Application of radix clematidis total saponins in preparation of medicine for treating acute gouty arthritis and improving uric acid renal injury

Abstract

The invention belongs to the technical field of biological medicines, and discloses an application of clematis total saponins in preparation of medicines for treating acute gouty arthritis and improving uric acid renal injury, which is characterized in that dried roots or rhizomes of clematis northeast of a clematis plant of ranunculaceae are crushed to extract the clematis total saponins; the extracted radix clematidis total saponins can reduce the uric acid level while relieving the symptoms of acute gouty arthritis, effectively relieve renal injury caused by high uric acid level, and have potential application in treating acute gouty arthritis and improving uric acid renal injury.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of total saponins of Clematis chinensis in preparing drugs for treating acute gouty arthritis and improving uric acid-induced kidney damage. Background Art

[0002] Uric acid is a weak organic acid that exists mainly in the form of monosodium urate under physiological conditions. It is the final oxidation product of human purine metabolism and is mainly produced in the liver, muscles and intestines. Uric acid is supersaturated in the blood or tissue fluid to form sodium urate crystals, which are precipitated in joints, synovium, renal tubules, etc. Crystals are formed and deposited locally in the joints, inducing local inflammatory reactions and tissue destruction, namely acute gouty arthritis. Deposition in the kidneys causes renal tissue damage and impaired function, and even kidney stones or chronic interstitial nephritis. After uric acid renal injury occurs, the body's uric acid excretion function further decreases, accelerating the progression of a variety of uric acid diseases. There is currently no expert guideline or consensus on uric acid renal injury, and clinical treatment is usually based on uric acid lowering and kidney disease treatment. Uric acid-lowering drugs include first-line drugs such as febuxostat and allopurinol. Although they can reduce uric acid levels to a certain extent, the recovery of kidney function remains to be seen. Therefore, it is still urgent to find new targeted therapeutic drugs.

[0003] Saponin compounds in Clematis chinensis are one of its main pharmacologically active ingredients. Although there are more than ten patents on total saponins of Clematis chinensis, there has been no report on its use in treating acute gouty arthritis and improving uric acid-induced kidney damage. Summary of the invention

[0004] The present invention aims at the technical problems existing in the above-mentioned prior art and provides the use of total saponins of Clematis chinensis in preparing a drug for treating acute gouty arthritis and improving uric acid-induced kidney damage.

[0005] The objective of the present invention is achieved through the following technical scheme: The present invention provides the use of total saponins of Clematis chinensis in the preparation of drugs for treating acute gouty arthritis and improving uric acid-induced kidney damage.

[0006] The application, the method for extracting total saponins of Clematis chinensis has the following specific steps:

[0007] (1) crushing the dried root or rhizome of Clematis mandshurica of the family Ranunculaceae, soaking it in 5 to 10 times the amount of 10% to 30% ethanol solution for 3 to 5 days, percolating it for 24 hours, ultrasonically extracting it for 30 minutes, or heating it under reflux at 70 to 100° C., and concentrating the extract under reduced pressure to obtain an extract without alcohol taste;

[0008] (2) dissolving the extract in water, filtering, and purifying by D101 macroporous resin column chromatography, eluting with water, 30% ethanol, 60% ethanol, and 95% ethanol in sequence until the effluent is colorless, collecting the 60% ethanol eluate, and recovering the solvent under reduced pressure to dryness to obtain a crude product of total saponins of Clematis chinensis;

[0009] (3) Dissolve the crude total saponins of Clematis chinensis in 5 to 10 times the amount of ethanol, filter, and recover the solvent from the filtrate under reduced pressure until dryness to obtain the total saponins of Clematis chinensis.

[0010] The total saponins of Clematis chinensis include 7 kinds of triterpenoid saponins of Clematis chinensis containing more than 6 sugars in the molecule, and the 7 kinds of triterpenoid saponins of Clematis chinensis are respectively: Clematichinenoside AR2, Clematomandshurica saponin F, Clematichinenoside AR, Clematomandshurica saponinC, Clematichinenoside C, Huzhangoside B, Clematichinenoside H, and the general structural formula of the 7 kinds of triterpenoid saponins of Clematis chinensis is shown in Formula I:

[0011]

[0012] When R1 is H, R2 is S2, R3 is S1, and R4 is OH, it is Clematichinenoside AR2.

[0013] When R1 is OH, R2 is S2, R3 is S1, and R4 is H, it is Clematomandshurica saponin F.

[0014] When R1 is H, R2 is S2, R3 is S1, and R4 is H, it is Clematichinenoside AR.

[0015] When R1 is H, R2 is S2, R3 is S3, and R4 is H, it is Clematomandshurica saponin C.

[0016] When R1 is H, R2 is S2, R3 is S4, and R4 is H, it is Clematichinenoside C.

[0017] When R1 is H, R2 is S2, R3 is H, and R4 is H, it is Huzhangoside B.

[0018] When R1 is H, R2 is H, R3 is S1, and R4 is H, it is Clematichinenoside H, wherein the structures of S1, S2, S3, and S4 are respectively:

[0019]

[0020] The total amount of the seven kinds of Clematis saponins is 93.02%.

[0021] The present invention has the following beneficial effects: the present invention establishes a rat model of uric acid renal injury combined with acute gouty arthritis induced by potassium oxonate, adenine, and monosodium urate crystals, and evaluates the therapeutic effect of total saponins of Clematis chinensis on acute gouty arthritis and uric acid renal injury. The degree of swelling of the rat paws, the arthritis index score, the gait score, and the synovial H&E staining results jointly confirm the occurrence of acute gouty arthritis, and show that total saponins of Clematis chinensis can adjust the increase of uric acid caused by the disease, effectively reduce the degree of swelling of the paws, improve gait, and alleviate the histopathological condition of the synovial membrane, thereby improving the symptoms of the disease. When the rats show a significant increase in uric acid levels, the results of renal tissue pathological sections and the detection of blood creatinine, urea nitrogen content in serum, and albumin level in 24h urine show that the kidneys of the experimental animals are also damaged, indicating that the animal model of uric acid renal injury combined with acute gouty arthritis is successfully established. The therapeutic effect of total saponins of Clematis chinensis was comprehensively evaluated by combining the above evaluation indicators. It was found that total saponins of Clematis chinensis can not only alleviate the symptoms of acute gouty arthritis, but also reduce uric acid levels and effectively relieve kidney damage caused by high uric acid levels. It has potential use in treating acute gouty arthritis and improving uric acid-induced kidney damage.

[0022] At the same time, it was confirmed that the NLRP3 / IL-1β signaling pathway and inflammatory response were involved in the renal function damage caused by high uric acid. The total saponins of Clematis chinensis extracted by the present invention not only reduced the uric acid level and improved renal function, but also effectively reduced the serum IL-1β level. It was revealed that the total saponins of Clematis chinensis inhibited the activation of NLRP3 inflammasomes and the secretion of IL-1β inflammatory factors, thereby reducing inflammatory infiltration and interstitial fibrosis, and played an improving role in uric acid nephropathy.

[0023] The invention uses Clematrs mandshusica, a plant of the genus Clematis in the family Ranunculaceae, for extraction to obtain total clematis saponins. The extract is mainly composed of 7 kinds of clematis triterpenoid saponins containing more than 6 sugars in the molecules. The total amount of the 7 kinds of clematis saponins in the extract is 93.02%, and the extract can significantly improve uric acid renal injury combined with acute gouty arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the serum uric acid concentration of rats in each group (***p<0.001 compared with the normal group, #p<0.05, ###p<0.001 compared with the model group);

[0025] Figure 2is the paw swelling rate of rats in each group (***p<0.001 compared with the normal group, ##p<0.01, ###p<0.001 compared with the model group);

[0026] Figure 3 is the rat joint inflammation score of rats in each group (***p<0.001 compared with the normal group, #p<0.05, ##p<0.01 compared with the model group);

[0027] Figure 4 is the gait score of rats in each group (***p<0.001 compared with the normal group, ##p<0.01 compared with the model group);

[0028] Figure 5 This is the H&E staining result of the rat ankle joint and synovial tissue (40x);

[0029] Figure 6 This is the result of H&E staining of rat kidney tissue (40x);

[0030] Figure 7 is the serum creatinine concentration of rats in each group (**p<0.01 compared with the normal group, #p<0.05 compared with the model group);

[0031] Figure 8 is the plasma urea nitrogen concentration of rats in each group (***p<0.001 compared with the normal group, ##p<0.01, #p<0.05 compared with the model group);

[0032] Fig. 9 is the 24-h urine protein concentration of rats in each group (**p<0.01 compared with the normal group, #p<0.05 compared with the model group);

[0033] Fig.10 is the serum IL-1β level of rats (**p<0.01 compared with the normal group, #p<0.05, ##p<0.01 compared with the model group). DETAILED DESCRIPTION

[0034] The present invention is further described and defined by the following examples but is not limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified. The present invention is further described below in conjunction with the accompanying drawings.

[0035] Extraction of total saponins from Clematis chinensis:

[0036] The dried root or rhizome of the plant Clematrs mandshusica of the genus Clematis of the Ranunculaceae family obtained from commercial channels is crushed, and extracted by cold soaking, percolation, ultrasonic or heating reflux with 5 to 10 times the amount of 10% to 30% ethanol solution, and the extract is concentrated under reduced pressure to obtain an extract without alcohol taste. The extract is dissolved in water, filtered, and purified by D101 macroporous resin column chromatography, and eluted with water, 30%, 60%, and 95% ethanol in sequence until the effluent is colorless. The 60% ethanol eluate is collected, and the solvent is recovered under reduced pressure to dryness to obtain a crude product of total saponins of Clematis chinensis. The crude product was dissolved with 5-10 times amount of ethanol or other organic solvent, filtered, and the filtrate was decompressed to recover the solvent to dryness, thus obtaining the total saponin extract of Clematis chinensis, which was mainly composed of 7 kinds of triterpenoid saponins of Clematis chinensis containing more than 6 sugars in the molecule, namely: Clematichinenoside AR2, Clematomandshurica saponin F, Clematichinenoside AR, Clematomandshurica saponinC, Clematichinenoside C, Huzhangoside B, Clematichinenoside H, and their structures were shown in the following formula and Table 1. The total amount of the 7 kinds of Clematis chinensis saponins in the extract was 93.02% as measured by HPLC.

[0037]

[0038] Table 1 Molecular formulas and structural formulas of seven Clematis saponins

[0039]

[0040] The following are the pharmacological experiments and results of the total saponins of Clematis chinensis according to the present invention.

[0041] 1. Experimental Methods

[0042] (1) Experimental animals and groups

[0043] 1.1 Experimental animals: SPF Wistar rats, male, 7-8 weeks old, weighing 170-180 g, were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. Rats were housed in separate cages in the clean animal experimental center of China Pharmaceutical University, with a temperature of 24±1℃, humidity of 50±10%, free access to water and food, and a 12-h light / dark cycle in the room. Rats were given normal growth-grade feed. Animal ethics and all experimental procedures were approved by the Animal Ethics Committee of China Pharmaceutical University.

[0044] 1.2 Grouping: After one week of adaptive feeding, the rats were randomly divided into a normal group (Control), a model group (Model), a Clematis chinensis total saponins intervention group (high-dose group TH 200 mg / kg / d; medium-dose group TM 100 mg / kg / d; low-dose group TL 50 mg / kg / d), a positive drug-allopurinol group (AP), and a positive drug-etoricoxib group (ET) according to their body weight, with 10 rats in each group.

[0045] (2) Reagent preparation

[0046] 2.1 Monosodium urate crystal solution: Add 5ml 1mol / L NaOH and 800mg sodium urate to 155ml pyrogen-free sterile water for injection and boil to dissolve the sodium urate completely. Cool naturally and stir. Add 1mol / L HCl dropwise to pH 7.0. When the solution turns milky white, centrifuge at 3000r / min for 2min. Centrifuge the supernatant repeatedly until crystals no longer precipitate. Finally, cool at 4℃ for 1h and centrifuge. Collect the crystals in a sterile EP tube, sterilize at 80℃, and store at 4℃. Before use, weigh 1g sodium urate, grind it thoroughly, and add 20mL sterile saline to make a 50g / L suspension.

[0047] 2.2 Clematis total saponin solution: Weigh an appropriate amount of Clematis total saponin powder (prepared by the above-mentioned Clematis total saponin extraction method), dissolve it in 0.9% sterile saline, and the prepared solution ensures that the dosage for rats is 200 mg / kg / d, 100 mg / kg / d and 50 mg / kg / d, respectively.

[0048] 2.3 Allopurinol solution: The contents of the allopurinol capsules were dissolved in 0.9% sterile saline to obtain a drug solution, and the prepared drug solution ensured that the dosage for rats was 25 mg / kg / d.

[0049] 2.4 Etoricoxib solution: 0.9% sterile saline was used to dissolve etoricoxib tablets to obtain a drug solution, and the prepared drug solution ensured that the dosage for rats was 6 mg / kg / d.

[0050] (3) Establishment of a rat model of uric acid-induced renal injury combined with acute gouty arthritis

[0051] The rats were given a mixture of adenine (0.1 g / kg / d) and potassium oxonate (1.5 g / kg / d) by oral gavage every day for 28 consecutive days to induce high uric acid levels in the rats. The control group was treated with normal saline by oral gavage in parallel.

[0052] On the 26th day of the experiment, rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (0.1 mL / 100 g). The posterior lateral side of the right ankle joint was selected as the puncture point. The needle mouth was inserted into the ankle joint cavity with the bevel facing forward and upward at a 45° angle to the tibia. 0.2 ml of MSU suspension was injected into the joint cavity, and the bulging of the contralateral joint capsule was used as the injection standard. Correspondingly, the control group was injected with an equal volume of sterile saline in parallel.

[0053] The drug was orally administered for 28 consecutive days starting from day 0 of the experiment. Each group was gavaged: the dose of the TH group was 200 mg / kg / d; the dose of the TM group was 100 mg / kg / d; the dose of the TL group was 50 mg / kg / d; the dose of the AP group was 25 mg / kg / d; and the dose of the ET group was 6 mg / kg / d. The control group and the model group were gavaged with an equal amount of normal saline. After the end of the drug administration, the animals were killed and samples were taken.

[0054] (4) Sample collection and processing

[0055] 4.1 Serum collection: After intraperitoneal injection of 20% urethane solution (1.5 ml / 200 g) for anesthesia, blood was collected from the abdominal aorta. The collected blood was allowed to stand for 30 min and then centrifuged (3000 rpm, 4°C, 20 min) to obtain serum, which was stored at -80°C for testing.

[0056] 4.2 Joint tissue collection: The right hind limb of the rat was cut off, and most of the tissue and calf bone were cut off, leaving 0.5 cm above and below the ankle joint, fixed in 4% paraformaldehyde fixative, and stained with H&E after decalcification.

[0057] 4.3 Collection of renal tissue: The abdominal cavity of the rat was opened and the renal tissue of the rat was completely separated. The left renal tissue was fixed in 4% paraformaldehyde fixative for H&E staining.

[0058] 4.4 Urine sample collection: At 8:00 a.m. on the 27th day of the experiment, the rats were placed in metabolic cages, urine was collected for 24 hours and the volume was measured. After mixing evenly, 2 mL was taken and stored at -80°C for testing.

[0059] (5) Determination and evaluation of indicators

[0060] 5.1 Measurement of paw swelling

[0061] Before injection of monosodium urate crystal solution (experimental day 26) and 24 hours after injection, the circumference of the ankle of each rat was measured by the winding method to calculate the degree of paw swelling. Paw swelling rate = (circumference 24 hours after injection - circumference before injection) / circumference before injection. The specific measurement method is: stretch the ankle to nearly 180°, wrap a wire under the root bone and record the circumference.

[0062] 5.2 Arthritis Index Score

[0063] The degree of joint lesions in rats was scored 24 hours after injection of monosodium urate crystal solution. The scoring criteria are as follows: 0 points, no redness or swelling; 1 point, slight redness or swelling of the paw with visible bone marks; 2 points, the bone marks disappeared, and obvious redness or swelling appeared, but limited to the ankle joint; 3 points, swelling of limbs other than the ankle joint, such as the sole of the foot and toes.

[0064] 5.3 Gait scoring

[0065] The gait of the rats was scored 24 hours after the injection of monosodium urate crystal solution. The scoring criteria are as follows: 0 points: normal walking; 1 point: slight lameness, with the lower limbs slightly bent; 2 points: moderate lameness, with the lower limbs just touching the ground; 3 points: severe lameness, with the lower limbs leaving the ground and walking on three feet.

[0066] 5.4 H&E staining of joints and kidney tissues

[0067] The removed joints and kidney tissues were fixed in 4% paraformaldehyde solution for 24 hours, and then the joints and decalcification solution were immersed in decalcification for one week and then embedded in paraffin; the kidney tissue was directly embedded in paraffin and sliced ​​after embedding. The slices were dewaxed in xylene, and then washed with anhydrous ethanol, 95% ethanol, 80% ethanol, 70% ethanol in sequence, and finally rinsed with distilled water. After that, they were stained in hematoxylin staining solution for 5-10 minutes, washed thoroughly with water for blueing, and then stained in eosin for 2 minutes, and then dehydrated in 80% ethanol, 95% ethanol, and anhydrous ethanol in sequence, and then immersed in xylene, and finally sealed with neutral gum. The slices were scanned and observed using a slice scanner.

[0068] 5.5ELISA to detect the content of IL-1β in serum

[0069] According to the instructions of the ELISA kit of Xinbosheng Biotechnology Co., Ltd., the IL-1β content in rat serum was detected: the required strips for the test were taken out from the sealed bag that had been equilibrated to room temperature, the blank wells were added with the universal diluent of standard and specimen, and the other corresponding wells were added with specimens or different concentrations of standard (100 μL / well), and incubated at 37°C in the dark for 90 minutes; the plate was washed 5 times; the blank wells were added with biotinylated antibody diluent, and the other wells were added with biotinylated antibody working solution (100 μL / well), and incubated at 37°C in the dark for 60 minutes; the plate was washed 5 times; the blank wells were added with enzyme conjugate diluent, and the other wells were added with enzyme conjugate working solution (100 μL / well), and incubated at 37°C in the dark for 30 minutes; the plate was washed 5 times; 100 μL / well of chromogenic substrate (TMB) was added, and the reaction stop solution was added, and the OD450 value was measured immediately after mixing. The IL-1β content in rat serum was calculated using the drawn standard curve.

[0070] 5.6 Colorimetric method for detection of uric acid, creatinine and urea nitrogen in rat serum

[0071] According to the kit instructions of Nanjing Jiancheng Bioengineering Institute, the content of uric acid, creatinine and urea nitrogen in rat serum was detected: a 96-well ELISA plate was taken, double distilled water was added to the blank wells, the given standard solution was added to the standard wells, and serum samples were added to the other corresponding wells. The reaction reagents were added in sequence according to the instructions, incubated at 37°C for 10 minutes, the color developer was added, and the OD value at the maximum absorption wavelength was measured immediately after mixing. The content of uric acid, creatinine and urea nitrogen in rat serum was calculated using the external standard one-point method.

[0072] 5.7 Colorimetric method for detecting protein content in rat 24h urine

[0073] According to the kit instructions of Nanjing Jiancheng Bioengineering Research Institute, the content of uric acid, creatinine and urea nitrogen in rat serum was detected: a 96-well ELISA plate was taken, double distilled water was added to the blank wells, a given 524 mg / L protein standard solution was added to the standard wells, and urine samples were added to the remaining corresponding wells, 50 μL each. 3 mL of CBB application solution was added, mixed thoroughly, allowed to stand for 5 minutes, and the OD595 nm value at the maximum absorption wavelength was measured. The protein content in rat urine for 24 hours was calculated using the external standard one-point method.

[0074] (6) Statistical analysis

[0075] Data were analyzed statistically using GraphPad Prism 9 software and expressed as mean ± SEM. The rat joint inflammation score and gait score data were analyzed using Kruskal-Wallis test, and the remaining data were analyzed using one-way ANOVA. p < 0.05 indicated a significant difference, and p < 0.01 indicated an extremely significant difference.

[0076] 2. Experimental results

[0077] (1) Effect of total saponins of Clematis chinensis on serum uric acid levels in rats

[0078] Compared with the normal group rats, the model group rats showed a significant increase in blood uric acid, and hyperuricemia occurred. In contrast, the serum uric acid levels of each drug-treated group decreased to varying degrees, and only the changes in the TH group and AP group showed significant differences. Figure 1 As shown. This indicates that total saponins from Clematis chinensis can reduce the serum uric acid level in rats with hyperuricemia.

[0079] (2) Effect of total saponins of Clematis chinensis on the swelling rate of rat paws

[0080] The paw swelling rate of rats in the model group was significantly higher than that in the normal group (p<0.001). The paw swelling rate of rats in each drug-treated group was reduced, and the TH group and ET group showed significant differences from the model group (p<0.01, p<0.001). Figure 2This indicates that total saponins from Clematis chinensis can effectively reduce the swelling of rat paws.

[0081] (3) Effects of total saponins from Clematis chinensis on joint inflammation scores in rats

[0082] The model group rats had obvious swelling and redness in the ankle joints, the bone marks disappeared, and the swelling spread to the soles and toes, which was significantly different from the normal group (p<0.001). The TH group and ET group rats mostly had slight swelling and redness, and some rats had disappearance of bone marks; the other three groups of rats had obvious swelling and redness, and some spread to other parts of the toes. The polynomial rank sum test analysis showed that the inflammatory index scores of the TH group and ET group were significantly different from those of the model group (p<0.05, p<0.01). The specific results are as follows Figure 3 As shown. This indicates that total saponins from Clematis chinensis play a role in alleviating the symptoms of acute gouty arthritis in rats.

[0083] (4) Effects of total saponins of Clematis chinensis on gait scores of rats

[0084] The gait of rats in the normal group was normal, while the gait of rats in the model group changed significantly, and most of them showed a tripod gait. The gait of rats in the TH group and ET group was significantly improved compared with that in the model group (p<0.05). Figure 4 This indicates that total saponins from Clematis chinensis play a role in alleviating the symptoms of acute gouty arthritis in rats and relieving pain.

[0085] (5) Effects of total saponins of Clematis chinensis on pathological changes of synovial tissue in rats

[0086] The synovium of the ankle joints of rats in the normal group was mildly edematous, and there was no obvious hyperplasia or necrosis of synovial cells. There was no obvious congestion or tissue hyperplasia in the underlying tissues, and only a small amount of inflammatory cell infiltration. The synovium of rats in the model group was severely thickened and moderately edematous, and cystic expansion, synovial cell degeneration, large-area tissue necrosis, accompanied by a large number of plasma cells and neutrophil infiltration, and MSU deposition was observed. Microscopically, the synovium in the TH group and the ET group was moderately thickened, mildly edematous, accompanied by neutrophil infiltration, and MSU deposition was observed. The overall situation was improved compared with the model group. The degree of synovial thickening, congestion, and edema in rats in the AP group, TM group, and TL group was only slightly weaker than that in the model group. Cystic expansion, synovial cell degeneration, large-area tissue necrosis, accompanied by a large number of plasma cells and neutrophil infiltration, and MSU deposition was observed. Compared with the model group, the improvement was not obvious. The specific results are as follows Figure 5 The above results show that total saponins of Clematis chinensis can effectively reduce the degree of synovial tissue lesions and improve the symptoms of acute gouty arthritis after treatment.

[0087] (6) Effects of total saponins of Clematis chinensis on pathological changes in rat renal tissue

[0088] The cortical-medullary boundary of the renal tissue of the rats in the normal group was relatively clear, the distribution of glomeruli was normal, no obvious lesions were found in the glomeruli, and the arrangement of renal tubules was normal. The rats in the model group showed obvious dilation of the renal tubular lumen, flattened epithelium, a small amount of inflammatory necrotic material was seen in the lumen, and yellow-brown urate particles were deposited in the renal tubular lumen and interstitium. Small foci of renal tubular necrosis, chronic inflammatory cell infiltration and fibrous tissue hyperplasia were seen in the interstitium. The renal tubular lumen of rats in the AP group was partially dilated, and local epithelial cell degeneration was observed. A small amount of inflammatory necrotic material was seen in some lumens, and chronic inflammatory cell infiltration was seen in the interstitium. No clear urate particle deposition was observed. The glomeruli and surrounding renal tubules of rats in the TH group were partially congested. The renal tubular lumen was partially dilated, inflammatory necrotic material was seen in some lumens, and a small amount of chronic inflammatory cell infiltration was seen in the interstitium. The renal tissues of rats in the ET, TM, and TL groups also showed dilation of the renal tubular lumen and flattened epithelium. There were inflammatory necrosis in the lumen, yellow-brown urate granules deposited in the lumen and interstitium of the renal tubules, chronic inflammatory cell infiltration and fibrosis, which were no significant differences from the model group.

[0089] In general, the overall condition of renal tissue sections in the TH group and AP group was improved compared with that in the model group. Figure 6 This indicates that total saponins of Clematis chinensis can effectively improve the renal tissue pathology of rats with uric acid kidney damage after treatment.

[0090] (7) Effects of total saponins of Clematis chinensis on serum creatinine, blood urea nitrogen, and 24-hour urine protein levels in rats

[0091] Creatinine and urea nitrogen are commonly used indicators for evaluating renal function. When renal function is impaired, the concentrations of the two in the blood increase. Kidney damage affects the glomerular filtration function and increases the protein level in the urine.

[0092] The levels of serum creatinine, blood urea nitrogen, and 24-hour urine protein in the model group were significantly higher than those in the normal group, indicating that high uric acid levels caused kidney damage. The levels of serum creatinine, blood urea nitrogen, and 24-hour urine protein in the TH group were significantly lower than those in the model group. Figure 7 , 8 , 9. This indicates that total saponins from Clematis chinensis improved renal damage caused by high uric acid levels in rats.

[0093] (8) Effect of total saponins of Clematis chinensis on serum IL-1β levels in rats

[0094] NLRP3 inflammasome and IL-1β are important inflammatory triggers during gout attacks. Mature IL-1β is considered to be the most important inflammatory mediator in acute gouty arthritis and the initiator of inflammation.

[0095] Compared with the normal group, the serum IL-1β of the rats in the model group was significantly upregulated. Compared with the model group, the serum IL-1β of the rats in each drug-treated group decreased to varying degrees, and the differences among the TH group, AP group, and ET group were significant. Fig.10 This indicates that total saponins from Clematis chinensis play a significant anti-inflammatory role in disease progression.

[0096] In summary, the present invention successfully established a reliable animal model of uric acid renal injury combined with acute gouty arthritis, and found that total saponins of Clematis chinensis can effectively alleviate the symptoms of acute gouty arthritis while also effectively alleviating renal damage caused by high uric acid levels, and has potential uses in the treatment of acute gouty arthritis and uric acid renal injury.

[0097] The above description is only a preferred embodiment of the present invention, but is not intended to limit the present invention. Those skilled in the art may make possible changes and modifications to the present invention using the technical contents disclosed above without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. Application of total saponins of Clematis chinensis in the preparation of drugs for acute gouty arthritis and improving uric acid-induced kidney damage.

2. The use according to claim 1, characterized in that: The specific steps of the method for extracting total saponins of Clematis chinensis are as follows: (1) crushing the dried root or rhizome of Clematis mandshurica of the family Ranunculaceae, cold soaking in 5 to 10 times the amount of 10% to 30% ethanol solution for 3 to 5 days, percolating for 24 hours, ultrasonically extracting for 30 minutes, or heating and refluxing at 70 to 100° C. for two times, and concentrating the extract under reduced pressure to obtain an extract without alcohol taste; (2) dissolving the extract in water, filtering, and purifying by D101 macroporous resin column chromatography, eluting with water, 30% ethanol, 60% ethanol, and 95% ethanol in sequence until the effluent is colorless, collecting the 60% ethanol eluate, and recovering the solvent under reduced pressure to dryness to obtain a crude product of total saponins of Clematis chinensis; (3) Dissolve the crude total saponins of Clematis chinensis in 5 to 10 times the amount of ethanol, filter, and recover the solvent from the filtrate under reduced pressure until dryness to obtain the total saponins of Clematis chinensis.

3. The use according to claim 2, characterized in that: The total saponins of Clematis chinensis include 7 kinds of triterpenoid saponins of Clematis chinensis containing more than 6 sugars in the molecule, and the 7 kinds of triterpenoid saponins of Clematis chinensis are respectively: Clematichinenoside AR2, Clematomandshurica saponin F, Clematichinenoside AR, ClematomandshuricasaponinC, Clematichinenoside C, Huzhangoside B, Clematichinenoside H, and the general structural formula of the 7 kinds of triterpenoid saponins of Clematis chinensis is shown in Formula I: When R1 is H, R2 is S2, R3 is S1, and R4 is OH, it is Clematichinenoside AR2. When R1 is OH, R2 is S2, R3 is S1, and R4 is H, it is Clematomandshurica saponin F. When R1 is H, R2 is S2, R3 is S1, and R4 is H, it is Clematichinenoside AR. When R1 is H, R2 is S2, R3 is S3, and R4 is H, it is Clematomandshurica saponin C. When R1 is H, R2 is S2, R3 is S4, and R4 is H, it is Clematichinenoside C. When R1 is H, R2 is S2, R3 is H, and R4 is H, it is Huzhangoside B. When R1 is H, R2 is H, R3 is S1, and R4 is H, it is Clematichinenoside H. Among them, the structures of S1, S2, S3, and S4 are respectively:

4. The use according to claim 3, characterized in that: The total amount of the seven kinds of Clematis saponins is 93.02%.

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