Traditional Chinese medicine composition for treating knee osteoarthritis as well as preparation method and application thereof

Abstract

The invention provides a traditional Chinese medicine composition for treating knee osteoarthritis as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-25 parts of radix cyathulae, 15-25 parts of prepared rehmannia root, 15-25 parts of cynomorium songaricum, 15-25 parts of obscured homalomena rhizome, 15-25 parts of snow lotus herb, 15-25 parts of rhizoma cibotii, 15-25 parts of common fenugreek seed, 15-25 parts of zaocys dhumnade, 10-20 parts of paniculate swallowwort root, 10-20 parts of pine nodular branch, 10-20 parts of corydalis tuber, 10-20 parts of combined spicebush root, 15-25 parts of Chinese angelica, 10-20 parts of ligusticum wallichii, 15-25 parts of Chinese yam, 15-25 parts of poria cocos, 10-20 parts of green tangerine peel, 15-25 The traditional Chinese medicine composition can effectively treat knee osteoarthritis.

Description

[0001] This application is a divisional application with an application date of May 12, 2023, application number 202310535145.0, and invention name "A Chinese medicine composition for treating knee osteoarthritis, its preparation method and application". Technical Field

[0002] The invention relates to a traditional Chinese medicine composition for treating knee osteoarthritis and a preparation method and application thereof, belonging to the technical field of traditional Chinese medicines. Background Art

[0003] Knee osteoarthritis (KOA) is a chronic joint disease characterized by degenerative changes in articular cartilage and secondary bone hyperplasia. The main symptoms are knee pain, swelling, limited mobility and even joint deformity. It is common in middle-aged and elderly people. Epidemiological surveys show that the prevalence of people over 55 years old is 44% to 70%, and the prevalence of men over 65 years old is about 60%, while that of women is as high as 70%. There are many treatments for knee osteoarthritis at present. Western medicine mostly uses non-steroidal anti-inflammatory drugs, which have many side effects after long-term use; surgical operations have certain limitations and risks, and poor long-term efficacy. Traditional Chinese medicine believes that knee osteoarthritis belongs to the category of traditional Chinese medicine arthralgia and bone arthralgia. The name of the disease first came from Sun Simiao's "Prescriptions for Emergencies". The cause of the disease is that the evil of wind, cold, dampness and heat blocks the meridians, and the qi and blood are not smooth, causing pain, soreness, swelling, heaviness and poor mobility in the affected joints. The pathogenesis is based on liver and kidney deficiency, and the invasion of external evils, meridian blockage, pain due to blockage, and qi and blood disorder are the symptoms. It is closely related to physical factors, climatic conditions, living environment, etc.

[0004] Before any drug can be used clinically, it must be made into a form suitable for medical or preventive use. Diseases can be either acute or chronic, and symptoms can be superficial or internal, so the requirements for dosage forms are also different. In order to better exert the efficacy of the drug, the requirements for dosage forms and preparations are also different according to the nature of the drug. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a Chinese medicine composition for treating knee osteoarthritis and a preparation method thereof. The Chinese medicine composition of the present application has significant efficacy in treating early and middle-stage knee osteoarthritis, has few side effects, and has stable efficacy, and can significantly improve pain and functional disorders of knee osteoarthritis, and improve the daily quality of life of patients.

[0006] The invention provides a traditional Chinese medicine composition for treating knee osteoarthritis. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-25 parts of Cynomorium root, 15-25 parts of Radix Rehmanniae Preparata, 15-25 parts of Cynomorium songaricum, 15-25 parts of Rhizoma Cibotii, 15-25 parts of Saussurea involucrata, 15-25 parts of Cibotium barometz, 15-25 parts of Fenugreek, 15-25 parts of Snakehead Snake, 10-20 parts of Cynomorium Changqing, 10-20 parts of Pinus tabulaeformis, 10-20 parts of Corydalis yanhusuo, 10-20 parts of Linderae umbellatae, 15-25 parts of Angelica sinensis, 10-20 parts of Rhizoma Chuanxiong, 15-25 parts of Dioscorea opposita, 15-25 parts of Poria, 10-20 parts of Pericarpium Citri Reticulatae, 15-25 parts of Orientalis and 10-20 parts of Licorice.

[0007] Preferably, the Chinese medicine composition is prepared from the following raw materials in parts by weight: 15 parts of Cynomorium Bidentatae, 15 parts of Rehmannia glutinosa, 15 parts of Cynomorium songaricum, 15 parts of Millennium Jian, 15 parts of Snow Lotus, 15 parts of Cibotium barometz, 15 parts of Fenugreek, 15 parts of Agkistrodon acutus, 10 parts of Cynomorium Changqing, 10 parts of Pinus tabulaeformis, 10 parts of Corydalis yanhusuo, 10 parts of Linderae scutellariae, 15 parts of Angelica sinensis, 10 parts of Ligusticum chuanxiong, 15 parts of Dioscorea opposita, 15 parts of Poria, 10 parts of Citrus aurantium, 15 parts of Orientalis and 10 parts of Licorice.

[0008] The present invention also provides a method for preparing the above-mentioned traditional Chinese medicine composition, which comprises mixing the raw materials in the formula and then soaking them in water until the drug tissue is penetrated, then adding water with a thickness of 30 to 35 mm thicker than the drug and boiling for 40 to 45 minutes, filtering the drug solution, adding water with a thickness of 25 to 30 mm thicker than the drug to the filter residue and boiling for 30 to 35 minutes, filtering and combining the filtrate.

[0009] The present invention also provides a preparation method of the above-mentioned traditional Chinese medicine composition, comprising the steps of mixing the raw materials of the formula amount and crushing them into fine powder, passing through a 120-mesh sieve so that the amount of fine powder that can pass through the sieve reaches 50%, and collecting the sieved fine powder; then extracting the coarse powder twice with water, combining the filtrates, heating the filtrates, concentrating and drying them, and grinding them into fine powder, mixing the two fine powders and loading them into a capsule shell to obtain a capsule.

[0010] The present invention also provides a method for preparing the above-mentioned traditional Chinese medicine composition, comprising: mixing the raw materials of the formula amount, adding 8 to 10 times the weight of water, soaking for 0.5 h, decocting twice, decocting for 1.5 h for the first time, and decocting for 1 h for the second time with 8 to 10 times the weight of water, combining the decoctions, filtering, concentrating to obtain an extract, drying and crushing into fine powder, adding auxiliary materials for granulation, and obtaining granules.

[0011] The present invention also provides application of the above-mentioned traditional Chinese medicine composition in preparing medicine for treating knee osteoarthritis.

[0012] The present invention also provides a medicine, the effective ingredients of which include the above-mentioned Chinese medicine composition.

[0013] Preferably, the medicine further comprises excipients; the excipients are excipients commonly used in pharmacy.

[0014] Preferably, the medicine includes decoction, capsule and granule.

[0015] The inventor of this application has conducted epidemiological studies on the constitution of knee osteoarthritis in Gansu Province and found that it is mainly yang deficiency constitution. On this basis, combined with the experience of treating knee osteoarthritis, the inventor of this application has summarized the Chinese medicine preparation of this application, and proposed that liver and kidney deficiency and malnutrition of tendons and veins are the root causes, and wind-cold-dampness arthralgia is an external factor. The syndrome belongs to liver and kidney deficiency, and the pathogenesis is the syndrome of "fundamental deficiency and superficial excess, mixed deficiency and excess". Malnutrition of joints makes them unable to move, and pain occurs when they are not nourished; wind-cold invades, tendons and veins are blocked, and pain occurs when they are not unblocked. The treatment is to nourish the liver and kidney, dispel cold and dampness, and relieve arthralgia and pain. The Chinese medicine preparation applied for is an empirical prescription summarized by the team through many years of clinical practice. The whole prescription is composed of 19 Chinese medicinal herbs, including Rhizoma Cynomorii, Radix Rehmanniae Preparata, Rhizoma Cynomorii, Rhizoma Cibotii, Rhizoma Fenugreek, Rhizoma Agaricuse, Rhizoma Cynomorii, Rhizoma Cynomorii, Rhizoma Cynomorii, Rhizoma Pinelliae, Rhizoma Corydalis, Rhizoma Linderae, Rhizoma Angelicae Sinensis, Rhizoma Chuanxiong, Rhizoma Dioscoreae, Poria, Rhizoma Cibotii, Rhizoma Alismatis, and Rhizoma Licorice. The prescription is properly matched and treats both the symptoms and the root causes. The combination of these herbs can nourish the liver and kidney, dispel cold and wind, and relieve numbness and relieve pain.

[0016] Explanation of the prescription: In the prescription, Radix Rehmanniae, Rhizoma Cynomorii, Rhizoma Cynomorii, and Rhizoma Cynomorii are used as the main drugs to replenish essence and marrow, nourish liver and kidney, soften meridians, and strengthen tendons and bones; Snow Lotus, Cibotium barometz, and Fenugreek help the main drugs to nourish liver and kidney, warm yang and relieve numbness; Snakehead Snake, Cynomorium longivora, and Pinus tabulaeformis dispel cold and dampness, relieve numbness and relieve pain, and expel evil; Corydalis and Linderae sibiricum promote blood circulation and qi, warm the meridians and relieve pain, and are collectively the ministerial drugs; Angelica sinensis and Rhizoma Chuanxiong nourish blood and promote blood circulation, dredge collaterals and relieve pain; Chinese Yam, Poria, and Citrus aurantium invigorate the spleen and promote qi and remove dampness, restrain the greasy and nourishing properties of Radix Rehmanniae, Rhizoma Cynomorii, and Rhizoma Cynomorii, and assist the efficacy of drugs; Alisma orientalis promotes water flow, infiltrates dampness and relieves swelling, and assists other drugs to be too warm and dry; Licorice relieves pain and harmonizes the drugs. The whole prescription is properly matched, rigorous and refined, and treats both the symptoms and the root causes. The drugs are combined to nourish the liver and kidney, dispel cold and dampness, relieve numbness and relieve pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0018] Figure 1 The diagram is an analysis of the changes in the diameter of the right hind knee joint of rats in each group at different stages (n=10);

[0019] Figure 2 The gross observation pictures of the knee joints of rats in each group;

[0020] Figure 3 The gross observation and scoring of the rat knee joints were performed;

[0021] Figure 4 HE staining of the right lower limb knee cartilage of rats in each group (×100);

[0022] Figure 5 It is the modified Mankin score;

[0023] Figure 6 The expression of IL-1β and TNF-α mRNA in cartilage tissue of rats in each group;

[0024] Figure 7 The expression of Aggrecan, CollagenⅡ, TIMP1 and MMP13 proteins in articular cartilage of each group;

[0025] Figure 8 The expression of Aggrecan, CollagenⅡ, TIMP1 and MMP13 mRNA in cartilage tissue of rats in each group;

[0026] Fig. 9 is the expression of autophagy-related proteins in articular cartilage of each group;

[0027] Fig.10 The expression of LC3 and P62 mRNA in cartilage tissue of rats in each group;

[0028] Fig.11 The expression of P-AMPK, P-mTOR, and P-ULK1 in articular cartilage of each group;

[0029] Fig.12 The expression of AMPK, mTOR and ULK1 mRNA in cartilage tissue of rats in each group. DETAILED DESCRIPTION

[0030] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0031] In the present invention, "Tongbi Jiangu Fang" refers to the Chinese medicine composition of the present invention.

[0032] A Chinese medicine composition for treating knee osteoarthritis of the present invention is composed of the following raw materials in parts by weight:

[0033] 15-25 parts of Cyathula officinalis, 15-25 parts of Rehmannia root, 15-25 parts of Cynomorium songaricum, 15-25 parts of Millennium jian, 15-25 parts of Snow lotus, 15-25 parts of Cibotium barometz, 15-25 parts of Fenugreek, 15-25 parts of Snakehead serpentine, 10-20 parts of Cynomorium longituba, 10-20 parts of Pinus tabulaeformis, 10-20 parts of Corydalis yanhusuo, 10-20 parts of Linderae umbellatae, 15-25 parts of Angelica sinensis, 10-20 parts of Ligusticum chuanxiong, 15-25 parts of Dioscorea opposita, 15-25 parts of Poria, 10-20 parts of Citrus aurantium, 15-25 parts of Orientalis and 10-20 parts of Liquorice.

[0034] The preparation method of the water decoction of the Chinese medicine composition for treating knee osteoarthritis of the present invention is as follows:

[0035] All raw medicinal materials are weighed according to the above weight portions and soaked in pure water until the medicine tissue is penetrated. The soaking time is generally half an hour. Then the soaked medicine and the clarified liquid of the soaked medicine are put into the medicine decocting machine together, and pure water is injected to exceed the medicine by 30 to 35 mm for the first decoction for 40 to 45 minutes. After filtering the medicine liquid, pure water is injected again to exceed the medicine by 25 to 30 mm for the second decoction for 30 to 35 minutes, and the water decoction is obtained by mixing.

[0036] The method of using the water decoction of the Chinese medicine composition of the present invention is to take orally, one dose per day, divided into two doses.

[0037] The preparation method of the Chinese medicine composition capsule for treating knee osteoarthritis of the present invention is as follows:

[0038] All raw medicinal materials are weighed according to the above weight portions, mixed and crushed into coarse powder, crushed again, passed through a 120-mesh sieve so that the amount of fine powder that can pass through the sieve reaches 50%, and the fine powder that can pass through the sieve is collected; the coarse powder that cannot pass through the sieve is put into a multifunctional Chinese medicine extraction tank for extraction twice, the first time 8 times the weight of water is added and boiled for 2 hours, the second time 3 to 5 times the weight of water is added and boiled for 1 hour, the two decoctions are filtered and combined, heated and concentrated to a paste, put into an oven at 70°C for drying, and then allowed to stand at room temperature, ground into fine powder, and the two fine powders are mixed and loaded into a capsule shell to obtain a capsule.

[0039] Due to the limitation of crushing technology, if all the Chinese medicines are crushed, some coarse powders cannot be crushed into fine powders to pass through 100-120 mesh sieves. Therefore, a multifunctional Chinese medicine extraction tank is used to extract with water to assist in crushing into fine powders without destroying the medicinal properties of the Chinese medicines.

[0040] The method of using the Chinese medicine composition capsule of the present invention is to take orally, twice a day, 4 to 6 capsules each time.

[0041] The preparation method of the Chinese medicine composition granules for treating knee osteoarthritis of the present invention is as follows:

[0042] Weigh all raw medicinal materials according to the above weight portions, mix them, add 8 to 10 times the weight of water, soak for 0.5 h, decoct twice, the first decoction is 1.5 h, and the second decoction is 8 to 10 times the weight of water and decoct for 1 h, combine the decoctions, filter, and concentrate the filtrate at normal pressure (100°C) and under reduced pressure (-70°C, -0.1 MPa) to an extract with a relative density of 1.25 (70°C), dry, grind into fine powder, add sucrose (the weight ratio of dry extract powder: sucrose = 1:2), stir for 10 min, mix well, granulate with 85% ethanol (the weight ratio of dry extract powder: 85% ethanol = 1:0.40), dry, granulate, and package to obtain the extract.

[0043] The method for using the Chinese medicine composition granules of the present invention is as follows: put the granules into a container, add boiling water and soak for 4 minutes, stir evenly and then take orally, 1 dose per day, 2 times per day.

[0044] To further illustrate the present invention, a Chinese medicine composition for treating knee osteoarthritis provided by the present invention, and its preparation method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] The formula of the Chinese medicine composition for treating knee osteoarthritis of the present invention is as follows:

[0047] 15g of Sichuan Cyathula, 15g of Rehmannia glutinosa, 15g of Cynomorium songaricum, 15g of Millennium Jian, 15g of Snow Lotus, 15g of Cibotium barometz, 15g of Fenugreek, 15g of Black-striped Snake, 10g of Cynomorium fortunei, 10g of Pinus tabulaeformis, 10g of Corydalis yanhusuo, 10g of Linderae scutellariae, 15g of Angelica sinensis, 10g of Chuanxiong, 15g of Dioscorea batatas, 15g of Poria, 10g of Citrus aurantium, 15g of Alisma orientalis and 10g of Licorice.

[0048] The preparation method of the water decoction of the Chinese medicine composition for treating knee osteoarthritis of the present invention is:

[0049] Purchase high-quality and clean raw medicinal materials according to the above-mentioned mass ratio, remove impurities and dust respectively, soak all the medicines in cold boiled water for half an hour to penetrate the medicine tissue, then put the soaked medicine and the clarified liquid of the soaked medicine into the medicine decocting machine, inject cold boiled water that has passed the medicine for 35mm for the first decoction for 40 minutes, filter out the medicine liquid, and inject pure water that has passed the medicine for 30mm again for the second decoction for 35 minutes. After mixing and concentrating into 400 ml of 50% traditional Chinese medicine liquid, put it in the refrigerator for freshness and standby use.

[0050] The method of using the water decoction of the Chinese medicine composition of this embodiment is: oral administration, one dose per day, divided into two doses.

[0051] Example 2

[0052] The formula of the Chinese medicine composition for treating knee osteoarthritis of the present invention is the same as that of Example 1.

[0053] The preparation method of the capsule of the Chinese medicine composition for treating knee osteoarthritis of the present invention is as follows:

[0054] All raw medicinal materials are weighed according to the weight ratio, crushed into coarse powder, further crushed, passed through a 120-mesh sieve so that the amount of fine powder that can pass through the sieve reaches 50%, and the fine powder that passes through the sieve is collected; the coarse powder that cannot pass through the sieve is put into a multifunctional Chinese medicine extraction tank for extraction twice, the first time 10 times the weight of water is added and boiled for 2 hours, the second time 5 times the weight of water is added and boiled for 1 hour, the two decoctions are filtered and combined, heated and concentrated to a paste, put into an oven at 70°C for drying, and then allowed to stand to room temperature, ground into fine powder, and the two fine powders are mixed and loaded into a capsule shell to obtain a capsule.

[0055] Example 3

[0056] The formula of the Chinese medicine composition for treating knee osteoarthritis of the present invention is the same as that of Example 1.

[0057] The preparation method of the Chinese medicine granules of the Chinese medicine composition of this embodiment is as follows:

[0058] Weigh all raw medicinal materials according to the above weight, mix them, add 8 times the weight of water to soak for 0.5h, decoct them twice, the first time for 1.5h, the second time with 8 times the weight of water for 1h, combine the decoctions, filter, concentrate the filtrate at normal pressure (100℃) and under reduced pressure (70℃, -0.1MPa) to an extract with a relative density of 1.25 (70℃), dry, grind into fine powder, add sucrose (weight ratio of dry extract powder: sucrose = 1:2), stir for 10min, mix well, granulate with 85% ethanol (weight ratio of dry extract powder: 85% ethanol = 1:0.40), dry, granulate, and package to obtain the extract.

[0059] The method of using the Chinese medicine composition granules of the present invention is as follows:

[0060] The granules prepared above are for one dose, which is taken twice a day, morning and evening.

[0061] Put the granules into a container, add 300 mL of boiling water, soak for 4 minutes, stir well and take orally.

[0062] The treatment cycle is 2 courses, each course lasts 7 days.

[0063] Example 4

[0064] The formula of the Chinese medicine composition for treating knee osteoarthritis of the present invention is as follows:

[0065] 25g of Sichuan Cyathula, 25g of Rehmannia glutinosa, 25g of Cynomorium songaricum, 25g of Millennium Jian, 25g of Snow Lotus, 25g of Cibotium barometz, 25g of Fenugreek, 25g of Black-striped Snake, 20g of Cynomorium macrostemon, 20g of Pinus tabulaeformis, 20g of Corydalis yanhusuo, 20g of Linderae scutellariae, 25g of Angelica sinensis, 20g of Chuanxiong, 25g of Dioscorea batatas, 25g of Poria, 20g of Citrus aurantium, 25g of Alisma orientalis and 20g of Licorice.

[0066] The preparation method of the water decoction of the Chinese medicine composition for treating knee osteoarthritis of the present invention is:

[0067] Purchase high-quality and clean raw medicinal materials according to the above-mentioned mass ratio, remove impurities and dust respectively, soak all the medicines in cold boiled water for half an hour to penetrate the medicine tissue, then put the soaked medicine and the clarified liquid of the soaked medicine into the medicine decocting machine, inject cold boiled water that has passed the medicine by 30mm for the first decoction for 45 minutes, filter out the medicine liquid, and inject pure water that has passed the medicine by 25mm for the second decoction for 30 minutes. After mixing and concentrating into 400 ml of 50% traditional Chinese medicine liquid, put it in the refrigerator for freshness and standby use.

[0068] Example 5

[0069] The formula of the Chinese medicine composition for treating knee osteoarthritis of the present invention is as follows:

[0070] 20g of Sichuan Cyathula, 20g of Rehmannia glutinosa, 20g of Cynomorium songaricum, 20g of Millennium Jian, 20g of Snow Lotus, 20g of Cibotium barometz, 20g of Fenugreek, 20g of Black-striped Snake, 15g of Cynomorium fortunei, 15g of Pinus tabulaeformis, 15g of Corydalis yanhusuo, 15g of Linderae scutellariae, 20g of Angelica sinensis, 15g of Ligusticum chuanxiong, 20g of Dioscorea batatas, 20g of Poria, 15g of Citrus aurantium, 20g of Alisma orientalis and 15g of Licorice.

[0071] The preparation method of the Chinese medicine granules of the Chinese medicine composition of the present invention is as follows:

[0072] Weigh all raw medicinal materials according to the above weight, mix them, add 10 times the weight of water, soak for 0.5h, decoct twice, the first time decoction is 1.5h, the second time add 9 times the weight of water and decoct for 1h, combine the decoctions, filter, and concentrate the filtrate under normal pressure (100℃) and under reduced pressure (70℃, -0.1MPa) to an extract with a relative density of 1.25 (70℃), dry, grind into fine powder, add sucrose (weight ratio of dry paste powder: sucrose = 1:2), stir for 10min, mix well, granulate with 85% ethanol (weight ratio of dry paste powder: 85% ethanol = 1:0.40), dry, granulate, and package to obtain the extract.

[0073] Example 6

[0074] Clinical efficacy of the Chinese medicine composition of the present invention in treating early and middle stages of KOA

[0075] 1Knee osteoarthritis KOA clinical test research subjects

[0076] 1.1 Case source

[0077] A total of 56 patients with early and middle-stage KOA who met the diagnostic criteria and visited the orthopedic outpatient and inpatient departments of Gansu Provincial Hospital of Traditional Chinese Medicine from November 2020 to January 2022 were collected.

[0078] 1.2 Develop a case report form

[0079] The case report form consists of: informed consent form for the subject, general information, and safety assessment test. General information includes the patient's gender, age, height, weight, current medical history, past medical history, allergy history, etc. Informed consent form for the subject. This project has passed the ethics review of the ethics committee, and all subjects are required to sign the informed consent form.

[0080] 1.3 Inclusion criteria

[0081] (1) Meet the diagnostic criteria of KOA in both traditional Chinese medicine and Western medicine;

[0082] (2) Meet the syndrome diagnosis criteria of the Guidelines for the Diagnosis and Treatment of Knee Osteoarthritis in Traditional Chinese Medicine (2020 Edition);

[0083] (3) Patients who meet the Kellgren-Lawrence (KL) radiological diagnostic classification of grade I, II, or III; grade I or II is early stage, and grade III is mid-stage.

[0084] (4) Meet the clinical staging criteria of traditional Chinese medicine;

[0085] (5) Age 50–70 years old;

[0086] (6) Patients who have not received systematic treatment for knee osteoarthritis in the past month and can complete the treatment as planned and cooperate with clinical observers and follow-up;

[0087] (7) The subject fully understands the nature of the present invention and the nature of his / her disease, and is able to understand and sign the informed consent form.

[0088] 2 Research Methods

[0089] 2.1 Clinical trial design methods

[0090] This trial adopted the simple random method commonly used in clinical research for grouping. The patients were randomly grouped according to the order of their visits. The first patient who met the inclusion criteria was admitted to Group A, the second patient who met the inclusion criteria was admitted to Group B, and so on. A total of 56 patients who met the inclusion criteria were collected, 28 cases in the Chinese medicine composition treatment group (Group A) prepared in Example 1 of the present invention, and 28 cases in the diclofenac sodium sustained-release capsules (control group B).

[0091] 2.2 Treatment options

[0092] Both groups received basic treatment mainly based on health education. In each group, group A was treated with the Chinese medicine composition prepared in Example 1 of the present invention; group B was treated with diclofenac sodium sustained-release capsules. Both groups were treated 7 times a week, one course of treatment was 7 days, and the treatment cycle was 2 courses.

[0093] 2.2.1 Basic treatment

[0094] Basic treatment is mainly based on health education, the specific contents are as follows:

[0095] (1) Inform the patient of the causes of KOA;

[0096] (2) KOA daily life prevention;

[0097] (3)KOA knee joint quadriceps extension and contraction exercise.

[0098] 2.2.2 Treatment plan of the Chinese medicine composition of the present invention:

[0099] The treatment method is shown in Example 1, one week is one course of treatment, and the treatment cycle is 2 courses of treatment.

[0100] 2.2.3 Treatment plan for the control group:

[0101] The patient was given diclofenac sodium sustained-release capsules (Xintaiqing), produced by Runhai Rundu Pharmaceutical Co., Ltd. Dosage: Oral, 2 capsules (100 mg) / time, once / day, taken with warm water 30 minutes after meals.

[0102] 2.3 General information of the case:

[0103] Includes the patient's basic information, including the patient's gender, age, disease course, and imaging Kellgren-Lawrence grade.

[0104] 2.4 Safety Assessment

[0105] Safety observation indicators:

[0106] (1) Blood and urine routine tests, liver function, kidney function, and electrocardiogram. Observe once before treatment and 2 weeks after treatment.

[0107] (2) Adverse reactions: The drug group was observed for gastrointestinal reactions during the study period, mainly stomach discomfort, abdominal pain, burning sensation, acid reflux, poor appetite, constipation, nausea, etc. When adverse reactions were found, intervention measures should be taken according to the situation, and it should be determined whether the adverse reaction was related to the treatment of this trial, and whether the trial should be terminated according to the condition. The degree, time and duration of adverse reactions during the study, the treatment taken and the outcome should be recorded truthfully.

[0108] 2.5 Statistical analysis

[0109] SPSS 26.0 statistical software was used to analyze the data. All data analysis results are presented in the table as “mean ± standard deviation”. " means. For measurement data such as age and course of disease between the two groups, if the test results have equal variance and conform to normal distribution, one-way analysis of variance is used for data processing. If the test does not conform to normal distribution and / or the variance is unequal, rank sum test is used for data analysis. For counting data such as gender, χ 2 The rank sum test was used for comparison, and the efficacy grade and other hierarchical data were compared by paired t test. The data within each group before and after treatment were compared by paired t test, and the data between groups were compared by one-way analysis of variance. The results were considered to be statistically insignificant when P>0.05, and statistically significant when P<0.05 or P<0.01.

[0110] 3 Clinical efficacy of the Chinese medicine composition of the present invention in treating early and middle stages of KOA

[0111] The experiment adopted a simple random method, and a total of 56 patients who met the inclusion criteria were collected: 28 cases in the Chinese medicine composition treatment group (Group A) prepared in Example 1 of the present invention, and 28 cases in the diclofenac sodium sustained-release capsules (control group B). Both groups adopted basic treatment with health education as the main method. In the case of basic treatment given to each group, Group A was intervened with the Chinese medicine composition treatment group prepared in Example 1 of the present invention; Group B was intervened with diclofenac sodium sustained-release capsules. Both groups were treated 7 times a week, one course of treatment was 7 days, and the treatment cycle was 2 courses.

[0112] Safety Assessment

[0113] There were 56 patients in the two groups. During the study, 3 patients in group A experienced mild gastrointestinal reactions such as stomach pain and indigestion; 2 patients experienced constipation; 8 patients in group B experienced stomach discomfort, abdominal pain, acid reflux, poor appetite, and constipation, but no ulcer, bleeding, or perforation occurred. The rest of the patients had normal blood, urine, and stool routine tests, liver and kidney function, and examinations before and after treatment, as shown in Table 1.

[0114] Table 1 Incidence of adverse reactions in each group

[0115] Adverse Reactions Group A Control group B Mild stomach pain 2 8 Mild constipation 1 0 ALT and AST increased 0 0 Incidence (%) 10.71 28.75

[0116] Example 7

[0117] The therapeutic effect of the Chinese medicine composition of the present invention on the KOA rat model and its influence on autophagy

[0118] Fifty SD rats were randomly divided into a control group, a model group, and a low-, medium-, and high-dose group of the Chinese medicine composition prepared in Example 1 of the present invention (hereinafter referred to as "Tongbi Jiangu Fang"), with 10 rats in each group. Except for the control group, the KOA model was established in the other groups by the modified Hulth method, and the corresponding drugs were gavaged for 4 weeks before anesthesia and sampling. During the treatment, the general condition of the rats was observed and the changes in the knee joint diameter were measured. When sampling, the changes in the joints of each group were roughly observed, and the degeneration of articular cartilage was evaluated by HE staining and modified Mankin score. The expressions of MMP-13, TIMP1, Aggrecan, Collagen II, LC3, P62, P-ULK1, P-AMPK, and P-mTOR in the cartilage of each group were detected by WB method. The expressions of MMP-13, TIMP1, Aggrecan, Collagen II, LC3, P62, ULK1, AMPK, mTOR, IL-1β, and TNF-α mRNA were detected by qRT-PCR.

[0119] 1 Experimental Materials

[0120] 1.1 Experimental animals

[0121] 50 SPF SD rats (half male and half female), weighing (160 g ± 20 g), 8 weeks old; purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., animal quality certificate number: SCXK (Beijing) 2019-0010. Animal breeding, modeling, and intervention were all carried out in the SPF Animal Experiment Center of Gansu University of Chinese Medicine SYXK (Gan) 2020-0009. Animal modeling, intervention, and sampling operations were in accordance with the provisions of the Ministry of Science and Technology's "Guiding Opinions on Treating Experimental Animals Well" (revised in 2006), and the entire experiment strictly followed the Animal Experiment Ethics Regulations of Gansu University of Chinese Medicine (Ethics Number: 2021-185).

[0122] 1.2 Experimental drugs

[0123] All medicinal materials in the Tongbi Jiangu recipe were purchased from the Pharmacy Department of Gansu Provincial Hospital of Traditional Chinese Medicine and meet the requirements of the 2020 edition of the "Pharmacopoeia of the People's Republic of China".

[0124] 2 Experimental methods

[0125] 2.1 Grouping and modeling

[0126] After 1 week of adaptive feeding, 50 SD rats were randomly divided into control group, model group, Tongbijiangufang low-dose group, Tongbijiangufang medium-dose group, and Tongbijiangufang high-dose group, with 10 rats in each group. Except for the 10 rats in the control group without any treatment, the remaining 40 SD rats were used to establish KOA rat model by modified Hulth method. Rats were anesthetized by intraperitoneal injection of 7% chloral hydrate 5ml / kg. After complete anesthesia, the skin was prepared, disinfected, and draped. The skin and joint capsule were cut open on the medial side of the right posterior knee patella, the medial collateral ligament and anterior cruciate ligament were cut, and the medial meniscus was completely removed, and care was taken not to damage the articular cartilage surface. The joint cavity was flushed three times with physiological saline using a disposable 10mL syringe. After a positive drawer test, the bleeding was stopped thoroughly, and the joint capsule and skin incision were sutured layer by layer. Erythromycin ointment was applied to the wound after the operation, and 200,000 units / d of penicillin were injected intramuscularly for 3 consecutive days to prevent infection. All modeling animals were driven away for 30 minutes / d one week after modeling. Rats in each group were allowed to drink water and eat freely under the same conditions. Subsequent experiments were carried out 4 weeks after modeling.

[0127] 2.2 Drug preparation

[0128] The Tongbi Jiangu Fang decoction was prepared according to the method of Example 1 of the present invention.

[0129] 2.3 Drug intervention

[0130] Four weeks after modeling, according to the "body surface area conversion method between humans and rats", the rat dosage (g·kg / d) = human dosage (g / d) / 60kg×0.018×5, the low, medium and high dose groups of Tongbi Jiangu Fang were 2, 4 and 8 mL / kg / d, respectively, and the control group and the model group were given an equal amount of normal saline by gavage. Before gavage, the Tongbi Jiangu Fang liquid prepared with appropriate concentration was heated to the appropriate temperature. During the gavage process, the movements should be gentle and slow to minimize unnecessary harm to the rats. After gavage, the rats were put back in the cage for observation. After 4 weeks of continuous treatment, relevant indicators were tested.

[0131] 2.4 General observation and measurement of rat knee joint diameter

[0132] The diet, drinking water, activity, sensitivity, mental state, knee joint deformity, swelling, activity, lower limb blood circulation and muscle fullness of each group of rats were observed; the right knee joint diameter was measured before and after modeling and 4 weeks after treatment. The measurement method was: the knee joint diameter was measured with a vernier caliper, repeated three times, and the average value was calculated.

[0133] 2.5 Collection of materials

[0134] After the rats were anesthetized, the muscles, ligaments and synovium around the knee joint were separated, and the articular cartilage tissue on the medial side of the right posterior knee joint of the rats was removed. A portion of the specimens in each group was marked and fixed in 4% formaldehyde for use in HE staining and other related experiments; another portion of the specimens in each group was marked and stored in a -80°C refrigerator for subsequent qRT-PCR, Western blot and other experiments.

[0135] 2.6 Gross Observation of Rat Knee Joint

[0136] When sampling, the knee joints of the modeling side of each group of rats were separated and the joint cavity was exposed. The articular cartilage was grossly observed and scored. The score was proportional to the degeneration of the knee articular cartilage. The higher the score, the more severe the degeneration of the articular cartilage. The scoring details are shown in Table 2.

[0137] Table 2 Knee cartilage gross observation score table

[0138] Scoring Criteria Fraction The joint surface is smooth and shiny, the cartilage is clear and transparent, and there are no obvious defects or neoplasms. 0 The joint surface is not smooth, the color is dull, the transparency of the cartilage is reduced, and there are no obvious defects or neoplasms 1 The joint surface is not smooth and shiny, the cartilage is opaque, and obvious defects and new organisms (osteophyte formation) can be seen. 2

[0139] 2.7 HE staining of knee cartilage tissue

[0140] ① Decalcification: Remove the specimen fixed in 4% formaldehyde, rinse with tap water for 4 to 6 hours, and then immerse in acetic acid for decalcification for 1 month.

[0141] ② Dehydration and transparency: Place the cartilage tissue in different concentrations of alcohol for dehydration. The higher the concentration, the shorter the time. After the cartilage tissue is dehydrated with alcohol, it must be transparent. The dehydrating agent is replaced with transparent agent xylene, and then paraffin embedding is performed. The specific steps are shown in Table 3.

[0142] Table 3 Cartilage tissue dehydration steps

[0143] Reagents time 75% alcohol 4h 85% alcohol 2h 90% alcohol 1.5h 95% alcohol 1h Anhydrous ethanol 1h Xylene I 1h

[0144] ③ After being wax-transparent, the cartilage tissue was wax-impregnated in 3 cylinders of paraffin (60°C) in sequence. The entire experiment was completed in a biological tissue dehydrator. See Table 4 for details.

[0145] Table 4 Experimental steps for wax immersion of cartilage tissue

[0146] frequency Paraffin bath temperature time Ⅰ paraffin 60℃ 1h Ⅱ paraffin 60℃ 1h Ⅲ paraffin 60℃ 1h

[0147] ④ Embedding: Wrap the wax-soaked cartilage tissue block in a paraffin block. The temperature of the embedding wax should be slightly higher than the wax soaking temperature to ensure that the tissue block and the embedding paraffin are completely integrated.

[0148] ⑤ Slicing and baking: Freeze the wax block section on a freezing table for a few minutes before slicing. Then fix the embedded block to be cut on the specimen clamp, adjust the required thickness of the slice (4μm) on the micro-motion device, move the knife table close to the specimen table, and start slicing.

[0149] The cut sections must be attached to the slide for further processing, but the sections often have fine horizontal lines and must be flattened before attachment, otherwise it will affect staining and observation. To retrieve the sections, first put the series of sections into a warm water bath at about 42°C, separate the sections with tweezers, and then use an APES or poly-lysine treated anti-detachment glass slide to tilt and insert it into the water surface to retrieve the sections, so that the sections are attached to the appropriate position on the slide, and then bake the sections in a 60°C oven for 3 hours.

[0150] ⑥ Dewaxing and hydration The dewaxing and hydration process of paraffin sections is shown in Table 5.

[0151] Table 5 Dewaxing process

[0152] Reagents time Xylene 20min Anhydrous ethanol 10min 95% alcohol 3min 90% alcohol 3min 80% alcohol 2min 70% alcohol 2min Distilled water rinsing 2min

[0153] ⑦ Dyeing Dyeing process, see Table 6.

[0154] Table 6 Dyeing process

[0155] Reagents time Hematoxylin staining 5min, rinse with tap water 1% hydrochloric acid alcohol differentiation 15s Tap water rinse turns blue 15min Distilled water rinse 2min 75% alcohol 2min 85% alcohol 2min 0.5% eosin staining 60s 95% alcohol 10min Anhydrous ethanol 10min Xylene 10min

[0156] ⑧ After the slides were air-dried, they were sealed with neutral gum and finally examined under a microscope. The knee cartilage of rats in each group was scored according to the modified Mankin score for knee cartilage tissue. The standards are shown in Table 7.

[0157] Table 7 Modified Mankin scoring criteria

[0158]

[0159]

[0160] 2.8 Western blot detection of related protein expression

[0161] ①Total protein extraction; ②Total protein concentration determination; ③SDS-PAGE protein gel electrophoresis; ④Transfer to membrane; ⑤Blocking; ⑥Incubation with primary antibody; ⑦Incubation with secondary antibody; ⑧Color imaging; ⑨Data analysis.

[0162] 2.9 Detection of relevant mRNA content by quantitative real-time PCR (qRT-PCR) technology

[0163] ① Extraction of total RNA from cartilage tissue (according to the instructions of Solarbio / Solarbio total RNA extraction kit).

[0164] ②Determination of total RNA concentration.

[0165] ③ Reverse transcription of total RNA in each group to synthesize cDNA According to the concentration of total RNA tested, dilute the total RNA sample concentration to 500ng / μL. According to the instructions of the reverse transcription kit, perform the following steps:

[0166] A. Reaction for removing genomic DNA: Add the following reaction components to an RNase-free centrifuge tube in an ice bath, see Table 8.

[0167] Table 8 Reaction system for removing genomic DNA

[0168] Reagent Volume(μL) 5×gDNAEraserBuffer 2.0 gDNAEraser 1.0 Total RNA 2.0 <![CDATA[RNasefreeddH2O]]> upto10

[0169] Reaction conditions: 42℃ for 2 min or room temperature for 5 min, can be extended to 30 min, terminated at 4℃.

[0170] B. Reverse transcription reaction: The reaction solution was prepared on ice. After gentle mixing, reverse transcription reaction was immediately performed. The specific reaction system is shown in Table 9. The synthesized cDNA was stored in a refrigerator at -20°C for later use.

[0171] Table 9 Reverse transcription reaction system

[0172] Reagent Volume(μL) Reaction step A reaction solution 10.0 PrimeScript RT Enzyme Mix I 1.0 RTPrimerMix 4.0 5×PrimeScriptBuffer2(forRealTime) 4.0 <![CDATA[RNasefreeddH2O]]> 1.0 Total 20.0

[0173] Reaction conditions: 37℃ for 15min, 85℃ for 5s, and terminate at 4℃.

[0174] C Real-time fluorescence quantitative PCR (quantitative real-time PCR, qRT-PCR)

[0175] Primers were designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd. The primer sequences are shown in Table 10. qRT-PCR detection was performed according to the instructions, and the specific reaction system is shown in Table 11.

[0176] Table 10 Gene amplification primer sequence list

[0177]

[0178]

[0179] Table 11 qRT-PCR detection reaction system

[0180] Reagent Volume(μL) cDNA 4.0 Forward Primer (10 μM) 0.4 Reverse Primer (10 μM) 0.4 SYBR Green Master Mix 10.0 50×ROXReferenceDye2 0.4 <![CDATA[RNasefreeddH2O]]> upto20.0

[0181] Reaction conditions: 50℃2min, 95℃10min; 95℃30sec, 60℃30sec, 40cycles. Draw the dissolution curve and the final data is 2 -△△Ct Conduct analysis.

[0182] 2.10 Statistical methods

[0183] The data obtained in this paper were processed using SPSS24.0 statistical software, and the quantitative data were It indicates that a normal distribution test was performed first. The t-test was used for comparison between groups that met the normal distribution, and one-way analysis of variance was used for comparison between multiple groups. The rank sum test was used for research data that did not meet the normal distribution and had uneven variances among groups. P < 0.05 was considered statistically significant.

[0184] 3 Results

[0185] 3.1 General Observations

[0186] During the experiment, the rats in the control group had normal diet and water intake, were sensitive, had good mental state, and were active as usual; the rats in the model group had reduced diet, normal water intake, reduced activity, and significantly worsened sensitivity and mental state. The model-side knee joint showed swelling and deformity, poor limb blood circulation, and muscle atrophy; compared with the model group, the rats in the low, medium, and high intervention groups of Tongbi Jiangu Fang had significantly improved diet, water intake, activity, sensitivity, and mental state. The degree of knee joint swelling, limb blood circulation, and muscle atrophy on the model side were significantly improved compared with the model group, among which the medium-dose group of Tongbi Jiangu Fang was the most significant.

[0187] 3.2 Changes in the diameter of the right hind knee joint of rats in each group

[0188] There was no difference among the groups before modeling (P>0.05); 4 weeks after modeling, compared with the control group, the diameter of the right hind knee joint of the rats in the model group and the low, medium and high dose groups of Tongbi Jiangu Fang was significantly increased (P<0.01), and there was no significant difference between the model group and the low, medium and high dose groups of Tongbi Jiangu Fang (P>0.05); after 4 weeks of treatment, compared with the control group, the diameter of the right hind knee joint of the rats in the model group was significantly increased (P<0.05); compared with the model group, the knee joint diameter of the medium dose group of Tongbi Jiangu Fang was significantly reduced (P<0.01), and there was no significant difference between the low and high dose groups of Tongbi Jiangu Fang and the model group (P>0.05), see Table 12, Figure 1 . Figure 1 The following is an analysis of the changes in the diameter of the right hind knee joint of rats in each group at different stages (n=10). * P<0.05, ** P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group.

[0189] Table 12 Changes in the diameter of the right hind knee joint of rats in each group at different stages ( mm, n = 10)

[0190] Group Before modeling After modeling (4 weeks) After treatment (4 weeks) Control group 9.79±0.68 10.27±0.648 11.10±0.55 Model Group 9.76±0.68 <![CDATA[12.22±0.869 ** ]]> <![CDATA[11.85±0.51 * ]]> Tongbi Jiangu Fang low dose group 9.69±0.78 <![CDATA[11.90±0.684 ** ]]> 11.70±0.14 Tongbi Jiangu Fang Medium Dose Group 9.65±0.85 <![CDATA[12.17±0.434 ** ]]> <![CDATA[11.24±0.44 ## ]]> Tongbi Jiangu Fang high dose group 9.93±0.70 <![CDATA[12.29±0.8023 ** ]]> 11.58±0.26

[0191] Note: *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group

[0192] 3.3 Gross observation of the right hind knee joint of rats in each group

[0193] The cartilage surface of the control group was smooth, the color was clear and transparent, there was no cartilage defect, and no neoplasm was formed; the cartilage surface of the knee joint in the model group was rough, the color was dull, and bone growths formed on the edge of the joint; the low, medium and high dose groups of Tongbi Jiangu Fang were improved compared with the model group, the cartilage surface was not smooth, the color was slightly dull, the transparency was reduced, the cartilage defect and osteophyte were reduced compared with the model group, among which the medium dose group of Tongbi Jiangu Fang showed the most significant improvement. Figure 2 After analyzing the gross observation scores of knee cartilage in each group, it was found that the score of the model group was significantly increased compared with the control group (P<0.01); after 4 weeks of treatment with Tongbi Jiangu Fang, the score of the medium-dose group of Tongbi Jiangu Fang was significantly reduced (P<0.05), and there was no statistically significant difference between the low-dose and high-dose groups of Tongbi Jiangu Fang and the model group, but the scores were lower than those of the model group, as shown in Table 13. Figure 3 . Figure 2 The gross observation pictures of the knee joints of rats in each group. Figure 3 The gross observation scores of the rat knee joints. *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group.

[0194] Table 13 Gross observation scores of knee joints of rats in each group ( , n = 10)

[0195] Group score Control group 0.40±0.52 Model Group <![CDATA[1.60±0.52 ** ]]> Tongbi Jiangu Fang low dose group 1.10±0.88 Tongbi Jiangu Fang Medium Dose Group <![CDATA[0.80±0.63 # ]]> Tongbi Jiangu Fang high dose group 1.00±0.82

[0196] Note: *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group

[0197] 3.4 HE staining and modified Mankin score of right posterior knee cartilage of rats in each group

[0198] The cartilage layer of the right hind knee joint of the rats in the control group was thicker, the surface was smooth and complete, without cracks, the chondrocytes were round or oval, arranged neatly horizontally, uniform in size and full, the matrix was evenly colored, the tide line was complete and clearly visible; the cartilage of the knee joint of the rats in the model group was rough and not smooth, the cartilage layer was thinner, the cartilage edge was severely damaged, and it showed degenerative changes. The number of chondrocytes was significantly reduced, the cells were disordered, the cells were clustered, the nucleus was condensed, the matrix staining became lighter, and the tide line was disordered and irregular; the cartilage of the medium-dose group of Tongbi Jiangu Fang was thicker than that of the model group, the cartilage surface was smoother, the staining was deeper and uniform, with occasional cracks, the number of chondrocytes increased significantly, the arrangement was slightly disordered, the cells were full, the morphology tended to be normal, the tide line was visible, and it was relatively complete; the cartilage structure of the low- and high-dose groups of Tongbi Jiangu Fang was relatively poor compared with the medium-dose group of Tongbi Jiangu Fang, the chondrocytes were unevenly distributed, the articular cartilage surface was not smooth, and the Figure 4 The modified Mankin score of the right posterior knee joint of rats in each group showed that compared with the control group, the modified Mankin score of the right posterior knee joint of rats in the model group was significantly increased (P<0.01); compared with the model group, the modified Mankin score of the right posterior knee joint of rats in the medium-dose group of Tongbi Jiangu Fang was significantly reduced (P<0.01), and there was no significant difference between the low-dose and high-dose groups of Tongbi Jiangu Fang and the model group (P>0.05). Figure 5 . Figure 4 HE staining of the right lower limb knee cartilage of rats in each group (×100). Among them, A: control group; B: model group; C: Tongbi Jiangu Fang low-dose group; D: Tongbi Jiangu Fang medium-dose group; E: Tongbi Jiangu Fang high-dose group. Figure 5 Modified Mankin score. Note: *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group.

[0199] 3.5 Expression of IL-1β and TNF-α mRNA in cartilage tissue of rats in each group

[0200] Compared with the control group, the model group had significantly higher IL-1β and TNF-α mRNA expressions (P<0.01). The low, medium and high dose groups of Tongbi Jiangu Fang had significantly lower IL-1β and TNF-α mRNA expressions (P<0.01). The medium dose group of Tongbi Jiangu Fang had the most significant effect. Figure 6 . Figure 6 The expression of IL-1β and TNF-α mRNA in cartilage tissue of rats in each group. A: IL-1β mRNA expression statistical chart; B: TNF-α mRNA expression statistical chart; *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group.

[0201] 3.6 Metabolism of extracellular matrix of knee cartilage in rats of each group

[0202] WB test results showed that compared with the control group, the protein expressions of Aggrecan, CollageⅡ and TIMP1 in the model group were significantly decreased (P<0.01, P<0.05), and the expression of MMP13 was significantly increased (P<0.05); compared with the model group, the protein expressions of Aggrecan, CollageⅡ and TIMP1 in the low- and medium-dose groups of Tongbi Jiangu Fang were significantly increased (P<0.01, P<0.05), among which the medium-dose group of Tongbi Jiangu Fang was the most significant, and there was no significant difference in the high-dose group of Tongbi Jiangu Fang (P>0.05). The expression of MMP13 in the medium-dose group of Tongbi Jiangu Fang was significantly decreased compared with the model group (P<0.05), and there was no significant difference in the low- and high-dose groups of Tongbi Jiangu Fang (P<0.05). Figure 7 The mRNA expression of Aggrecan, CollagenⅡ, TIMP1, and MMP13 was consistent with the protein expression trend. Figure 8 . Figure 7 The expression of Aggrecan, CollagenⅡ, TIMP1 and MMP13 proteins in articular cartilage of each group. A: control group; B: model group; C: low-dose group of Tongbi Jiangu Fang; D: medium-dose group of Tongbi Jiangu Fang; E: high-dose group of Tongbi Jiangu Fang; F: relative expression statistics of Aggrecan protein; G: relative expression statistics of CollagenⅡ protein; H: relative expression statistics of TIMP1 protein; I: relative expression statistics of MMP13 protein; *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group. Figure 8 The expression of Aggrecan, CollagenⅡ, TIMP1, and MMP13 mRNA in the cartilage tissue of rats in each group. A: Aggrecan mRNA expression statistics; B: CollagenⅡ mRNA expression statistics; C: TIMP1 mRNA expression statistics; D: MMP13 mRNA expression statistics; *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group.

[0203] 3.7 Expression of autophagy-related proteins LC3 and P62 in the knee cartilage of rats in each group

[0204] Compared with the control group, the LC3Ⅱ / LC3Ι ratio in the model group was significantly decreased, and the expression of P62 protein was significantly increased (P<0.01); compared with the model group, the LC3Ⅱ / LC3Ι ratio in the medium-dose group of Tongbi Jiangu Fang was significantly increased, and the expression of P62 protein was significantly decreased (P<0.01), and there was no significant difference between the low-dose group and the high-dose group of Tongbi Jiangu Fang and the model group (P>0.05). Fig. 9 The expression trend of LC3 and P62 genes is consistent with that of protein. Fig.10 . Fig. 9 The expression of autophagy-related proteins in articular cartilage of each group. A: control group; B: model group; C: low-dose group of Tongbi Jiangu Fang; D: medium-dose group of Tongbi Jiangu Fang; E: high-dose group of Tongbi Jiangu Fang; F: LC3Ⅱ / LC3Ι ratio statistics; G: P62 protein relative expression statistics; *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group. Fig.10 The expression of LC3 and P62 mRNA in cartilage tissue of rats in each group. A: LC3 mRNA expression statistical chart; B: P62 mRNA expression statistical chart; *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group.

[0205] 3.8 Expression of P-AMPK, P-mTOR, and P-ULK1 in the knee cartilage of rats in each group

[0206] Compared with the control group, the expression of P-AMPK and P-ULK1 proteins in the model group was significantly decreased (P<0.01, P<0.05), and the expression of P-mTOR was significantly increased (P<0.01). Compared with the model group, the expression of P-AMPK protein in the medium-dose group of Tongbi Jiangu Fang was significantly increased (P<0.05), and there was no significant difference between the low-dose and high-dose groups of Tongbi Jiangu Fang (P>0.05). The expression of P-ULK1 protein in the low-dose and medium-dose groups of Tongbi Jiangu Fang was significantly increased (P<0.01), among which the medium-dose group of Tongbi Jiangu Fang was the most significant, and there was no significant difference between the high-dose group of Tongbi Jiangu Fang (P>0.05). The expression of P-mTOR protein in the medium-dose group of Tongbi Jiangu Fang was significantly decreased (P<0.05), and there was no significant difference between the low-dose and high-dose groups of Tongbi Jiangu Fang (P>0.05). Fig.11 The gene expression of AMPK, mTOR, and ULK1 was consistent with the protein expression trend, see Fig.12 . Fig.11The expression of P-AMPK, P-mTOR, and P-ULK1 in articular cartilage of each group. Among them, A: control group; B: model group; C: low-dose group of Tongbi Jiangu Fang; D: medium-dose group of Tongbi Jiangu Fang; E: high-dose group of Tongbi Jiangu Fang; F: P-AMPK protein relative expression statistical chart; G: P-mTOR protein relative expression statistical chart; H: P-ULK1 protein relative expression statistical chart; *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group. Fig.12 The expression of AMPK, mTOR, and ULK1 mRNA in the cartilage tissue of rats in each group. A: AMPK mRNA expression statistical chart; B: mTOR mRNA expression statistical chart; C: ULK1 mRNA expression statistical chart; *P<0.05, **P<0.01, vs. control group; # P<0.05, ## P<0.01, vs. model group.

[0207] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Chinese medicine composition for treating knee osteoarthritis, characterized in that: The Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-25 parts of Cynomorium Bidentatae, 15-25 parts of Rehmannia glutinosa, 15-25 parts of Cynomorium songaricum, 15-25 parts of Millennium scutellariae, 15-25 parts of Snow Lotus, 15-25 parts of Cibotium barometz, 15-25 parts of Fenugreek, 15-25 parts of Agkistrodon acutus, 10-20 parts of Cynomorium Changqing, 10-20 parts of Pinus tabulaeformis, 10-20 parts of Corydalis yanhusuo, 10-20 parts of Linderae scutellariae, 15-25 parts of Angelica sinensis, 10-20 parts of Ligusticum chuanxiong, 15-25 parts of Dioscorea opposita, 15-25 parts of Poria, 10-20 parts of Citrus aurantium, 15-25 parts of Orientalis and 10-20 parts of Licorice. The pharmaceutical dosage form of the pharmaceutical composition is a decoction, a capsule or a granule.

2. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is prepared from the following raw materials in parts by weight: 15 parts of Cyathula officinalis, 15 parts of Rehmannia root, 15 parts of Cynomorium songaricum, 15 parts of Millennium scutellaria, 15 parts of Snow Lotus, 15 parts of Cibotium barometz, 15 parts of Fenugreek, 15 parts of Snakehead acanthus, 10 parts of Cynomorium longituba, 10 parts of Pinus tabulaeformis, 10 parts of Corydalis yanhusuo, 10 parts of Linderae scutellariae, 15 parts of Angelica sinensis, 10 parts of Ligusticum chuanxiong, 15 parts of Dioscorea opposita, 15 parts of Poria, 10 parts of Citrus aurantium, 15 parts of Orientalis and 10 parts of Licorice.

3. The method for preparing the Chinese medicine composition according to claim 1 or 2, characterized in that: The preparation method of the water decoction comprises the following steps: mixing the raw materials in the formula and soaking them in water until the drug tissue is penetrated, then adding water with a thickness of 30 to 35 mm thicker than the drug and boiling for 40 to 45 minutes, filtering the drug solution, adding water with a thickness of 25 to 30 mm thicker than the drug to the filter residue and boiling for 30 to 35 minutes, filtering and combining the filtrate.

4. The method for preparing the Chinese medicine composition according to claim 1 or 2, characterized in that: The preparation method of the capsule comprises the following steps: mixing the raw materials of the formula amount and crushing them into fine powder, passing through a 120-mesh sieve so that the amount of fine powder that can pass through the sieve reaches 50%, and collecting the sieved fine powder; then extracting the coarse powder twice with water, combining the filtrate, heating the filtrate to concentrate and dry it, and grinding it into fine powder, mixing the two fine powders and filling them into a capsule shell to obtain the capsule.

5. The method for preparing the Chinese medicine composition according to claim 1 or 2, characterized in that: The preparation method of the granules comprises the following steps: mixing the raw materials in the formula, adding 8 to 10 times the weight of water, soaking for 0.5 h, decocting twice, decocting for 1.5 h for the first time, and decocting for 1 h for the second time with 8 to 10 times the weight of water, combining the decoctions, filtering, concentrating to obtain an extract, drying and crushing into fine powder, adding auxiliary materials for granulation, and obtaining granules.

6. Use of the Chinese medicine composition according to claim 1 or 2 in the preparation of a medicament for treating knee osteoarthritis.

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