A Chinese medicine composition for resisting gouty arthritis

Abstract

The present invention provides a Chinese medicine composition for anti-gouty arthritis. The raw materials of the composition are composed of chicory, Poria cocos, Smilax glabra, Sophora japonica, and Gentiana macrophylla, which provides a new drug option for removing uric acid crystals in joints and anti-gouty arthritis. The Chinese medicine composition of the present invention can significantly reduce uric acid crystals in joints, reduce the level of uric acid in joint fluid, and at the same time, significantly improve the swelling degree of quail joints with gouty arthritis, reduce the level of inflammatory factor interleukin-1β in serum, and have no effect on liver and kidney function, and has a pharmacological effect of anti-gouty arthritis.

Description

Technical Field

[0001] The invention relates to a Chinese medicine composition, in particular to a new Chinese medicine combination which is prepared by combining chicory, tuckahoe, smilax glabra, sophora japonica seeds and gentiana macrophylla for removing uric acid crystals in joints and resisting gouty arthritis. Background Art

[0002] Gouty arthritis is a progressive, disabling metabolic disease closely related to uric acid crystals, and it is also showing a high incidence and younger trend worldwide. Currently, the commonly used drugs for gouty arthritis include three categories: nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, and glucocorticoids. Among them, NSAIDs mainly inhibit the activity of cyclooxygenase (COX) to block the production of prostaglandins (PGE), colchicine mainly prevents neutrophil chemotaxis, and glucocorticoids mainly inhibit the release of arachidonic acid to reduce the production of inflammatory substances, thereby exerting anti-inflammatory and analgesic effects. Although the above drugs have clear targets and strong pharmacological activity, they are mostly limited to interventions in a single pathological stage, and are often used in combination in clinical practice to meet the comprehensive treatment needs of gout. This not only increases the medication burden of patients, but also poses a potential safety risk for the combined use of multiple drugs.

[0003] At present, there is no radical cure for acute gouty arthritis, and most of them are mainly anti-inflammatory and analgesic. However, most drugs have side effects such as gastrointestinal reactions, which affect the treatment effect. Traditional surgical treatment of gouty arthritis is generally traumatic, and it cannot accurately judge the degree of lesions in the joints. At the same time, it is very easy to get infected during surgical treatment, and there are many adverse reactions and complications, and the clinical effect is not good. The "Chinese Multidisciplinary Expert Consensus on the Diagnosis and Treatment of Hyperuricemia-Related Diseases" points out that "gout is the deposition of urate crystals in patients with hyperuricemia, leading to gouty arthritis, uric acid nephropathy and kidney stones", correcting the limited understanding that gout is only defined as inflammatory joint disease, and clarifying the pathological characteristics of gout with urate deposition. This also shows that the cause of gouty arthritis lies in the appearance of uric acid crystals in the joints, and how to remove uric acid crystals in the joints to fight gouty arthritis has become a problem that needs to be solved urgently in the clinic.

[0004] Traditional Chinese medicine has advantages in the prevention and treatment of progressive diseases related to uric acid metabolism disorders such as gouty arthritis. This combination has been found to remove uric acid crystals in the joints, thereby playing an anti-gouty arthritis role. Chicory and Smilax glabra can strengthen the spleen, eliminate dampness and turbidity, clear the joints, and regulate uric acid metabolism; Poria cocos and Gentiana macrophylla can eliminate rheumatism and pain, and intervene in gouty inflammation and swelling; Sophora japonica seeds can clear away heat and cool blood, and relieve inflammation and pain. The whole formula can strengthen the spleen, eliminate dampness and turbidity, reduce acid, reduce swelling and relieve pain. Modern research shows that some drugs can prevent and treat gouty arthritis. However, there are no research reports on the use of the above-mentioned drug combination to remove uric acid crystals in the joints and fight gouty arthritis.

[0005] In addition, the chicory, tuckahoe and sophora japonica in this Chinese medicine compound have the characteristics of being both medicine and food, and are highly safe. Among them, chicory, tuckahoe and sophora japonica are all included in the "List of Items That Are Both Food and Medicine" published by the Ministry of Health, and Chinese yam is included in the "List of Items That Can Be Used as Health Foods". Summary of the invention

[0006] The present invention aims to provide a Chinese medicine composition for removing uric acid crystals from joints and resisting gouty arthritis, and at the same time provide a new drug option for the prevention and treatment of gouty arthritis.

[0007] As one aspect of the present invention, the present invention provides a traditional Chinese medicine composition for resisting gouty arthritis, wherein the traditional Chinese medicine composition is prepared from the following raw materials: chicory, tuckahoe, smilax glabra, sophora japonica seed, and gentiana macrophylla.

[0008] Preferably, the traditional Chinese medicine composition is made of the following raw materials: 6-18 parts by weight of chicory, 5-15 parts by weight of Poria cocos, 10-60 parts by weight of Smilax glabra, 5-10 parts by weight of Sophora japonica seeds, and 3-10 parts by weight of Gentiana macrophylla.

[0009] Further preferably, the traditional Chinese medicine composition is made of the following raw materials: 6-10 parts by weight of chicory, 10-15 parts by weight of Poria cocos, 20-40 parts by weight of Smilax glabra, 5-10 parts by weight of Sophora japonica seeds, and 3-10 parts by weight of Gentiana macrophylla.

[0010] Most preferably, the Chinese medicine composition is made of the following raw materials: 9 parts by weight of chicory, 10 parts by weight of Poria cocos, 30 parts by weight of Smilax glabra, 6 parts by weight of Sophora japonica seeds, and 9 parts by weight of Gentiana macrophylla.

[0011] In the above technical scheme, the Chinese medicine composition can be any form composed or made of the above raw materials, including: a composition formed by mixing the above raw materials separately after crushing; or, a composition obtained by mixing the above raw materials and then crushing; or, the above raw materials are mixed and then extracted according to a conventional extraction method to obtain an extract, the extract is further subjected to a refining and purification process to obtain an effective part, and the above extract and effective part are further prepared according to a conventional preparation process to obtain a conventional oral dosage form.

[0012] The above-mentioned conventional extraction methods include immersion extraction, decoction extraction, reflux extraction, percolation extraction, ultrasonic extraction, microwave extraction, etc.; the extraction solvent includes water or conventional organic solvents, such as ethanol, methanol, ethyl acetate, petroleum ether, isopropanol, etc.; the refining and purification process includes extraction, column chromatography separation, high performance liquid chromatography separation, etc.

[0013] The above conventional oral dosage forms include tablets, capsules, granules, pills, powders, and oral liquids. When preparing the above dosage forms, it is necessary to add commonly used pharmaceutically acceptable excipients, including: fillers, disintegrants, lubricants, suspending agents, adhesives, sweeteners, flavoring agents, preservatives, bases, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include: polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; adhesives include starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.

[0014] In addition to being fed in the form of raw materials, the Chinese medicine composition of the present invention can also be fed in the form of extracts or granules. Therefore, as another aspect of the present application, the present application further provides a Chinese medicine composition for anti-gouty arthritis, the Chinese medicine composition is made of the following raw materials: 6-18 parts by weight of chicory extract, 5-15 parts by weight of Poria cocos extract, 10-60 parts by weight of Smilax glabra extract, 5-10 parts by weight of Sophora japonica extract, and 3-10 parts by weight of Gentiana macrophylla extract; wherein the extracts are water extracts of the raw materials, or granules made of the water extracts according to conventional processes.

[0015] As another aspect of the present invention, the present invention provides the use of the traditional Chinese medicine composition in the preparation of a drug for removing uric acid crystals in joints and resisting gouty arthritis.

[0016] Pharmacological studies have shown that the Chinese medicine composition of different concentrations can significantly reduce uric acid crystals in joints and reduce the level of uric acid in joint fluid. At the same time, it can significantly improve the swelling of quails with gouty arthritis, reduce the level of interleukin-1β, an inflammatory factor in serum, and has no effect on liver and kidney functions, thus having a pharmacological effect of resisting gouty arthritis.

[0017] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a new drug option for the prevention and treatment of gouty arthritis, and the Chinese medicine composition can significantly reduce uric acid crystals in joints. Experimental studies have shown that the various prescriptions after the prescriptions are separated do not have this effect, indicating that the specific compatibility of the raw materials of the Chinese medicine composition produces a synergistic effect, and has a specific technical effect in removing uric acid crystals in joints and resisting gouty arthritis. In addition, the Chinese medicine composition of the present invention has a simple taste and contains a dual-purpose medicine for medicine and food, has the characteristics of rich drug sources and high safety, and has good clinical application prospects and economic utilization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The present invention is a flow chart of the extraction method of a Chinese medicine composition.

[0019] Figure 2 These are the uric acid crystallization diagrams of the quail joints in each group in Example 2; wherein, 2A is the joint lavage fluid of the normal group, 2B is the suspension of the positive control group, 2C is the joint lavage fluid of the model group, 2D is the joint lavage fluid of the high-dose Chrysanthemum and Poria group, 2E is the joint lavage fluid of the medium-dose Chrysanthemum and Poria group, and 2F is the joint lavage fluid of the low-dose Chrysanthemum and Poria group.

[0020] Figure 3 This is a graph of uric acid levels in the joint fluid of quails in each group in Example 2.

[0021] Figure 4 The diagram shows the swelling degree of the left wrist joint of each group of quails at different time points in Example 2.

[0022] Figure 5 The diagram shows the swelling degree of the right wrist joint of each group of quails at different time points in Example 2.

[0023] Figure 6 The diagram is a diagram of the swelling degree of the left paw joints of quails in each group at different time points in Example 2.

[0024] Figure 7 The diagram is a diagram of the swelling degree of the right paw joints of quails in each group at different time points in Example 2.

[0025] Figure 8 This is a graph showing the inhibition rate of left wrist joint swelling in each group of quails at different time points in Example 2.

[0026] Fig. 9 This is a graph showing the inhibition rate of right wrist joint swelling in each group of quails at different time points in Example 2.

[0027] Fig.10 This is a graph showing the inhibition rate of left paw joint swelling in each group of quails at different time points in Example 2.

[0028] Fig.11 This is a graph showing the inhibition rate of right paw joint swelling in each group of quails at different time points in Example 2.

[0029] Fig.12 These are the appearance morphology pictures of the joints of the quail in the model group in Example 2; 12A is a side morphology picture, and 12B and 12C are front morphology pictures.

[0030] Fig.13 The figures are the appearance morphology of the paws of quails in each group in Example 2, wherein 13A is a comparison between the model group (left) and the normal group (right), 13B is a comparison between the high-dose Chrysanthemum Poria group (left) and the model group (right), 13C is a comparison between the medium-dose Chrysanthemum Poria group (left) and the model group (right), and 13D is a comparison between the low-dose Chrysanthemum Poria group (left) and the model group (right).

[0031] Fig.14This is a graph of serum IL-1β levels in each group of quails in Example 2.

[0032] Fig.15 These are the hematoxylin-eosin stained pathological images of the quail joint tissues in each group in Example 2.

[0033] Fig.16 This is a graph of AST and ALT activity in serum of quail in each group in Example 2.

[0034] Fig.17 This is a graph of the serum CRE and BUN levels of quails in each group in Example 2. DETAILED DESCRIPTION

[0035] The present invention will be further described below by means of specific implementation methods. The following implementation methods are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following implementation methods.

[0036] Example 1 Study on the Optimal Formulation of Anti-Gouty Arthritis

[0037] 1. Experimental purpose: To explore the pharmacological effects of different Chinese herbal medicine combinations in lowering uric acid and treating gouty arthritis, and to optimize the effective formula.

[0038] 2. Experimental Materials

[0039] 2.1 Experimental system

[0040] 2.1.1 Experimental animals: Kunming mice, male, weighing 28±2g, 80 mice in total.

[0041] 2.1.2 Source of animals: provided by Sibeifu (Beijing) Biotechnology Co., Ltd. [Experimental Unit Use License Number: SYXK (Beijing) 2020-0033, License Number: SCXK (Beijing) 2019-0010, Experimental Animal Ethics Number: BUCM-4-2023030901-1033].

[0042] 2.1.3 Rearing conditions: Beijing University of Chinese Medicine experimental animal barrier system, room temperature 20-25℃, relative humidity, humidity 35-75%, 12h standard light.

[0043] 2.1.4 Feed: provided by Spef Biotech Co., Ltd.

[0044] 2.2 Experimental drugs and reagents

[0045] Modeling agent: MSU, Sigma, USA, batch number: BCBQ2650V;

[0046] Experimental preparations:

[0047]

[0048] Positive control drug: colchicine, Kunming Pharmaceutical Group Co., Ltd., 210603-01.

[0049] Solvent: Deionized water

[0050] Reagents:

[0051] 0.9% sodium chloride injection, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., batch number: 2208102011;

[0052] Universal tissue fixative, Wuhan Sewell Biotechnology Co., Ltd., batch number: G1101-500ML; Chloral hydrate, Shanghai McLean Biochemical Technology Co., Ltd., batch number: C12204202

[0053] 2.3 Detection kit

[0054] Mouse interleukin-1β (IL-Iβ) ELISA kit, Jiangsu Kete Biotechnology Co., Ltd., batch number: 20230240;

[0055] Creatinine (CRE) assay kit, Nanjing Jiancheng Bioengineering Institute, batch number: 20230129;

[0056] Urea nitrogen (BUN) assay kit, Nanjing Jiancheng Bioengineering Institute, batch number: 20230129;

[0057] Alanine aminotransferase (GPT) kit, Nanjing Jiancheng Bioengineering Institute, batch number: 20230111;

[0058] Aspartate aminotransferase (GOT) kit, Nanjing Jiancheng Bioengineering Institute, batch number: 20230111.

[0059] 2.4 Experimental instruments and equipment

[0060] HH-2 digital constant temperature water bath, Ronghua Instrument Manufacturing Co., Ltd., Jintan City, Jiangsu Province;

[0061] DH5-3 low-speed desktop centrifuge, Beijing Shidai Beili Centrifuge Co., Ltd.;

[0062] MTW120 Lemei electronic balance, Shenzhen Meifu Electronics Co., Ltd.;

[0063] TLE303E / 02 electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.;

[0064] KQ5200E ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.;

[0065] SPECROstar Nano microplate reader, BMG, Germany;

[0066] Reichert Histo STAT paraffin tissue slicer, AO Company, USA;

[0067] Olmpus BX53 microscope, DP72CCD camera, Olympus, Japan;

[0068] RE-501 rotary evaporator, Beijing Shentai Weiye Instrument Equipment Co., Ltd.;

[0069] D2F-6050ABF Electric Heating Vacuum Drying Oven, Shanghai Kuntian Laboratory Instrument Co., Ltd.

[0070] 3 Experimental methods

[0071] 3.1 Experimental design basis

[0072] The experimental plan and dosage were determined by referring to the "Technical Guidelines for Pharmaceutical Research at Various Stages of New Chinese Medicine Research (Trial)" issued by the Drug Evaluation Center of the State Food and Drug Administration and the results of preliminary laboratory research.

[0073] 3.2 Preparation of test drugs

[0074] 3.2.1 Julingfang Extract

[0075] Juling Fang prescription: chicory 9g, tuckahoe 10g, smilax glabra 30g, sophora japonica seed 6g, gentiana macrophylla 9g;

[0076] Preparation method of Julingfang extract: according to the prescription ratio of Julingfang, weigh an appropriate amount of medicinal materials, add 12 times the amount of deionized water to soak for 30 minutes, use decoction method, extract for 1.5 hours, collect the extract, repeat 3 times, and combine the extract. Evaporate and concentrate in a rotary evaporator to a compound decoction concentrate containing 1 mg / ml of crude drug, place the compound concentrate in an 80°C water bath and steam until it has no fluidity, then place it in a vacuum drying oven for drying, keep the drying temperature at 80°C, continue drying for 7 days, take it out, put it at room temperature and crush it to obtain the compound extract powder.

[0077] 3.2.2 Preparation of extracts from the formula without qinjiao, without huaimi, without fuling, and without tufuling

[0078] The extract of the formula without Qinjiao: based on the Juling formula, the Qinjiao is removed from the Juling formula, and the remaining medicinal materials are extracted according to the original proportion and preparation process of the Juling formula; the extract of the formula without Sophora japonica: the Sophora japonica in the Juling formula is removed, and the remaining medicinal materials are extracted according to the original proportion and preparation process of the Juling formula; the extract of the formula without Poria: the Poria in the Juling formula is removed, and the remaining medicinal materials are extracted according to the original proportion and preparation process of the Juling formula; the extract of the formula without Smilax glabra: the Smilax glabra in the Juling formula is removed, and the remaining medicinal materials are extracted according to the original proportion and preparation process of the Juling formula.

[0079] 3.2.3 Colchicine solution

[0080] Colchicine was selected as the positive control drug in this experiment. Before use, take an appropriate amount of colchicine and dissolve it in deionized water to make the corresponding dose.

[0081] 3.3 Grouping and Dosage:

[0082] This experiment was divided into 8 groups. They were normal group, model group, colchicine group, Julingfang group, Gentiana macrophylla-free group, Sophora japonica-free group, Poria cocos-free group, and Smilax glabra-free group, with 10 Kunming mice in each group. Each drug-administered group was given preventive and therapeutic drugs by gavage, and the dosage was calculated in proportion to the amount of crude drugs in each group, and the dosage was given as shown in Table 1.

[0083] Table 1 Design of the dosage for each group of mice during the experiment

[0084] Group drug Dosage Gavage volume (ml / 100g) Normal group —— —— 1 Model Group —— —— 1 Colchicine group Colchicine 1mg / kg 1 Juling Fang Group Juling Fang 12.86g / kg 1 Gentiana macrophylla group Gentiana macrophylla prescription 11.05g / kg 1 Sophora japonica group Sophora japonica 11.65g / kg 1 Poria cocos group Recipe for removing Poria cocos 10.85g / kg 1 Group without Smilax glabra Recipe for removing smilax glabra 6.83g / kg 1

[0085] 3.4 Administration method:

[0086] Route of administration: intragastric administration (consistent with the intended route of clinical administration in humans).

[0087] Dosing volume: 1ml / 100g.

[0088] Administration time: gavage once a day in the morning according to body weight.

[0089] Dosage duration: 7 consecutive days.

[0090] 3.5 Preparation of test drugs: Prepare before use. Weigh a certain amount of test drugs and add them into quantitative aqueous solution. Mix by ultrasonic and set aside.

[0091] 3.6 Preparation and administration of modeling agent: Prepare and use immediately. Take a certain amount of MSU crystals and appropriate physiological saline and irradiate them under ultraviolet light for 6 hours. Then dissolve MSU in sterile physiological saline to prepare a 50 mg / ml MSU suspension.

[0092] 3.7 Modeling method: 1 hour after administration on the 5th day of the experiment, 25 μl of MSU suspension was injected into the ankle joint cavity of the model group and each administration group mice using a 1 ml sterile insulin syringe, and 25 μl of 0.9% sterile saline was injected into the ankle joint cavity of the normal group mice. The standard for successful ankle joint injection was bulging on the opposite side of the joint cavity.

[0093] 3.8 Sample collection and processing: The foot thickness and ankle circumference of mice were measured before (0h) and 4h, 8h, 12h, 24h, and 48h after MSU injection, and the inflammation score was performed. Samples were collected on the 7th day of the experiment. The eyeballs were removed to collect blood, and the serum was separated for the detection of various biochemical indicators and stored at -20°C; joint tissues were taken and fixed in 4% paraformaldehyde. After fixation for 48h, 10% formic acid solution was used for decalcification, paraffin embedded, sliced ​​(thickness 4μm), spread, and fished out, and stored at 37°C overnight.

[0094] 3.9 Detection indicators

[0095] 3.9.1 General status indicators: ankle joint swelling, foot thickness swelling, inflammation score.

[0096] 1) Ankle / foot swelling: Vernier calipers were used to measure the ankle circumference and foot thickness of the mice before (0 h) and 4 h, 8 h, 12 h, 24 h, and 48 h after MSU injection, and the results were repeated three times. The swelling at each time point was calculated according to formula (1).

[0097]

[0098]

[0099] 2) Inflammation score

[0100] The grading standard of the inflammation index score is as follows: 0 points, normal; 2 points, erythema of the skin of the tested joint, mild swelling, and visible bone marks; 4 points, obvious redness and swelling of the tested joint, disappearance of bone marks, but swelling is limited to the joint; 6 points, swelling of the limbs other than the tested joint.

[0101] 3.9.2 Inflammation-related indicators: Interleukin-1β (IL-1β) level, the kit was used to detect serum IL-1β level.

[0102] 3.9.3 Renal function related indicators: serum creatinine (SCre) and blood urea nitrogen (BUN) levels. The kit was used to detect serum Cre and BUN levels.

[0103] 3.9.4 Liver function related indicators: serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The test kit was used to detect serum ALT and AST levels.

[0104] 4. Results Statistics and Analysis

[0105] SPSS 27.0 software was used for statistical analysis, and the data were expressed as mean ± standard deviation. The data between multiple groups were compared by one-way analysis of variance or Kruskal-Wallis H nonparametric test according to whether each group was normal or not. The two-way comparison between groups of normally distributed data was performed by LSD test or Dunnett's T3 test according to whether the variances were equal or not; the Kruskal-Wallis H test was used for multiple comparison of mean ranks for non-normally distributed data, and P < 0.05 was considered statistically significant.

[0106] 5 Results

[0107] 5.1 General status indicators

[0108] 5.1.1 Ankle joint swelling

[0109] In this experiment, the ankle joint swelling was calculated using formula (1).

[0110] Compared with the normal group, the ankle swelling of mice in the model group was significantly increased at each observation time point (P<0.01).

[0111] Compared with the model group, the ankle swelling of the mice in the colchicine group was significantly reduced at 24h and 48h after modeling (P<0.05), and there were no significant differences at other time points; the ankle swelling of the mice in the Julingfang group was significantly reduced at 24h and 48h after modeling (P<0.05), and there were no significant differences at other time points; the ankle swelling of the mice in the Gentiana removal group was significantly reduced at 24h and 48h after modeling (P<0.05), and there were no significant differences at other time points; the ankle swelling of the mice in the Sophora japonica removal group was significantly reduced at 24h after modeling (P<0.05), and there were no significant differences at other time points; there were no significant differences in the ankle swelling of the mice in the Poria removal group and the Smilax glabra removal group at each observation point.

[0112] Compared with the Julingfang group, there was no significant difference in ankle swelling of mice in the Qinjiao group at each observation point; the ankle swelling of mice in the Sophora japonica group increased significantly at 4h and 48h after modeling (P<0.05), and there was no significant difference at other time points; the ankle swelling of mice in the Fuling group increased significantly at 48h after modeling (P<0.05), and there was no significant difference at other time points; the ankle swelling of mice in the Tusmilax group increased significantly at 48h after modeling (P<0.05), and there was no significant difference at other time points. See Table 2 below.

[0113] Table 2 Ankle joint swelling of mice in each group at each time point (% n=10)

[0114]

[0115]

[0116] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05; compared with the Julingfang group, & P<0.05.

[0117] 5.1.2 Foot Thickness and Swelling

[0118] In this experiment, formula (2) was used to calculate the swelling degree of the foot thickness.

[0119] Compared with the normal group, the paw swelling of the mice in the model group was significantly increased at each observation time point (P < 0.05 or P < 0.01).

[0120] Compared with the model group, there was no significant difference in the paw thickness swelling degree of the mice in the colchicine group at any observation time point; the paw thickness swelling degree of the mice in the Juling Fang group was significantly reduced 4 hours after modeling (P < 0.05), and there was no significant difference at the other time points; the paw thickness swelling degree of the mice in the Sophora japonicae removal group was significantly increased 12 hours after modeling (P < 0.01), and there was no significant difference at the other time points; there was no significant difference in the paw thickness swelling degree of the mice in the Gentiana removal group, Poria removal group, and Smilax glabra removal group at any observation time point.

[0121] Compared with the Julingfang group, the group without Qinjiao showed no significant difference at each observation time point; the group without Sophora japonica significantly increased at 48h after modeling (P < 0.05), and there was no significant difference at other time points; the group without Fuling significantly increased at 8h and 48h after modeling (P < 0.05 or P < 0.01), and there was no significant difference at other time points; the group without Tusmilax glabra showed no significant difference at each observation time point. See Table 3.

[0122] Table 3 Paw thickness swelling of mice in each group at each time point (% n=10)

[0123]

[0124] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with the Julingfang group, & P<0.05, && P<0.01.

[0125] 5.1.3 Mouse inflammation score

[0126] Compared with the normal group, the inflammation scores of mice in the model group were significantly increased at each observation time point (P<0.01).

[0127] Compared with the model group, the inflammatory scores of mice in the colchicine group were significantly reduced 12h after modeling (P<0.05), and there were no significant differences at other time points; the inflammatory scores of mice in the Julingfang group were significantly reduced 4h and 12h after modeling (P<0.05 or P<0.01), and there were no significant differences at other time points; the inflammatory scores of mice in the Gentiana removal group, Sophora japonica removal group, Poria removal group, and Smilax glabra removal group had no significant differences at any observation time point.

[0128] Compared with the Julingfang group, the inflammatory scores of mice in the Qinjiao group without Qinjiao were not significantly different at any observation time point; the inflammatory scores of mice in the Sophora japonica group without Qinjiao were significantly increased at 12h after modeling (P < 0.05), and there were no significant differences at other time points; the inflammatory scores of mice in the Fuling group without Qinjiao were significantly increased at 12h after modeling (P < 0.05), and there were no significant differences at other time points; the inflammatory scores of mice in the Smilax glabra group without Qinjiao were significantly increased at 12h after modeling (P < 0.05), and there were no significant differences at other time points. See Table 4.

[0129] Table 4 Inflammation scores of mice in each group at each time point ( n=10)

[0130]

[0131] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with the Julingfang group, & P<0.05, && P<0.01.

[0132] 5.2 Mouse serum interleukin-1β (IL-1β) levels

[0133] Compared with the normal group, the IL-1β level in the serum of mice in the model group was significantly increased (P<0.01).

[0134] Compared with the model group, the IL-1β level in the serum of mice in each drug-treated group was significantly decreased (P<0.01 or P<0.05).

[0135] Compared with the Julingfang group, the serum IL-1β levels of mice in the Qinjiao group, Fuling group, and Smilax glabra group were significantly increased (P < 0.01 or P < 0.05), and the serum IL-1β level of mice in the Sophora japonica group showed a downward trend, but no significant difference was observed. See Table 5.

[0136] Table 5 Serum IL-1β content (ng / L, )

[0137]

[0138] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with the Julingfang group, & P<0.05.

[0139] 5.3 Pathological changes in the ankle joint of mice

[0140] The results of pathological sections showed that the ankle joints of mice in the normal group had clear structures, smooth joint surfaces, and no significant pathological changes in the synovial tissue; the synovial tissue of the ankle joints of mice in the model group showed significant hyperplasia, accompanied by a large number of inflammatory cell infiltrations; the ankle joints of mice in the colchicine group had clear structures, and no significant pathological changes in the synovial tissue; the ankle joints of mice in the Julingfang group had clear structures, smooth joint surfaces, and no significant pathological changes in the synovial tissue; the ankle joints of mice in the Qinjiao removal group had clear structures, smooth joint surfaces, and a small amount of hyperplasia in the synovial tissue; the ankle joints of mice in the Sophora japonica removal group had clear structures, and a small amount of hyperplasia in the synovial tissue; the synovial tissue of the ankle joints of mice in the Poria removal group had a small amount of hyperplasia, accompanied by a small amount of inflammatory cell infiltrations; the synovial tissue of the ankle joints of mice in the Smilax glabra removal group had a small amount of hyperplasia, accompanied by a small amount of inflammatory cell infiltrations.

[0141] 5.4 Serum creatinine (Cre) and blood urea nitrogen (BUN) levels

[0142] Compared with the normal group, there were no significant differences in the levels of Cre and BUN in the serum of the model group; there were no significant differences in the levels of Cre and BUN in the serum of each drug-treated group.

[0143] 5.5 Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) expression levels

[0144] Compared with the normal group, 48 hours after modeling, there was no significant difference in the activities of serum ALT and AST in the model group mice; there was no significant difference in the activities of serum ALT and AST in the colchicine group, Juling prescription group, Qinjiao-removed group, Sophora japonica seed-removed group, Poria-removed group, and Smilax glabra-removed group.

[0145] 6 Discussions

[0146] The Juling compound prescription is the crystallization of Professor Zhang Bing's experience in treating arthralgia and acute and chronic gouty arthritis. Combined with more than 20 years of research by our research group, the whole prescription has the effects of dredging collaterals and relieving pain, clearing away heat and resolving phlegm, and strengthening the spleen and removing dampness. The purpose of this study is to observe and compare the effects of the Juling prescription and its separated prescriptions on the mouse model of acute gouty arthritis.

[0147] In terms of overall animal characterization, this experiment used the swelling of the mouse ankle joint and the swelling of the foot thickness as the main evaluation indicators, and the inflammation score as an auxiliary evaluation indicator.

[0148] Compared with the normal group, at each observation time point of the experiment, the ankle swelling, foot thickness swelling, inflammation score and serum IL-1β level of the mice in the model group were significantly increased (P < 0.05 or P < 0.01).

[0149] Compared with the model group, the serum IL-1β levels of mice in each drug-treated group were significantly decreased (P<0.05 or P<0.01); the ankle swelling of mice in the colchicine group was significantly decreased at 24h and 48h after modeling (P<0.05), and the inflammation score was significantly decreased at 12h after modeling (P<0.05), and there were no significant differences in the other time points; the ankle swelling of mice in the Julingfang group was significantly decreased at 24h and 48h after modeling (P<0.05), the foot swelling was significantly decreased at 4h after modeling (P<0.05), and the inflammation score was significantly decreased at 4h and 1h after modeling. The ankle swelling of the mice in the genus-free group was significantly reduced 24h and 48h after modeling (P<0.05), and there were no significant differences in the other time points; the ankle swelling of the mice in the sophora japonica group was significantly reduced 24h after modeling (P<0.05), and the paw thickness swelling was significantly increased 12h after modeling (P<0.05), and there were no significant differences in the other time points; the other indicators of the mice in the fuling group and the smilax glabra group had no significant differences at all observation time points of the experiment.

[0150] Compared with the Julingfang group, the regulatory effects of the Qinjiao group, the Fuling group and the Smilax group on the serum IL-1β level of mice were decreased (P<0.05 or P<0.01), and there was no significant difference in the Sophora japonica group; the other indicators of the mice in the Qinjiao group had no significant differences at all observation time points of the experiment. The ankle swelling of the mice in the Sophora japonica group was significantly higher than that in the Julingfang group at 4h and 48h after modeling (P<0.05), the swelling of the foot thickness was significantly higher than that in the Julingfang group at 48h after modeling (P<0.05), and the inflammation score was significantly higher than that in the Julingfang group at 12h after modeling (P<0.05); the ankle swelling of the mice in the Fuling group was significantly higher than that in the Julingfang group at 48h after modeling (P<0.05), and the swelling of the foot thickness was significantly higher than that in the Julingfang group at 12h after modeling. The swelling of ankle joints of mice in the group without Smilax glabra was significantly higher than that in the group with Juling Fang at 48h after modeling (P<0.05); the swelling of ankle joints of mice in the group without Smilax glabra was significantly higher than that in the group with Juling Fang at 12h after modeling (P<0.05); the swelling of inflammation score was significantly higher than that in the group with Juling Fang at 12h after modeling (P<0.05), and there were no significant differences in the indicators at other time points.

[0151] Compared with the normal group, the model group showed significant hyperplasia of the ankle synovial tissue, accompanied by a large number of inflammatory cell infiltration. Compared with the model group, the colchicine group and the Juling Fang group had clear ankle joint structures, and no significant pathological changes in the synovial tissue; the qinjiao group, the bud group, the fuling group, and the smilax group had clear ankle joint structures, and a small amount of hyperplasia of the synovial tissue.

[0152] During the experiment, there was no significant difference in the levels of CRE, BUN, ALT and AST in the serum of each group of experimental animals, indicating that the Chinese medicine test substance had no obvious effect on the liver and kidney function of mice.

[0153] The above results show that colchicine, as a commonly used anti-gout drug in clinical practice, played an anti-inflammatory effect as a positive drug in this experiment, proving that the mouse model of acute gouty arthritis was successfully established. Juling Fang played a good role in inhibiting the swelling of the ankle joints and the thickness of the foot of mice, and each split-prescription group could also inhibit the swelling of the ankle joints and the thickness of the foot of mice. The results of IL-1β in serum confirmed that Juling Fang and its split-prescriptions had good anti-acute gouty arthritis effects, and the anti-gout effect of Juling Fang was significantly better than that of each split-prescription group.

[0154] In summary, Juling prescription and its separate prescriptions can improve acute gouty arthritis in mice induced by MSU, and Juling prescription has the best efficacy, which shows that the Chinese herbal compound is the result of the synergistic effect of its components.

[0155] Example 2 Study on the efficacy of Chinese medicine composition against gouty arthritis

[0156] 1. Experimental purpose: Based on Example 1, the pharmacological effect of the Chinese medicine composition against gouty arthritis was explored to provide a new drug option for the prevention and treatment of gouty arthritis.

[0157] 2. Experimental methods

[0158] 2.1 Animal grouping

[0159] A total of 72 40-day-old Difak quails were selected and adaptively fed for 3 days. They were randomly divided into 6 groups according to body weight, namely, normal group, model group, benzbromarone group (20.00 mg / kg), high-dose Chrysanthemum Poria extract group (equivalent to 10 g / kg of crude drug), medium-dose Chrysanthemum Poria extract group (equivalent to 7.5 g / kg of crude drug), and low-dose Chrysanthemum Poria extract group (equivalent to 3.75 g / kg of crude drug), with 12 birds in each group.

[0160] 2.2 Model establishment

[0161] From day 1 to day 50 of the experiment, the model group and each drug-treated group were given a high-purine feed (basic feed: yeast extract powder was 8:2); from day 51 to day 83 of the experiment, the model group and each drug-treated group were given a high-protein and high-calcium feed (basic feed: yeast extract powder: bone meal was 5:2:3) combined with 10% fructose water (drinking was restricted to 15 mL / animal / d). During the experiment, the normal group was given sufficient basic feed and clean water. The room temperature of the animal room was controlled at 20-25°C and the humidity was 40-60%.

[0162] 2.3 Preparation method of composition (Chrysanthemum and Poria extract)

[0163] Prescription: Chicory 9g, Poria 10g, Smilax glabra 30g, Sophora japonica seed 6g, Gentiana macrophylla 9g;

[0164] Preparation method: Add deionized water with a solid-liquid ratio of 12 times the mass and soak for 30 minutes. Use the decoction method to extract for 1.5 hours, collect the extract, repeat 3 times, and combine the extracts. Evaporate and concentrate in a rotary evaporator to a concentration of 10g / kg, 7.5g / kg, and 3.75g / kg, and store at 4℃ for future use. See the attached for the specific operation process. Figure 1 .

[0165] 2.4 Experimental drug administration

[0166] The concentrations of the high, medium and low dose groups of the composition were 10g / kg, 7.5g / kg and 3.75g / kg, respectively. Benzbromarone was selected as the positive control drug for lowering uric acid in this experiment. Before use, an appropriate amount of benzbromarone was dissolved in deionized water to the corresponding dose. The concentration of benzbromarone was 20mg / kg. The administration method was intragastric administration, and the normal group and the model group were intragastrically administered with an equal volume of ultrapure water every day.

[0167] 2.5 Collection of materials

[0168] 1.0-1.5 mL of blood was collected from the jugular vein of each group of quails, and they were fasting but not drinking water for 12 hours before blood collection. The serum samples were centrifuged at 3500r / min for 10 minutes at room temperature, and the supernatant was taken for the detection of serum biochemical indicators. 0.2 mL of normal saline was used to rinse the left wrist joint cavity of each group of quails, and the obtained wrist joint rinsing fluid was applied to a slide, and uric acid crystals in the wrist joint rinsing fluid were observed under a polarized light microscope. The joint tissues of the quails in each group were collected for HE staining to observe the inflammatory infiltration.

[0169] 2.6 Collection of joint swelling index

[0170] On the 50th, 60th, 70th and 80th days of the experiment, the circumferences of the left and right wrist joints and the left and right claw joints of the quails in each group were measured with a soft tape measure. The measurement results on the 50th day of the experiment were used as the circumferences of the wrist and claw joints at the beginning of the experiment, and the swelling degrees of the wrist and claw joints of the quails in each group on the 60th, 70th and 80th days of the experiment were calculated according to formula (1), and the swelling inhibition rates of the wrist and claw joints of the quails in each group on the 60th, 70th and 80th days of the experiment were calculated according to formula (2).

[0171]

[0172]

[0173] 2.7 Collection and processing of joint lavage fluid

[0174] On day 83 of the experiment, 0.2 mL of normal saline was used to flush the left wrist joint cavity of quail in each group. The obtained wrist joint flushing fluid was applied on a glass slide, and uric acid crystals in the wrist joint flushing fluid were observed under a polarized light microscope.

[0175] 2.8 Pathological observation of joint tissue

[0176] The joint tissue fixed with 10% formalin was used to prepare paraffin sections, which were then stained with hematoxylin and eosin. Images were collected using an inverted microscope for histopathological observation.

[0177] 3. Data Analysis

[0178] GraphPad Prism 7.0 software was used for statistical analysis, and all data were expressed as mean ± standard deviation. The data among multiple groups were compared by one-way analysis of variance or Kruskal-Wallis test according to whether the groups were normal and had equal variances. The data among two groups were compared by Dunnett-t test or Dunnett's T3 test according to whether the variances were equal. P < 0.05 was considered statistically significant.

[0179] 4. Main instruments and equipment (see Table 6 below)

[0180] Table 6 Main instruments and equipment

[0181] name model factory induction cooker HY-221 Guangdong Hemisphere Industrial Group Corporation Rotary evaporator RE-501 Beijing Shentai Weiye Instrument Equipment Co., Ltd. Circulating water multi-purpose vacuum pump SHB-III Beijing Shentai Weiye Instrument Equipment Co., Ltd. Electronic balance TLE303E Mettler-Toledo Instruments (Shanghai) Co., Ltd. Benchtop centrifuge DT5-3 Beijing Shidai Beili Centrifuge Co., Ltd. Cryoembedding machine KH-BL Hubei Xiaogan Kuohai Medical Technology Co., Ltd. Paraffin tissue slicer Reichert Histo STAT AO Corporation microscope Olmpus BX53 Olympus Japan camera DP72CCD Olympus Japan Electric heating blast drying oven Type 101-1AB Tianjin Test Instrument Co., Ltd. ELISA reader sunrise Swiss TECAN Water bath HH-1 High-end Jintan Chengxi Zhengrong Experimental Instrument Factory

[0182] 5. Main reagents and drugs (see Table 7 below)

[0183] Table 7 Main reagents and drugs

[0184]

[0185]

[0186] 6. Results

[0187] 6.1 Effect of Chinese herbal medicine combination on uric acid crystallization in joints

[0188] A 0.375% sodium urate suspension was prepared as a positive control and applied to a glass slide. The positive control and each group of quail wrist joint washing fluid were placed under a 200x polarized light microscope for observation. Figure 2 .

[0189] In the black field of view, no needle-shaped urate crystals were found in the normal group. Compared with the normal group, bright, blue or yellow urate crystals were seen in the suspension of the positive control group, mostly in the form of crystal clusters aggregated into "spherical" shapes, with large needle-shaped crystals scattered around.

[0190] Compared with the normal group, the model group had more scattered, bright, blue or yellow urate crystals, which were shaped like small needles with a large head and a small tail or sharp ends. Occasionally, they were aggregated into "feather-like" crystal clusters, and the length of a single needle crystal was about 1 to 20 μm.

[0191] Compared with the model group, scattered needle-shaped urate crystals were rare in the high (10g / kg), medium (7.5g / kg), and low (3.75g / kg) dose groups of Juling, as well as a small number of needle-shaped crystal bundles arranged in parallel, or needle-shaped crystal bundles crossed and superimposed in an "X-shape". No crystal clusters were found.

[0192] 6.2 Joint fluid uric acid level

[0193] The quail joint cavity was flushed with 0.2 ml of normal saline, the ankle joint fluid of each group was collected, and the uric acid level of the joint fluid of each group was tested. Figure 3 Compared with the normal group, the uric acid level in the joint fluid of the quail in the model group was significantly increased (P<0.01).

[0194] Compared with the model group, the uric acid levels in the joint fluid of quails in the high (10 g / kg) and medium (7.5 g / kg) dose groups of Chrysanthemum Poria extract were significantly reduced (P<0.05).

[0195] 6.3 Joint swelling

[0196] 6.3.1 Swelling of left wrist joint

[0197] In this experiment, the left wrist joint swelling was calculated using formula (1). The results are shown in Table 8. Figure 4 Compared with the normal group, the swelling of the left wrist joint of the quail in the model group increased significantly at 60d, 70d, and 80d of the experiment (P<0.01).

[0198] Compared with the model group, on day 70 of the experiment, the swelling of the left wrist joint of quails in the high (10 g / kg), medium (7.5 g / kg), and low (3.75 g / kg) dose groups of Chrysanthemum Poria extract was significantly reduced (P<0.05 or P<0.01).

[0199] Table 8 Swelling degree of left wrist joint of quail in each group at each time point (%, n=12, )

[0200]

[0201] Note: Compared with the normal group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0202] 6.3.2 Swelling of right wrist joint

[0203] In this experiment, the right wrist joint swelling was calculated using formula (1). The results are shown in Table 9. Figure 5 Compared with the normal group, the swelling of the right wrist joint of the quail in the model group increased significantly at 60d, 70d, and 80d of the experiment (P<0.01).

[0204] Compared with the model group, on the 70th day of the experiment, the swelling degree of the right wrist joint of the quails in the medium dose (7.5g / kg) of Chrysanthemum and Poria extract group was significantly reduced (P<0.05), and the swelling degree of the right wrist joint of the quails in the high dose (10g / kg) and low dose (3.75g / kg) of Chrysanthemum and Poria extract groups showed a decreasing trend (P=0.059, P=0.052); on the 80th day of the experiment, the swelling degree of the right wrist joint of the quails in the high dose (10g / kg) of Chrysanthemum and Poria extract group showed a decreasing trend (P=0.092).

[0205] Table 9 Swelling degree of right wrist joint of quail in each group at different time points (%, n=12, )

[0206]

[0207] Note: Compared with the normal group, **P<0.01; compared with the model group, #P<0.05.

[0208] 6.3.3 Swelling of the left paw joint

[0209] In this experiment, the left paw joint swelling was calculated using formula (1). See Table 10. Figure 6 Compared with the normal group, the swelling degree of the left paw joints of the quails in the model group increased at 60 days (P=0.069); at 70 and 80 days, the swelling degree of the left paw joints of the quails in the model group increased significantly (P<0.05 or P<0.01).

[0210] Compared with the model group, on the 60th day of the experiment, the swelling degree of the left paw joints of the quails in the benzbromarone group and the low-dose (3.75 g / kg) group of Chrysanthemum extract showed a decreasing trend (P=0.076, P=0.064); on the 70th day of the experiment, the swelling degree of the left paw joints of the quails in the benzbromarone group and the medium-dose (7.5 g / kg) group of Chrysanthemum extract was significantly reduced (P<0.05); on the 80th day of the experiment, the swelling degree of the left paw joints of the quails in the benzbromarone group and the high-dose (10 g / kg), medium-dose (7.5 g / kg), and low-dose (3.75 g / kg) groups of Chrysanthemum extract was significantly reduced (P<0.05 or P<0.01).

[0211] Table 10 Swelling degree of left paw joints of quails in each group at different time points (%, n=12, )

[0212]

[0213] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0214] 6.3.4 Swelling of right paw joint

[0215] In this experiment, the right paw joint swelling was calculated using formula (1). The results are shown in Table 11. Figure 7 Compared with the normal group, the swelling degree of the right paw joint of the quail in the model group increased significantly on the 80th day of the experiment (P<0.01).

[0216] Compared with the model group, on day 80 of the experiment, the swelling of the right paw joints of quails in the benzbromarone group, high (10 g / kg), medium (7.5 g / kg), and low (3.75 g / kg) dose groups of Juling was significantly reduced (P<0.01).

[0217] Table 11 Swelling degree of right paw joints of quails in each group at different time points (%, n=12, )

[0218]

[0219]

[0220] Note: Compared with the normal group, **P<0.01; Compared with the model group, ## P<0.01.

[0221] 6.3.5 Left wrist joint swelling inhibition rate

[0222] In this experiment, the left wrist joint swelling inhibition rate was calculated using formula (2). The results are shown in Table 12. Figure 8On the 60th day of the experiment, the inhibition rate of swelling degree of the left wrist joint of quail in the medium dose group (7.5g / kg) of Chrysanthemum Poria extract was the highest at 65.77%, and the inhibition rate in the low dose group (3.75g / kg) and the high dose group (10g / kg) decreased successively, but they were all higher than the benzbromarone group.

[0223] On the 70th day of the experiment, the inhibition rate of swelling of the left wrist joint of quail in the medium dose (7.5g / kg) of Chrysanthemum Poria extract was the highest at 77.95%, and the high (10g / kg) and low (3.75g / kg) doses were comparable, but both were higher than the benzbromarone group.

[0224] On day 80 of the experiment, the inhibition rate of swelling of the left wrist joint of quails in the medium dose (7.5 g / kg) group of Chrysanthemum Poria extract was the highest at 24.61%, followed by the benzbromarone group, and the medium (7.5 g / kg) and low (3.75 g / kg) dose groups were weaker.

[0225] Table 12 Inhibition rate of left wrist joint swelling in quails of each drug administration group at each time point (%, n = 12)

[0226]

[0227] 6.3.6 Swelling inhibition rate of right wrist joint

[0228] In this experiment, the right wrist joint swelling inhibition rate was calculated using formula (2). The results are shown in Table 8. Fig. 9 On day 60 of the experiment, the inhibition rates of swelling degree of the right wrist joint of quails in the high (10 g / kg), medium (7.5 g / kg), and low (3.75 g / kg) dose groups of Chrysanthemum Poria extract were all higher than that in the benzbromarone group, and the highest inhibition rate of swelling degree of the left wrist joint of quails in the high (10 g / kg) dose group was 28.86%.

[0229] On day 70 of the experiment, the inhibition rates of swelling in the right wrist joints of quails in the high (10 g / kg), medium (7.5 g / kg), and low (3.75 g / kg) dose groups of Chrysanthemum Poria extract were all higher than those in the benzbromarone group. The highest inhibition rate of swelling in the left wrist joints of quails in the medium (7.5 g / kg) dose group was 57.87%.

[0230] On day 80 of the experiment, the inhibition rates of swelling in the right wrist joints of quails in the high (10 g / kg), medium (7.5 g / kg), and low (3.75 g / kg) dose groups of Chrysanthemum Poria extract were all higher than those in the benzbromarone group. The highest inhibition rate of swelling in the left wrist joints of quails in the high (10 g / kg) dose group was 28.40%.

[0231] Table 8 Inhibition rate of right wrist joint swelling at different time points in each group of quails (%, n = 12)

[0232]

[0233] 6.3.7 Inhibition rate of left paw joint swelling

[0234] In this experiment, the left paw joint swelling inhibition rate was calculated using formula (2). The results are shown in Table 9. Fig.10 On the 60th day of the experiment, the inhibition rate of left paw joint swelling of quail in the low-dose group (3.75 g / kg) of Chrysanthemum Poria extract was the highest at 36.73%, which was higher than that in the benzbromarone group.

[0235] On the 70th day of the experiment, the inhibition rate of left paw joint swelling in the quail group with the medium dose of Chrysanthemum Poria extract (7.5 g / kg) was the highest at 34.23%, which was higher than that in the benzbromarone group.

[0236] On the 80th day of the experiment, the inhibition rate of joint swelling of the left paw of quail in the Chrysanthemum Poria extract (7.5 g / kg) group was comparable to that in the benzbromarone group, which were 38.66% and 39.78% respectively.

[0237] Table 9 Inhibition rate of left paw joint swelling in each group of quails at each time point (%, n = 12)

[0238]

[0239] 6.3.8 Inhibition rate of right paw joint swelling

[0240] In this experiment, the right paw joint swelling inhibition rate was calculated using formula (2). The results are shown in Table 10. Fig.11 On day 60 of the experiment, the inhibition rate of swelling in the right paw joint of quail in the medium-dose group (7.5 g / kg) of Chrysanthemum Poria extract was the highest at 32.59%, and the high-dose group (10 g / kg) of Chrysanthemum Poria extract was comparable to the benzbromarone group, at 28.81% and 28.97% respectively. The low-dose group (3.75 g / kg) had the lowest swelling.

[0241] On day 70 of the experiment, the inhibition rate of swelling in the right paw joints of quails in the medium-dose group (7.5 g / kg) of Chrysanthemum and Poria extract was the highest at 32.82%, which was higher than that in the benzbromarone group. The high-dose group (10 g / kg) and the low-dose group (3.75 g / kg) of Chrysanthemum and Poria extract were comparable, at 25.52% and 27.63%, respectively.

[0242] On day 80 of the experiment, the results of the low-dose (3.75 g / kg) group of Chrysanthemum Poria extract were comparable to those of the benzbromarone group, which were 50.19% and 52.03% respectively, while the medium- and low-dose groups were inferior.

[0243] Table 10 Inhibition rate of right paw joint swelling in each group of quails at each time point (%, n = 12)

[0244]

[0245] 6.4 Changes in the appearance of wrist joints and paws

[0246] During the experiment, the wrist joints and paws of the quails in the normal group were normal in shape and flexible in movement. Compared with the normal group, the wrist joints of the quails in the model group showed varying degrees of swelling and local erythema; obvious swelling or several swollen nodules of varying sizes (gouty stones) were seen at the paw and toe joints. The swollen paws of some quails showed purple bruises, pus and ulcers. Fig.12 .

[0247] Compared with the model group, the wrist and paw joints of the quails in the high, medium and low dose groups of Chrysanthemum extract showed slight swelling. Fig.13 .

[0248] 6.5 Expression levels of inflammatory factors

[0249] Results Fig.14 Compared with the normal group, the serum IL-1β level of the quails in the model group was significantly increased on the 83rd day of the experiment; compared with the model group, the serum IL-1β level of the quails in the high, medium and low dose groups of Chrysanthemum jelly was significantly decreased on the 83rd day of the experiment (P<0.01).

[0250] 6.6 Pathological observation of joint tissue

[0251] Results Fig.15 . HE staining of quail wrist joint tissue showed that the wrist joint tissue structure of the quail in the normal group was clear, the joint surface was smooth and flat, and the synovial cells were loosely arranged and normal in shape; the wrist joint tissue of the model group showed synovial fibrous tissue hyperplasia, pannus formation, and a large number of inflammatory cell infiltration. Compared with the model group, the wrist joint tissue of the high-dose Juling group and the medium-dose Juling group showed significantly reduced inflammatory cell infiltration and reduced synovial fibrous tissue hyperplasia.

[0252] 6.7 Liver and kidney function indicators

[0253] 6.7.1 Serum AST and ALT activity

[0254] The results are shown in Table 11. Fig.16 Compared with the normal group, no significant difference was found in the serum AST and ALT activities of the quails in the model group on day 83 of the experiment.

[0255] Compared with the model group, on the 83rd day of the experiment, the serum AST activity of quails in the high-dose (10 g / kg) Chrysanthemum Poria extract group showed a downward trend but no significant difference (P=0.052). No significant differences were found in the serum AST and ALT activities of quails in the other drug-treated groups.

[0256] Table 11 Serum AST and ALT activities of quail in each group (U / L, n=12, )

[0257]

[0258] 6.7.2 Serum CRE and BUN Content

[0259] The results are shown in Table 12. Fig.17 Compared with the normal group, on the 83rd day of the experiment, there was no significant difference in the serum CRE content of the quails in the model group, and the serum BUN content was significantly increased (P<0.05). There was no significant difference in the serum CRE and BUN contents of the quails in the drug administration group.

[0260] Compared with the model group, on the 83rd day of the experiment, the serum CRE content of quails in the Juling high (10g / kg) dose group was significantly reduced (P<0.05), while there was no significant difference in the serum CRE content of quails in the other drug-treated groups; the serum BUN content of quails in the Juling high (10g / kg), medium (7.5g / kg), and low (3.75g / kg) dose groups was significantly reduced (P<0.01), and showed a dose-dependent manner, while there was no significant difference in the serum BUN content of quails in the benzbromarone group.

[0261] Table 12 Serum CRE and BUN levels in quail of each group (n=12, )

[0262]

[0263] Note: Compared with the normal group, *P<0.05; compared with the model group, # P<0.05, ## P<0.01.

[0264] 7. Results Analysis

[0265] This study explored the effect of Chinese herbal medicine composition on removing uric acid crystals from joints and fighting gouty arthritis. The results showed that the high, medium and low dose groups of Chinese herbal medicine composition could significantly reduce uric acid crystals in the joints of quails in the model group and reduce the level of uric acid in the joint fluid. At the same time, it significantly improved the swelling of the joints of quails with gouty arthritis and reduced the level of interleukin-1β in the serum, which clarified the efficacy of Chinese herbal medicine composition in removing uric acid crystals from joints and fighting gouty arthritis.

[0266] In terms of uric acid crystals in the joints, this experiment used polarized light microscopy to observe the wrist joint wash fluid of each group of quails, and found that needle-shaped, bright, blue-yellow joint uric acid crystals were visible in the wrist joint wash fluid of the model group quails. "Uric acid crystals in synovial fluid" is the "gold indicator" for the clinical diagnosis of gouty arthritis, which can effectively prove the successful creation of the gouty arthritis model. The results of this experiment showed that the uric acid crystals deposited in the wrist joint wash fluid of the quail in the model group were scattered needle-shaped or aggregated into feathers, and had the unique morphological and optical characteristics of uric acid crystals under polarized light, namely "needle-shaped, blue / yellow, and strong negative birefringence".

[0267] In terms of inflammation expression, this experiment used joint swelling as the main evaluation index and joint appearance as the auxiliary evaluation index. It was found that the local wrist and paw joints of the quail in the model group showed typical inflammation manifestations of "swelling and redness" during acute attacks of gouty arthritis. The results of joint swelling showed that compared with the normal group, the swelling of the left and right wrist joints of the quail in the model group at different time points was significantly increased. Observation of joint appearance showed that compared with the normal joints in the normal group, several swollen nodules of varying sizes were visible in the toe joints of the quail in the model group. In severe cases, ulceration and pus discharge could be seen at the swollen part; the wrist joints were also obviously swollen, and the skin at the swollen part was reddish purple.

[0268] This experiment evaluated the anti-gout efficacy of Chrysanthemum Poria extract from the aspects of wrist joint swelling, paw joint swelling and the shape of uric acid crystals deposited in joint washing fluid. First, in terms of anti-articular urate deposition: compared with the large scattered or "feather-like" urate crystal clusters in the wrist joint washing fluid of quail in the model group, the urate crystals present in the wrist joint washing fluid of quail in the high, medium and low dose groups of Chrysanthemum Poria extract were mostly scattered, single needle crystals, or two or three parallel arrangements or cross-overlapping "X-shaped" crystal bundles. Second, in terms of anti-articular urate deposition-induced inflammation: Chrysanthemum Poria extract can significantly reduce the swelling of wrist and paw joints at a dose of 3.75-10g / kg, indicating that Chrysanthemum Poria extract has a good anti-gouty arthritis effect.

[0269] In summary, this study concludes that the Chinese medicine composition of the present invention has a significant anti-gouty arthritis effect, and its efficacy may be related to the removal of uric acid crystals in the joints and the improvement of joint damage in model animals, providing a new drug option for the prevention and treatment of gouty arthritis.

[0270] Example 3 Chinese medicine compound composition and preparation method thereof

[0271] The traditional Chinese medicine composition of the present invention is prepared by water extraction of the following raw materials: 9g of chicory, 10g of tuckahoe, 30g of smilax glabra, 6g of sophora japonica seed and 9g of gentiana macrophylla.

[0272] Extraction method: S1, add 12 times the weight of deionized water according to the weight ratio, use the decoction method, extract for 1 hour, collect the extract, repeat 3 times, and combine the extracts;

[0273] S2, concentration: the obtained mixed liquid is concentrated under reduced pressure at -0.09 MPa and 80°C to a thick paste;

[0274] S3, preparing medicinal powder: drying the obtained thick paste under reduced pressure at -0.09 MPa and 80° C. to obtain a dry extract; taking the dry extract, crushing it, and passing it through a 60-mesh sieve to obtain a Chinese medicine composition.

[0275] Excipients: lactose; starch; dextrin; sorbitol; mannitol

[0276] The dry paste powder of the above-mentioned Chinese herbal combination extract is added with appropriate amounts of auxiliary materials and prepared into dosage forms such as granules, capsules or oral liquids by conventional methods.

[0277] Example 4 Chinese medicine compound composition and preparation method thereof

[0278] The traditional Chinese medicine composition of the present invention is prepared by water extraction of the following raw materials: 7g of chicory, 13g of tuckahoe, 20g of smilax glabra, 9g of sophora japonica seed and 5g of gentiana macrophylla.

[0279] The extraction method is the same as in Example 3

[0280] Excipients: lactose; starch; dextrin; sorbitol; mannitol

[0281] The dry paste powder of the above-mentioned Chinese herbal combination extract is added with appropriate amounts of auxiliary materials and prepared into dosage forms such as granules, capsules or oral liquids by conventional methods.

[0282] Example 5 Chinese medicine compound composition and preparation method thereof

[0283] The traditional Chinese medicine composition of the present invention is prepared by water extraction of the following raw materials: 15g of chicory, 6g of tuckahoe, 50g of smilax glabra, 5g of sophora japonica seed and 8g of gentiana macrophylla.

[0284] The extraction method is the same as in Example 3

[0285] Excipients: lactose; starch; dextrin; sorbitol; mannitol

[0286] The dry paste powder of the above-mentioned Chinese herbal combination extract is added with appropriate amounts of auxiliary materials and prepared into dosage forms such as granules, capsules or oral liquids by conventional methods.

Claims

1. Use of a Chinese medicine composition in the preparation of a drug for treating gouty arthritis, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials: 6-18 parts by weight of chicory, 5-15 parts by weight of tuckahoe, 10-60 parts by weight of smilax glabra, 5-10 parts by weight of sophora japonica seeds, and 3-10 parts by weight of gentiana macrophylla.

2. The use according to claim 1, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials: 6-10 parts by weight of chicory, 10-15 parts by weight of tuckahoe, 20-40 parts by weight of smilax glabra, 5-10 parts by weight of sophora japonica seeds, and 3-10 parts by weight of gentiana macrophylla.

3. The use according to claim 2, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials: 9 parts by weight of chicory, 10 parts by weight of Poria cocos, 30 parts by weight of Smilax glabra, 6 parts by weight of Sophora japonica seeds, and 9 parts by weight of Gentiana macrophylla.

4. The use according to any one of claims 1 to 3, characterized in that: The Chinese medicine composition is: a composition obtained by mixing raw materials after crushing them separately; or a composition obtained by mixing raw materials and crushing them; or an extract obtained by extracting the mixed raw materials according to a conventional extraction method, the extract is further subjected to a refining and purification process to obtain an effective part, and the above extract and effective part are further prepared according to a conventional preparation process to obtain a conventional oral dosage form.

5. The use according to claim 4, characterized in that: The conventional oral dosage forms are tablets, capsules, granules, pills, powders, and oral liquids.

6. The use according to any one of claims 1 to 3, characterized in that: The Chinese medicine composition is prepared according to the following method: S1, take each raw material drug in proportion and add water to extract; S2, concentrating the extract into a thick paste; S3, drying the thick paste under reduced pressure to obtain a dry extract; The mixture is crushed and sieved to obtain a Chinese medicine composition.

7. The use according to any one of claims 1 to 3, characterized in that: The Chinese medicine composition is used in preparing a medicine for removing uric acid crystals from joints.

8. The use according to any one of claims 1 to 3, characterized in that: The Chinese medicine composition is used in preparing a medicine for inhibiting inflammatory response of gouty arthritis.

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