A drug for treating knee osteoarthritis, its preparation method and application

Abstract

The present invention belongs to the technical field of drug preparation, and discloses a drug for treating knee osteoarthritis, a preparation method and an application thereof. It is composed of 20 g of Rehmanniae Radix, 15 g of Epimedii Folium, 15 g of Drynariae Rhizoma, 12 g of Angelicae Sinensis Radix, 12 g of Polygoni Cuspidati Rhizoma et Radix, and 10 g of Achyranthis Bidentatae Radix by weight. In the present invention, Rehmanniae Radix for tonifying the kidney and generating essence is used as the monarch drug, Epimedii Folium and Drynariae Rhizoma for tonifying the liver and kidney and strengthening the bones and muscles are used as the ministerial drugs, Angelicae Sinensis Radix and Polygoni Cuspidati Rhizoma et Radix for promoting blood circulation to remove stasis and relieve pain are used as the adjuvant drugs, and Achyranthis Bidentatae Radix for conducting the drugs downward to reach the affected site is used as the messenger drug. The combination of various drugs exerts the effects of tonifying the liver and kidney, strengthening the bones and muscles, and activating blood circulation and dredging collaterals. The various drugs work together, each performing its own function and synergistically exerting the curative effect. On the basis of long-term clinical practice, the present invention puts forward the pathogenesis concept of kidney deficiency and bone erosion, prolonged illness invading collaterals, and finally resulting in the coexistence of kidney deficiency and stasis. Through clinical research and animal experiment verification, the drug formula has significant curative effect and high safety in the treatment of knee osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and particularly relates to a drug for treating knee osteoarthritis, a preparation method and an application thereof. Background Art

[0002] Knee osteoarthritis (KOA) is a chronic inflammatory disease, and its clinical manifestations are mainly knee joint pain and functional limitation. Cartilage lesions are its main pathological changes. The high-incidence population is mainly concentrated in the middle-aged and elderly, and it is also closely related to gender and body weight. At present, Western medicine drugs for treating KOA include non-steroidal anti-inflammatory drugs (NSAIDs), opioid analgesics, glucosamine with certain cartilage protection effects, chondroitin sulfate, and locally applied intra-articular injection drugs, etc. Although certain curative effects have been achieved clinically, and most treatment methods lack evidence-based medicine evidence support, the clinical curative effects are limited, and there are many related adverse reactions, resulting in serious joint damage and even disability in many patients in the late stage. At present, there is still no drug specifically for KOA among the Chinese patent medicines for treating osteoarthritis. Therefore, finding an effective, economical and practical treatment plan for KOA has become the focus of attention of the current medical community and patients.

[0003] KOA belongs to the categories of "Bi syndrome" and "Bone Bi" in traditional Chinese medicine. Combining the records of ancient Chinese physicians and traditional Chinese medicine ancient books, it is considered that the pathological nature of KOA is deficiency in origin and excess in superficiality. As mentioned in the "Inner Canon of Huangdi", after "Tiankui exhaustion", the functions of the zang-fu organs decline, the vital qi is insufficient, and the body is invaded by wind-cold-damp pathogens. The exogenous pathogens stay in the body for a long time and turn into stasis. Its pathology is closely related to "deficiency", "pathogens" and "stasis". The research group believes through long-term clinical practice that kidney deficiency is the basis for the onset of KOA, and long-term Bi syndrome turning into stasis is an important factor in its pathological process. Kidney deficiency and blood stasis run through the entire process of the onset of KOA. The method of tonifying the kidney and activating blood circulation should run through the entire process of treating KOA. On this basis, the "method of tonifying the kidney and activating blood circulation" is summarized, and the Gu Bi formula is composed, and good curative effects have been obtained through preliminary clinical observations. Therefore, the research group designed the clinical curative effect and safety of Gu Bi granules in treating KOA, analyzed the action targets of Gu Bi granules in treating KOA by network pharmacology methods, and verified its effective active ingredients and their targets by molecular docking technology.

[0004] To make a breakthrough in the research of traditional Chinese medicine, it is necessary to first make a breakthrough in the research of the pathogenesis. In view of the characteristics of the inconsistent traditional Chinese medicine pathogenesis, insufficient understanding, and poor clinical efficacy of knee osteoarthritis, based on long-term clinical practice and through repeated clinical verification, the inventor proposed that the traditional Chinese medicine pathogenesis of knee osteoarthritis is caused by "kidney deficiency and blood stasis", and the treatment should be based on the method of "tonifying the kidney and strengthening tendons, promoting blood circulation and dredging collaterals". In the prescription, cooked rehmannia root is the monarch drug for tonifying the kidney and generating essence, epimedium and drynaria rhizome are the ministerial drugs for tonifying the liver and kidney and strengthening tendons and bones, angelica sinensis and polygonum cuspidatum are the adjuvant drugs for promoting blood circulation to remove blood stasis and relieve pain, and achyranthes bidentata is the guiding drug for leading the medicine downward to reach the affected part. The combination of these drugs can achieve the effects of tonifying the liver and kidney, strengthening tendons and bones, and promoting blood circulation and dredging collaterals, and has achieved good clinical efficacy.

[0005] Through the above analysis, the problems and defects of the existing technology are as follows: There is no proprietary Chinese medicine specifically for the treatment of knee osteoarthritis on the current market. The traditional Chinese medicine treatment is mainly based on syndrome differentiation and treatment. Although the curative effect is good, it is not convenient for long-term use, and it is not easy to carry, making it difficult for patients to take medicine for a long time, resulting in reduced compliance and difficulty in maintaining the curative effect. Summary of the Invention

[0006] In view of the problems existing in the existing technology, the present invention provides a drug, a preparation method and an application for the treatment of knee osteoarthritis.

[0007] The present invention is realized as follows: A drug for the treatment of knee osteoarthritis is composed of 5-30 g of cooked rehmannia root, 5-30 g of epimedium, 5-30 g of drynaria rhizome, 5-30 g of angelica sinensis, 5-30 g of polygonum cuspidatum, and 5-30 g of achyranthes bidentata by weight.

[0008] Another object of the present invention is to provide a preparation method of a drug for the treatment of knee osteoarthritis, and the preparation method of the drug for the treatment of knee osteoarthritis includes:

[0009] Step 1: Weigh 20 g of cooked rehmannia root, 15 g of epimedium, 15 g of drynaria rhizome, 12 g of angelica sinensis, 12 g of polygonum cuspidatum, and 10 g of achyranthes bidentata by weight;

[0010] Step 2: Mix the above components, decoct with water, filter, boil, concentrate, freeze-dry with an ultra-low temperature freeze-dryer, pulverize into powder, and package, with each bag weighing 10 g.

[0011] Another object of the present invention is to provide an application of the drug for the treatment of knee osteoarthritis in the preparation of a drug for the treatment of degenerative joint disease with bone destruction in the knee joint.

[0012] Another object of the present invention is to provide an application of the drug for the treatment of knee osteoarthritis in the preparation of a drug for the treatment of inflammatory pain of knee osteoarthritis.

[0013] Another object of the present invention is to provide a capsule prepared by using the drug for the treatment of knee osteoarthritis.

[0014] Another object of the present invention is to provide an granule prepared by using the medicine for treating knee osteoarthritis.

[0015] Furthermore, the method for using the granules prepared from the medicine for treating knee osteoarthritis is to take it three times a day.

[0016] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0017] First, the present invention is guided by the theory of "kidney deficiency and blood stasis" in traditional Chinese medicine, and believes that the key to the onset of knee osteoarthritis is kidney deficiency and bone erosion, and long-term illness enters the collaterals, which eventually leads to the concept of kidney deficiency and blood stasis. The treatment should be to tonify the kidney and strengthen the tendons, activate blood circulation and dredge the collaterals, and use Rehmannia glutinosa and Herba Osmanthi as the main medicine to tonify the kidney and replenish essence, Epimedium and Herba Drynariae as the minister medicine to tonify the liver and kidney, strengthen the tendons and bones, and Angelica sinensis and Polygonum cuspidatum as the adjuvant medicine to activate blood circulation, remove blood stasis and relieve pain, and Achyranthes bidentata as the guiding medicine to lead the medicine downward and directly reach the diseased site. All the medicines are used together to play the functions of tonifying the liver and kidney, strengthening the tendons and bones, and activating blood circulation and dredge the collaterals. All the medicines are used together, each performing its own duties, and synergistically exerting the therapeutic effect.

[0018] Rehmannia root can nourish the kidney and replenish essence. Modern pharmacological research has found that Rehmannia root contains many effective chemical components such as verbascoside and rehmannia glutinosa, which have anti-inflammatory, antioxidant and anti-tumor immunomodulatory effects. It can regulate immune function by reducing the expression of pro-inflammatory factors, enhancing the expression of anti-inflammatory factors, and enhancing the phagocytic function of monocytes.

[0019] Epimedium can nourish kidney yang, strengthen tendons and bones, and eliminate rheumatism. Epimedium contains a variety of chemical components such as icariin, flavonoids, polysaccharides, etc., which have important regulatory effects on the immune system, bone development, and reproductive system. Modern pharmacological studies have confirmed that icariin can repair cartilage damage. Its mechanism of action is manifested in its regulatory effect on chondrocytes, reducing extracellular matrix degradation, and its effect on bone marrow mesenchymal stem cells; icariin can also reduce the expression of interleukin-1β, tumor necrosis factor α and inhibit the monocyte protein receptor axis in the knee joint.

[0020] Drynaria enters the kidney meridian and has the functions of nourishing the kidney and strengthening bones, promoting blood circulation and relieving pain. Its main chemical components include flavonoids, triterpenes, phenylpropanoids, etc. Modern pharmacological research and analysis have found that Drynaria can regulate bone metabolism signaling pathways and inhibit the expression of inflammatory factors; Drynaria can regulate the balance of OPG / RANKL axis, increase bone density in rats, reduce pain after fracture surgery, and prevent recurrence of fractures. Drynaria total flavonoids can interfere with IL-1β-mediated SW1353 cell proliferation, apoptosis and cell morphology.

[0021] Angelica sinensis enters the liver, heart, and spleen meridians, and functions to enrich blood and promote blood circulation, regulate menstruation and relieve pain. Modern pharmacological research shows that Angelica sinensis has anti-inflammatory, anti-tumor, antidepressant, cardio-cerebrovascular enhancing, diabetes prevention and treatment, liver and kidney protection effects. It can regulate inflammatory factors through multiple signaling pathways, inhibit the production of pro-inflammatory factor IL-17 and the expression of NF-κB, thereby improving osteoarthritis. It can also inhibit the secretion of inflammatory factors through the OPG / RANKL / RANK signaling pathway and affect the expression of bone metabolism-related regulatory factors.

[0022] Polygonum cuspidatum has the functions of promoting blood circulation to relieve pain, clearing heat and promoting diuresis to detoxify. Pharmacological research has found that the extract of Polygonum cuspidatum can effectively improve blood lipid, reduce blood sugar, and has anti-inflammatory effects. Its mechanism of treating osteoarthritis is to regulate the level of inflammatory factors through the TLR4 / MyD88 / NF-κB signaling pathway, MAPKs signaling pathway, and vascular endothelial growth factor signaling pathway.

[0023] Achyranthes bidentata enters the liver and kidney meridians, and has the effects of tonifying the liver and kidney, strengthening tendons and bones, and removing blood stasis and dredging meridians. Modern pharmacological research shows that Achyranthes bidentata contains a variety of chemical components, among which the effective components such as sterones, triterpenoid saponins, and polysaccharides have strong inhibitory effects on cell proliferation and anti-inflammatory effects. It can regulate the metabolism of chondrocytes to prevent and treat osteoarthritis by mediating multiple signaling pathways such as NF-κB, Wnt / β-catenin, PI3K / AKT, Caspase-3 / 9-CASP3, and p38MAPK to regulate inflammatory factors and bone metabolism markers in chondrocytes.

[0024] Second, modern medical research has confirmed that knee osteoarthritis is a non-infectious inflammatory disease. Its pathogenesis is due to the attack on the autoimmune function, resulting in immune dysfunction, secretion of inflammatory factors, causing chronic synovitis of the joint, and forming inflammatory joint degeneration and bone destruction. All the drugs in the composition of this prescription have immune regulation and bone and joint protection effects, which is consistent with the modern medical treatment concept.

[0025] The traditional view holds that the TCM etiology and pathogenesis of knee osteoarthritis are liver and kidney deficiency. However, after years of clinical research and repeated verification, the therapeutic effect of simply tonifying the liver and kidney is not good. Based on long-term clinical practice, the present invention proposes the pathogenesis concept that kidney deficiency is the root, and long-term illness leads to blood stasis, and finally kidney deficiency and stasis coexist. After clinical research and animal experiment verification, this drug formula has significant curative effect and high safety in the treatment of knee osteoarthritis.

[0026] According to the pathogenesis characteristics of knee osteoarthritis, the present invention composes a drug formula for treating knee osteoarthritis, Gubi Formula, based on the theory of "kidney deficiency and blood stasis". Clinical application shows that it has a good curative effect on improving clinical symptoms such as knee osteoarthritis and joint function, and it has been verified in animal experiments.

[0027] Third, as the auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0028] (1) The expected benefits and commercial value after the transformation of the technical solution of the present invention are:

[0029] The transformation of the achievements of the present invention or the joint research and development with enterprises of Chinese patent medicines for treating knee osteoarthritis can solve the current situation of poor clinical efficacy of knee osteoarthritis and difficulty in maintaining long-term treatment, bring new therapeutic drugs to tens of millions of knee osteoarthritis patients, delay the disease progression of knee osteoarthritis patients, and improve the quality of life of patients.

[0030] (2) The technical solution of the present invention fills the technical gaps in the domestic and international industries:

[0031] There is no Chinese patent medicine specifically targeted at treating knee osteoarthritis on the market. Traditional Chinese medicine treatment is mainly based on syndrome differentiation and treatment. Although the curative effect is good, it is not convenient for long-term use, and coupled with inconvenient carrying, it makes it difficult for patients to take medicine for a long time, the compliance is reduced, and the curative effect is difficult to maintain. Therefore, the present invention can fill the technical gaps in the treatment of knee osteoarthritis by traditional Chinese medicine at home and abroad through the transformation of achievements.

[0032] (3) The technical solution of the present invention solves the technical problems that people have been eager to solve but have never succeeded in:

[0033] According to the estimation of the World Health Organization, the prevalence rate of osteoarthritis in the elderly is about 50% before the age of 50; about 70% at the age of 60; about 80%, even 90% for those over 65 years old. The prevalence rate of knee arthritis in China is about 8%, and the prevalence rate of people over 75 years old reaches 80%. If the condition is not well controlled and joint disability is caused, only joint replacement surgery can be performed in the late stage, which brings a serious economic burden to patients and their families. Therefore, the present invention can solve the technical problem that there has been no medicine available for knee osteoarthritis patients for a long time through the transformation of achievements.

[0034] Fourth, the significant technical progress achieved by the drug for treating knee osteoarthritis of the present invention and its preparation method mainly includes:

[0035] 1) Effective combination of components: Through the selected combination of traditional Chinese medicinal materials (Rehmannia glutinosa, Epimedium brevicornu, Drynaria fortunei, Angelica sinensis, Polygonum cuspidatum, Achyranthes bidentata), this solution can effectively relieve the symptoms of knee osteoarthritis. The combination of these medicinal materials utilizes the principles of traditional Chinese medicine pharmacology to exert a synergistic effect and improve the therapeutic effect.

[0036] 2) Innovation in drug preparation: Ultra-low temperature freeze-drying technology is adopted in the preparation process. This method can better retain the active ingredients in the drug and avoid the loss of active ingredients during the traditional heat drying process.

[0037] 3) Convenience and stability: The freeze-dried drug powder has a long shelf life and is easy to store and transport. In addition, the powdered drug is convenient for patients to take as needed, improving the convenience of use.

[0038] 4) Precise dosage control: Through precise weight ratio and packaging, each bag of the drug can ensure a consistent dosage, which is crucial for ensuring the therapeutic effect and safety.

[0039] 5) Reduced side effects: Compared with some chemically synthesized drugs, the drug formulation of the present invention uses natural herbal ingredients and has a lower risk of side effects, making it suitable for long-term use.

[0040] In summary, based on the traditional treatment of knee osteoarthritis, the drug formulation and preparation method of the present invention have improved the effectiveness, stability and safety of the drug through the application of modern scientific and technological means, showing obvious technological progress and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments of the present invention. Obviously, the following described drawings are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts.

[0042] Figure 1 is a flowchart of the preparation method of the drug for treating knee osteoarthritis provided by the embodiment of the present invention;

[0043] Figure 2 is a Venn diagram of database disease targets provided by the embodiment of the present invention;

[0044] Figure 3 is a Venn diagram of drug-disease intersection targets provided by the embodiment of the present invention;

[0045] Figure 4 is a drug-component-target network diagram provided by the embodiment of the present invention;

[0046] Figure 5 is a PPI network diagram provided by the embodiment of the present invention;

[0047] Figure 6 is a core target diagram provided by the embodiment of the present invention;

[0048] Figure 7 is a GO functional enrichment analysis diagram provided by the embodiment of the present invention;

[0049] Figure 8 is a KEGG bubble diagram provided by the embodiment of the present invention;

[0050] Figure 9 It is the key target-signaling pathway network diagram provided by the embodiments of the present invention;

[0051] Figure 10 It is the binding mode diagram of kaempferol and AKT1 provided by the embodiments of the present invention;

[0052] Figure 11 It is the binding mode diagram of quercetin and AKT1 provided by the embodiments of the present invention;

[0053] Figure 12 It is the binding mode diagram of luteolin and AKT1 provided by the embodiments of the present invention;

[0054] Figure 13 It is the binding mode diagram of sitosterol and AKT1 provided by the embodiments of the present invention;

[0055] Figure 14 It is the binding mode diagram of stigmasterol and AKT1 provided by the embodiments of the present invention;

[0056] Figure 15 It is the pathological picture result diagram (inverted microscope ×200) provided by the embodiments of the present invention;

[0057] Figure 16 It is the expression diagram of β-catenin protein in cartilage tissues of each group provided by the embodiments of the present invention. Detailed implementation manners

[0058] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0059] Example 1: Treatment of patients with chronic knee osteoarthritis

[0060] 1) Ingredient ratio: According to the drug formula of the present invention, weigh 25 g of Rehmannia glutinosa, 20 g of Epimedium brevicornu, 20 g of Drynaria fortunei, 15 g of Angelica sinensis, 15 g of Polygonum cuspidatum, and 12 g of Achyranthes bidentata.

[0061] 2) Drug preparation: After mixing the above medicinal materials, add an appropriate amount of water and decoct them. After decocting until the medicinal liquid is concentrated to a certain extent, filter to remove the residue.

[0062] 3) Freeze-drying treatment: Put the filtered medicinal liquid into an ultra-low temperature freeze-dryer for freeze-drying treatment to obtain drug powder.

[0063] 4) Packaging: Pack the drug powder into small packages of 10 g per bag for convenient use by patients as needed.

[0064] 5) Usage instructions: Instruct patients to take it twice a day, 1 bag each time, and take it continuously for 4 weeks.

[0065] Example 2: Prevention of Early Knee Osteoarthritis

[0066] 1) Ingredient Ratio: According to the drug formula of the present invention, weigh 20 g of Rehmannia glutinosa, 15 g of Epimedium brevicornu, 15 g of Drynaria fortunei, 12 g of Angelica sinensis, 12 g of Polygonum cuspidatum, and 10 g of Achyranthes bidentata.

[0067] 2) Drug Preparation: Mix the above medicinal materials, add an appropriate amount of water for decoction, and filter after decocting until the medicinal liquid is concentrated.

[0068] 3) Freeze-drying Treatment: Freeze-dry the filtered medicinal liquid in an ultra-low temperature freeze-dryer to convert it into powder.

[0069] 4) Packaging: Package the drug powder into small packages of 10 g per bag.

[0070] 5) Usage Instructions: Instruct the patient to take it once a day, one bag each time, for daily health care use, and continue to take it for 2 weeks.

[0071] In these two examples, the differences in the drug formula mainly lie in the ratio and usage frequency of the medicinal materials to meet the needs of patients with knee osteoarthritis at different degrees. In this way, the dosage and treatment cycle of the drug can be adjusted more precisely to achieve the best treatment effect.

[0072] In view of the problems existing in the prior art, the present invention provides a drug for treating knee osteoarthritis, a preparation method and an application thereof.

[0073] A drug for treating knee osteoarthritis, the drug for treating knee osteoarthritis is composed of 5 - 30 g of Rehmannia glutinosa, 5 - 30 g of Epimedium brevicornu, 5 - 30 g of Drynaria fortunei, 5 - 30 g of Angelica sinensis, 5 - 30 g of Polygonum cuspidatum, and 5 - 30 g of Achyranthes bidentata by weight.

[0074] Another object of the present invention is to provide a preparation method of a drug for treating knee osteoarthritis, the preparation method of the drug for treating knee osteoarthritis includes:

[0075] S101, weigh 20 g of Rehmannia glutinosa, 15 g of Epimedium brevicornu, 15 g of Drynaria fortunei, 12 g of Angelica sinensis, 12 g of Polygonum cuspidatum, and 10 g of Achyranthes bidentata by weight;

[0076] S102, mix the above components, decoct with water, filter, boil, concentrate, then freeze-dry with an ultra-low temperature freeze-dryer (aiming to fully retain the effective components of the drug), crush into powder, and package, 10 g per bag.

[0077] Another object of the present invention is to provide an application of the drug for treating knee osteoarthritis in the preparation of drugs for treating degenerative joint diseases with bone destruction of the knee joint.

[0078] Another object of the present invention is to provide a drug for treating knee osteoarthritis for use in preparing a drug for treating inflammatory pain caused by knee osteoarthritis.

[0079] Another object of the present invention is to provide a capsule prepared using the drug for treating knee osteoarthritis.

[0080] Another object of the present invention is to provide an granule prepared by using the medicine for treating knee osteoarthritis.

[0081] Furthermore, the method for using the granules prepared from the medicine for treating knee osteoarthritis is to take it three times a day.

[0082] In order to verify the technical value of the invention, the inventor designed a clinical study in the early stage, and allocated 120 patients with knee osteoarthritis in a 1:1 ratio. The subjects were divided into a Gubi granule observation group of 60 cases and a meloxicam control group of 60 cases using a random number table method. Statistical tests were performed on the age, gender, body mass index (BMI), course of disease, and affected site of the two groups of patients to ensure that there was no statistical difference in the baseline level of the required samples. The treatment results showed that Gubi granules can effectively improve the inflammatory indicators ESR, CRP, and IL-6 in patients with knee osteoarthritis of kidney deficiency and blood stasis type, and reduce VAS scores, WOMAC scores, Lequesne index and TCM syndrome scores (P<0.05), which is equivalent to the meloxicam control group. The improvement of TCM syndrome efficacy scores in the Gubi granule group was better than that in the meloxicam control group, and the comparison between the two groups was statistically significant (P<0.05). This shows that the invention is clinically effective for patients with knee osteoarthritis and has good drug safety.

[0083] The embodiments of the present invention have achieved some positive effects during the development or use process, and indeed have great advantages over the prior art. The following content is described in conjunction with data, charts, etc. of the test process.

[0084] (I) Clinical efficacy and safety of Gubi granules in the treatment of knee osteoarthritis

[0085] The inventors allocated 120 patients in a 1:1 ratio, and used a random number table method to divide the subjects into a Gubi granule observation group of 60 cases and a meloxicam control group of 60 cases. Statistical tests were performed on the age, gender, body mass index (BMI), course of disease, and site of disease of the two groups of patients to ensure that there was no statistical difference in the required sample baseline level.

[0086] 1. Treatment Methods

[0087] Gubi Granule Observation Group: Oral administration of Chinese medicine Gubi Granule (Chinese medicine formula granule, produced by Sinopharm Holding Company). The drug consists of 20g of Rehmannia glutinosa, 15g of Epimedium, 15g of Drynariae Rhizoma, 15g of Angelicae Sinensis, 10g of Polygonum Cuspidatum, and 10g of Achyranthes bidentata. Take it twice a day, morning and evening, for 4 consecutive weeks. Meloxicam Control Group: Meloxicam Tablets (production batch number: 2018 / 07423, Yangtze River Pharmaceutical Group Co., Ltd., specification: 7.5mg×20 tablets) were administered 7.5mg each time, once a day after meals, for 4 consecutive weeks.

[0088] 2. Treatment outcomes

[0089] (1) The invention has obvious clinical efficacy. The results of Gubi Granule treatment showed that 7 cases were clinically effective, 47 cases were effective, and the total effective rate was 96.61%. The efficacy of TCM syndrome showed that 15 cases were effective, 37 cases were effective, and the total effective rate was 96.61%.

[0090] (2) The invention can significantly improve the pain score (VAS) of patients with knee osteoarthritis

[0091] Table 1 VAS scores of the two groups of patients before and after treatment (points, )

[0092]

[0093] Note: t1 / P1 value is the comparison of data between the two groups after treatment. ## P<0.01. Comparison between before and after treatment, P<0.01.

[0094] (2) This invention can improve the WOMAC score of joint function in patients with knee osteoarthritis

[0095] Intra-group comparison of the two groups showed that the WOMAC pain, stiffness, joint function and total scores after treatment were lower than those before treatment (P < 0.01). Inter-group comparison of the WOMAC pain score after treatment showed that the observation group was better than the control group (P < 0.01). There was no significant difference in the stiffness score, joint function score and total score between the two groups, as shown in Table 2.

[0096] Table 2 Comparison of WOMAC scores between the two groups before and after treatment (points, )

[0097]

[0098] Note: t1 / P1 value is the comparison of data between the two groups after treatment. ## P<0.01. Comparison between before and after treatment, P<0.01.

[0099] (4) The invention can improve the Lequesne index score of patients with knee osteoarthritis

[0100] After the within-group comparison of the two groups, the Lequesne pain discomfort, walking ability, living ability and total score were lower than those before treatment, and the difference was statistically significant (P < 0.01); after treatment, there was no statistically significant difference in the Lequesne scores between the two groups. See Table 3.

[0101] Table 3 Comparison of Lequesne scores of two groups of patients before and after treatment (points, )

[0102]

[0103] Note: The t1 / P1 value is the data comparison after treatment. Compared with before treatment, P < 0.01.

[0104] (5) The invention can improve the inflammatory indexes of patients with knee osteoarthritis

[0105] After the within-group comparison of the two groups, ESR, CRP, and IL6 were all lower than those before treatment, and the difference was statistically significant (P < 0.01). After the between-group comparison, there was no statistically significant difference in ESR, CRP, IL6, and TNF-α after treatment, indicating that the two groups of drugs can both reduce the three inflammatory indexes of ESR, CRP, and IL6, and the curative effects of the two groups are equivalent. The comparison of TNF-α is meaningless. See Table 4.

[0106] Table 4 Inflammatory factors of two groups of patients before and after treatment

[0107]

[0108] Note: The t1 / P1 value is the data comparison after treatment. Compared with before treatment, P < 0.01.

[0109] 2. Network pharmacology analysis and molecular docking analysis of the mechanism of action of the invention in the treatment of knee osteoarthritis

[0110] (1) Research methods

[0111] Retrieve the disease targets of KOA by searching the five major databases of GeneCards, OMIM, TTD, DrugBank, and PharmGkb with the keyword "Kneeosteoarthritis". Remove duplicates between the active ingredient targets of Gubi Granule and the KOA disease targets, draw a Venn diagram, and take the intersection genes to obtain the key targets of Gubi Granule in the treatment of KOA. Import the active ingredients and action targets of Gubi Granule into Cytoscape 3.9.1 software for drawing. Import the intersection targets of drugs and diseases into the Metascape platform for GO and KEGG enrichment analysis. According to the significance of the analysis results, import them into Bioinformatics to obtain bar charts and bubble charts. Determine the compound names, molecular weights, and 3D structures of the active ingredients from the PubChem database, and then download the corresponding 3D structures of the active ingredients from the RCSB PDB database. Use AutoDock software to prepare the ligands and proteins required for molecular docking. For the target protein, remove water molecules, add hydrogen, modify amino acids, optimize energy, and adjust force field parameters for its crystal structure, and then obtain the low-energy conformation that meets the ligand structure. Finally, perform molecular docking on the structures of 5 key targets and 5 key active ingredients. The Affinity (kcal / mol) value represents the binding ability of the two. The lower the binding ability, the more stable the binding of the ligand to the receptor. Finally, use Discovery Studio to analyze and observe the docking results, select the top 5 results of the compounds with the best binding energy, and plot and analyze them.

[0112] (2) Active ingredients and action targets of Gubi Granule

[0113] The research results show that there are 3 active ingredients of Rehmannia glutinosa in this invention, with 26 action targets; 23 active ingredients of Epimedium brevicornu, with 510 action targets; 20 active ingredients of Achyranthes bidentata, with 443 action targets; 10 active ingredients of Polygonum cuspidatum, with 319 action targets; 18 active ingredients of Drynaria fortunei, with 301 action targets; and 2 active ingredients of Angelica sinensis, with 69 action targets. After removing invalid and duplicate targets, 145 active ingredient targets of Gubi Granule are obtained, as shown in Table 6. There are 1424 in the GeneCards database, 30 in the OMIM database, 62 in the DrugBank database, 4 in the PharmGkb database, and 2 in the TTD database, for a total of 1522 related targets. After removing duplicates, 1367 disease-related targets are obtained. Import the targets of each database into the DrawVenn Diagram website to draw a Venn diagram, as shown in Figure 2 .

[0114] Screen out the intersection targets of 145 drugs and 1367 diseases, use Draw Venn Diagram to draw a Venn diagram, and obtain 126 intersection targets, as shown in Figure 3。Construct a drug-component-target network diagram, as shown in Figure 4 Figure 1. In the figure, GSB, HZ, NX, SD, YYH, and DG represent Drynaria rhizome, Polygonum cuspidatum, Achyranthes bidentata, Rehmannia glutinosa, Epimedium brevicornu, and Angelica sinensis respectively. A, B, C, D, E, F, and G are the common components between drugs and also the key components for treating KOA. The larger the node, the higher the degree value (Degree value). The specific connections are shown in Table 5. The rectangular distribution icons connected to the components are drug targets.

[0115] Table 5 Common components of drugs

[0116]

[0117] The PPI network diagram of the intersection targets for treating KOA with the invention's Gubi Granules is as shown in Figure 5 Figure 2. Import 126 intersection targets into the STRING platform to obtain a TSV file, and then import it into Cytoscape 3.9.1 software. After hiding 17 free nodes, 109 nodes and 477 edges are obtained. Then, adjust according to the degree value. The larger the degree value, the darker the color and the larger the node. The results show that nodes such as TP53, AKT1, TNF, IL-6, and EGFR are the core targets for treating KOA with Gubi Granules. According to the median of Betweenness, Clossness, and Degree (Betweenness > 74.192, Clossness > 0.377, Degree ≥ 7), 35 core targets such as TP53, AKT1, TNF, IL6, and EGFR are obtained, as shown in Figure 6 。

[0118] Use the Metascape database to perform GO functional analysis on 126 intersection targets. A total of 1758 biological processes, 84 cellular components, and 148 molecular functions are enriched. The top 10 enriched ones are plotted as a bar chart, as shown in Figure 7 Figure 3. The biological processes of Gubi Granules in treating KOA are mainly reflected in the response to peptides, the response of cells to lipids, inflammatory responses, responses to foreign biological stimuli, and positive regulation of cell death, etc.; molecular functions include DNA transcription factor binding, cytokine receptor binding, protease binding, oxidoreductase, etc.; cellular components include membrane rafts, vesicular cavities, transcriptional regulatory complexes, extracellular matrix, and serine-type peptidase complexes, etc. KEGG enrichment analysis yields 196 pathways, and the top 20 pathways with high significance are plotted as a bubble chart, as shown in Figure 8 Figure 4.

[0119] Import the top 20 signaling pathways and their related targets into Cytoscape 3.9.1 software to construct a "key target-signaling pathway" diagram, as shown in Figure 9, in the figure, the red diamond nodes are signal pathways, the larger the volume, the higher the degree value; the light purple circles are key targets, the darker the color, the larger the node, and the higher the degree value. The results show that nodes such as TP53, AKT1, TNF, IL6, and EGFR are the core targets of Gubi Granules in the treatment of KOA, and the significantly related pathways are mainly Pathways in cancer, Lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, Fluid shear stress and atherosclerosis, and TNF signaling pathway.

[0120] (3) Molecular docking and visualization analysis of the invention results

[0121] According to the above results, the top 5 components and 5 targets were selected as ligands and receptors for docking, and 25 binding energies were calculated. As shown in Table 6, the lower the binding energy, the better the binding ability. The results show that the 5 ligands and 5 receptors have good binding ability to each other. The data shows that the binding energies of AKT1 with kaempferol, quercetin, luteolin, sitosterol, and stigmasterol rank among the top 5, and 3D visualization analysis was performed.

[0122] Table 6 Binding energies (kcal / mol) of the core components and core targets in docking

[0123]

[0124] Kaempferol forms Conventional Hydrogen Band interactions with SER205, THR211, and ASN54 of AKT1; forms Pi-Alkyl interactions with LEU210 and LEU264; forms Pi-Anion interactions with ASP292; forms Pi-Sigma interactions with VAL270; forms Pi-Stacked interactions with TRP80.

[0125] As Figure 10Quercetin forms Conventional Hydrogen Bond interactions with ILE290, THR211, SER205, VAL271, GLN79 of AKT1; forms Pi-Sigma interaction with VAL270; forms Pi-Alkyl interactions with IEU210, LEU264; forms Pi-Anion interaction with ASP292; and forms Pi-Stacked interaction with TRP80. As Figure 11 。

[0126] Luteolin forms Conventional Hydrogen Bond interactions with THR211, SER205, ASN54, GLN79 of AKT1; forms Pi-Sigma interaction with VAL270; forms Pi-Alkyl interactions with IEU210, LEU264; forms Pi-Anion interaction with ASP292; and forms Pi-Stacked interaction with TRP80. As Figure 12 。

[0127] Sitosterol forms Alkyl interactions with IEU210, LEU264, VAL270, ILE84 of AKT1; forms Pi-Alkyl interactions with TRP80, TYR272. As Figure 13 。

[0128] Stigmasterol forms Pi-Sigma interaction with TRP80 of AKT1; forms Alkyl interactions with IEU210, LEU264, VAL270, ILE84, LYS268, ARG273; forms Pi-Alkyl interactions with TRP80, TYR263, TYR272. As Figure 14 。

[0129] Results of animal experiments: Gubi Granules can effectively improve the inflammatory indexes of the knee osteoarthritis (KOA) model and reduce the expression of genes related to the Wnt / β-catenin signaling pathway

[0130] The inventors randomly divided 36 New Zealand white rabbits into a blank control group, a model control group, high, medium, and low treatment groups of Gubi Granules, and a meloxicam control group, with 6 rabbits in each group. Except for the blank control group, the other groups were used to construct a KOA animal model by injecting papain into the joint cavity. After 4 weeks of modeling, the rabbits in the treatment groups were given intragastric administration of the aqueous solution of Gubi Granules, and the positive control group was given intragastric administration of the aqueous solution of meloxicam, once a day. After continuous administration for 2 weeks, the rabbits were sacrificed by air injection, the knee joints were exposed, and the synovium and articular cartilage of the joints were dissected for experiments. The results are as follows.

[0131] (1) Gubi Granules can effectively improve the pathological morphology of the model rabbits

[0132] The pathological examination results showed that the synovial tissue of the knee joint was congested and edematous, with lymphocyte infiltration and exudation. The surface of the cartilage was rough and the articular surface was eroded. No obvious lymphocytes were seen in the blank control group, and the articular surface was smooth. In the Guobi Granule treatment group and the meloxicam control group, the synovial tissue congestion was mild, with a small amount of lymphocyte infiltration and no obvious exudation. The surface of the cartilage was slightly rough, but there was no erosion on the articular surface. See Figure 15 。

[0133] (2) Guobi Granule can effectively reduce the expression level of β-catenin protein

[0134] As Figure 16 shown, the WB research results showed that the expression level of β-catenin in the cartilage tissue of the model group was the lowest, that of the blank control group was the highest, and the expression levels of each treatment group were lower than those of the blank control group and higher than those of the model group (P<0.05) or (P<0.01); in the comparison between groups, compared with the blank control group, P<0.05; compared with the model group, P<0.01 for the medium-dose, high-dose and positive control groups, and P<0.05 for the low-dose group; in the comparison within the treatment groups, there was no difference in the expression of β-catenin between the medium-dose and high-dose groups, and the differences in the other groups were statistically significant (P<0.05).

[0135] (3) Guobi Granule can reduce the expression levels of the upstream genes Wnt 3a, Wnt5a, Wnt7a and the downstream genes MMP-13, c-myc mRNA of the Wnt / β-catenin signaling pathway

[0136] Real-time fluorescence PCR showed that each amplification curve was smooth, with a significant exponential amplification period, and a single melting peak was visible in each melting curve, without specific amplification. The expression levels of Wnt 3a, Wnt5a, Wnt7a and the downstream genes MMP-13, c-myc genes were as follows: the expression levels of Wnt 3a, Wnt5a, Wnt7a mRNA in the cartilage tissue of the model group were the highest, those of the blank group were the lowest, and the expression levels of mRNA in each treatment group were higher than those of the blank group and lower than those of the model group (P<0.05); in the comparison within the treatment groups, there was no statistical difference between the low-dose and medium-dose groups (P>0.05), and the differences in the other groups were statistically significant (P<0.05). The expression levels of MMP-13, c-myc mRNA in the cartilage tissue of the model group were the highest, those of the blank group were the lowest, and the expression levels of mRNA in each treatment group were higher than those of the blank group and lower than those of the model group (P<0.05); in the comparison within the treatment groups, there was no statistical difference between the low-dose and medium-dose groups (P>0.05), and the differences in the other groups were statistically significant (P<0.05), as shown in Tables 1 and 2

[0137] Table 1 Expression levels of Wnt3a, Wnt5a, Wnt7a mRNA expression

[0138] Group Number of cases (n) <![CDATA Wnt 3a]]> Wnt5a Wnt7a Blank group 6 1.04±0.17 0.97±0.16 0.95±0.05 Model group 6 <![CDATA[3.50±0.18 a > <![CDATA[3.39±0.26 a > <![CDATA[4.17±0.35 a > Low dose of Gubi granule 6 <![CDATA[2.62±0.70 ac > <![CDATA[2.48±0.59 ac > <![CDATA[2.32±0.53 ac > Medium dose of Gubi granule 6 <![CDATA[1.75±0.39 ac > <![CDATA[1.82±0.21 ac > <![CDATA[1.90±0.43 ac > High dose of Gubi granule 6 <![CDATA[1.67±0.32 c > <![CDATA[1.38±0.24 c > <![CDATA[1.45±0.21 c > Cataplasm group 6 <![CDATA[1.44±0.34 c > <![CDATA[1.53±0.28 c > <![CDATA[1.42±0.22 c >

[0139] Note: Compared with the blank control group, there were no significant differences in the expressions of Wnt3a, Wnt5a, and Wnt7a between the high-dose group and the cataplasm control group, and the differences in the other groups were statistically significant, aP < 0.05; compared with the model group, cP < 0.05; within the treatment group, there were no significant differences in the expressions in the low- and medium-dose groups, and the differences in the other groups were statistically significant (P < 0.05).

[0140] Table 2 MMP-13, c-myc mRNA expression levels

[0141] Group Number of cases (n) MMP-13 <![CDATA c-myc > Blank group 6 1.08±0.14 1.02±0.06 Model group 6 <![CDATA[5.45±0.49 a > <![CDATA[6.03±0.40 a > Low dose of Gubi granule 6 <![CDATA[3.20±1.09 ac > <![CDATA[3.89±0.91 ac > Medium dose of Gubi granule 6 <![CDATA[2.21±0.46 ac > <![CDATA[2.75±0.63 ac > High dose of Gubi granule 6 <![CDATA[1.81±0.35 c > <![CDATA[2.30±0.28 ac > Cataplasm group 6 <![CDATA[1.59±0.26 c > <![CDATA[1.75±0.38 ac >

[0142] Note: Compared with the blank control group, there was no significant difference in the expression of MMP-13 between the high-dose group and the cataplasm control group, and the differences in the other groups were statistically significant, aP < 0.05; compared with the model group, cP < 0.05; within the treatment group, there were no significant differences in the expressions in the low- and medium-dose groups, and the differences in the other groups were statistically significant (P < 0.05).

[0143] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention shall be covered by the protection scope of the present invention.

Claims

1. A drug for treating knee osteoarthritis, characterized in that: The medicine for treating knee osteoarthritis is prepared from 5-30g of Rehmannia glutinosa, 5-30g of Epimedium, 5-30g of Drynariae Rhizoma, 5-30g of Angelicae Sinensis, 5-30g of Polygonum cuspidatum and 5-30g of Achyranthes bidentata by weight.

2. A method for preparing a drug for treating knee osteoarthritis, characterized in that: The preparation method of the drug for treating knee osteoarthritis comprises: Step 1: weigh 20g of Rehmannia glutinosa, 15g of Epimedium, 15g of Drynaria fortunei, 12g of Angelica sinensis, 12g of Polygonum cuspidatum, and 10g of Achyranthes bidentata; Step 2, mixing the above components, decocting with water, filtering, boiling, concentrating, freeze-drying with an ultra-low temperature freeze dryer, crushing into powder, and packaging, 10g per bag.

3. Use of the drug for treating knee osteoarthritis as claimed in claim 1 in the preparation of a drug for treating inflammatory pain caused by knee osteoarthritis.

4. A capsule prepared using the drug for treating knee osteoarthritis according to claim 1.

5. An granule prepared using the medicine for treating knee osteoarthritis according to claim 1.

Images