA[38.73±2.02 #### > <![CDATA[199.40±11.21 #### > Low-dose group 29.68±1.23**** 108.0±11.68**** Medium-dose group 19.51±1.70**** 47.30±6.10**** High-dose group 12.00±2.03**** 30.88±7.71****

[0107] Note: # indicates a significant difference compared with the blank control group (p < 0.0001); **** indicates a relatively significant difference compared with the model group (p < 0.0001).

[0108] As can be seen from Table 5, moracin can effectively inhibit the increase in the levels of inflammatory factors in articular cartilage caused by osteoarthritis, and shows a dose-dependent trend.

Claims

1. An application of mulberry orange pigment in the preparation of a product for treating osteoarthritis.

2. The use according to claim 1, characterized in that The osteoarthritis is osteoarthritis and / or cartilage degeneration induced by aging factors.

3. The use according to claim 1, characterized in that The treatment of osteoarthritis is to enhance the toughness of articular cartilage, reduce articular cartilage damage, and delay the course of osteoarthritis.

4. The use according to claim 3, characterized in that The product enhances the toughness of articular cartilage and reduces articular cartilage damage by increasing the metabolic activity of chondrocytes, promoting chondrocyte proliferation and extracellular matrix secretion.

5. The use according to claim 3, 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.

6. The use according to claim 1, characterized in that The product comprises moringin or a pharmaceutically acceptable salt thereof.

7. The use according to claim 1, characterized in that The product is a medicine, health food, functional food or food additive.

8. The use according to claim 7, characterized in that The medicine, health food or functional food consists of mulberry orange extract and a pharmaceutically acceptable carrier or a food acceptable carrier.

9. The use according to claim 7, characterized in that The medicine, health food or functional food also contains one or more of disintegrants, wetting agents, adhesives, fillers, absorption promoters, solvents, lubricants, buffers, surfactants, flavoring agents, sweeteners, antioxidants, preservatives and pigments, ointment bases and transdermal enhancers.

10. The use according to claim 7, characterized in that The medicines include tablets, capsules, injections, granules and suspensions; the health foods include tablets, hard capsules, soft capsules, oral liquids, milk powder, biscuits, candies, beverages and wine; the functional foods include functional dairy products, bread and beverages.

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