Extract with osteoarthritis prevention and treatment effect and application thereof

Abstract

The invention relates to the technical field of medicine and food, in particular to an extract with an osteoarthritis prevention and treatment effect and application thereof, the extract is a mulberry polyphenol extract obtained by taking mulberry as a raw material and extracting with water and an alcohol solvent or combining with a refining process and a monomer of the mulberry polyphenol extract. The mulberry polyphenol extract is screened and evaluated by using an in-vitro model of cartilage cell injury induced by hydrogen peroxide (H2O2) and an osteoarthritis animal model induced by D-galactose subcutaneous injection, and the mulberry polyphenol extract is found to be capable of effectively improving cartilage cell injury, reducing proteoglycan secretion, obviously inhibiting joint osteomalacia change and improving osteoarthritis activity. The level of articular cartilage inflammatory factors is reduced, and the effect and the total polyphenol content are in a definite dose-effect relationship. The mulberry total polyphenol is an effective part for intervening the osteoarthritis, so that the mulberry total polyphenol has a wide application prospect in the aspect of preparing products for preventing and treating the osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine and food, and particularly relates to an extract with the function of preventing and treating osteoarthritis and its application. Background Art

[0002] Osteoarthritis (OA) has become one of the top ten disabling diseases globally. Its pathogenic risk factors are complex, involving genetics, inflammation, obesity, and age, among which the most prominent risk factor is the aging factor. A large number of studies have revealed that senescent cells exist 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 generation of reactive oxygen species (ROS). More and more studies have shown that the drug development for intervening in the senescence of various types of cells, such as cartilage, subchondral bone, synovium, and infrapatellar fat pad, has great development prospects (Cross-talk of inflammation and cellular senescence: a new insight into the occurrence and progression of osteoarthritis, Bone Research, 2024).Meanwhile, various methods of inducing senescence have been used in the research on the mechanisms, prevention, and control of osteoarthritis. For example, a subcutaneous injection of d-galactose was used to establish an animal model of osteoarthritis under senescence 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 senescence 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, the current treatment drugs for osteoarthritis are still hormones, analgesics, non-steroidal anti-inflammatory drugs, etc. These drugs mostly relieve pain and inflammation temporarily, are difficult to effectively prevent and control the development of the disease, and bring many side effects when used for a long time. Therefore, based on the rich evidence-based medicine evidence of traditional Chinese medicine, exploring new functional factors that can intervene in the senescence of articular chondrocytes and enhance the performance of articular cartilage is a very promising direction for the prevention and control of osteoarthritis.

[0004] Classical medical books such as Compendium of Materia Medica and Huangdi Neijing record that mulberry "enters the kidney meridian and benefits the five internal organs and joints", and "the kidney governs the bones and produces marrow", suggesting the potential value of mulberry in the treatment of osteoarthritis. However, the effective material basis, exact efficacy and mechanism of mulberry in the treatment of osteoarthritis are still unclear. Although the research paper "Mulberry water extract regulates miR-139 / MMP-14 to promote chondrogenic differentiation of BMSCs" reports that mulberry water extract can promote the differentiation of mesenchymal stem cells into chondrocytes. However, the purpose of this study was to explore the differentiation of mesenchymal stem cells promoted by mulberry extract to promote the application of cartilage tissue engineering, and there was no relevant research on chondrocyte injury and osteoarthritis. At the same time, the extract was prepared by a simple water-boiling process according to classical decoctions, and the effective part of mulberry was not determined. Therefore, in the face of the complex etiology and pathogenesis of osteoarthritis and the complex components of the water extract, it is difficult to determine the exact material basis of mulberry in the treatment of osteoarthritis from this, and it is also difficult to deduce its exact efficacy in the treatment of osteoarthritis. Summary of the Invention

[0005] Aiming at the problems of limited therapeutic effect of existing osteoarthritis drugs and many side effects of long-term use, the present invention provides an extract with the function of preventing and treating osteoarthritis and its application. The present invention uses mulberry as raw material, and obtains mulberry polyphenol extract with total polyphenol content of 3.8%-63.4% through different preparation processes. Through screening and evaluation with cell and animal osteoarthritis models, it is found that there is a clear dose-effect relationship between the total polyphenol content of mulberry and the intervention effect of osteoarthritis, indicating that the total polyphenol of mulberry is an effective part for intervening in osteoarthritis induced by aging and has broad application prospects.

[0006] The specific technical solutions are as follows:

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

[0008] Further, the total polyphenol content of the above extract is 5%-90%.

[0009] Further, the active ingredients of the above extract include phenolic acids, polyphenol derivatives, flavonoids, isoflavonoids, tannin substances.

[0010] Further, the active ingredients of the above extract include pelargonidin-3-O-rutinoside chloride, protocatechuic acid, cyanidin-3-O-galactoside, 8-prenylnaringenin, quercetin, kaempferol, procyanidin A2, luteolin, caffeic acid, chlorogenic acid, resveratrol, myricetin, gallic acid, catechol, epigallocatechin gallate, vanillic acid.

[0011] Further, the preparation method of the above extract comprises the following steps:

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

[0013] (2) Dissolve the mulberry powder in ethanol with a volume fraction of 70%, stir well, heat under ultrasonic assistance, and centrifuge to collect the supernatant; then add ethanol with a volume fraction of 70% to the residue, heat under ultrasonic assistance and centrifuge, and collect the supernatant;

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

[0015] (4) Load pretreated D101 macroporous adsorption resin into a glass chromatography column, dilute the concentrated solution, load it onto the glass chromatography column, after adsorption is completed, first elute with distilled water to remove some impurities, and then elute successively with ethanol solutions with volume fractions of 10%, 30%, and 60%, and collect the eluate;

[0016] (5) Concentrate the eluate under reduced pressure to remove ethanol, dilute it, and obtain mulberry extract by spray drying.

[0017] In a second aspect, the present invention provides an application of the above extract in the preparation of a product for preventing and treating osteoarthritis.

[0018] Further, the above product can achieve one or more of the following effects:

[0019] (1) Inhibit chondrocyte damage;

[0020] (2) Improve the reduction of proteoglycan secretion;

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

[0022] (4) Improve the abnormal pyknosis and distribution of cell nuclei in the hypertrophic zone of articular cartilage;

[0023] (5) Improve cartilage toughness;

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

[0025] Further, the above extract can be used alone or in combination to prepare a product for preventing and treating osteoarthritis.

[0026] Further, the above product is one of a drug, a health food, a functional food, and a food additive.

[0027] Furthermore, the above-mentioned drugs include tablets, capsules, injections, granules and suspensions; the above-mentioned health foods include tablets, hard capsules, soft capsules, oral liquids, milk powders, biscuits, candies, beverages and wines; the above-mentioned functional foods include functional dairy products, breads and beverages.

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

[0029] 1. The present invention provides an extract with the function of preventing and treating osteoarthritis. Using mulberry as the raw material, it is prepared by three processes: water extraction method, water boiling and alcohol precipitation, and alcohol extraction and refining, and a mulberry polyphenol extract with a total polyphenol content of 3.8%-63.4% is obtained. Then, an in vitro model of chondrocytes induced by hydrogen peroxide (H2O2) and an osteoarthritis animal model of subcutaneous injection of D-galactose are used to screen and evaluate the mulberry polyphenol extract, and it is found that the mulberry polyphenol extract can effectively improve chondrocyte damage and reduce proteoglycan secretion, significantly inhibit the pathological changes of articular cartilage, reduce the level of inflammatory factors in articular cartilage, and there is a clear dose-effect relationship between the effect and the total polyphenol content. It is revealed that the total polyphenols in mulberry are an effective part for the intervention of osteoarthritis, indicating that it has broad application prospects in the preparation of products for the prevention and treatment of osteoarthritis.

[0030] 2. The present invention also provides a preparation method of mulberry polyphenol extract and its application in the preparation of products for preventing and treating arthritis. The total polyphenol content of the mulberry extract obtained by ethanol extraction and macroporous adsorption resin refining is as high as 634.9 mg gallic acid / g, laying a practical foundation for the application of total polyphenols in mulberry in the fields of drugs, health foods and functional foods. Description of the Drawings

[0031] 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, other drawings can also be obtained based on these drawings without creative efforts.

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

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

[0034] Figure 3 It is the experimental result graph 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 4It is a graph showing the experimental results of the improvement effect of mulberry extracts (A, B, and C) with different total polyphenol contents in Example 6 on the reduction of proteoglycan caused by hydrogen peroxide.

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

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

[0038] Figure 7 It is a graph showing the experimental results of the improvement effect of different doses of mulberry extract C in Example 10 on the reduction of cartilage toughness caused by osteoarthritis. Detailed implementation manners

[0039] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention 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 of the embodiments. Based on the embodiments in 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.

[0040] 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 special restrictive 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.

[0041] 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.

[0042] The sources of the materials, reagents, and experimental equipment used in the present invention are as follows:

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

[0044] Gallic acid was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.;

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

[0046] The reagents used for LC-MS are all of chromatographic grade and were purchased from Sinopharm Chemical Reagent Co., Ltd.;

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

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

[0049] The CCK-8 solution was purchased from Wuhan Solarbio Science & Technology Co., Ltd.;

[0050] The toluidine blue staining solution was purchased from Wuhan Solarbio Science & Technology Co., Ltd.;

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

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

[0053] The EDTA decalcifying solution was purchased from Senbeijia Biotechnology Co., Ltd.;

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

[0055] Liquid chromatography ultra-high resolution mass spectrometer, model Orbitrap Exploris 480;

[0056] Nikon microscope, model ECLIPSE Ts2;

[0057] Electron universal material testing machine, model Instron 5943.

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

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

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

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

[0062] (3) Boil twice, 20 min each time, and combine the medicinal liquids.

[0063] (4) Filter the medicinal liquid to remove the residues to obtain the filtrate.

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

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

[0066] (1) Dry the mulberries at 60 °C and crush them using a multi-functional grinder.

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

[0068] (3) Boil for 2 h.

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

[0070] (5) Use an ethanol solution with a volume fraction of 50% to perform alcohol precipitation on the concentrated solution, and centrifuge (5000 rpm, 5 min), remove the supernatant, and collect the precipitate.

[0071] (6) Add deionized water to the alcohol-precipitated product to dilute it 15 times and centrifuge (3000 rpm, 5 min). Collect the supernatant and perform spray drying (inlet air temperature: 180 °C, needle passing: 5 s / time, peristaltic pump: 30%) to obtain mulberry extract B.

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

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

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

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

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

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

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

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

[0080] Referring to the "Method for Determination of Total Phenol Content" on page 69 of "Pharmacopoeia of the People's Republic of China: Volume I, 2020 Edition" and "National Standard of the People's Republic of China (GB / T 44349-2024)", the total polyphenol content in mulberry extracts A, B, and C prepared in Examples 1-3 was determined.

[0081] (1) Preparation of reference solution

[0082] An appropriate amount of gallic acid reference substance was accurately weighed and dissolved in water to make a solution containing 50 μg per 1 mL, that is, the reference solution was obtained.

[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, respectively, place them in 10 mL volumetric flasks, add 6 mL of water, shake well, then add 0.5 mL of Folin-Ciocalteu reagent B, shake well, and add 1.5 mL of 20% sodium carbonate solution within 0.5 - 8 min, and add water to the scale and shake well. Place it in a water bath at 75 °C for 10 min, use the corresponding reagent as a blank, and according to the ultraviolet-visible spectrophotometry, measure the absorbance at a wavelength of 760 nm. Taking the absorbance as the ordinate and the concentration as the abscissa, plot the standard curve (see Figure 1 ), and the regression equation is: Y = 0.0572X + 0.0731, R 2 = 0.9991.

[0085] (3) Take 2 mg of the extract, weigh it accurately, place it in a 2 mL centrifuge tube, add deionized water to the scale to obtain a dilution solution, and take 1.4 mL of the dilution solution into a 10 mL volumetric flask. According to the method in step (2), starting from "add 6 mL of water", measure the absorbance according to law, read the concentration of gallic acid equivalent in the test solution from the standard curve, and the calculation results are shown in Table 1.

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

[0087] Group Total polyphenol content (mg gallic acid / g) 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. Use liquid chromatography-mass spectrometry (LC-MS) to identify the components in the mulberry extract C of Example 3

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

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

[0091] (3) Filter the supernatant with a 0.22 μm filter membrane to obtain the test sample solution. Use a C18 chromatographic column, the column temperature is 40 °C, and the mobile phase is selected as acidified water and acetonitrile; use an electrospray ionization source (ESI) in the positive ion mode to capture the secondary spectrogram.

[0092] (4) Data analysis:

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

[0094] S2. Compare the obtained retention time and mass spectrometry data with the retention time and mass spectrometry information in the MS-Dail spectral library to identify the main polyphenol components in the extract;

[0095] S3. For unknown peaks, combine the literature data and mass spectrometry fragment information for structural analysis and identification.

[0096] Experimental results: Through LC-MS analysis, the main components of phenolic substances include phenolic acids, polyphenol derivatives, flavonoids, isoflavonoids, tannins and other phenolic substances, as Figure 2 shown. Further, combine the literature data and mass spectrometry fragment information for structural analysis and identification, and identify 16 components of phenolic substances and their derivatives. The specific information is shown in Table 2.

[0097] Table 2 Determination results of total polyphenol components in mulberry extract C

[0098]

[0099]

[0100] Example 5 Experiment on the Improvement Effect of Different Mulberry Extracts on Hydrogen Peroxide-Induced Chondrocyte Injury

[0101] (1) Accurately weigh appropriate amounts of mulberry extracts A, B, and C obtained in Examples 1, 2, and 3, and dissolve them in double-distilled water to prepare 20 mg / mL stock solutions, which are stored at -20 °C for later use.

[0102] (2) Add cell culture medium to a 96-well plate and culture primary chondrocytes, which are divided into a blank control group, a model group, an Example 1 group, an Example 2 group, and an Example 3 group. The control group only adds ordinary culture medium, and the Example 1 group, Example 2 group, and Example 3 group are respectively added with 0.06 mg / L of mulberry extract A solution, mulberry extract B solution, and mulberry extract C solution, with 3 replicates set for each group. After culturing for 24 h, the model group, Example 1 group, Example 2 group, and Example 3 group are added with a hydrogen peroxide solution with a final concentration of 0.1% and stimulated for 3 h. The OD value in the 96-well plate is measured using an enzyme-linked immunosorbent assay (ELISA) at an absorbance of 450 nm. The results are as Figure 3 shown.

[0103] It can be Figure 3 seen that 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 Example 1 group added with mulberry extract had no significant improvement effect, while the OD values of the Example 2 group and Example 3 group increased significantly, indicating that mulberry extract has an improvement effect on hydrogen peroxide-induced chondrocyte injury, and there is an obvious dose-effect relationship with the increase in the total polyphenol content.

[0104] Example 6 Improvement Effect of Different Mulberry Extracts on the Reduction of Proteoglycan Caused by Hydrogen Peroxide

[0105] (1) Accurately weigh appropriate amounts of mulberry extracts A, B, and C obtained in Examples 1, 2, and 3, and dissolve them in double-distilled water to prepare 20 mg / mL stock solutions, which are stored at -20 °C for later use.

[0106] (2) Add cell culture medium to a 24-well plate and culture primary chondrocytes, divided into a blank control group, a model group, an Example 1 group, an Example 2 group, and an Example 3 group. The control group only adds ordinary culture medium. The Example 1 group, the Example 2 group, and the Example 3 group are respectively added with 0.06 mg / L of mulberry extract A solution, mulberry extract B solution, and mulberry extract C solution, and each group is set with 3 replicates. After culturing for 24 h, the model group, the Example 1 group, the Example 2 group, and the Example 3 group are added with a hydrogen peroxide solution with a final concentration of 0.1% and stimulated for 3 h, and then fixed with 4% paraformaldehyde. Toluidine staining is used to measure the secretion of proteoglycan in each group, and the area of the positive staining region is measured by ImageJ software. The results are as Figure 4 shown.

[0107] It can be Figure 4 seen that compared with the blank control group, the secretion of proteoglycan by chondrocytes in the model group was significantly reduced. Compared with the model group, there was no significant improvement effect in the Example 1 group added with mulberry extract. Both the Example 2 group and the Example 3 group could significantly improve the reduction of proteoglycan secretion caused by hydrogen peroxide injury, increase the level of chondrocyte extracellular matrix, and there was an obvious dose-effect relationship with the increase of the total polyphenol content.

[0108] Improvement effect of different doses of mulberry extract C in Example 7 on hydrogen peroxide-induced chondrocyte injury

[0109] (1) Accurately weigh an appropriate amount of mulberry extract C obtained in Example 3, dissolve it with double-distilled water to prepare a 20 mg / mL mother liquor, and store it at -20 °C for later use.

[0110] (2) Dilute the mother liquor into mulberry extract C solutions of 3.125 μg / mL, 6.25 μg / mL, and 12.5 μg / mL. Add cell culture medium to a 96-well plate and culture primary chondrocytes, divided into a blank control group, a model group, an Example 3-1 group, an Example 3-2 group, and an Example 3-3 group. The Example 3-1 group, the Example 3-2 group, and the Example 3-3 group are respectively added with 3.125 μg / mL, 6.25 μg / mL, and 12.5 μg / mL of mulberry extract C solution, and each group is set with 3 replicates. After culturing for 24 h, the model group, the Example 3-1 group, the Example 3-2 group, and the Example 3-3 group are added with a hydrogen peroxide solution with a final concentration of 0.1% and stimulated for 3 h. The OD value in the 96-well plate is measured by an enzyme-linked immunosorbent assay at an absorbance of 450 nm. The results are as Figure 5 shown.

[0111] It can be Figure 5 seen that compared with the model group, mulberry extract C could significantly improve the injury caused by hydrogen peroxide, and with the increase of the dose, this promoting effect gradually increased, showing an obvious dose-effect relationship.

[0112] Example 8 Improvement effect of mulberry extract C at different doses on pathological changes of articular cartilage caused by osteoarthritis

[0113] (1) Weigh an appropriate amount of mulberry extract C obtained in Example 3 accurately, dissolve it with double-distilled water to prepare a 20 mg / mL stock solution, and store it at -20 °C for later use.

[0114] (2) SD rats aged 6 - 8 weeks 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:

[0115] ① Blank control group: Without any intervention;

[0116] ② Model group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily;

[0117] ③ Low-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 25 mg of mulberry extract C daily;

[0118] ④ Medium-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 50 mg of mulberry extract C daily;

[0119] ⑤ High-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 100 mg of mulberry extract C daily.

[0120] (3) After the experiment, the rats were sacrificed, and the cartilage tissue was taken out. After fixation, dehydration and paraffin embedding were carried out, and HE pathological section staining was performed. The results are as Figure 6 shown.

[0121] It can be Figure 6 seen that in the blank control group, the cells in the hypertrophic zone were evenly distributed and had regular morphology, and the extracellular matrix structure was clear and uniform, showing typical normal histological characteristics; in the model group, nuclear pyknosis and significant abnormal distribution of cells appeared in the hypertrophic zone, the extracellular matrix structure was damaged, and a large number of inflammatory cells were visible; 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 amount 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, mulberry extract C can improve the pathological changes of articular cartilage caused by osteoarthritis.

[0122] Example 9 Improvement effect of mulberry extract C at different doses on the increase in cartilage water content caused by osteoarthritis

[0123] (1)Accurately weigh an appropriate amount of the mulberry extract C obtained in Example 3, dissolve it with double-distilled water to prepare a 20 mg / mL stock solution, and store it at -20 °C for later use.

[0124] (2)SD rats aged 6 - 8 weeks 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:

[0125] ①Blank control group: Without any intervention;

[0126] ②Model group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily;

[0127] ③Low-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 25 mg of mulberry extract C daily;

[0128] ④Medium-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 50 mg of mulberry extract C daily;

[0129] ⑤High-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 100 mg of mulberry extract C daily.

[0130] (3)After the experiment, the rats were sacrificed, and the hip joint cartilage was immediately taken out. Use filter paper to carefully absorb the moisture on its surface, and accurately weigh the wet weight of the cartilage. Subsequently, the cartilage was placed in acetone for dehydration treatment for 24 hours. After dehydration, the cartilage was vacuum-dried at room temperature for 1 week until the weight was constant, and then the dry weight of the cartilage was accurately weighed. Finally, calculate the water content according to the formula. The calculation formula is: Water content = (wet weight - dry weight) ÷ wet weight × 100%, and the results are shown in Table 3.

[0131] Table 3 Measurement results of the water content of the articular cartilage of rats in each group

[0132] Group Moisture content (%) 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**

[0133] Note: # indicates a significant difference compared with the blank control group (p < 0.05); ** indicates a relatively significant difference compared with the model group (p < 0.01).

[0134] As can be seen from Table 3, compared with the blank control group, the water content of the articular cartilage of the rats in the model group increased significantly, indicating that osteoarthritis under aging conditions will show an increase in the water content of the articular cartilage. Compared with the model group, the water content of the articular cartilage of the rats in the low-dose group, the medium-dose group, and the high-dose group decreased significantly, showing a dose-dependent trend, indicating that low, medium, and high doses of mulberry extract C can effectively improve the increase in the water content of cartilage caused by osteoarthritis.

[0135] Example 10 Improvement effect of mulberry extract C at different doses on the reduced cartilage toughness caused by osteoarthritis

[0136] (1) Accurately weigh an appropriate amount of mulberry extract C obtained in Example 3, dissolve it with double-distilled water to prepare a 20 mg / mL stock solution, and store it at -20 °C for later use.

[0137] (2) SD rats aged 6 - 8 weeks are 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:

[0138] ① Blank control group: Without any intervention;

[0139] ② Model group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily;

[0140] ③ Low-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 25 mg of mulberry extract C daily;

[0141] ④ Medium-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 50 mg of mulberry extract C daily;

[0142] ⑤ High-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 100 mg of mulberry extract C daily.

[0143] (3) After the experiment, the rats are sacrificed, and the hip joint cartilage is taken out. Using a specifically calibrated mold, the cartilage is processed into tensile specimens that meet the standard size requirements. Use a high-precision measuring tool to measure and record the specimen size. Firmly install the prepared specimen at the center position of the calibrated tensile testing machine fixture, adjust the fixture to ensure that the specimen is uniformly stressed and axially consistent during the stretching process. Set the loading rate of the tensile testing machine to 1 mm / min, start the equipment, and during the stretching process, use the supporting data acquisition system to continuously record the load-displacement data in real time. Continue stretching until the specimen breaks, and stop the test. According to the recorded load-displacement data, calculate the elastic modulus of the articular cartilage according to the relevant mechanical calculation formula, and the results are as Figure 7 shown.

[0144] It can be Figure 7 seen that mulberry extract C can significantly increase the elastic modulus of articular cartilage, improve the reduced cartilage toughness caused by osteoarthritis, and with the increase of the dose, the improvement effect increases, showing an obvious dose-effect relationship.

[0145] Example 11 Improvement effect of mulberry extract C at different doses on the local inflammatory response caused by osteoarthritis

[0146] (1) Weigh an appropriate amount of the mulberry extract C obtained in Example 3 accurately, dissolve it with double-distilled water to prepare a 20 mg / mL stock solution, and store it at -20 °C for later use.

[0147] (2) SD rats aged 6 - 8 weeks 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:

[0148] ① Blank control group: Without any intervention;

[0149] ② Model group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily;

[0150] ③ Low-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 25 mg of mulberry extract C daily;

[0151] ④ Medium-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 50 mg of mulberry extract C daily;

[0152] ⑤ High-dose group: Subcutaneously inject D-galactose at a dose of 250 mg / kg daily, and intragastrically administer 100 mg of mulberry extract C daily.

[0153] (4) After the experiment, the rats were sacrificed, the hip joint cartilage was taken out, and the levels of tumor necrosis factor (TNF-α) and interleukin 6 (IL-6) in the articular cartilage were measured by enzyme-linked immunosorbent assay. The results are shown in Table 4.

[0154] Table 4 Levels of inflammatory factors in the articular cartilage of rats in each group

[0155] 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*

[0156] Note: # indicates significant difference compared with the blank control group (p < 0.05); * indicates significant difference compared with the model group

[0157] (p < 0.05)

[0158] As can be seen from Table 4, mulberry extract C can effectively inhibit the increase in the levels of inflammatory factors in articular cartilage caused by osteoarthritis, and with the increase in dose, the inhibitory effect is enhanced, showing an obvious dose-effect relationship.

[0159] Although the present invention has been described in detail by referring to the accompanying drawings and in conjunction with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. An extract having osteoarthritis prevention and treatment effects, characterized in that: The extract is a mulberry polyphenol extract and its monomer obtained by taking mulberry as raw material, adopting water and alcohol solvent extraction, or combining with 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 according to claim 1, characterized in that The active ingredients of the extract include phenolic acid, polyphenol derivatives, flavonoids, isoflavones and tannic acid substances.

4. The extract according to claim 3, characterized in that The active ingredients of the extract include pelargonidin-3-O-rutin chloride, protocatechuic acid, cyanidin-3-O-galactoside, 8-isopentenylnaringenin, quercetin, kaempferol, proanthocyanidin A2, luteolin, caffeic acid, chlorogenic acid, resveratrol, myricetin, gallic acid, catechol, epigallocatechin gallate, and vanillic acid.

5. The extract according to claim 1, characterized in that The preparation method of the extract comprises the following steps: (1) drying and crushing mulberries to prepare mulberry powder; (2) dissolving mulberry powder in 70% ethanol by volume, stirring thoroughly, heating under ultrasound assistance, and centrifuging to collect the supernatant; then adding 70% ethanol by volume to the residue, heating under ultrasound assistance, and centrifuging to collect the supernatant; (3) combining the collected supernatants and concentrating under reduced pressure to obtain a concentrate; (4) loading the pretreated D101 macroporous adsorption resin into a glass chromatography column, diluting the concentrated solution, and loading the sample into the glass chromatography column. After the adsorption is completed, firstly eluting with distilled water to remove some impurities, and then eluting with 10% by volume ethanol solution, 30% by volume ethanol solution, and 60% by volume ethanol solution in sequence, and collecting the eluate; (5) The eluate is concentrated under reduced pressure to remove ethanol, diluted and spray-dried to obtain a mulberry extract.

6. Use of the extract according to any one of claims 1 to 5 in the preparation of products for preventing and treating osteoarthritis.

7. The use according to claim 6, characterized in that The product can achieve one or more of the following effects: (1) Inhibit chondrocyte damage; (2) Improve the reduction of proteoglycan secretion; (3) Inhibit the increase of cartilage water content; (4) Improve the pyknosis and abnormal distribution of nuclei in the hypertrophic area of ​​articular cartilage; (5) Improve cartilage toughness; (6) Reduce inflammatory cells in articular cartilage and lower the levels of inflammatory factors.

8. The use according to claim 6, characterized in that The extracts can be used alone or in combination to prepare products for preventing and treating osteoarthritis.

9. The use according to claim 6, characterized in that The product is one of medicine, health food, functional food and food additive.

10. The use according to claim 9, 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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