Extract with preventive and therapeutic effect against osteoarthritis and application thereof

Abstract

An extract with a preventive and therapeutic effect against osteoarthritis and an application thereof. The extract is a mulberry polyphenol extract and a monomer thereof obtained by using mulberry as a raw material and performing extraction with water and an alcohol solvent, or in combination with a purification process. The extract can be used for preparing a product for preventing and treating osteoarthritis.

Description

An extract with preventive and therapeutic effects on osteoarthritis and its applications Technical Field

[0001] This invention relates to the field of pharmaceutical and food technology, specifically to an extract with preventive and therapeutic effects on osteoarthritis and its applications. Background Technology

[0002] Osteoarthritis (OA) has become one of the top ten disabling diseases worldwide. Its risk factors are complex, involving genetics, inflammation, obesity, and age, with aging being the most prominent. Numerous studies have revealed the presence of senescent cells in multiple tissues of OA (including cartilage, subchondral bone, synovium, and infrapatellar fat pad), exhibiting common aging-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). Increasing research shows that drug development targeting the senescence of various cell types, including cartilage, subchondral bone, synovium, and infrapatellar fat pad, holds great promise (Cross-talk of inflammation and cellular senescence: a new insight into the occurrence and progression of osteoarthritis, Bone Research, 2024).Meanwhile, various aging-inducing methods have been used in research on the mechanisms and prevention of osteoarthritis. For example, subcutaneous injection of d-galactose has been 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), (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 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).

[0003] However, current treatments for osteoarthritis still primarily involve hormones, analgesics, and nonsteroidal anti-inflammatory drugs (NSAIDs). These medications mostly provide temporary relief from pain and inflammation, failing to effectively control disease progression, and long-term use leads to numerous side effects. Therefore, leveraging the rich evidence-based medicine of traditional Chinese medicine, research into novel functional factors that can intervene in the aging of articular cartilage cells and enhance articular cartilage performance represents a highly promising direction for the prevention and control of osteoarthritis.

[0004] Classic medical texts such as the *Compendium of Materia Medica* and the *Huangdi Neijing* record that mulberries "enter the kidney meridian, benefiting the five internal organs and joints," and that "the kidney governs bones and produces marrow," suggesting the potential value of mulberries in the treatment of osteoarthritis. However, the effective material basis, exact efficacy, and mechanism of mulberry treatment for osteoarthritis remain unclear. Although the research paper "Mulberry Aqueous Extract Regulates miR-139 / MMP-14 to Promote BMSCs Chondrogenic Differentiation" reports that mulberry aqueous extract can promote the differentiation of mesenchymal stem cells into chondrocytes, this study aimed to explore the application of mulberry extract in promoting mesenchymal stem cell differentiation to facilitate cartilage tissue engineering, and did not include any research on chondrocyte damage and osteoarthritis. Furthermore, the extract was prepared using a simple boiling process similar to classic decoctions, and the effective components of mulberries are not definitively identified. Therefore, given the complex etiology and pathogenesis of osteoarthritis, as well as the complex composition of aqueous extracts, it is difficult to determine the exact material basis of mulberry treatment for osteoarthritis, and it is also difficult to deduce its exact therapeutic efficacy. Summary of the Invention

[0005] To address the limitations of existing osteoarthritis drug treatments and the numerous side effects associated with long-term use, this invention provides an extract with preventative and therapeutic effects on osteoarthritis and its applications. Using mulberry as raw material, this invention obtains mulberry polyphenol extracts with a total polyphenol content of 3.8%-63.4% through different preparation processes. Screening and evaluation using cell and animal osteoarthritis models revealed a clear dose-response relationship between the total polyphenol content of mulberry and its interventional efficacy in osteoarthritis, indicating that total mulberry polyphenols are an effective component for intervening in age-induced osteoarthritis and have broad application prospects.

[0006] The specific technical solution is as follows:

[0007] In a first aspect, the present invention provides an extract with the function of preventing and treating osteoarthritis, wherein the extract is a mulberry polyphenol extract and its monomers obtained by extracting mulberry with water and alcohol solvents, or by combining a refining process.

[0008] Furthermore, the total polyphenol content of the above extracts is 5% to 90%.

[0009] Furthermore, the active ingredients of the above extracts include phenolic acids, polyphenol derivatives, flavonoids, isoflavones, and tannins.

[0010] Furthermore, the active ingredients of the above extracts include pelargonidin-3-O-chlororutin, protocatechuic acid, cyanidin-3-O-galactoside, 8-isopentenyl naringenin, quercetin, kaempferol, proanthocyanidin A2, luteolin, caffeic acid, chlorogenic acid, resveratrol, myricetin, gallic acid, catechin, epigallocatechin gallate, and vanillic acid.

[0011] Furthermore, the preparation method of the above-mentioned extract includes the following steps:

[0012] (1) Dry and pulverize the mulberries to make mulberry powder;

[0013] (2) Dissolve mulberry powder in 70% ethanol, stir thoroughly, heat under ultrasonic assistance, centrifuge and collect the supernatant; then add 70% ethanol to the residue, heat under ultrasonic assistance and centrifuge, collect the supernatant.

[0014] (3) Combine the collected supernatants, concentrate under reduced pressure to obtain concentrated solution;

[0015] (4) Pack the pretreated D101 macroporous adsorption resin into the glass chromatography column, dilute the concentrate, and load the sample into the glass chromatography column. After the adsorption is completed, first wash with distilled water to remove some impurities, and then elute with 10% ethanol solution, 30% ethanol solution and 60% ethanol solution in sequence, and collect the eluent.

[0016] (5) The eluent was concentrated under reduced pressure to remove ethanol, diluted and then spray-dried to obtain mulberry extract.

[0017] Secondly, the present invention provides an application of the above-mentioned extract in the preparation of products for the prevention and treatment of osteoarthritis.

[0018] Furthermore, the above-mentioned products can achieve one or more of the following effects:

[0019] (1) Inhibits chondrocyte damage;

[0020] (2) Improves reduced proteoglycan secretion;

[0021] (3) Inhibits the increase of cartilage water content;

[0022] (4) Improves nuclear pyknosis and abnormal distribution in hypertrophic areas of articular cartilage;

[0023] (5) Improve cartilage toughness;

[0024] (6) Reduce inflammatory cells in articular cartilage and lower the level of inflammatory factors.

[0025] Furthermore, the above-mentioned extracts can be used alone or in combination to prepare products for the prevention and treatment of osteoarthritis.

[0026] Furthermore, the aforementioned products are one of the following: pharmaceuticals, health foods, functional foods, and food additives.

[0027] Furthermore, the aforementioned drugs include tablets, capsules, injections, granules, and suspensions; the aforementioned health foods include tablets, hard capsules, soft capsules, oral liquids, milk powder, biscuits, candies, beverages, and alcohol; and the aforementioned functional foods include functional dairy products, bread, and beverages.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. This invention provides an extract with preventive and therapeutic effects on osteoarthritis. Using mulberry as raw material, it is prepared through three processes: water extraction, water-boiling and alcohol precipitation, and alcohol extraction and purification, yielding mulberry polyphenol extracts with a total polyphenol content of 3.8%-63.4%. The mulberry polyphenol extract was then screened and evaluated using an in vitro model of chondrocytes induced by hydrogen peroxide (H2O2) and an animal model of osteoarthritis induced by subcutaneous injection of D-galactose. The results showed that the mulberry polyphenol extract can effectively improve chondrocyte damage, reduce proteoglycan secretion, significantly inhibit pathological changes in articular cartilage, and reduce the level of inflammatory factors in articular cartilage. Furthermore, the effect showed a clear dose-response relationship with the total polyphenol content. This reveals that total mulberry polyphenols are an effective component for intervention in osteoarthritis, indicating its broad application prospects in the preparation of products for the prevention and treatment of osteoarthritis.

[0030] 2. This invention also provides a method for preparing mulberry polyphenol extract and its application in the preparation of products for the prevention and treatment of arthritis. The total polyphenol content of the mulberry extract obtained by ethanol extraction and purification with macroporous adsorption resin is as high as 634.9 mg gallic acid / g, which lays a practical foundation for the application of mulberry total polyphenols in the fields of pharmaceuticals, health foods, and functional foods. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0032] Figure 1 is a standard curve diagram for the determination of total polyphenol content in Example 4.

[0033] Figure 2 is a qualitative composition diagram of the main components of phenolic substances and their derivatives in Example 4.

[0034] Figure 3 shows the experimental results of the improvement effect of mulberry extracts (A, B, C) with different total polyphenol contents on hydrogen peroxide-induced chondrocyte damage in Example 5.

[0035] Figure 4 shows the experimental results of the improvement effect of mulberry extracts (A, B, C) with different total polyphenol contents on the reduction of proteoglycans caused by hydrogen peroxide in Example 6.

[0036] Figure 5 shows the experimental results of the improvement effect of different doses of mulberry extract C on hydrogen peroxide-induced chondrocyte damage in Example 7.

[0037] Figure 6 shows the experimental results of the improvement effect of different doses of mulberry extract C on the pathological changes of articular cartilage caused by osteoarthritis in Example 8.

[0038] Figure 7 shows the experimental results of the improvement effect of different doses of mulberry extract C on the reduction of cartilage toughness caused by osteoarthritis in Example 10. Embodiments of the present invention

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The materials, reagents, and experimental equipment used in this invention are sourced from the following sources:

[0043] The mulberries were purchased from Qingdao Dingkang Yipin Life Science and Technology Development Co., Ltd.

[0044] Gallic acid was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0045] The elution and extraction solvents were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0046] All reagents used in the LC-MS were of chromatographic grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0047] The cell culture medium was DMEM / F12 medium supplemented with 10% FBS and 1% penicillin antibiotics, purchased from Thermo Fisher Scientific.

[0048] The calcium ion probe was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0049] CCK-8 solution was purchased from Wuhan Solarbio Biotechnology Co., Ltd.

[0050] Toluidine blue staining solution was purchased from Wuhan Solarbio Biotechnology Co., Ltd.

[0051] The tissue fixative was purchased from White Shark Biotechnology Co., Ltd.

[0052] HE staining solution was purchased from White Shark Biotechnology Co., Ltd.

[0053] EDTA decalcification solution was purchased from Senbeijia Biotechnology Co., Ltd.

[0054] SD rats were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0055] Liquid chromatography-ultra-high resolution mass spectrometry system, model OrbitrapExploris 480;

[0056] Nikon microscope, model ECLIPSE Ts2;

[0057] Electronic universal testing machine, model Instron 5943.

[0058] The D101 macroporous adsorption resin used in the examples was pretreated according to the relevant methods in the "Pretreatment Method of Ion Exchange Resins" in the "National Standard of the People's Republic of China (GB / T 5476-2013)".

[0059] Example 1: Preparation of mulberry extract by water extraction

[0060] (1) The mulberries were dried at 60°C and then pulverized using a multi-functional pulverizer to obtain mulberry powder.

[0061] (2) Take 50g of mulberry powder and add 500mL of deionized water.

[0062] (3) Boil twice, 20 minutes each time, and combine the liquid.

[0063] (4) Filter the medicine to remove the dregs and obtain the filtrate.

[0064] (5) Dilute the filtrate 1.5 times with deionized water and spray dry (inlet air temperature: 180℃, needle: 5s / time, peristaltic pump: 30%) to obtain mulberry extract A.

[0065] Example 2: Preparation of mulberry extract by water boiling and alcohol precipitation method

[0066] (1) Dry the mulberries at 60℃ and then crush them using a multi-functional pulverizer.

[0067] (2) Take 50g of crushed mulberry and add 500mL of deionized water.

[0068] (3) Boil for 2 hours.

[0069] (4) Filter to remove filter residue and concentrate the filtrate under reduced pressure (temperature: 80℃, speed: 90rpm, vacuum: 0.09MPa).

[0070] (5) The concentrate was precipitated with 50% ethanol solution and centrifuged (5000 rpm, 5 min) to remove the supernatant and collect the precipitate.

[0071] (6) Dilute the alcohol precipitate 15 times with deionized water and centrifuge (3000 rpm, 5 min). Collect the supernatant and spray dry (inlet air temperature: 180℃, needle: 5s / time, peristaltic pump: 30%) to obtain mulberry extract B.

[0072] Example 3: Preparation of mulberry extract by alcohol extraction and purification method

[0073] (1) Dry the mulberries at 60°C and then pulverize them using a multi-functional pulverizer to obtain mulberry powder.

[0074] (2) Take 50g of mulberry powder, dissolve it in 500mL of 70% ethanol, stir thoroughly, and then perform ultrasonic ethanol extraction. The extraction temperature is 60℃, the time is 6h, the ultrasonic power is 150W, centrifuge (5min, 5000rpm), and collect the supernatant. Then, add 500mL of 70% ethanol to the residue, and continue ultrasonic ethanol extraction. The extraction temperature is 60℃, the time is 1h, the ultrasonic power is 150W, centrifuge (5min, 5000rpm), and collect the supernatant. Repeat twice.

[0075] (3) Combine the supernatants from the three times and concentrate them under reduced pressure (temperature: 60℃, rotation speed: 90rpm, vacuum degree: 0.09MPa) to concentrate the volume to one column volume of a macroporous resin column (diameter 1.6cm, height 50cm).

[0076] (4) A glass chromatography column with a diameter of 1.6 cm and a height of 50 cm was packed with pretreated D101 macroporous adsorption resin. The concentrated mulberry polyphenol solution diluted 4 times with distilled water was loaded into the glass chromatography column at a flow rate of 1 mL / min at 60 °C. After adsorption was completed, 100 mL of distilled water was used to remove some impurities. Then, 100 mL of 10% ethanol solution, 200 mL of 30% ethanol solution and 300 mL of 60% ethanol solution were used to elute in sequence. Finally, the eluent was collected.

[0077] (5) First, the eluent is concentrated under reduced pressure (temperature: 80℃, rotation speed: 90rpm, vacuum degree: 0.09MPa) to remove ethanol, and then the eluent concentrate is diluted 10 times with deionized water and spray dried (inlet air temperature: 180℃, needle: 5s / time, peristaltic pump: 30%) to obtain mulberry polyphenol extract C.

[0078] Example 4: Determination of total polyphenol content and identification of components in mulberry extract

[0079] 1. Determination of total polyphenol content in mulberry extract

[0080] Referring to page 69 of the Pharmacopoeia of the People's Republic of China (2020 Edition, Part I) and the "Determination Method of Total Phenolic Content" in the National Standard of the People's Republic of China (GB / T44349-2024), the total polyphenol content of mulberry extracts A, B, and C prepared in Examples 1-3 was determined.

[0081] (1) Preparation of reference solution

[0082] Take an appropriate amount of gallic acid reference standard, accurately weigh it, and add water to prepare a solution containing 50 μg per 1 mL to obtain the reference standard solution.

[0083] (2) Preparation of standard curve

[0084] Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 1.4 mL of the reference solution into separate 10 mL volumetric flasks. Add 6 mL of water, shake well, then add 0.5 mL of Folin-Ciocalteu solution B, shake well, and add 1.5 mL of 20% sodium carbonate solution over 0.5–8 min. Dilute to the mark with water and shake well. Incubate in a 75°C water bath for 10 min. Using the corresponding reagents as blanks, measure the absorbance at 760 nm using UV-Vis spectrophotometry. Plot a standard curve (see Figure 1) with absorbance as the ordinate and concentration as the abscissa. The regression equation is: Y = 0.0572X + 0.0731, R0 2 =0.9991.

[0085] (3) Weigh 2 mg of the extract accurately and place it in a 2 mL centrifuge tube. Add deionized water to the mark to obtain a diluted solution. Take 1.4 mL of the diluted solution into a 10 mL volumetric flask. Following the method in step (2), starting from "add 6 mL of water", determine the absorbance according to the method. Read the concentration of gallic acid in the test solution from the standard curve. The calculation results are shown in Table 1.

[0086] Table 1. Determination of total polyphenol content in mulberry extract

[0087] Total polyphenol content (mg gallic acid / g) by group: Example 1 (Extract A) 38.5±1.9; Example 2 (Extract B) 127.5±2.9; Example 3 (Extract C) 634.9±5.1

[0088] 2. The components in Mulberry Extract C from Example 3 were identified using liquid chromatography-mass spectrometry (LC-MS).

[0089] (1) Accurately weigh 1g of mulberry extract, dissolve it in 50mL of methanol, and place it in an ultrasonic cleaner for ultrasonic extraction for 30min.

[0090] (2) After centrifugation (10000 rpm, 10 min), take the supernatant.

[0091] (3) Filter the supernatant using a 0.22 μm filter membrane to obtain the sample solution to be tested. Use a C18 column at a column temperature of 40 °C, and select acidified water and acetonitrile as the mobile phase; use an electrospray ionization source (ESI) in positive ion mode to capture the secondary spectrum.

[0092] (4) Data analysis:

[0093] S1. The mulberry extract was analyzed by LC-MS to obtain the retention time of the chromatographic peaks and mass spectrometry data.

[0094] S2. The acquired retention time and mass spectrometry data are compared with the retention time and mass spectrometry information in the MS-Dail spectral library to identify the main polyphenolic components in the extract;

[0095] S3. For unknown peaks, structural analysis and identification are performed by combining literature data and mass spectrometry fragment information.

[0096] Experimental Results: LC-MS analysis revealed that the main components of phenolic compounds include phenolic acids, polyphenol derivatives, flavonoids, isoflavones, tannins, and other phenolic substances, as shown in Figure 2. Further structural analysis and identification, combining literature data and mass spectrometry fragment information, identified 16 components of phenols and their derivatives, as detailed in Table 2.

[0097] Table 2. Results of determination of total polyphenols in mulberry extract C

[0098] Component Name Molecular Ion Peak (m / z) Geraniol-3-O-chlororutin [M+H]^+ 509 Protocatechuic acid [MH]^- 169 Cyanidin-3-O-galactoside [M+H]^+ 4658-Isopentenylnaringerone [M+H]^+ 345 Quercetin [MH]^- 301 Kaempferol [MH]^- 150 Proanthocyanidins A2 [MH]^- 577 Luteolin [M+H]^+ 284 Caffeic acid [MH]^- 353 Chlorogenic acid [MH]^- 353 Resveratrol [MH]^- 227 Myricetin [MH]^- 289 Gallic acid [MH]^- 169 Catechol [MH]^- 109 Epigallocatechin gallate [MH]^- 609 Vanillic acid [MH]^- 165

[0099] Example 5: Experimental study on the ameliorative effects of different mulberry extracts on hydrogen peroxide-induced chondrocyte damage.

[0100] (1) Accurately weigh appropriate amounts of mulberry extracts A, B, and C obtained in Examples 1, 2, and 3, dissolve them in double-distilled water to prepare 20 mg / mL stock solutions, and store them at -20℃ for later use.

[0101] (2) Cell culture medium was added to 96-well plates to culture primary chondrocytes, which were divided into a blank control group, a model group, Example 1 group, Example 2 group, and Example 3 group. The control group was only given ordinary culture medium. Example 1 group, Example 2 group, and Example 3 group were given 0.06 mg / L mulberry extract A solution, mulberry extract B solution, and mulberry extract C solution, respectively, with 3 replicates for each group. After 24 h of culture, the model group, Example 1 group, Example 2 group, and Example 3 group were stimulated with 0.1% hydrogen peroxide solution for 3 h. The OD value in the 96-well plates was measured using an ELISA reader at an absorbance of 450 nm. The results are shown in Figure 3.

[0102] As shown in Figure 3, compared with the blank control group, the OD value of the model group decreased, indicating that hydrogen peroxide has a significant damaging effect on chondrocytes. Compared with the model group, the OD value of the Example 1 group with added mulberry extract showed no significant improvement, while the OD values ​​of the Example 2 and Example 3 groups increased significantly, indicating that mulberry extract has an ameliorative effect on hydrogen peroxide-induced chondrocyte damage, and a clear dose-response relationship is shown with the increase of total polyphenol content.

[0103] Example 6: The effect of different mulberry extracts on improving the reduction of proteoglycans induced by hydrogen peroxide.

[0104] (1) Accurately weigh appropriate amounts of mulberry extracts A, B, and C obtained in Examples 1, 2, and 3, dissolve them in double-distilled water to prepare 20 mg / mL stock solutions, and store them at -20℃ for later use.

[0105] (2) Cell culture medium was added to 24-well plates to culture primary chondrocytes, which were divided into a blank control group, a model group, Example 1 group, Example 2 group, and Example 3 group. The control group was only given ordinary culture medium. Example 1 group, Example 2 group, and Example 3 group were given 0.06 mg / L mulberry extract A solution, mulberry extract B solution, and mulberry extract C solution, respectively, with 3 replicates for each group. After 24 h of culture, the model group, Example 1 group, Example 2 group, and Example 3 group were stimulated with 0.1% hydrogen peroxide solution for 3 h and then fixed with 4% paraformaldehyde. Toluidine staining was used to determine the secretion of proteoglycans in each group, and the area of ​​positive staining regions was calculated using ImageJ software. The results are shown in Figure 4.

[0106] As shown in Figure 4, compared with the blank control group, the proteoglycan secretion of chondrocytes in the model group was significantly reduced. Compared with the model group, the mulberry extract-added Example 1 group showed no significant improvement, while Example 2 and Example 3 groups significantly improved the reduction in proteoglycan secretion caused by hydrogen peroxide damage, increased the extracellular matrix level of chondrocytes, and showed a clear dose-response relationship with the increase of total polyphenol content.

[0107] Example 7: The effect of different doses of mulberry extract C on improving hydrogen peroxide-induced chondrocyte damage.

[0108] (1) Accurately weigh an appropriate amount of mulberry extract C obtained in Example 3, dissolve it in double-distilled water to prepare a 20 mg / mL stock solution and store it at -20℃ for later use.

[0109] (2) The mother liquor was diluted to prepare mulberry extract C solutions of 3.125 μg / mL, 6.25 μg / mL, and 12.5 μg / mL. Cell culture medium was added to 96-well plates to culture primary chondrocytes, which were divided into a blank control group, a model group, and groups of Example 3-1, Example 3-2, and Example 3-3, respectively. Mulberry extract C solutions of 3.125 μg / mL, 6.25 μg / mL, and 12.5 μg / mL were added to groups of Example 3-1, Example 3-2, and Example 3-3, respectively, with three replicates for each group. After culturing for 24 h, hydrogen peroxide solution of 0.1% final concentration was added to the model group, group of Example 3-1, group of Example 3-2, and group of Example 3-3 to stimulate for 3 h. The OD values ​​in the 96-well plates were measured using an ELISA reader at an absorbance of 450 nm. The results are shown in Figure 5.

[0110] As shown in Figure 5, compared with the model group, mulberry extract C can significantly improve the damage caused by hydrogen peroxide, and the promoting effect gradually increases with increasing dose, showing a clear dose-response relationship.

[0111] Example 8: The effect of different doses of mulberry extract C on improving pathological changes in articular cartilage caused by osteoarthritis.

[0112] (1) Accurately weigh an appropriate amount of mulberry extract C obtained in Example 3, dissolve it in double-distilled water to prepare a 20 mg / mL stock solution and store it at -20℃ for later use.

[0113] (2) Six- to eight-week-old SD rats were randomly divided into five groups of four rats each: a blank control group, a model group, a low-dose group, a medium-dose group, and a high-dose group. The treatment methods for each group were as follows, and the experimental period lasted for 14 days:

[0114] ① Blank control group: No intervention was performed;

[0115] ② Model group: D-galactose was administered subcutaneously daily at a dose of 250 mg / kg;

[0116] ③ Low-dose group: D-galactose was injected subcutaneously at a dose of 250 mg / kg daily, and mulberry extract C was administered by gavage at a dose of 25 mg daily;

[0117] ④ Medium-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and mulberry extract C was administered by gavage daily at a dose of 50 mg.

[0118] ⑤ High-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and mulberry extract C was administered by gavage daily at a dose of 100 mg.

[0119] (3) After the experiment, the rats were euthanized and the cartilage tissue was removed. After fixation, it was dehydrated and embedded in paraffin, and then stained with HE pathological sections. The results are shown in Figure 6.

[0120] As shown in Figure 6, the hypertrophic cells in the control group were evenly distributed and regularly morphologically regular, with a clear and uniform extracellular matrix structure, exhibiting typical normal histological characteristics. In the model group, the hypertrophic cells showed nuclear pyknosis and significantly abnormal distribution, with disrupted extracellular matrix structure and a large accumulation of inflammatory cells. 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 control group, with a near-normal extracellular matrix structure, but a small number of inflammatory cells were visible. The high-dose group had a hypertrophic cell morphology similar to the control group, with a near-normal extracellular matrix structure. In conclusion, mulberry extract C can improve the pathological changes in articular cartilage caused by osteoarthritis.

[0121] Example 9: The effect of different doses of mulberry extract C on improving the increased cartilage water content caused by osteoarthritis.

[0122] (1) Accurately weigh an appropriate amount of mulberry extract C obtained in Example 3, dissolve it in double-distilled water to prepare a 20 mg / mL stock solution and store it at -20℃ for later use.

[0123] (2) Six- to eight-week-old SD rats were randomly divided into five groups of four rats each: a blank control group, a model group, a low-dose group, a medium-dose group, and a high-dose group. The treatment methods for each group were as follows, and the experimental period lasted for 14 days:

[0124] ① Blank control group: No intervention was performed;

[0125] ② Model group: D-galactose was administered subcutaneously daily at a dose of 250 mg / kg;

[0126] ③ Low-dose group: D-galactose was injected subcutaneously at a dose of 250 mg / kg daily, and mulberry extract C was administered by gavage at a dose of 25 mg daily;

[0127] ④ Medium-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and mulberry extract C was administered by gavage daily at a dose of 50 mg.

[0128] ⑤ High-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and mulberry extract C was administered by gavage daily at a dose of 100 mg.

[0129] (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 wet weight of the cartilage was accurately measured. Subsequently, the cartilage was placed in acetone for dehydration treatment for 24 hours. After dehydration, the cartilage was vacuum dried at room temperature for one week until the weight was constant. At this time, 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%. The results are shown in Table 3.

[0130] Table 3. Results of water content measurement of articular cartilage in rats of each group

[0131] Moisture content (%) of each group: Blank control group 59.85±3.70; Model group 69.20±3.50; Low-dose group 63.50±3.60**; Medium-dose group 61.40±2.70**; High-dose group 60.20±3.40**

[0132] Note: # indicates a significant difference compared to the blank control group (p<0.05); ** indicates a relatively significant difference compared to the model group (p<0.01).

[0133] Table 3 shows that, compared with the blank control group, the water content of articular cartilage in the model group rats was significantly increased, indicating that osteoarthritis under aging conditions leads to an increase in articular cartilage water content. Compared with the model group, the water content of articular cartilage in the low-dose, medium-dose, and high-dose groups of rats was significantly decreased, showing a dose-dependent trend, indicating that low, medium, and high doses of mulberry extract C can effectively improve the increase in cartilage water content caused by osteoarthritis.

[0134] Example 10: The effect of different doses of mulberry extract C on improving cartilage toughness reduction caused by osteoarthritis.

[0135] (1) Accurately weigh an appropriate amount of mulberry extract C obtained in Example 3, dissolve it in double-distilled water to prepare a 20 mg / mL stock solution and store it at -20℃ for later use.

[0136] (2) Six- to eight-week-old SD rats were randomly divided into five groups of four rats each: a blank control group, a model group, a low-dose group, a medium-dose group, and a high-dose group. The treatment methods for each group were as follows, and the experimental period lasted for 14 days:

[0137] ① Blank control group: No intervention was performed;

[0138] ② Model group: D-galactose was administered subcutaneously daily at a dose of 250 mg / kg;

[0139] ③ Low-dose group: D-galactose was injected subcutaneously at a dose of 250 mg / kg daily, and mulberry extract C was administered by gavage at a dose of 25 mg daily;

[0140] ④ Medium-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and mulberry extract C was administered by gavage daily at a dose of 50 mg.

[0141] ⑤ High-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and mulberry extract C was administered by gavage daily at a dose of 100 mg.

[0142] (3) After the experiment, the rats were euthanized and the hip cartilage was removed. Using a specific mold calibrated by metrology, the cartilage was processed into tensile specimens that met the standard size requirements. The specimen dimensions were measured and recorded using high-precision measuring instruments. The prepared specimens were securely installed in the center position of the clamps of the calibrated tensile testing machine. The clamps were adjusted to ensure that the specimens were subjected to uniform and axial stress during the tensile process. The loading rate of the tensile testing machine was set to 1 mm / min, and the equipment was started. During the tensile process, the load-displacement data was recorded continuously in real time using the matching data acquisition system. The tensile test was continued until the specimens broke, and then the test was stopped. Based on the recorded load-displacement data, the elastic modulus of the articular cartilage was calculated according to the relevant mechanical calculation formulas. The results are shown in Figure 7.

[0143] As shown in Figure 7, mulberry extract C can significantly increase the elastic modulus of articular cartilage and improve the cartilage toughness reduction caused by osteoarthritis. Moreover, the improvement effect increases with increasing dosage, showing a clear dose-response relationship.

[0144] Example 11: Effects of different doses of mulberry extract C on improving local inflammatory response caused by osteoarthritis

[0145] (1) Accurately weigh an appropriate amount of mulberry extract C obtained in Example 3, dissolve it in double-distilled water to prepare a 20 mg / mL stock solution and store it at -20℃ for later use.

[0146] (2) Six- to eight-week-old SD rats were randomly divided into five groups of four rats each: a blank control group, a model group, a low-dose group, a medium-dose group, and a high-dose group. The treatment methods for each group were as follows, and the experimental period lasted for 14 days:

[0147] ① Blank control group: No intervention was performed;

[0148] ② Model group: D-galactose was administered subcutaneously daily at a dose of 250 mg / kg;

[0149] ③ Low-dose group: D-galactose was injected subcutaneously at a dose of 250 mg / kg daily, and mulberry extract C was administered by gavage at a dose of 25 mg daily;

[0150] ④ Medium-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and mulberry extract C was administered by gavage daily at a dose of 50 mg.

[0151] ⑤ High-dose group: D-galactose was injected subcutaneously daily at a dose of 250 mg / kg, and mulberry extract C was administered by gavage daily at a dose of 100 mg.

[0152] (4) 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 4.

[0153] Table 4. Levels of inflammatory factors in the articular cartilage of rats in each group

[0154] Group TNF-α (ng / mL) IL-6 (ng / mL) Blank Control Group 10.25±0.78 6.12±1.92 Model Group 30.15±2.86# 180.25±38.62# Low-dose Group 22.34±1.15* 100.45±8.23* Medium-dose Group 18.23±0.89* 70.32±5.21* High-dose Group 12.56±1.88* 20.11±3.65*

[0155] Note: # indicates a significant difference compared to the blank control group (p<0.05); * indicates a significant difference compared to the model group (p<0.05).

[0156] As shown in Table 4, mulberry extract C can effectively inhibit the increase in the level of inflammatory factors in articular cartilage caused by osteoarthritis, and the inhibitory effect is enhanced with increasing dose, showing a clear dose-response relationship.

[0157] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. An extract with preventive and therapeutic effects on osteoarthritis, characterized in that, The extract is a mulberry polyphenol extract and its monomers obtained by extracting mulberries with water and alcohol solvents, or by combining a refining process.

2. The extract according to claim 1, characterized in that, The total polyphenol content of the extract is 5% to 90%.

3. The extract as described in claim 1, characterized in that, The active ingredients of the extract include phenolic acids, polyphenol derivatives, flavonoids, isoflavones, and tannins.

4. The extract as described in claim 3, characterized in that, The active ingredients of the extract include pelargonidin-3-O-chlororutin, protocatechuic acid, cyanidin-3-O-galactoside, 8-isopentenyl naringenin, quercetin, kaempferol, proanthocyanidin A2, luteolin, caffeic acid, chlorogenic acid, resveratrol, myricetin, gallic acid, catechin, epigallocatechin gallate, and vanillic acid.

5. The extract as described in claim 1, characterized in that, The method for preparing the extract includes the following steps: (1) Dry and pulverize the mulberries to make mulberry powder; (2) Dissolve mulberry powder in 70% ethanol, stir thoroughly, heat under ultrasonic assistance, centrifuge and collect the supernatant; then add 70% ethanol to the residue, heat under ultrasonic assistance and centrifuge, collect the supernatant. (3) Combine the collected supernatants, concentrate under reduced pressure to obtain concentrated solution; (4) Pack the pretreated D101 macroporous adsorption resin into the glass chromatography column, dilute the concentrate, and load the sample into the glass chromatography column. After the adsorption is completed, first wash with distilled water to remove some impurities, and then elute with 10% ethanol solution, 30% ethanol solution and 60% ethanol solution in sequence, and collect the eluent. (5) The eluent was concentrated under reduced pressure to remove ethanol, diluted and then spray-dried to obtain mulberry extract.

6. The use of the extract as described in any one of claims 1-5 in the preparation of products for the prevention and treatment of osteoarthritis.

7. The application as described in claim 6, characterized in that, The product can achieve one or more of the following effects: (1) Inhibits chondrocyte damage; (2) Improves reduced proteoglycan secretion; (3) Inhibits the increase of cartilage water content; (4) Improves nuclear pyknosis and abnormal distribution in hypertrophic areas of articular cartilage; (5) Improve cartilage toughness; (6) Reduce inflammatory cells in articular cartilage and lower the level of inflammatory factors.

8. The application as described in claim 6, characterized in that, The extract can be used alone or in combination to prepare products for the prevention and treatment of osteoarthritis.

9. The application as described in claim 6, characterized in that, The product is one of the following: medicine, health food, functional food, or food additive.

10. The application as described in claim 9, characterized in that, The drugs include one or more of tablets, capsules, injections, granules, and suspensions; the health foods include one or more of tablets, hard capsules, soft capsules, oral liquids, milk powder, biscuits, candies, beverages, and alcohol; the functional foods include one or more of functional dairy products, bread, and beverages.

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