Medicine for preventing and treating rheumatoid arthritis and / or osteoarthritis and application

Abstract

The invention discloses an application of at least one of notopterol, clematidoside or osthole in preparation of a medicine for treating or / and preventing rheumatoid arthritis and / or osteoarthritis. The invention also discloses a composition for treating or / and preventing rheumatoid arthritis and / or osteoarthritis. The composition takes a combination of at least two components selected from notopterygium alcohol, clematidoside and osthole as an effective component. The composition is a combination of notopterygium alcohol and clematidoside, or a combination of notopterygium alcohol and cnidium lactone, or a combination of clematidoside and cnidium lactone, or a combination of notopterygium alcohol, clematidoside and cnidium lactone. Cell experiments and animal experiments prove that the composition composed of at least two of notopterol, clematidoside and osthole can prevent and / or treat rheumatoid arthritis and osteoarthritis, and the treatment effect of the composition is obviously better than that of a single component.

Description

[0001] This application is a divisional application in accordance with the single defect pointed out by the examiner in the first notice of the first review on April 18, 2025 for the invention name "A medicine for preventing and treating rheumatoid arthritis and / or osteoarthritis and its application", application number "2023111317226", the invention name "A medicine for preventing and treating rheumatoid arthritis and / or osteoarthritis and its application", application number "2023111317226" is a divisional application submitted on November 23, 2022 with the invention name "A medicine for preventing and treating rheumatoid arthritis and / or osteoarthritis and its application", application number 2022114704048. TECHNICAL FIELD

[0002] The present application relates to a medicine with anti-inflammatory function, in particular to a medicine for treating or / and preventing rheumatoid arthritis and / or osteoarthritis and its application. BACKGROUND

[0003] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease mainly manifested by erosive, symmetrical and polyarticular arthritis. Studies have shown that RA mainly involves small joints such as wrists and feet in the early stage, and gradually invades large joints and extra-articular connective tissue as the disease progresses, and can cause systemic complications in the eyes, heart, lungs and nervous system of the body, seriously affecting the patient's mobility and reducing the patient's quality of life, causing a heavy economic burden to the patient's family and even the society. Epidemiology shows that rheumatoid arthritis accounts for about 0.5-1% of the total global population. At the same time, the incidence of RA is related to gender and age, and the incidence of men is about three times that of women, while the incidence of men increases with age.

[0004] Osteoarthritis (OA), also known as degenerative arthritis, is the most common joint disease characterized by damage to articular cartilage and involvement of the entire joint tissue. The main pathological features of OA in clinical practice are degeneration and destruction of articular cartilage, osteosclerosis under the cartilage, hyperplasia of joint margin bone, hyperplasia and inflammation of synovial membrane, etc. Epidemiological studies have shown that the incidence of OA is closely related to age, gender, body mass index, etc., especially in postmenopausal women. With the progression of the disease, OA patients show joint deformation, pain, stiffness and swelling, and can even be severely disabled, seriously affecting the patient's quality of life.

[0005] Currently, patients with osteoarthritis and rheumatoid arthritis are often encountered in clinical medicine, and clinical medicine research shows that there is some similarity between the pathogenesis of rheumatoid arthritis in the early stage and the pathogenesis of osteoarthritis. Osteoarthritis is a pathological change of joint cartilage degeneration, softening, disappearance, and new bone formation at the joint edge. It may be due to congenital joint anatomical abnormalities, damaged bone avascular necrosis, intra-articular infection, intra-articular deposition of crystals, etc. Rheumatoid arthritis is a chronic autoimmune disease characterized by progressive joint disease. Its characteristics are symmetrical polyarthritis, with the most common involvement of the hands, wrist joints, elbow joints, knee joints, ankle joints and foot joints, and other joints of the whole body can also be involved. Clinically, rheumatoid arthritis and osteoarthritis both show joint inflammation, and belong to two diseases with similar clinical characteristics. At present, there are many drugs used to treat RA and OA in clinical practice: such as non-steroidal anti-inflammatory drugs with antipyretic, analgesic and anti-inflammatory effects; anti-rheumatic drug methotrexate; adrenal cortex hormone, etc., but all have some shortcomings, such as non-steroidal anti-inflammatory drugs are metabolized quickly in the body, and the maintenance time is short; long-term use of adrenal cortex hormone may exacerbate the symptoms, and two kinds of diseases need to be given different drugs for treatment, and the combination of different types of drugs may have potential adverse reactions or toxic side effects. SUMMARY

[0006] The corner medicine is based on the basic theory of traditional Chinese medicine, based on the premise of syndrome differentiation and treatment, and based on the compatibility principle of the smell, performance and seven emotions of traditional Chinese medicine. Three kinds of traditional Chinese medicine are used together, which can play a role in reducing toxicity and increasing efficacy in clinical application. Notopterygium and Angelica dahurica have the functions of relieving the surface and dispelling wind and cold, and dispelling wind and dampness, among which Notopterygium has strong dispersing and relieving surface, and Angelica dahurica has strong dispelling wind and dampness. Radix Stephaniae Tetrandrae has the functions of dispelling wind and dampness and freeing joints, and the three are used as corner medicine to reflect the efficacy of treating from "cold and dampness". Notopterygium alcohol is one of the chemical components of Notopterygium or Notopterygium forrestii, and Stephaniae Tetrandrae New Glycoside and Cusparidin are chemical components of Radix Stephaniae Tetrandrae and Angelica dahurica, respectively. The component traditional Chinese medicine has the advantages of clear composition and clear ratio, and is an important development direction of traditional Chinese medicine compound medicine. The efficacy is consistent with or more significant than that of traditional Chinese medicine, and the quality is controllable, which can be used for industrialization and popularization. Notopterygium alcohol, Stephaniae Tetrandrae New Glycoside and Cusparidin are from Notopterygium, Radix Stephaniae Tetrandrae and Angelica dahurica, which are dispelling wind and dampness. At least two compounds selected from Notopterygium alcohol, Stephaniae Tetrandrae New Glycoside and Cusparidin are used to form a component medicine, so as to better play the synergistic effect of multi-component and multi-target, improve the immune capacity of the body, and be used for the treatment of rheumatoid arthritis, osteoarthritis and the two diseases, and construct a new treatment scheme of component combination medicine based on traditional Chinese medicine compound for treating complex diseases and their comorbidities.

[0007] The purpose of this invention is to address the disadvantages of existing drugs used clinically to treat rheumatoid arthritis (RA) and osteoarthritis (OA), which have varying degrees of toxic side effects or adverse reactions. The inventors have combined at least two of the following ingredients—Notopterygium alcohol, Lingxianxin glycoside, and Osthol—into a novel composition. Notopterygium alcohol and Lingxianxin glycoside excel in analgesia and anti-inflammation, while Osthol enhances the inhibitory effects on the central nervous system through its immune-boosting, sedative, and analgesic properties. This multi-component synergistic effect is used to improve pain, exert anti-inflammatory functions, and treat rheumatoid arthritis, osteoarthritis, and comorbidities. This invention provides a pharmaceutical composition derived from traditional Chinese medicine, combining active ingredients to achieve synergistic effects. This composition can more safely and effectively prevent or treat rheumatoid arthritis and / or osteoarthritis, or comorbidities, offering patients a safer, more effective, and quality-controlled innovative combination drug option to improve their pathological condition and quality of life.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] The use of at least one of gentianol, lingxianxin glycoside or osthol in the preparation of medicaments for the treatment and / or prevention of rheumatoid arthritis and / or osteoarthritis.

[0010] The application described is the use of notopterygium alcohol in the preparation of drugs for the treatment and / or prevention of rheumatoid arthritis and / or osteoarthritis;

[0011] Or the application described herein is the use of lecithin in the preparation of drugs for the treatment and / or prevention of rheumatoid arthritis and / or osteoarthritis;

[0012] Or the application described is the use of osthol in the preparation of drugs for the treatment and / or prevention of rheumatoid arthritis and / or osteoarthritis;

[0013] Or the application described herein is the use of notopterygium alcohol and lingxianxin glycoside as active ingredients in the preparation of drugs for the treatment and / or prevention of rheumatoid arthritis and / or osteoarthritis;

[0014] Or the application described herein is the use of notopterygium alcohol and osthol as active ingredients in the preparation of drugs for the treatment and / or prevention of rheumatoid arthritis and / or osteoarthritis;

[0015] Alternatively, the application may be the use of clematisin and osthol as active ingredients in the preparation of drugs for the treatment and / or prevention of rheumatoid arthritis and / or osteoarthritis.

[0016] The application described is the use of gentianol, lingxianxin glycoside and osthol as active ingredients in the preparation of drugs for the treatment and / or prevention of rheumatoid arthritis and / or osteoarthritis.

[0017] The medicine is in pharmaceutically acceptable dosage form prepared with at least one of effective components selected from the group consisting of Ziyang alcohol, Lingxian neoside and Cnidium seed element and pharmaceutically acceptable pharmaceutical adjuvant.

[0018] Another object of the present application is to provide a composition for treating or / and preventing rheumatoid arthritis and / or osteoarthritis, which is prepared with a combination of at least two components selected from the group consisting of Ziyang alcohol, Lingxian neoside and Cnidium seed element.

[0019] Preferably, the composition is a combination of Ziyang alcohol and Lingxian neoside, or a combination of Ziyang alcohol and Cnidium seed element, or a combination of Lingxian neoside and Cnidium seed element, or a combination of Ziyang alcohol, Lingxian neoside and Cnidium seed element.

[0020] When the composition is a combination of Ziyang alcohol and Lingxian neoside, the molar ratio of Ziyang alcohol and Lingxian neoside is 5.5:1-1:1.5, and specifically can be 5.1:1 (i.e. the mass ratio of Ziyang alcohol and Lingxian neoside is 1:1), 7:3, 1:1, 1:1.5, and preferably 5.1:1, 1:1.

[0021] When the composition is a combination of Ziyang alcohol and Cnidium seed element, the molar ratio of Ziyang alcohol and Cnidium seed element is 7:3-1:1.5, and specifically can be 7:3, 1:1, 1:1.45 (i.e. the mass ratio of Ziyang alcohol and Cnidium seed element is 1:1), 1:1.5, and preferably 1:1, 1:1.45.

[0022] When the composition is a combination of Lingxian neoside and Cnidium seed element, the molar ratio of Lingxian neoside and Cnidium seed element is 7:3-1:7.5, and specifically can be 7:3, 1:1, 1:1.5, 1:7.4 (i.e. the mass ratio of Lingxian neoside and Cnidium seed element is 1:1), and preferably 1:1, 1:7.4.

[0023] When the composition is a combination of Ziyang alcohol, Lingxian neoside and Cnidium seed element, the molar ratio of Ziyang alcohol, Lingxian neoside and Cnidium seed element is (1-5.5):1:(1-7.5), and specifically can be 1:1:1, 5.1:1:7.4 (i.e. the mass ratio of Ziyang alcohol, Lingxian neoside and Cnidium seed element is 1:1:1).

[0024] Another object of the present application is to provide the use of the composition in the preparation of a medicine, health care product or functional food for preventing or / and treating rheumatoid arthritis and / or osteoarthritis.

[0025] The composition is prepared with pharmaceutically acceptable pharmaceutical adjuvant into ointment, liniment, patch, lotion, solution, powder, spray, syrup, suppository, tablet, pill, granule, capsule, injection or gastro-soluble controlled-release preparation.

[0026] The rheumatoid arthritis and / or osteoarthritis is rheumatoid arthritis, osteoarthritis, rheumatoid arthritis and osteoarthritis.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] (1) Each component of the present application is from a commonly used natural medicine for dispelling wind and removing dampness, and the two or three are used in combination, and are systematically matched to achieve the effects of dispelling wind and removing dampness, warming the meridians and unblocking the collaterals, and freeing the joints.

[0029] (2) Each composition of the present application is composed of natural medicine-derived active monomers of traditional Chinese medicine, has small toxic and side effects, the components are relatively clear, the mechanism is relatively clear, the curative effect is consistent with or more significant than that of traditional Chinese medicine, and the quality is controllable.

[0030] (3) Each composition of the present application has no obvious toxicity, and in vitro cell experiments show that it can significantly improve the survival rate of RAW264.7 cells and synovial cells.

[0031] (4) The results of the RAW264.7 cell in vitro inflammatory cell model show that each composition in the present application can down-regulate the inflammatory factor NO level of LPS-induced RAW264.7 cells, and has good anti-inflammatory activity; different compositions can inhibit FLS cell proliferation and induce apoptosis.

[0032] (5) The composition of the present application can reduce the levels of inflammatory factors in the serum of CIA rats and OA rats, and increase the level of anti-inflammatory factors, and has significant anti-rheumatoid arthritis, osteoarthritis and the possibility of co-morbidity of the two diseases. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The contents of TNF-α, IL-1β, IL-6 and VEGF in the serum of rats in different treatment groups; wherein, Con: normal group, Mod: model group, Ibu: positive control group (ibuprofen), Tri: positive control group (Tripterygium glycoside tablets), Not: notopterol, Cle: lindleyin, Ost: osthol, N+C: notopterol+osthol, N+O: notopterol+lindleyin, C+O: lindleyin+osthol, N+C+O: notopterol+lindleyin+osthol; compared with the blank group, ### P<0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0034] Figure 2HE staining results of ankle joints of type II collagen-induced arthritis rats in different treatment groups; among them, A: normal group, B: model group, C: positive control group (ibuprofen), D: positive control group (Tripterygium glycosides tablets), E: patchouli alcohol, F: lindley neoside, G: cnidium lactone, H: patchouli alcohol + cnidium lactone, I: patchouli alcohol + lindley neoside, J: lindley neoside + cnidium lactone, K: patchouli alcohol + lindley neoside + cnidium lactone. DETAILED DESCRIPTION

[0035] The technical solutions of the present application will be further described below in combination with specific embodiments.

[0036] Example 1

[0037] RAW264.7 cell activity verification

[0038] The causes of arthritis are complex, mainly related to autoimmune response, infection, metabolic disorder, trauma, degenerative disease and other factors. According to the cause, arthritis can be divided into bone, rheumatoid arthritis and the like. RAW264.7 (mouse monocyte macrophage leukemia cells) is the main immune effector cell, which is widely used in inflammation, immunity and apoptosis research. Therefore, effectively inhibiting the activation and proliferation of RAW264.7 cells and reducing the level of inflammatory factors is the key to anti-inflammatory. The present application uses lipopolysaccharide (LPS) induced RAW264.7 cells to establish an in vitro inflammatory cell model, and uses CCK-8 method and Griess experiment to investigate the influence of the composition of the present application on cell activity and inflammatory factor NO.

[0039] Positive control drug: dexamethasone.

[0040] 1 Experimental method

[0041] 1.1 CCK-8 method for detecting cell activity

[0042] Preparation of LPS solution: weigh 4 mg of LPS powder, dissolve in 40 mL of DMEM medium to obtain a 100 μg / mL stock solution, divide into 1.5 mL EP tubes, each tube contains 200 μL, and store at -80℃.

[0043] Take RAW264.7 cells in logarithmic growth phase, inoculate in 96-well plates, and the cell inoculation density is 5×10 3 / mL, 100 μL cell suspension per well, and incubated in an incubator for 24 h; a blank group, a model group (LPS), a positive control group (dexamethasone), and a drug administration group (according to Table 1, administered with Notopterygium alcohol, Lingxianoside, Osthole, Notopterygium alcohol + Lingxianoside, Notopterygium alcohol + Osthole, Lingxianoside + Osthole, Notopterygium alcohol + Lingxianoside + Osthole) were set up; the positive control group and the drug administration group were first stimulated with LPS for 10 h, and then 10 μL of the sample to be tested and the cell suspension were added to each well; the blank group was not added with the sample but only with the culture solution; 5 replicate wells were set up for each group; then the 96-well plate was placed in an incubator for continuous incubation for 24 h, 10 μL of CCK-8 reagent was added to each well in the dark, and the incubation was continued for 1 h. The OD 450 was measured by an enzyme marker, and the cell survival rate was calculated.

[0044] (1) The blank group (CON): DMEM culture medium;

[0045] (2) The model group (MOD): DMEM culture medium + LPS (1 μg / mL);

[0046] (3) The positive control group: DMEM culture medium + dexamethasone (20 μM, final concentration) + LPS (1 μg / mL);

[0047] (4) The drug administration group: DMEM culture medium + a solution of different compound combinations (30 μM, final concentration) + LPS (1 μg / mL).

[0048] 1.2 Griess experiment

[0049] Logarithmic growth period RAW264.7 cells were taken, inoculated in a 96-well plate, the cell inoculation density was 2×10 5 / mL, 100 μL cell suspension per well, and incubated in an incubator for 24 h, and the supernatant was discarded; a blank group, a model group (LPS), a positive control group (dexamethasone), and a drug administration group were set up, and LPS was first added to the positive control group and the drug administration group for stimulation for 10 h, and then the sample to be tested and the cell suspension were added to each well; the blank group was not added with the sample but only with the culture solution; 5 replicate wells were set up for each group. After incubation in an incubator for 24 h, 100 μL of the supernatant was taken from each well to a clean 96-well plate, 100 μL of Griess reagent was added to each well, mixed thoroughly, and developed in the dark for 10 min, and the OD 540 was measured. The measured OD 540 was substituted into the standard equation drawn by the standard sodium nitrite solution, and the content of the released NO in the supernatant of the RAW264.7 cells was calculated.

[0050] The NO inhibition rate was calculated according to the following formula:

[0051] NO inhibition rate (%) = (C 模型 -C给药 ) / (C 模型 -C 空白 )×100%

[0052] wherein, C 模型 represents the concentration value of the model group, C 给药 represents the concentration value of the positive control group or the administration group, and C 空白 represents the concentration value of the blank group.

[0053] 2 Experimental results

[0054] Table 1 shows that the administration of cnidilide, neohesperidoside, osthole, cnidilide + neohesperidoside, cnidilide + osthole, neohesperidoside + osthole, and cnidilide + neohesperidoside + osthole can inhibit the production of NO, indicating that they have certain anti-inflammatory activity in vitro, and the effect of the composition is significantly better than that of single drug.

[0055] Table 1. Effects of different compositions on the activity and NO release of LPS-induced RAW264.7 cells (n = 5, x ± SD)

[0056]

[0057] Note: The dosage ratio of the compound is molar ratio; compared with the model group, ** P < 0.01, *** P < 0.001, compared with the positive control group, && P < 0.01, there is a significant difference.

[0058] Example 2

[0059] Effect of drugs on the proliferation of FLS-RA cells stimulated by TNFα

[0060] One of the main pathological manifestations of RA is the proliferation of synovial tissue, and the apoptosis defect of synovial cells is an important pathological mechanism of RA. Synovial cells can respond to and mediate inflammatory reactions, directly destroy cartilage and bone tissue and mediate the destruction of other cells to soft tissue, and thus become an important target for the treatment of arthritis. Synovial fibroblasts (FLS) are an important component of synovial tissue. Therefore, the present application uses a synovial fibroblast proliferation model and a synovial fibroblast apoptosis model induced by tumor necrosis factor α (TNFα) to investigate the effect of the composition of the present application on the proliferation and apoptosis of synovial fibroblasts.

[0061] 1 Experimental method

[0062] 1.1 CCK-8 method for detecting the proliferation inhibition effect of drugs on FLS cells

[0063] Logarithmic growth phase FLS cells were harvested, digested with trypsin, and seeded into 96-well culture plates at a density of 1 × 10⁶ cells / well. 4 / well, 200 μL per well, incubated at 37℃ for 24 h, supernatant discarded, and divided into: blank group (DMEM medium containing 0.01% DMSO), model group (DMEM medium containing 0.01% DMSO + 10 ng / mL TNFα), and drug treatment group (DMEM medium containing 0.01% DMSO + 10 ng / mL TNFα; the drug treatment group was stimulated with TNFα for 12 h, and then administered according to Table 2: notopterygium alcohol, lingxianxin glycoside, osthol, notopterygium alcohol + lingxianxin glycoside, lingxianxin glycoside + osthol, notopterygium alcohol + lingxianxin glycoside + osthol), with 5 replicates for each group. Incubated for 48 h, and then incubated for 2 h at a CCK8 reagent:DMEM medium volume ratio of 1:10. OD value at 450 nm was measured using a microplate reader.

[0064] 1.2 Flow cytometry detection of FLS cell apoptosis

[0065] Log-phase FLS cells were seeded into 6-well plates at a density of 2.5 × 10⁶ cells / well. 4 / well, cells were cultured overnight, washed with pre-warmed (37℃) PBS and then allowed to rest for 6 hours. The grouping and drug administration were the same as in "1.1 CCK-8 assay for the inhibitory effect of drugs on the proliferation of FLS cells". 48 hours after drug administration, cell apoptosis was examined by flow cytometry.

[0066] Annexin V-FITC apoptosis detection was performed according to the kit instructions: Cells were transferred to centrifuge tubes, washed twice with PBS, digested with trypsin, centrifuged at 1000g for 5 min, the supernatant was discarded, and the cells were resuspended in 195 μL of Annexin V-FITC binding buffer. Then, 5 μL of Annexin V-FITC was added, mixed well, and 10 μL of propidium iodide staining solution was added, followed by mixing. The cells were incubated at room temperature in the dark for 20 min, resuspending them three times during incubation to improve staining results. Finally, the fluorescence intensity was detected by flow cytometry (Annexin V-FITC is green fluorescence, PI is red fluorescence).

[0067] 2 Experimental Results

[0068] The results are shown in Table 2: Both single drugs and different combinations could inhibit the proliferation of FLS cells, especially the different combinations, which were more effective than single drugs, suggesting possible anti-inflammatory activity. Simultaneously, the different combinations also induced FLS cell apoptosis, and their effects were significantly better than those of single drugs, consistent with the results regarding their anti-inflammatory activity.

[0069] Table 2. Effects of different compositions on inhibition of TNFα-induced FLS cell proliferation and induction of FLS cell apoptosis (n=5, x±SD)

[0070]

[0071] Note: The dosage of the compound is molar ratio; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0072] Example 3

[0073] Effects of different compositions on collagen type II-induced arthritis (CIA) rat models

[0074] 1. Experimental method

[0075] 1.1 Experimental grouping

[0076] SPF level healthy Wistar rats of about 8 weeks old, weighing 130-150 g, were randomly divided into 9 groups, one cage for each group: normal group, model group, positive drug group (two groups of positive drug group, each group was given ibuprofen and tripterygium glycosides tablets, respectively) and drug administration group (given n- butylphthalide, linxian neoside, osthole, n-butylphthalide+linxian neoside, n-butylphthalide+osthole, linxian neoside+osthole, n-butylphthalide+linxian neoside+osthole). Except for the normal group, the rest of the groups were induced to construct arthritis rat models with collagen type II. The specific modeling method was as follows: first immunization, collagen type II was dissolved in 0.05M glacial acetic acid to prepare a collagen solution with a concentration of 2mg / mL, which was placed at 4℃ overnight; the collagen solution and CFA (complete Freund's adjuvant) were mixed at a volume ratio of 1:1 and emulsified on ice, and each rat was injected with 200μg of collagen type II subcutaneously in the tail; one week later, the second immunization was performed using the same method to emulsify the collagen solution and IFA (incomplete Freund's adjuvant), and each rat was injected with 200μg of collagen type II subcutaneously, for a total of two weeks (the first day of immunization was day 1), to obtain collagen type II-induced arthritis (CIA) rat models.

[0077] Based on this model, continuous gavage was started on day 15 for 21 days. The dosage of ibuprofen was 6.75mg / kg / d, and the dosage of tripterygium glycosides tablets was 18.90mg / kg / d. In each drug administration group, the total dosage was 20mg / kg / d. In the compositions, the mass ratio of n-butylphthalide+linxian neoside, n-butylphthalide+osthole, and linxian neoside+osthole was 1:1, and the mass ratio of n-butylphthalide+linxian neoside+osthole was 1:1:1.

[0078] 1.2 HE staining observation

[0079] According to the grouping of "1.1 Experimental grouping", the rats were administered for 21 days, and the ankle joints of the rats in the blank group, model group, positive control group and different administration groups were taken respectively for frozen section, and then HE staining was performed.

[0080] The rat ankle joints were fixed with 4% paraformaldehyde for 24 h, then decalcified with 10% EDTA at 37°C, and transferred into phosphate buffer for gradient dehydration. After dehydration, the sections were cut by a freezing microtome, attached to gelatin-treated glass slides and dried. After washing with water for 1-2 s, 95% ethanol for 1-2 s, anhydrous ethanol for 1-2 s, xylene for 2-3 s, and sealing with neutral balsam, the sections were observed under a microscope and photographed.

[0081] 1.3 ELISA method for detecting the concentrations of TNF-α, IL-1β, IL-6 and VEGF in serum

[0082] According to the grouping of "1.1 Experimental grouping", the rats were administered for 21 days, and the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the abdominal aorta was used to take blood, and the blood was centrifuged at 3000 r·min -1 After centrifugation for 10 min, the serum was taken. The levels of TNF-α, IL-1β, IL-6 and VEGF in the serum of the rats in the blank group, model group, positive control group and different administration groups were detected by ELISA method.

[0083] 2 Experimental results

[0084] During the modeling stage, compared with the normal group, the rats in the model group had red and swollen feet and swollen ankle joints. After the model was established, the swelling degree decreased and the inflammation subsided after administration. Inflammatory scores were performed from the 14th day after modeling, and the results showed that there was a significant difference between the model group and the normal group, indicating that the modeling was successful.

[0085] As shown in Figure 1 , the levels of TNF-α, IL-1β, IL-6 and VEGF in the serum of the rats in the model group were significantly increased. After administration of positive drugs (ibuprofen or tripterygium glycoside tablets), cnidilide, lindleyin, osthole, cnidilide + lindleyin, cnidilide + osthole, lindleyin + osthole, cnidilide + lindleyin + osthole, the positive drugs, single drugs and different combinations all showed inhibitory effects on TNF-α, IL-1β, IL-6 and VEGF in the serum of the rats, and the inhibitory effects of the combinations were significantly better than those of the single compounds.

[0086] The results of HE staining are shown in Figure 2 , which shows that the synovial cell proliferation in the model group is obvious, and there are visible tissue hyperemia and inflammatory cell infiltration. The positive drug group and different administration groups all show different degrees of pathological changes, but compared with the model group, the pathology is improved, and the effect of the combination is better than that of the single drug, indicating that they may have anti-rheumatoid arthritis activity.

[0087] Example 4

[0088] Effects of different compositions on rat models of osteoarthritis

[0089] 1. Experimental method

[0090] 1.1 Experimental grouping

[0091] Male SPF healthy Wistar rats weighing 130-150 g were randomly divided into 9 groups, one cage for each group: a normal group, a model group, a positive control group (glucosamine hydrochloride capsules), and different administration groups (administered with n-Butylidenephthalide, Lingxianoside, Ostruthin, n-Butylidenephthalide + Lingxianoside, n-Butylidenephthalide + Ostruthin, Lingxianoside + Ostruthin, n-Butylidenephthalide + Lingxianoside + Ostruthin). Except for the normal group, the rest of the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the hair around the knee joint of the model side was removed clean with a hair clipper. The rat knees were flexed, and 50 μL of physiological saline containing 3 mg of sodium iodoacetate was injected into the knee joint cavity of the rest of the rats in each group once a day for 14 consecutive days to induce rat osteoarthritis models. The normal group was injected with the same volume of normal saline. After the last injection of sodium iodoacetate solution into the knee joint cavity, the positive control group (glucosamine hydrochloride capsules) and each administration group were given the corresponding concentration of the test drug by gavage once a day, for a total of 4 weeks of administration.

[0092] The administration dose of glucosamine hydrochloride capsules (GH) was 25 mg / kg / d; the total administration dose of the rest of the administration groups was 20 mg / kg / d. In the administration of the composition, the mass ratio of the two drugs in n-Butylidenephthalide + Lingxianoside, n-Butylidenephthalide + Ostruthin, and Lingxianoside + Ostruthin was 1:1, and the mass ratio of the three drugs in n-Butylidenephthalide + Lingxianoside + Ostruthin was 1:1:1.

[0093] 1.2 Macroscopic observation and scoring of the knee joint

[0094] The luster, integrity, and presence or absence of fissures, erosion, and defects on the surface of the knee joint cartilage were observed with the naked eye, and the femoral condyle and tibial plateau of the rat knee joint were scored using the Pelletier scoring method.

[0095] Scoring criteria: 0 points, joint surface is complete and smooth, color is normal; 1 point, joint surface is rough, has small fissures, and color is dark; 2 points, joint surface is eroded, and cartilage erosion extends to the surface or middle layer; 3 points, joint surface ulceration, cartilage erosion reaches the deep layer of cartilage; 4 points, cartilage is detached, and subchondral bone is exposed.

[0096] 1.3 HE staining observation

[0097] According to the grouping of "1.1 Experimental grouping", the rats were administered for 4 weeks, and the knee joints of the rats in the blank group, model group, positive control group and different administration groups were taken, respectively. The knee joints of the rats were fixed with 4% paraformaldehyde for 24 h, then decalcified with 10% EDTA at 37℃, and transferred into phosphate buffer for gradient dehydration. After dehydration, the samples were sectioned by a freezing microtome, attached to gelatin-treated glass slides and dried. After washing with water for 1-2 s, 95% ethanol for 1-2 s, anhydrous ethanol for 1-2 s, xylene for 2-3 s, and sealing with neutral balsam, the samples were observed and photographed under a microscope.

[0098] 1.4 ELISA method for detecting the concentrations of IL-1β, TNF-α and IL-10 in serum

[0099] The rats were anesthetized by intraperitoneal injection of 10% (v / v) chloral hydrate, and blood was taken from the abdominal aorta. The blood was centrifuged at 3000 r·min -1 After centrifugation for 10 min, the serum was taken. The levels of IL-1β, TNF-α and IL-10 in the serum of the rats in the blank group, model group, positive control group and different administration groups were detected by ELISA.

[0100] 2 Experimental results

[0101] Table 3. Macroscopic observation score table of the femoral condyle and tibial plateau cartilage of the rats n = 6

[0102]

[0103] Note: ### P < 0.001, indicating that the model group had a significant difference compared with the normal group; compared with the model group, *P < 0.05, **P < 0.01 and ***P < 0.001, the different administration groups had significant differences, and compared with the positive control group, &&& P < 0.001, indicating a significant difference.

[0104] According to the macroscopic score results in Table 3, it was found that compared with the normal group, the knee joints of the rats in the model group were swollen during the modeling stage; the articular cartilage surface of the knee joints of the rats in the normal group was complete and smooth, and the articular cartilage of the rats in the model group was exfoliated, and the subchondral bone was exposed. After administration of the positive drug, n-jojoba alcohol, lirioresin, cnidim, n-jojoba alcohol + lirioresin, n-jojoba alcohol + cnidim, lirioresin + cnidim, n-jojoba alcohol + lirioresin + cnidim, the swelling degree of the knee joints of the rats decreased; compared with the model group, the positive control group and different administration groups could alleviate the cartilage damage to different degrees, especially the rats in the different composition administration groups only showed slight damage to the articular cartilage. In addition, the articular cartilage damage in the model group was severe, and the subchondral bone was exposed. The cartilage damage degree in the different administration groups was reduced, the knee joints of the rats showed slight cartilage changes, and the joint space almost returned to normal. Moreover, the effect of the composition was better than that of the single drug, suggesting that it might have an active effect against rheumatoid arthritis.

[0105] The results of HE staining of the knee joint showed that the cartilage surface of the normal group of rats was smooth and complete, and the joint space was normal. The knee joint cartilage of the model group of rats was severely damaged, and the joint space was enlarged. The knee joints of each drug administration group and the model group of rats had similar pathological changes, but the cartilage damage was reduced, and the joint space was restored. Among them, the knee joint cartilage of rats in different drug administration groups was relatively complete, and the joint space was basically restored to normal. The isolated knee femoral condyle tissue was taken for HE staining to observe the changes of articular cartilage. The cartilage surface of the femoral condyle tissue of the normal group of rats was smooth. The femoral condyle cartilage surface of the model group was rough and irregular, and the cartilage was severely damaged. The femoral condyle cartilage of the positive control group of rats was hyperplastic, accompanied by cartilage damage, and the surface was uneven. After administration of Qianghuayu alcohol, Lingsuaxin or Cnidim, the cartilage of the femoral condyle tissue of rats was severely damaged, but it was lighter than that of the model group. The cartilage damage of Qianghuayu alcohol + Lingsuaxin, Qianghuayu alcohol + Cnidim, Lingsuaxin + Cnidim and Qianghuayu alcohol + Lingsuaxin + Cnidim groups was lighter, especially the cartilage surface of Qianghuayu alcohol + Cnidim group was relatively smooth. It showed that different drug administration groups could improve the pathological changes of cartilage, and the effect of the combination was better than that of single component administration group.

[0106] As shown in Table 4, compared with the normal group of rats, the levels of TNF-α, IL-1β and IL-10 in the serum of the model group of rats were significantly increased. The administration of positive drugs and single drugs and different combinations showed inhibitory effect on TNF-α, IL-1β and IL-10 in the serum of rats, and the effect of combination was better than that of single drug, which might have anti-osteoarthritis activity.

[0107] Table 4. Determination of TNF-α, IL-1β and IL-10 levels in the serum of rats n = 6

[0108]

[0109] Note: ### P < 0.001, indicating that the model group was significantly different from the normal group; compared with the model group, *P < 0.05, **P < 0.01 and ***P < 0.001, there were significant differences, compared with the positive control group, && P < 0.01 and && P < 0.001, there were significant differences.

[0110] In summary, the inventors established the in vitro models of RAW264.7 cells and synovial cells by cell experiments. The results show that the single compound or different combinations of the compounds can down-regulate the level of inflammatory factor NO of LPS-induced RAW264.7 cells; different combinations can inhibit the proliferation of TNFα-induced FLS cells and can induce the apoptosis of FLS cells to a certain extent, showing certain anti-inflammatory activity. The results of cell experiments show that the single traditional Chinese medicine active ingredient or the combination thereof can effectively prevent and treat rheumatoid arthritis and osteoarthritis, and the effect of the combination is significantly better than that of the single ingredient.

[0111] In the exploration of the effects of single compounds and combinations on the inflammation of collagen-induced rheumatoid arthritis rats, Wistar rats were randomly divided into a normal group, a model group, a positive drug group (ibuprofen and tripterygium glycosides tablets), and a drug administration group of cnidium alcohol, neolignan and cnidium seed, and different combinations of the above three compounds. Except for the normal group, the rest of the groups were induced to construct the arthritis rat model by using self-made chicken type II collagen. On this basis, the rats were continuously administered by gavage for 21 days starting from the 15th day. Similarly, Wistar rats were divided into a normal group, a model group, a positive drug group (glucosamine hydrochloride), and a drug administration group of cnidium alcohol, neolignan and cnidium seed, and different combinations of the above three compounds. Except for the normal group, the rest of the groups of rats were induced to construct the osteoarthritis rat model by injecting 50 μL of 3 mg of sodium iodoacetate physiological saline into the knee joint cavity of the rats once a day for 14 consecutive days. After the last injection of sodium iodoacetate solution into the knee joint cavity, the positive control group and each drug administration group were administered with the corresponding concentration of the test drug once a day for a total of 4 weeks.

[0112] The results show that the swelling degree, arthritis score, serum TNF-α, IL-1β, and IL-10 levels of the CIA rat model group are significantly increased. The pathological results show that the synovial cells of the model group of rats proliferate, the interstitial edema is accompanied by inflammatory cell infiltration, and the above-mentioned indicators are improved to different degrees after being treated with the positive drug, cnidium alcohol, neolignan and cnidium seed, and different combinations of the above three compounds. In the sodium iodoacetate-induced rat OA model, the knee joint swelling degree, serum TNF-α, IL-1β, and IL-10 levels of the model group are significantly increased, and the above-mentioned indicators are improved to different degrees after being treated with the positive drug, cnidium alcohol, neolignan and cnidium seed, and different combinations of the above three compounds. In addition, the inventors found that the efficacy of the combination is better than that of the single drug, which can significantly improve the symptoms of the animal model and has the activity of resisting rheumatoid arthritis and osteoarthritis.

[0113] Therefore, the inventors found that the combination of the three compounds, especially the combination of two of the three compounds, can replace multiple drugs with a single drug composition, reduce the physical burden of patients with comorbidities due to the simultaneous administration of multiple drugs, reduce costs, and to some extent, avoid the toxic side effects or adverse reactions caused by the combination of drugs. While the combination of the three compounds can produce synergistic effects, the drug composition of the present application can also reduce the types and doses of drugs.

Claims

1. The use of a combination of effective components of Ziyol, Lingxian Neixi and Shechuangzi in the preparation of a medicine for treating or / and preventing rheumatoid arthritis and / or osteoarthritis; the molar ratio of Ziyol, Lingxian Neixi and Shechuangzi is (1-5.5):1:(1-7.5); or the use of a combination of effective components of Lingxian Neixi and Shechuangzi in the preparation of a medicine for treating or / and preventing rheumatoid arthritis and / or osteoarthritis; the molar ratio of Lingxian Neixi and Shechuangzi is 7:3-1:7.

5. The composition is a combination of Ziyol, Lingxian Neixi and Shechuangzi, or a combination of Lingxian Neixi and Shechuangzi; when the composition is a combination of Ziyol, Lingxian Neixi and Shechuangzi, the molar ratio of Ziyol, Lingxian Neixi and Shechuangzi is (1-5.5):1:(1-7.5); when the composition is a combination of Lingxian Neixi and Shechuangzi, the molar ratio of Lingxian Neixi and Shechuangzi is 7:3-1:7.

5.

2. A composition for the treatment or / and prevention of rheumatoid arthritis and / or osteoarthritis, characterized in that:

3. The use of the composition of claim 2 in the preparation of a medicine for preventing or / and treating rheumatoid arthritis and / or osteoarthritis with anti-inflammatory function. The composition is prepared into ointment, liniment, patch, lotion, solution, powder, spray, syrup, suppository, tablet, pill, granule, capsule, injection or gastro-soluble controlled-release preparation with pharmaceutically acceptable pharmaceutical adjuvant.

4. Use according to claim, characterized in that: ​

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