CN116808128A

CN116808128A - Traditional Chinese medicine composition for treating knee osteoarthritis and preparation method and application thereof

Info

Publication number: CN116808128A
Application number: CN202310535145.0A
Authority: CN
Inventors: 周明旺; 王晓萍; 王玉磊; 李盛华; 柳海平; 吉星; 魏长浩; 张朋威; 何帮靖
Original assignee: GANSU PROVINCIAL HOSPITAL OF TCM
Current assignee: GANSU PROVINCIAL HOSPITAL OF TCM
Priority date: 2023-05-12
Filing date: 2023-05-12
Publication date: 2023-09-29

Abstract

The invention provides a traditional Chinese medicine composition for treating knee osteoarthritis, which is prepared from the following raw materials in parts by weight: 15-25 parts of medicinal cyathula root, 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 fenugreek, 15-25 parts of zaocys dhumnade, 10-20 parts of paniculate swallowwort root, 10-20 parts of Chinese pine, 10-20 parts of rhizoma corydalis, 10-20 parts of combined spicebush root, 15-25 parts of Chinese angelica, 10-20 parts of szechuan lovage rhizome, 15-25 parts of Chinese yam, 15-25 parts of poria cocos, 10-20 parts of green tangerine peel, 15-25 parts of rhizoma alismatis and 10-20 parts of liquorice. The traditional Chinese medicine composition disclosed by the invention can be used for effectively treating knee osteoarthritis.

Description

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

Technical Field

The invention relates to a traditional Chinese medicine composition for treating knee osteoarthritis and a preparation method and application thereof.

Background

Knee Osteoarthritis (KOA) is a chronic joint disorder characterized by degenerative changes of articular cartilage and secondary hyperosteogeny, with pain, swelling, limited movement and even joint deformity of the knee joint as the principal symptoms, common in the middle-aged and elderly. Epidemiological investigation shows that the prevalence of people over 55 years old is 44% -70%, and the prevalence of men in people over 65 years old is about 60%, and the prevalence of women is as high as 70%. At present, the knee osteoarthritis is treated by a plurality of methods, western medicines are mostly non-steroidal anti-inflammatory drugs, and side effects are numerous after long-term use; surgical operations have certain limitations and risks and have poor long-term curative effects. The traditional Chinese medicine considers that the knee osteoarthritis belongs to the category of arthralgia syndrome and osteoarthritis in traditional Chinese medicine, the disease name comes from the first-aid Qianjin Fang of Sunshengao, the etiology is pathogenic wind-cold-damp-heat, the meridian is blocked, and qi-blood is unsmooth, so that joint pain, soreness, swelling, heavy attachment and movement are caused at the affected part. The pathogenesis is based on deficiency of liver and kidney, invasion of exogenous evil, blockage of channels and collaterals, pain due to obstruction, and qi and blood disorder. And is closely related to physical factors, climate conditions, living environments and the like.

The Chinese medicinal preparation is any medicine which must be prepared into a form suitable for medical or preventive application before clinical use. The disease is mild and urgent, and the disease is superficial and internal, so the requirements on the dosage form are different. In order to exert the therapeutic effect of the drug better, the requirements on the dosage form and the preparation are also different according to the nature of the drug.

Disclosure of Invention

In order to solve the problems in the prior art, the application provides a traditional Chinese medicine composition for treating knee osteoarthritis and a preparation method thereof. The traditional Chinese medicine composition disclosed by the application has the advantages of remarkable curative effect on early-stage and medium-stage knee osteoarthritis, small side effect and stable curative effect, and can be used for remarkably improving pain and dysfunction of knee osteoarthritis and improving the quality of daily life. The traditional Chinese medicine composition mainly takes the formulation granule formulation as a main component in clinical application, overcomes the defects of long decoction time, difficult preservation, inconvenient carrying and the like of the traditional Chinese medicine, ensures the characteristic of dialectical treatment of the traditional Chinese medicine to a certain extent, and fully exerts the treatment effect of the traditional Chinese medicine. The traditional Chinese medicine composition granule of the application extracts and separates the medicinal components of the traditional Chinese medicine decoction pieces by a modernization technology to prepare the traditional Chinese medicine preparation, thereby avoiding longer decoction time and effectively preventing the problems of mildew, worm-eating and the like caused by improper storage of the traditional Chinese medicine decoction pieces.

The invention provides a traditional Chinese medicine composition for treating knee osteoarthritis, which is prepared from the following raw materials in parts by weight: 15-25 parts of medicinal cyathula root, 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 fenugreek, 15-25 parts of zaocys dhumnade, 10-20 parts of paniculate swallowwort root, 10-20 parts of Chinese pine, 10-20 parts of rhizoma corydalis, 10-20 parts of combined spicebush root, 15-25 parts of Chinese angelica, 10-20 parts of szechuan lovage rhizome, 15-25 parts of Chinese yam, 15-25 parts of poria cocos, 10-20 parts of green tangerine peel, 15-25 parts of rhizoma alismatis and 10-20 parts of liquorice.

Preferably, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15 parts of medicinal cyathula root, 15 parts of prepared rehmannia root, 15 parts of cynomorium songaricum, 15 parts of obscured homalomena rhizome, 15 parts of snow lotus herb, 15 parts of rhizoma cibotii, 15 parts of fenugreek, 15 parts of zaocys dhumnade, 10 parts of paniculate swallowwort root, 10 parts of Chinese pine node, 10 parts of rhizoma corydalis, 10 parts of combined spicebush root, 15 parts of Chinese angelica, 10 parts of szechuan lovage rhizome, 15 parts of Chinese yam, 15 parts of poria cocos, 10 parts of green tangerine peel, 15 parts of rhizoma alismatis and 10 parts of liquorice.

The invention also provides a preparation method of the traditional Chinese medicine composition, which comprises the steps of mixing the raw materials in the formula amount, soaking the raw materials in water until the raw materials penetrate into tissue, adding water with the thickness of 30-35mm for decocting for 40-45 minutes, filtering the liquid medicine, adding water with the thickness of 25-30mm into filter residues for decocting for 30-35 minutes, filtering and combining the filtrates.

The application also provides a preparation method of the traditional Chinese medicine composition, which comprises the steps of mixing the raw materials in the formula amount, crushing the mixture into fine powder, sieving the fine powder through a 120-mesh sieve to ensure that the amount of the fine powder passing through the sieve reaches 50%, and collecting the sieved fine powder; extracting coarse powder with water twice, mixing filtrates, heating, concentrating, drying, grinding into fine powder, mixing the two fine powders, and encapsulating to obtain capsule.

The application also provides a preparation method of the traditional Chinese medicine composition, which comprises the steps of mixing the raw materials in the formula amount, adding water with the weight being 8-10 times that of the raw materials for soaking for 0.5h, decocting for 2 times, 1.5h, 2 times, 8-10 times that of the raw materials for 1h, mixing decoctions, filtering, concentrating to obtain extract, drying, pulverizing into fine powder, adding auxiliary materials for granulating, and obtaining the granule.

The application also provides application of the traditional Chinese medicine composition in preparing medicines for treating knee osteoarthritis.

The application also provides a medicine, and the active ingredients of the medicine comprise the traditional Chinese medicine composition.

Preferably, the medicament further comprises an auxiliary material; the auxiliary materials are auxiliary materials commonly used in pharmacy.

Preferably, the medicament comprises decoction, capsules and granules.

The inventor of the application is based on epidemiology discovery on knee osteoarthritis physique in Gansu region, mainly based on yang deficiency physique, and on the basis, the traditional Chinese medicine preparation is summarized by combining experience of treating knee osteoarthritis, and provides the idea that liver and kidney deficiency and malnutrition of tendons and vessels are root causes, wind-cold-dampness arthralgia is an external factor, the symptoms belong to liver and kidney deficiency, and pathogenesis is the symptoms of "deficiency and excess of principal deficiency and excess inclusion". Malnutrition of the joints, inability to exercise, and pain; wind-cold invasion, blockage of tendons and vessels, and pain due to obstruction. Is used for treating liver and kidney invigorating, cold dispelling, dampness eliminating, arthralgia relieving, and pain relieving. The Chinese medicinal preparation is an proved recipe summarized in clinical practice of a team for many years, and the whole recipe consists of 19 Chinese medicaments of medicinal cyathula root, prepared rehmannia root, cynomorium songaricum, obscured homalomena rhizome, snow lotus herb, east Asian tree fern rhizome, fenugreek, zaocys dhumnade, paniculate swallowwort root, chinese pine node, corydalis tuber, combined spicebush root, chinese angelica, szechuan lovage rhizome, chinese yam, indian buead, green tangerine peel, oriental waterplantain rhizome and liquoric root, has proper compatibility, treats both principal and secondary aspect of disease, combines the medicaments, and has the effects of tonifying liver and kidney, dispelling cold and dispelling wind, and activating arthralgia and relieving pain.

And (3) square solution: in the formula, prepared rehmannia root, medicinal cyathula root, obscured homalomena rhizome and cynomorium songaricum are taken as monarch drugs for replenishing essence and nourishing marrow, tonifying liver and kidney, softening meridians and strengthening tendons and bones; herba Saussureae Involueratae, rhizoma Cibotii, and semen Trigonellae are used as principal drugs for invigorating liver and kidney, warming yang and dispersing arthralgia, zaocys, radix Cynanchi Paniculati, herba Pini nodi dispelling cold and dampness, relieving arthralgia and pain, eliminating pathogenic factors, promoting blood circulation and activating qi-flowing, warming channels and relieving pain; the Chinese angelica and the szechuan lovage rhizome have the effects of nourishing blood, promoting blood circulation, dredging collaterals and relieving pain, the Chinese yam, the poria cocos and the green Pi Jian have the effects of promoting qi circulation and removing dampness, restricting the suspicion of greasiness such as prepared rehmannia root, medicinal cyathula root, cynomorium songaricum and the like, assisting the exertion of the efficacy of the medicine, and the alisma rhizome has the effects of excreting dampness and relieving swelling, and assisting other medicines to be excessively warm and dry; the licorice root, radix Glycyrrhizae Praeparata is used as a guiding drug for relieving pain and harmonizing various drugs. The whole formula is proper in compatibility, strict in refining and capable of treating both principal and secondary aspect of disease. The medicines are used for tonifying liver and kidney, dispelling cold and dampness, and relieving arthralgia and pain.

The inventor of the present application conducts modern network pharmacological research on the traditional Chinese medicine preparation, and discovers that quercetin, kaempferol and beta-sitosterol are core active ingredients. Quercetin is a flavonoid compound widely existing in traditional Chinese medicinal materials, and has effects of resisting tumor, oxidation, inhibiting inflammation and virus, and regulating immune response. Modern pharmacological research shows that quercetin can induce RAW264.7 cells to release and can remarkably inhibit secretion of nitric oxide, tumor necrosis factor-alpha (tumor necrosis factor-alpha, TNF-alpha), interleukin (IL) -6 and IL-1 beta, and is a potential anti-inflammatory analgesic drug for treating KOA. Kaempferol and quercetin have similar effects, and have antioxidant, antiinflammatory, immunity regulating and estrogen-like pharmacological activities, and can be used for preventing and treating OA, RA and osteoporosis. Beta-sitosterol has various biological activities such as cholesterol reduction, antioxidation, anti-inflammatory, bacteriostasis, hormone-like function and the like. In conclusion, the traditional Chinese medicine preparation has the effects of resisting inflammation, easing pain and regulating immunity in the treatment of KOA.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:

fig. 1 is a graph showing the analysis of the change in diameter of the right hind knee joint at different stages in each group of rats (n=10).

Fig. 2 is a view of the knee joint of each group of rats.

Fig. 3 is a general observation score for rat knee joints.

Fig. 4 is HE staining (×100) of the knee cartilage of the right lower limb of each group of rats.

Fig. 5 is a modified Mankin score.

FIG. 6 shows the expression of IL-1. Beta. And TNF-. Alpha.mRNA in cartilage tissue of each group of rats.

FIG. 7 shows the expression of Aggrecan, collagen II, TIMP1 and MMP13 proteins in various articular cartilage groups.

FIG. 8 shows the expression of Aggrecan, collagen II, TIMP1 and MMP13 mRNA in cartilage tissue of rats in each group.

FIG. 9 shows the expression of autophagy-related proteins in various groups of articular cartilage.

FIG. 10 shows the expression of LC3 and P62 mRNA in cartilage tissue of rats in each group.

FIG. 11 shows the expression of P-AMPK, P-mTOR, and P-ULK1 in various articular cartilage groups.

FIG. 12 shows the expression of AMPK, mTOR, ULK mRNA in cartilage tissue of rats in each group.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagents. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

In the invention, the "arthralgia-relieving and bone-strengthening prescription" refers to the traditional Chinese medicine composition.

The traditional Chinese medicine composition for treating knee osteoarthritis consists of the following raw materials in parts by weight:

15-25 parts of medicinal cyathula root, 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 fenugreek, 15-25 parts of zaocys dhumnade, 10-20 parts of paniculate swallowwort root, 10-20 parts of Chinese pine, 10-20 parts of rhizoma corydalis, 10-20 parts of combined spicebush root, 15-25 parts of Chinese angelica, 10-20 parts of szechuan lovage rhizome, 15-25 parts of Chinese yam, 15-25 parts of poria cocos, 10-20 parts of green tangerine peel, 15-25 parts of rhizoma alismatis and 10-20 parts of liquorice.

The preparation method of the traditional Chinese medicine composition decoction for treating knee osteoarthritis comprises the following steps:

weighing all the raw materials according to the parts by weight, soaking in purified water until the tissue of the medicine is permeated, wherein the soaking time is generally half an hour, then putting the soaked medicine and the clear liquid of the soaked medicine into a medicine decocting machine, pouring purified water with the thickness of 30-35mm into the medicine decocting machine for first decoction for 40-45 minutes, filtering the liquid medicine, pouring purified water with the thickness of 25-30mm into the medicine decocting machine for second decoction for 30-35 minutes, and mixing to obtain the water decoction.

The application method of the traditional Chinese medicine composition decoction comprises the following steps: orally taken, one dose is taken every day, and twice is taken separately.

The preparation method of the traditional Chinese medicine composition capsule for treating knee osteoarthritis comprises the following steps:

weighing all the raw materials according to the weight parts, mixing, crushing into coarse powder, crushing again, sieving with a 120-mesh sieve to ensure that the amount of fine powder passing through the sieve reaches 50%, and collecting fine powder passing through the sieve; extracting the coarse powder with 8 times of water for 2 hr, decocting with 3-5 times of water for 1 hr, filtering, mixing decoctions, heating, concentrating to paste, oven drying at 70deg.C, standing to room temperature, grinding into fine powder, mixing the fine powder, and encapsulating.

If all the Chinese medicines are crushed by the crushing technology, part of coarse powder cannot be crushed into fine powder and pass through a 100-120-mesh sieve, so that the multifunctional Chinese medicine extracting tank is used for extracting with water to assist in crushing into fine powder, and the medicine property of the Chinese medicines cannot be destroyed.

The application method of the traditional Chinese medicine composition capsule comprises the following steps: orally taken, 2 times a day, once for 4-6 granules.

The preparation method of the traditional Chinese medicine composition granules for treating knee osteoarthritis comprises the following steps:

Weighing all the raw materials according to the weight parts, mixing, adding 8-10 times of water for soaking for 0.5h, decocting for 2 times, decocting for 1.5h for 1 st time, adding 8-10 times of water for 1h for 2 times, mixing decoctions, filtering, sequentially concentrating the filtrate under normal pressure (100 ℃) and vacuum concentration (-70 ℃ and 0.1 MPa) to obtain extractum with the relative density of 1.25 (70 ℃), drying, pulverizing into fine powder, adding sucrose (dry extract powder: sucrose weight ratio=1:2), stirring for 10min, mixing, granulating with 85% ethanol (dry extract powder: 85% ethanol weight ratio=1:0.40), drying, granulating, and packaging.

The application method of the traditional Chinese medicine composition granule comprises the following steps: placing the granule into a container, soaking in boiling water for 4min, stirring, and orally administering 1 dose per day for 2 times per day.

Example 1

The formula of the traditional Chinese medicine composition for treating knee osteoarthritis is as follows:

15g of medicinal cyathula root, 15g of prepared rehmannia root, 15g of cynomorium songaricum, 15g of obscured homalomena rhizome, 15g of snow lotus herb, 15g of rhizoma cibotii, 15g of fenugreek seed, 15g of zaocys dhumnade, 10g of paniculate swallowwort root, 10g of Chinese pine node, 10g of rhizoma corydalis, 10g of combined spicebush root, 15g of Chinese angelica, 10g of szechuan lovage rhizome, 15g of Chinese yam, 15g of poria cocos, 10g of green tangerine peel, 15g of rhizoma alismatis and 10g of liquorice.

The preparation method of the water decoction of the traditional Chinese medicine composition for treating knee osteoarthritis comprises the following steps:

According to the mass ratio, the raw material medicines with high quality and cleanness are purchased, after impurities and dust are removed respectively, the medicines are placed into cold boiled water for soaking for half an hour, the soaked medicines and clear liquid for soaking the medicines are placed into a medicine decocting machine together, cold boiled water with the thickness of 35mm is injected for carrying out first decoction for 40 minutes, after medicine liquid is filtered, purified water with the thickness of 30mm is injected for carrying out second decoction for 35 minutes, and after the medicines are mixed and concentrated into 400 milliliters of 50% of traditional Chinese medicine liquid, the traditional Chinese medicine liquid is put into a refrigerator for fresh keeping for standby.

Example 2

The formulation of the traditional Chinese medicine composition for treating knee osteoarthritis of the present invention is the same as that of example 1.

The preparation method of the capsule of the traditional Chinese medicine composition for treating knee osteoarthritis comprises the following steps:

weighing all the raw materials according to the weight portion ratio, crushing into coarse powder, further crushing, sieving with a 120-mesh sieve to ensure that the amount of the fine powder passing through the sieve reaches 50%, and collecting the fine powder passing through the sieve; extracting the coarse powder which cannot pass through the capsule with a multifunctional Chinese medicine extracting tank for 2 times, adding 10 times of water for 2 hours for the first time, adding 5 times of water for 1 hour for the second time, filtering, combining the two decoctions, heating and concentrating to paste, putting into a baking oven for drying at 70 ℃, standing to room temperature, grinding into fine powder, mixing the two fine powder, and filling into a capsule shell to obtain the capsule.

Example 3

The preparation method of the traditional Chinese medicine granule of the traditional Chinese medicine composition comprises the following steps:

weighing all the raw materials according to the weight, mixing, adding 8 times of water to soak for 0.5h, decocting for 2 times, decocting for 1 st time for 1.5h, adding 8 times of water for 1h, mixing decoctions, filtering, sequentially concentrating the filtrate under normal pressure (100 ℃) and concentrating under reduced pressure (70 ℃ and minus 0.1 MPa) to obtain extract with the relative density of 1.25 (70 ℃), drying, pulverizing into fine powder, adding sucrose (dry extract powder: sucrose weight ratio=1:2), stirring for 10min, mixing, granulating with 85% ethanol (dry extract powder: 85% ethanol weight ratio=1:0.40), drying, granulating, and packaging.

The application method of the traditional Chinese medicine composition granule comprises the following steps:

the granule is 1 dose, and is taken twice a day in the morning and evening.

Placing the granule into a container, adding 300mL of boiling water, soaking for 4min, stirring, and oral administration.

The treatment period is 2 courses of treatment, and each course of treatment is 7 days.

Example 4

radix cyathulae 25g, prepared rehmannia root 25g, cynomorium songaricum 25g, obscured homalomena rhizome 25g, snow lotus herb 25g, rhizoma cibotii 25g, fenugreek 25g, zaocys dhumnade 25g, paniculate swallowwort root 20g, chinese pine node 20g, corydalis tuber 20g, lindera root 20g, chinese angelica 25g, ligusticum wallichii 20g, chinese yam 25g, poria cocos 25g, green tangerine peel 20g, alisma orientale 25g and licorice root 20g.

according to the mass ratio, the raw material medicines with high quality and cleanness are purchased, after impurities and dust are removed respectively, the medicines are placed into cold boiled water for soaking for half an hour, the soaked medicines and clear liquid for soaking the medicines are placed into a medicine decocting machine together, cold boiled water with the thickness of 30mm is injected for carrying out first decoction for 45 minutes, after medicine liquid is filtered, purified water with the thickness of 25mm is injected for carrying out second decoction for 30 minutes, and after the medicines are mixed and concentrated into 400 milliliters of 50% of traditional Chinese medicine liquid, the traditional Chinese medicine liquid is put into a refrigerator for fresh keeping for standby.

Example 5

20g of medicinal cyathula root, 20g of prepared rehmannia root, 20g of cynomorium songaricum, 20g of obscured homalomena rhizome, 20g of snow lotus herb, 20g of rhizoma cibotii, 20g of fenugreek seed, 20g of zaocys dhumnade, 15g of paniculate swallowwort root, 15g of Chinese pine node, 15g of rhizoma corydalis, 15g of combined spicebush root, 20g of Chinese angelica, 15g of szechuan lovage rhizome, 20g of Chinese yam, 20g of poria cocos, 15g of green tangerine peel, 20g of rhizoma alismatis and 15g of liquorice.

weighing all the raw materials according to the weight, mixing, adding 10 times of water to soak for 0.5h, decocting for 2 times, decocting for 1 st time for 1.5h, adding 9 times of water for 1h, mixing decoctions, filtering, sequentially concentrating the filtrate under normal pressure (100 ℃) and concentrating under reduced pressure (70 ℃ and minus 0.1 MPa) to obtain extract with the relative density of 1.25 (70 ℃), drying, pulverizing into fine powder, adding sucrose (dry extract powder: sucrose weight ratio=1:2), stirring for 10min, mixing, granulating with 85% ethanol (dry extract powder: 85% ethanol weight ratio=1:0.40), drying, granulating, and packaging.

Example 6 discovery of the mechanism of action of the Chinese medicinal composition of the application for treating osteoarthritis of the knee based on network pharmacology and molecular docking techniques

1 screening of active ingredients and targets of the Chinese medicinal composition

The inventor discovers that the core active ingredients of the traditional Chinese medicine composition are quercetin, kaempferol and beta-sitosterol through modern network pharmacological research. See in particular table 1.

TABLE 1 basic information of the major active ingredients of the Chinese medicinal composition of the application

2 molecular docking verification

The key targets IL-1 beta, MMP-3 and TNF-alpha with higher degree value are selected as receptors, and the core active ingredients of the traditional Chinese medicine composition are used as ligands for molecular docking verification. See in particular table 2.

TABLE 2 Butt score of core active ingredients and key target molecules of the inventive herbal compositions

3 effects of the Chinese medicinal composition of the application on IL-1 beta, MMP-3, TNF-alpha expression in the supernatant of KOA chondrocytes

The experiment is set to 4 groups, namely a blank control group, a low concentration group, a medium concentration group and a high concentration group of the traditional Chinese medicine composition. The Chinese medicinal composition prepared in the embodiment 1 of the application is centrifuged for 10min (the rotating speed is 2000 r.min) ^-1 Centrifugation radius 125 mm), taking supernatant; freeze drying with freeze drier to obtain freeze dried powder with yield of 25%. Precisely weighing a proper amount of lyophilized powder, dissolving and thawing the dried powder with dimethyl sulfoxide, filtering and sterilizing with microporous filter, and preparing into medicine with mass concentration of 4g.L ^-1 The reference mother liquor of the arthralgia-relieving and bone-strengthening prescription is preserved in a refrigerator at the temperature of minus 20 ℃ for standby. When in use, the mother solution is thawed in advance, and diluted by a complete culture medium to obtain the product with the high, medium and low mass concentrations of 600,400,200mg.L calculated by crude drug ^-1 The working fluids of the high concentration group, the medium concentration group and the low concentration group of the traditional Chinese medicine composition.

The experimental procedure was the same as in example 8.

Compared with a blank control group, the levels of IL-1 beta, MMP-3 and TNF-alpha in the supernatant of each concentration group of the traditional Chinese medicine composition are reduced (P < 0.05); the high concentration group was lower and the IL-1β, MMP-3 and TNF- α levels were decreased to a dose-dependent extent compared to the medium and low concentration groups, and the results are shown in Table 3.

TABLE 3 Effect of the Chinese medicinal composition of the invention on IL-1 beta, MMP-3, TNF-alpha expression in the supernatant of KOA chondrocytes

Note that: comparison with blank group ¹⁾ P<0.05; compared with the high concentration group ²⁾ P<0.05。

4 influence of the Chinese medicinal composition on the expression of NF- κ B p65mRNA and IκB-alpha mRNA of KOA chondrocyte

The experimental group is the same as step 3.

The experimental procedure was the same as in example 8.

Compared with blank groups, the NF-kappa B P mRNA level of each group of the traditional Chinese medicine composition is reduced (P < 0.05), and the I kappa B-alpha mRNA level is increased (P < 0.05); compared with the middle and low concentration groups, the high concentration group has lower NF-kappa B p mRNA, higher I kappa B-alpha m RNA level and lower NF-kappa B p mRNA level, and the increase degree of I kappa B-alpha mRNA level is dose-dependent with the concentration of the traditional Chinese medicine composition, and the result is shown in Table 4.

TABLE 4 Effect of the Chinese medicinal composition of the invention on the expression of KOA chondrocyte NF- κ B p65, IκB- αmRNA

The main action mechanism of the traditional Chinese medicine composition is to apply active ingredients such as quercetin, kaempferol, beta-sitosterol and the like, and to use IL-1 beta, MMP-3 and TNF-alpha as key targets to play a role in treating knee osteoarthritis through NF- κB signaling pathway.

EXAMPLE 7 clinical efficacy of the Chinese medicinal composition of the invention for treating early-mid-term KOA

1 subject for clinical test of osteoarthritis KOA in knee

1.1 case Source

56 early and medium-term KOA patients meeting the diagnosis standard in orthopaedics outpatient service and hospitalization department of the department of hospitalization in Gansu province are collected from 11 months in 2020 to 1 month in 2022.

1.2 formulation of case report forms

The composition of the case report form comprises: subjects informed consent, general data, VAS score, WOMAC score, time-to-rise walking test, 6-minute walking test, joint active-passive activity, safety assessment detection. General data include patient gender, age, height, weight, current medical history, past history, allergic history, etc. Subjects informed consent, the project was ethically approved by the ethical committee and all subjects were required to sign informed consent.

1.3 inclusion criteria

(1) Meets the KOA diagnosis standard of traditional Chinese medicine and Western medicine;

(2) Meets the standard of diagnosis and waiting for the symptoms of the traditional Chinese medicine diagnosis and treatment guidelines for knee osteoarthritis (2020 edition);

(3) Radiology diagnosis conforming to Kellgren-Lawrence (K-L) is graded as grade I, II or III patients; stage I or II is early stage and stage III is medium stage.

(4) Meets the clinical staging standard of traditional Chinese medicine;

(5) Age 50-70 years old;

(6) Patients who have not been treated by the knee osteoarthritis system within 1 month can adhere to the schedule to complete the treatment, and can cooperate with clinical observers and follow-up visits;

(7) The subjects were fully aware of the nature of the study, the nature of the disease, and were able to understand and sign informed consent.

2. Research method

2.1 Clinical trial design method

The experiment adopts a simple random method commonly used in clinical study to group, the patients meeting the inclusion standard are randomly grouped according to the patient treatment sequence, the patients meeting the inclusion standard are 1 in group A, the patients meeting the inclusion standard are 2 in group B, and the like, 56 patients meeting the inclusion standard are collected altogether, 28 treatment groups (group A) of the traditional Chinese medicine composition prepared in the embodiment 1 of the invention, and 28 treatment groups (group B) of the diclofenac sodium sustained-release capsules (group B) of the traditional Chinese medicine composition prepared in the embodiment 1 of the invention.

2.2 treatment regimen

Both groups used basic treatments based on health propaganda and education. In the case of administration of the basal treatment to each group, group a intervened in the traditional Chinese medicine composition prepared in example 1 of the present invention; group B diclofenac sodium sustained release capsules, both groups are treated 7 times per week, one treatment course is 7 days, and the treatment period is 2 treatment courses.

2.2.1 basal therapy

The basic treatment mainly takes health propaganda and education as main materials, and the specific contents are as follows:

(1) Informing the patient of the cause of KOA onset;

(2) KOA daily life prevention;

(3) KOA knee joint quadriceps muscle stretching exercise.

2.2.2A treatment scheme of the traditional Chinese medicine composition group of the invention:

the treatment method is shown in example 1, one treatment course is one week, and the treatment period is 2 treatment courses.

2.2.3 control treatment protocol:

the diclofenac sodium sustained release capsule (Xintaiqing) is given to the production enterprises: sea wetting is available from pharmaceutical Co., ltd. The administration method comprises the following steps: is orally administered 2 (100 mg) granules per 1 time, 1 time per day, 30min after meal with warm water.

2.3 efficacy observation index

2.3.1 case general data:

including patient profile including patient gender, age, course of disease and imaging Kellgren-Lawrence classification.

2.3.2 observations index:

2.3.2.1VAS score:

a facial makeup pain scoring list is adopted, a long line is drawn between painless and severe pain, 1-10 scales are marked on the line, and a facial makeup from smiling face to bitter face is matched, and no special mark or word is made on the line so as not to influence the evaluation result. The smiling face end represents no pain, and the bitter face (crying) end represents severe pain, so that the patient can select the facial makeup or the number which can most describe the pain degree of the patient. 0 point: no pain; the 3-point method comprises the following steps: there is a slight pain; 4-6: the patients are painful and influence sleep, and can endure; 7-10: patients have stronger pain, which is hard to feel, and the appetite and sleep are affected.

2.3.2.2WOMAC osteoarthritis index score:

the questionnaire is divided into three modules: (1) pain: containing 5 problems; the higher the pain degree, the higher the score; (2) degree of stiffness, including 2 problems; the higher the stiffness, the higher the score; (3) difficulty level of daily activity sensation, including 17 questions; the higher the difficulty, the higher the score. The total fraction is more than or equal to 40 and is 1 grade, 20-39 is 2 grade, 10-19 is 3 grade, and 0-9 is 4 grade. The lower the total score, the higher the ranking, and the lesser the extent to which KOA affects the patient's daily life.

2.3.2.3 timing standing walking test:

a method for rapidly and quantitatively assessing functional walking ability. The patient sits on the armchair with armrests (chair seat height about 45cm, armrests height about 20 cm) with the body resting on the back and with both hands resting on the armrests during assessment. If a walker (e.g., cane, walker) is used, the walker is held in the hand. A color bar or a visible thick line is attached or a marked mark is placed on the ground 3 meters away from the seat. When the tester issues a "start" instruction, the patient stands up from the back rest chair. After standing, the user walks forward for 3 meters according to the gait of walking at ordinary times, turns around after passing through a thick line or a marker, then walks back to the front of the chair, turns around again and sits down, and leans against the chair back. No help is given to any body during the test. The tester records the time (in seconds) it takes for the back of the patient to leave the seat back and sit down again (against the seat back), and the risk of possible falls during the completion of the test. A time of less than 10 seconds indicates that the activity is normal. 11-20 seconds is normal Fan for the elderly and disabled, with time exceeding 20 seconds indicating the need for assistance, further inspection and thousand pre-treatment.

2.3.2.4 The 6-minute walk test tests the distance in meters walked by the test subject within 6 minutes.

2.3.2.5 active-passive articulation: the active and passive activities of the affected knee were measured (israel KAPRO open-circuit 992 electronic digital ruler protractor).

2.4 efficacy assessment criteria

According to the disease curative effect judging standard in the clinical research guidelines (2002 edition) of new traditional Chinese medicines, the clinical curative effect is classified into four grades of clinical control, obvious effect, effective and ineffective according to different manifestations of the symptom relief and the physical sign improvement degree of the subjects after treatment, and the method is as follows:

(1) clinical control: pain disappears, the joint movement is recovered to be normal, and the integral reduction is more than or equal to 95%;

(2) the effect is shown: pain disappears, the joint movement range is not limited, the joint weight-bearing movement and the durability are slightly deficient, and the integral reduction is more than or equal to 70 percent and less than 95 percent;

(3) the method is effective: pain is basically eliminated, the joint movement range is slightly limited, and the integral is reduced by more than or equal to 30 percent and is less than 70 percent;

(4) invalidation: pain and joint movement are not improved obviously, and the integral reduction is less than 30%.

Note that: calculation formula (nimodipine method): effective rate = [ (WOMAC integral before treatment-WOMAC integral after treatment)/(WOMAC integral before treatment ] ×100%).

2.5 Security assessment

Safety observation index:

(1) Routine blood and urine, liver function, kidney function, and electrocardiogram. One observation was performed before and after 2 weeks of treatment.

(2) Adverse reaction: the medicine group observes whether gastrointestinal tract reaction is caused during the research period, mainly including stomach discomfort, abdominal pain, burning sensation, acid regurgitation, anorexia, constipation, nausea and the like; when an adverse reaction is found, whether to intervene or not is determined according to the situation, whether the adverse reaction is related to the treatment of the test is determined, and whether to stop the test is determined according to the state of the illness. The extent, time and duration of adverse reactions occurring during the study, the treatments taken and how well they were counted were faithfully recorded.

2.6 statistical treatment

Statistical analysis is carried out on the data by uniformly applying statistical software of version SPSS26.0, and all data analysis results are uniformly in a table by mean value plus or minus standard deviation"means. And if the test results are consistent with normal distribution and/or inconsistent with normal distribution, selecting rank sum test for data analysis. For the gender and other counting data, X is selected ² The test is used for comparison, and the rank and the test are selected for comparison, wherein the curative effect grade and the like belong to grade data. Data comparison between groups before and after treatment is performed by paired t test, data comparison between groups is performed by single factor analysis of variance, and the result is represented by P >There is no statistical difference between 0.05 and P < 0.05 or between P < 0.01.

3 clinical efficacy of the Chinese medicinal composition for treating early and middle stage KOA

The trial was performed using a simple randomized procedure to collect 56 patients meeting inclusion criteria: 28 cases of the traditional Chinese medicine composition prepared in the embodiment 1 of the invention are treated (group A) and 28 cases of diclofenac sodium slow-release capsules (group B). Both groups used basic treatments based on health propaganda and education. In the case of administration of the basic treatment to each group, group a intervened in the treatment group of the Chinese medicinal composition prepared in example 1 of the present invention; group B intervenes diclofenac sodium sustained release capsule, and both groups are treated 7 times a week, one treatment course is 7 days, and the treatment period is 2 treatment courses.

3.1 comparison of VAS score before and after treatment

The VAS score was statistically significant for the comparisons between the groups before and after treatment (p < 0.01), and not for the comparisons between groups A and B (p > 0.05). Indicating that both groups had efficacy in treating early and mid-term KOA pain. See in particular table 5.

Table 5 comparison of the two sets of pre-treatment and post-treatment VAS scores

Note that: in comparison with the pre-treatment period of this group, ^a p＜0.05。

3.2 WOMAC score comparison before and after treatment

The WOMAC total score was statistically significant (p < 0.01) for comparisons within the pre-treatment and post-treatment groups. The comparison of group a with group B is not statistically significant. See in particular table 6.

Table 6 total score comparison of WOMAC scores before and after treatment for two groups of patients

3.3 comparison of timing of the up-and-down walk test before and after treatment

Functional walking ability assessment of the comparisons within the pre-and post-treatment groups were statistically significant (p < 0.01). Group A has no statistical significance (p > 0.05) compared to group B. It is demonstrated that both groups have an effect of improving the functional walking ability of early and medium stage KOA patients. See in particular table 7.

Table 7 comparison of time to start-to-stop walk test time(s) before and after treatment for two groups of patients

3.4 comparison of walking test 6 minutes before and after treatment

The 6-minute walk test was statistically significant (p < 0.01) for the comparisons in the pre-and post-treatment groups. Group a has no statistical significance (p > 0.05) compared to group B. The two groups of patients with early and middle stage KOA have the effect of improving walking ability. See in particular table 8.

Table 8 comparison of 6 minute walk test distance (m) before and after treatment for two groups of patients

3.5 active and passive flexion Activity comparison of knee joints before and after treatment

The knee joint active and passive flexion activities are statistically significant (p < 0.01) in the groups before and after treatment. Group a has no statistical significance (p > 0.05) compared to group B. See in particular table 9.

Table 9 comparison of active flexion mobility of knee joints before and after treatment for two groups of patients

And (3) injection: in comparison with the pre-treatment period of this group, ^a p＜0.05。

3.6 active and passive extension Activity comparison of knee joints before and after treatment

The active and passive extension activities of the knee joint are statistically significant (p < 0.05) in the groups before and after treatment. Group a has no statistical significance (p > 0.05) compared to group B. See in particular table 10.

Table 10 active and passive extension Activity comparison of knee joints before and after treatment of two groups of patients

3.7 clinical effective rate

The efficacy of each of the two groups after treatment was compared, 75.0% for group A and 89.2% for group B, as shown in Table 11.

Table 11 comparison of clinical efficacy between two groups

3.8 Security assessment

Two groups of 56 patients, and during the study period, group A shows gastrointestinal reactions such as mild gastralgia, dyspepsia and the like in 3 cases; constipation occurs in 2 cases; group B showed 8 cases of gastric discomfort, abdominal pain, acid regurgitation, anorexia, constipation, and no ulcers, bleeding, and perforation. The other patients had normal blood, urine and feces, liver and kidney functions, and the examination before and after treatment were all normal, see in Table 12.

TABLE 12 incidence of adverse reactions in each group

Adverse reactions	Group A	Control group B
			Slight stomach ache	2	8
Mild constipation	1	0
			ALT and AST increases	0	0
Incidence (%)	10.71	28.75

EXAMPLE 8 therapeutic Effect of the Chinese medicinal composition of the invention on KOA rat model and Effect on autophagy

Randomly dividing 50 SD rats into a control group, a model group and a low, medium and high dose group of the traditional Chinese medicine composition (hereinafter referred to as a "arthralgia-syndrome-relieving and bone-strengthening prescription") prepared in the embodiment 1 of the invention, wherein 10 SD rats in each group are respectively subjected to anesthesia and material drawing after the corresponding medicines are infused into the stomach by adopting an improved Hulth method to establish a KOA model except the control group; observing the general condition of the rat and the diameter change of the knee joint during the treatment period, and observing the joint change of each group when the material is obtained, and carrying out HE staining and improving Mankin score to evaluate the degeneration condition of the joint cartilage; the WB method detects the expression of MMP-13, TIMP1, aggrecan, collagen II, LC3, P62, P-ULK1, P-AMPK and P-mTOR in each group; qRT-PCR method detects MMP-13, TIMP1, aggrecan, collagenII, LC3, P62, ULK1, AMPK, mTOR, IL-1 beta, TNF-alpha mRNA expression.

1. Experimental materials

1.1 Experimental animal

50 SPF SD rats (male and female halves), body weight (160 g.+ -. 20 g), 8 weeks old; purchased from si Bei Fu (beijing) biotechnology limited, animal quality certification No.: SCXK (Beijing) 2019-0010 (see appendix 1). Animal feeding, modeling and intervention are all carried out in the SPF-grade animal experiment center SYXK 2020-0009 of Gansu university of Chinese medicine (see appendix 2). The operations of animal modeling, intervention, material taking and the like accord with the regulations of the Ministry of science and technology (the guidance opinion on animals to be tested (revised in 2006), the whole course of the experiment strictly follows the ethical regulations (ethical number: 2021-1 85) of animal experiments of Gansu university of Chinese medicine, see annex 3.

1.2 Experimental drugs

All the medicinal materials in the arthralgia-relieving and bone-strengthening prescription are purchased from pharmaceutical departments of Chinese hospitals in Gansu province, and meet the requirements of pharmacopoeia of the people's republic of China in 2020 edition.

2. Experimental method

2.1 Grouping and modeling

After 50 SD rats are adaptively fed for 1 week, the rats are randomly divided into a control group and a model group, a low dose group of a general formula for treating arthralgia and strengthening bones, a medium dose group of a general formula for treating arthralgia and strengthening bones, and a high dose group of the general formula for treating arthralgia and strengthening bones, wherein 10 rats in each group are subjected to no treatment except 10 rats in the control group, and the other 40 SD rats are subjected to an improved Hulth method to establish a KOA rat model. The rat anesthesia adopts 5ml/kg of 7% chloral hydrate for intraperitoneal injection, after complete anesthesia, skin preparation, disinfection and towel spreading are carried out, the incision on the inner side of the right knee patella is taken for cutting the skin and joint capsule, the inner collateral ligament and the anterior cruciate ligament are cut off, the inner meniscus is completely resected, and the articular cartilage surface is not damaged. After the positive test is carried out, the joint cavity is thoroughly hemostatic, joint bags and skin incisions are sewn layer by layer, erythromycin ointment is smeared on the wound after operation, penicillin is continuously injected for 3 days for 20 ten thousand units/d, infection is prevented, all model animals are driven for 30min/d 1 week after model building, all groups of rats are subjected to free drinking water and ingestion under the same condition, and after model building for 4 weeks, the subsequent experiment is carried out.

2.2 pharmaceutical preparation

The decoction of the arthralgia-relieving and bone-strengthening prescription is prepared according to the method of the embodiment 1 of the invention.

2.3 pharmaceutical intervention

After molding for 4 weeks, according to the "body surface area change algorithm of human and rat" the dosage of the murine drug (g.kg/d) =the dosage of the human drug (g/d)/60 kg×0.018×5, the low, medium and high dosage groups of the general arthralgia-syndrome-relieving and bone-strengthening prescription are respectively 2, 4 and 8mL/kg/d, the control group and the model group are subjected to equivalent physiological saline gastric lavage, and the general arthralgia-relieving and bone-strengthening prescription liquid medicine with proper concentration is heated to proper temperature before gastric lavage. The action is gentle and slow in the stomach irrigation process, so that unnecessary damage to rats is reduced as much as possible. After gastric lavage, rats were returned to the cage for observation. Relevant index detection was performed 4 weeks after continuous treatment.

2.4 general case observations and measurements of rat knee diameter

Observing diet, drinking water, activity, sensitivity, mental state, knee joint deformity, swelling and activity of each group of rats, and blood circulation and muscle fullness of the lower limbs; the right knee joint diameters were measured before and after molding and after 4 weeks of treatment for each group, measuring method: knee joint diameter was measured with vernier calipers and repeated three times to calculate an average.

2.5 materials selection

After the rat is anesthetized, separating the muscles, ligaments and synovium around the knee joint, taking down cartilage tissue of the inner side joint of the right rear knee joint of the rat, and placing a part of marked samples in 4% formaldehyde for fixation for related experiments such as HE staining and the like; and the other part of the labeled samples of each group are stored in a refrigerator at the temperature of minus 80 ℃ and then subjected to experiments such as qRT-PCR, western blot and the like.

2.6 general observations of rat knee joints

When the materials are obtained, the knee joints of the modeling sides of the rats are separated completely, the joint cavities are exposed, and the joint cartilages are generally observed and scored. The score was proportional to knee cartilage degeneration, with higher scores representing more severe cartilage degeneration and details of the scores are shown in table 13.

TABLE 13 general observations and scores for knee cartilage

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2.7 HE staining of cartilage tissue of knee

(1) Decalcification the specimen fixed in 4% formaldehyde was taken out, washed with tap water for 4-6 hours, and then decalcified by immersing in acetic acid for 1 month.

(2) Dewatering and transparent placing cartilage tissue into alcohol with different concentration gradients, dewatering, and shortening the time when the concentration is higher; after the cartilage tissue is dehydrated by alcohol, the cartilage tissue is subjected to transparency, a dehydration agent is replaced by xylene as a transparency agent, and paraffin embedding is carried out, and the specific steps are shown in Table 14.

TABLE 14 cartilage tissue dehydration step

(3) The cartilage tissue after wax dipping is sequentially subjected to wax dipping by 3-cylinder paraffin (60 ℃), and the whole experiment process is completed in a biological tissue dehydrator, and the details are shown in table 15.

Table 15 experimental procedure for wax infusion of cartilage tissue

(4) Embedding the paraffin blocks encapsulate the wax-impregnated cartilage tissue blocks. The temperature of the embedding wax is slightly higher than the wax dipping temperature, so that the tissue block and the embedding paraffin are ensured to be completely fused into a whole.

(5) Before slicing and baking, the wax block is frozen on a freezing table for several minutes, then the embedded block to be cut is fixed on a specimen holder, the thickness (4 μm) required by slicing is regulated on a micro-motion device, and a knife table is moved to a position close to the specimen table for slicing.

The cut sections must be attached to the slide for further processing, but the sections often have fine transverse lines that must be flattened before attachment, otherwise staining and viewing are affected. The slice is fished, firstly, a series of slices are put into a warm water bath slice unfolding pot with the temperature of about 42 ℃, the slices are separated by tweezers, then, an anti-drop slide treated by APES or polylysine is obliquely inserted into the water surface to scoop the slices, the slices are attached to the proper positions of the slide, and the slices are baked for 3 hours in a 60 ℃ oven.

(6) Dewaxing and hydration paraffin sections were dewaxed and hydrated as shown in table 16.

TABLE 16 dewaxing process

(7) The dyeing process is shown in Table 17.

TABLE 17 dyeing Process

(8) And (3) after the sealing sheets are air-dried, sealing the sealing sheets with neutral resin, and finally performing microscopic examination, wherein the knee joint cartilage of each group of rats is scored according to the knee joint cartilage tissue improvement Mankin score, and the standard is shown in a table 18.

Table 18 improved Mankin scoring criteria

2.8Western blot method for detecting related protein expression

(1) Extracting total protein; (2) measuring the total protein concentration; (3) SDS-PAGE protein gel electrophoresis; (4) transferring a film; (5) closing; (6) incubating the primary antibody; (7) incubating the secondary antibody; (8) color development imaging; (9) and (5) data analysis.

2.9 real-time fluorescent quantitative PCR (quantitative real-time PCR, qRT-PCR) technique to detect the related mRNA content (1) cartilage tissue total RNA extraction (according to Solarbio/Soxhibao total RNA extraction kit instructions).

(2) Total RNA concentration determination.

(3) Total RNA reverse transcription synthesis cDNA for each group was diluted to a total RNA sample concentration of 500ng/μl according to the total RNA concentration detected. According to the instruction of the reverse transcription kit, the following operation steps are carried out:

a genomic DNA removal reaction: the following reaction components were added to an RNase-free centrifuge tube in an ice bath, as shown in Table 19.

TABLE 19 genomic DNA removal reaction System

Reaction conditions: the temperature can be 42 ℃ for 2min or room temperature for 5min, and can be prolonged to 30min and terminated at 4 ℃.

B reverse transcription reaction: the reaction solution was prepared on ice. Reverse transcription was performed immediately after gentle mixing, and the specific reaction system is shown in Table 20. The synthesized cDNA was stored in a refrigerator at-20℃for use.

TABLE 20 reverse transcription reaction system

The reaction conditions are as follows: the reaction was terminated at 37℃for 15min,85℃for 5s and 4 ℃.

C real-time fluorescent quantitative PCR (quantitative real-time PCR, qRT-PCR)

Primers were designed and synthesized by Shanghai Biotechnology Inc., and the primer sequences are shown in Table 21; qRT-PCR was performed according to the instructions, and the specific reaction system is shown in Table 22.

Table 21 Gene amplification primer sequence listing

Table 22qRT-PCR detection reaction system

Reaction conditions: 2min at 50 ℃ and 10min at 95 ℃;95℃for 30sec,60℃for 30sec,40cycles. The dissolution curve was plotted and the final data was analyzed at 2- Δct.

2.10 statistical method

The data obtained in this study were all processed using SPSS24.0 statistical software to meter the dataThe method comprises the steps of firstly, carrying out normal distribution test, adopting t test for group comparison conforming to normal distribution, adopting single-factor analysis of variance for group comparison, adopting rank sum test for research data which does not conform to normal distribution and has uneven variance of data of each group, and adopting P<0.05 is statistically significant.

3 results

3.1 general Condition observations

During the experiment, rats in the control group have normal diet and water, sensitive response, good mental state and normal activities; the model group rats have reduced diet, normal drinking water, reduced activity, obviously poor sensitivity and mental state, and the knee joint on the modeling side has swelling, deformity, poor blood circulation of limbs and muscle atrophy; compared with the model group, the low, medium and high intervention groups of the general arthralgia-relieving and bone-strengthening prescription have the advantages that the diet, drinking water, activity, sensitivity and mental state of rats are obviously improved, the swelling degree of knee joints at the modeling side, the blood circulation of limbs and the muscular atrophy degree are obviously improved compared with the model group, and the dosage group of the general arthralgia-relieving and bone-strengthening prescription is most obvious.

3.2 changes in diameter of the right hind knee in rats of each group

The groups have no difference (P > 0.05) before molding; after molding for 4 weeks, compared with a control group, the diameters of the right rear knee joints of rats in the model group, the low, medium and high dose groups of the general arthralgia-relieving and bone-strengthening formula are obviously increased (P < 0.01), and the model group has no obvious difference (P > 0.05) compared with the low, medium and high dose groups of the general arthralgia-relieving and bone-strengthening formula; after 4 weeks of treatment, the right hind knee joint diameter was significantly greater in model rats compared to control (P < 0.05); compared with the model group, the knee joint diameter of the dose group in the general formula for dredging arthralgia and strengthening bones is obviously reduced (P < 0.01), and the low dose group and the high dose group in the general formula for dredging arthralgia and strengthening bones have no obvious difference (P > 0.05) compared with the model group, as shown in table 23 and figure 1.

TABLE 23 diameter variation of the right hind knee at different stages for each group of rats，mm，n＝10)

Note that: * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs. model group

Fig. 1 is a graph showing the analysis of the change in diameter of the right hind knee joint at different stages in each group of rats (n=10). Wherein, the liquid crystal display device comprises a liquid crystal display device, ^* P<0.05， ^** P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

3.3 general observations of the right hind knee joints in groups of rats

The cartilage of the control group has smooth surface, clear and transparent color, no defect and no new organism formation; the knee joint cartilage of the model group has rough surface, dull luster and no light luster, and the joint edge forms bone hyperplasia; the low, medium and high dose groups of the general formula for treating arthralgia and strengthening bones are improved compared with the model group, the cartilage surface is less smooth, the color is slightly dull, the transparency is reduced, the cartilage defect and the bone tag are reduced compared with the model group, wherein the dose group in the general formula for treating arthralgia and strengthening bones is improved most remarkably, as shown in figure 2. Analysis of the general observation scores of knee cartilage from each group revealed a significant increase in model group score (P < 0.01) compared to the control group; after 4 weeks of treatment with the general formula, the score of the dose group in the general formula was significantly reduced (P < 0.05), and the score of the low and high dose groups was statistically less significant than that of the model group, but lower than that of the model group, as shown in table 24, fig. 3.

Fig. 2 is a view of the knee joint of each group of rats.

TABLE 24 general observation score of knee joints of rats of each groupn＝10)

Note that: * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01,vsmodel set

Fig. 3 is a general observation score for rat knee joints. Wherein P<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

3.4 Right hind knee articular cartilage HE staining and improved Mankin scoring results for groups of rats

The cartilage layers of the right rear knee joint of the rats in the control group are thicker, the surfaces are smooth and complete, no cracks exist, the cartilage cells are round or oval, are orderly arranged horizontally, are uniform and full in size, are uniformly colored, and have complete and clear tide lines; the knee joint cartilage of the model group rat is rough and not smooth, the cartilage layer is thinned, the cartilage edge is seriously damaged, the cartilage is in degenerative change, the chondrocyte is obviously reduced, the cell arrangement is disordered, the cell clusters are clustered, the nucleus is concentrated, the matrix is shallowly dyed, and the tide line is disordered and irregular; compared with the cartilage of the model group, the dosage group in the arthralgia-relieving and bone-strengthening prescription has thicker cartilage, smoother cartilage surface, deepened dyeing, even coloring, even fissures, obviously increased chondrocyte quantity, slightly disordered arrangement, plump cells, normal morphology, visible tide line and relative integrity; the low and high dose groups of the general formula for treating arthralgia and strengthening bones have relatively poorer cartilage structure than the dose groups in the general formula for treating arthralgia and strengthening bones, the chondrocytes are unevenly distributed, and the surface of the articular cartilage is unsmooth, as shown in figure 4. The results of the improved Mankin score of the right hind knee joint of the rats in each group show that the improved Mankin score of the right hind knee joint of the rats in the model group is obviously improved (P < 0.01) compared with the control group; compared with the model group, the dose group of rats in the general arthralgia-syndrome-relieving and bone-strengthening prescription has significantly reduced score of the improved Mankin of the right rear knee joint, (P < 0.01), and the low dose group and the high dose group of the general arthralgia-relieving and bone-strengthening prescription have no significant difference (P > 0.05) compared with the model group, as shown in figure 5.

Fig. 4 is HE staining (×100) of the knee cartilage of the right lower limb of each group of rats. Wherein A: a control group; b: a model group; c: low dose group of arthralgia-relieving and bone-strengthening prescription; d: dose group in the general arthralgia-relieving and bone-strengthening prescription; e: high dosage group of the arthralgia-relieving and bone-strengthening prescription.

Fig. 5 is a modified Mankin score. And (3) injection: * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

3.5 expression of IL-1 beta, TNF-alpha mRNA in cartilage tissue of groups of rats

The expression of IL-1 beta and TNF-alpha mRNA in the model group is obviously increased (P < 0.01) compared with the control group, and the expression of IL-1 beta and TNF-alpha mRNA in the low, medium and high dose groups of the general arthralgia-relieving and bone-strengthening prescription is obviously reduced (P < 0.01) compared with the model group, wherein the dose group in the general arthralgia-relieving and bone-strengthening prescription is most obvious, as shown in figure 6.

FIG. 6 shows the expression of IL-1. Beta. And TNF-. Alpha.mRNA in cartilage tissue of each group of rats. Wherein A: IL-1 βmrna expression statistics; b: TNF- α mRNA expression statistics; * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

3.6 metabolism of the cartilage matrix in the knee joints of rats of the groups

WB detection results show that compared with the control group, the model group Aggrecan, collage ii and TIMP1 protein expression are significantly reduced (P <0.01, P < 0.05), and MMP13 expression is significantly increased (P < 0.05); compared with the model group, the low and medium dose groups Aggrecan, collage II and TIMP1 protein expression are obviously increased (P <0.01 and P < 0.05), wherein the dose group in the general formula is most obvious, the high dose group in the general formula has no obvious difference (P > 0.05), the dose group in the general formula has obviously reduced MMP13 expression (P < 0.05) compared with the model group, and the low and high dose groups in the general formula have no obvious difference (P < 0.05), as shown in figure 7.Aggrecan, collagen II, TIMP1, MMP13 mRNA expression and protein expression tendencies were consistent, see FIG. 8.

FIG. 7 shows the expression of Aggrecan, collagen II, TIMP1 and MMP13 proteins in various articular cartilage groups. Wherein A: a control group; b: a model group; c: low dose group of arthralgia-relieving and bone-strengthening prescription; d: dose group in the general arthralgia-relieving and bone-strengthening prescription; e: high dose group of arthralgia-relieving and bone-strengthening prescription; f, aggrecan protein relative expression statistical graph; g, a Collagen II protein relative expression statistical graph; h: a statistical graph of TIMP1 protein relative expression; i: a statistical map of relative MMP13 protein expression; * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

FIG. 8 shows the expression of Aggrecan, collagen II, TIMP1 and MMP13 mRNA in cartilage tissue of rats in each group. Wherein A: an agglecan mRNA expression statistical map; b: a Collagen II mRNA expression profile; c: TI MP1 mRNA tableReaching a statistical graph; d: MMP13 mRNA expression statistics; * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

3.7 expression of autophagy-related protein LC3, P62 in rat knee cartilage of each group

Compared with the control group, the LC3 II/LC 3I ratio of the model group is obviously reduced, and the P62 protein expression is obviously increased (P < 0.01); compared with the model group, the LC3 II/LC 3I ratio of the dosage group in the general formula for treating arthralgia-syndrome and strengthening bones is obviously increased, the expression of P62 protein is obviously reduced (P < 0.01), and the low and high dosage groups in the general formula for treating arthralgia-syndrome and strengthening bones have no obvious difference (P > 0.05) compared with the model group, as shown in figure 9.LC3, P62 gene expression and protein expression tendencies were consistent, see figure 10.

FIG. 9 shows the expression of autophagy-related proteins in various groups of articular cartilage. Wherein A: a control group; b: a model group; c: low dose group of arthralgia-relieving and bone-strengthening prescription; d: dose group in the general arthralgia-relieving and bone-strengthening prescription; e: high dose group of arthralgia-relieving and bone-strengthening prescription; f: a LC3 II/LC 3I ratio statistical graph; g: relative expression statistics of P62 protein; * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

FIG. 10 shows the expression of LC3 and P62 mRNA in cartilage tissue of rats in each group. Wherein A: LC3 mRNA expression statistics; b: p62 mRNA expression statistics; * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

3.8 cases of P-AMPK, P-mTOR, and P-ULK1 expression in rat knee articular cartilage of each group

The expression of the P-AMPK and P-ULK1 proteins in the model group is obviously reduced (P <0.01, P < 0.05) and the expression of P-mTOR is obviously increased (P < 0.01) compared with the control group; compared to the model group, the P-AMPK protein expression in the dose group of the general formula was significantly increased (P < 0.05), the low-dose group of the general formula was not significantly different (P > 0.05), the low-dose group of the general formula was significantly increased (P < 0.01), the P-ULK1 protein expression in the general formula was significantly increased (P < 0.01), the most significant dose group was in the general formula, the high-dose group was not significantly different (P > 0.05), the P-mTOR protein expression in the general formula was significantly reduced (P < 0.05), the low-dose group was not significantly different (P > 0.05), and the high-dose group was not significantly different (P > 0.05), see fig. 11.AMPK, mTOR, ULK1 gene expression was consistent with protein expression trend, see figure 12.

FIG. 11 shows the expression of P-AMPK, P-mTOR, and P-ULK1 in various articular cartilage groups. Wherein A: a control group; b: a model group; c: low dose group of arthralgia-relieving and bone-strengthening prescription; d: dose group in the general arthralgia-relieving and bone-strengthening prescription; e: high dose group of arthralgia-relieving and bone-strengthening prescription; f: P-AMPK protein relative expression statistical graph; g: relative expression profile of P-mTOR protein; h: relative expression statistics of P-ULK1 protein; * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

FIG. 12 shows the expression of AMPK, mTOR, ULK mRNA in cartilage tissue of rats in each group. Wherein A: AM PK mRNA expression statistics; b: mTOR mRNA expression statistics; c: ULK1 mRNA expression statistics; * P (P)<0.05,**P<0.01, vs. control group; ^# P<0.05, ^## P<0.01, vs.

Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating knee osteoarthritis is characterized in that: the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-25 parts of medicinal cyathula root, 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 fenugreek, 15-25 parts of zaocys dhumnade, 10-20 parts of paniculate swallowwort root, 10-20 parts of Chinese pine, 10-20 parts of rhizoma corydalis, 10-20 parts of combined spicebush root, 15-25 parts of Chinese angelica, 10-20 parts of szechuan lovage rhizome, 15-25 parts of Chinese yam, 15-25 parts of poria cocos, 10-20 parts of green tangerine peel, 15-25 parts of rhizoma alismatis and 10-20 parts of liquorice.

2. The traditional Chinese medicine composition according to claim 1, wherein: the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15 parts of medicinal cyathula root, 15 parts of prepared rehmannia root, 15 parts of cynomorium songaricum, 15 parts of obscured homalomena rhizome, 15 parts of snow lotus herb, 15 parts of rhizoma cibotii, 15 parts of fenugreek, 15 parts of zaocys dhumnade, 10 parts of paniculate swallowwort root, 10 parts of Chinese pine node, 10 parts of rhizoma corydalis, 10 parts of combined spicebush root, 15 parts of Chinese angelica, 10 parts of szechuan lovage rhizome, 15 parts of Chinese yam, 15 parts of poria cocos, 10 parts of green tangerine peel, 15 parts of rhizoma alismatis and 10 parts of liquorice.

3. The method for preparing the traditional Chinese medicine composition as claimed in claim 1 or 2, which is characterized in that: mixing the raw materials according to the formula, soaking in water until the medicinal tissue is penetrated, adding water with the thickness of 30-35mm, decocting for 40-45 min, filtering to obtain medicinal liquid, adding water with the thickness of 25-30mm into the filter residue, decocting for 30-35 min, filtering, and mixing filtrates.

4. The method for preparing the traditional Chinese medicine composition as claimed in claim 1 or 2, which is characterized in that: mixing the raw materials according to the formula, pulverizing into fine powder, sieving with a 120-mesh sieve to enable the amount of the fine powder passing through the sieve to reach 50%, and collecting the sieved fine powder; extracting coarse powder with water twice, mixing filtrates, heating, concentrating, drying, grinding into fine powder, mixing the two fine powders, and encapsulating to obtain capsule.

5. The method for preparing the traditional Chinese medicine composition as claimed in claim 1 or 2, which is characterized in that: mixing the raw materials according to the formula, soaking in 8-10 times of water for 0.5h, decocting for 2 times, decocting for 1.5h, decocting for 2 times with 8-10 times of water for 1h, mixing decoctions, filtering, concentrating to obtain extract, drying, pulverizing into fine powder, adding adjuvants, and granulating to obtain granule.

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

7. A medicament, the active ingredient of which comprises the Chinese medicinal composition of claim 1 or 2.

8. The medicament according to claim 7, wherein: the medicine also comprises auxiliary materials.

9. A medicament according to claim 8, characterized in that: the medicine comprises decoction, capsules and granules.