Application of 1-O-sinapyl-beta-glucose in preparation of medicine for treating hyperuricemia or improving kidney injury caused by hyperuricemia

Abstract

The invention belongs to the technical field of natural medicines, and particularly relates to application of 1-O-sinapyl-beta-glucose in preparation of a medicine for treating hyperuricemia or improving kidney injury caused by hyperuricemia. It is found for the first time that 1-O-sinapyl-beta-glucose can significantly reduce serum uric acid for a mouse with hyperuricemia, improve renal function impairment and renal structure injury caused by hyperuricemia and promote excretion of renal uric acid. Therefore, the 1-O-sinapyl-beta-glucose can be used for preparing the medicine for treating the hyperuricemia and the medicine for treating the kidney injury caused by the hyperuricemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural medicines, and in particular relates to the use of 1-O-sinapoyl-β-glucose in the preparation of medicaments for treating hyperuricemia or improving renal injury caused by hyperuricemia. Background Art

[0002] Hyperuricemia (HUA) is a chronic metabolic disease mainly caused by abnormal purine metabolism, characterized by an elevated level of serum uric acid (UA). Hyperuricemia is the main cause of gout and can also damage the kidneys through multiple mechanisms, including urate deposition, endothelial dysfunction, RAS activation, inflammatory response, metabolic disorders, and tubular dysfunction, etc.

[0003] Medicaments for treating hyperuricemia can be classified into the following categories according to their mechanisms of action: drugs that inhibit uric acid production (such as allopurinol, febuxostat), drugs that promote uric acid excretion (such as benzbromarone, probenecid), drugs that promote uric acid decomposition (such as pegloticase, rasburicase), and adjuvant therapeutic drugs (such as sodium bicarbonate, potassium citrate), etc. In clinical medication, it is usually necessary to make an individualized selection of drugs according to the patient's uric acid metabolism type (excessive production or reduced excretion), renal function status, and comorbidities. Therefore, it is necessary to continue to explore drugs that can be used to treat hyperuricemia and relieve the symptoms of patients, so as to provide more choices for clinical medication of different patients. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides the use of 1-O-sinapoyl-β-glucose in the preparation of medicaments for treating hyperuricemia or improving renal injury caused by hyperuricemia. The present invention discovers for the first time the effects of 1-O-sinapoyl-β-glucose in reducing serum uric acid, improving renal function injury and renal structure injury caused by hyperuricemia, and promoting renal uric acid excretion. Therefore, it can be used to prepare medicaments for treating hyperuricemia and medicaments for renal injury caused by hyperuricemia.

[0005] To achieve the above invention object, the present invention adopts the following technical scheme: The first aspect of the present invention provides the use of 1-O-sinapoyl-β-glucose in the preparation of medicaments for treating hyperuricemia, and the active ingredient of the medicaments includes 1-O-sinapoyl-β-glucose.

[0006] 1-O-sinapoyl-β-glucose is a sinapic acid ester compound mainly present in cruciferous plants such as Arabidopsis thaliana and Brassica campestris. In existing research, there are few reports on the physiological activity of 1-O-sinapoyl-β-glucose on the body, especially no research on reducing the serum uric acid level of the body with 1-O-sinapoyl-β-glucose has been found. Although there are studies on the effect of sinapic acid on uric acid levels, the research results show that sinapic acid will increase the uric acid level in obese mice, so it is impossible to speculate that 1-O-sinapoyl-β-glucose can reduce uric acid.

[0007] Surprisingly, through research, the present invention finds that 1-O-sinapoyl-β-glucose can significantly reduce the serum uric acid in hyperuricemic mice, improve the renal function damage and renal structure damage caused by hyperuricemia, and promote the excretion of uric acid by the kidneys. For hyperuricemic patients, reducing the serum uric acid content, improving renal structure damage and promoting the excretion of uric acid by the kidneys are of great significance: by reducing the serum uric acid level, the risk of acute gout attacks can be effectively reduced, the progression of renal function damage can be delayed, and the incidence of chronic kidney disease and urinary tract stones can be reduced; by improving the renal structure damage in hyperuricemic patients, renal fibrosis and apoptosis can be significantly reduced, renal function can be improved, and the progression of chronic kidney disease can be delayed; promoting the excretion of uric acid by the kidneys can directly reduce the serum uric acid content and reduce the degree of hyperuricemia. Therefore, 1-O-sinapoyl-β-glucose can be used to prepare drugs for treating hyperuricemia.

[0008] Preferably, the drug is a drug for promoting uric acid excretion.

[0009] Preferably, the active ingredient of the drug further includes other ingredients that can inhibit uric acid production, promote uric acid excretion, promote uric acid decomposition and / or alkalize urine. The above active ingredients can be combined according to the clinical medication requirements.

[0010] Preferably, the dosage form of the drug is an oral preparation.

[0011] Optionally, the oral preparation includes but is not limited to oral liquid, powder, granule, capsule, tablet, etc.

[0012] Preferably, the drug further contains pharmaceutically acceptable excipients for preparing oral preparations. The active ingredient and the excipients can be made into corresponding products according to the conventional preparation methods of each oral preparation, and the present invention does not limit the types of excipients and the preparation methods.

[0013] The second aspect of the present invention provides the use of 1-O-sinapoyl-β-glucose in the preparation of a drug for improving renal damage caused by hyperuricemia, and the active ingredient of the drug includes 1-O-sinapoyl-β-glucose.

[0014] Preferably, the renal damage is renal function damage.

[0015] Preferably, the kidney injury is kidney structural injury.

[0016] Preferably, the active ingredient of the drug further includes other ingredients that can improve kidney injury.

[0017] Preferably, the dosage form of the drug is an oral preparation.

[0018] Optionally, the oral preparation includes but is not limited to oral liquid, powder, granule, capsule, tablet, etc.

[0019] Preferably, the drug further contains pharmaceutically acceptable excipients for preparing oral preparations. The active ingredient and the excipients can be made into corresponding products according to the conventional preparation methods of each oral preparation, and the types of excipients and preparation methods are not limited in the present invention.

[0020] The beneficial effects of the present invention are as follows: The present invention discovers for the first time that 1-O-sinapoyl-β-glucose has the effects of reducing serum uric acid, improving renal function injury and kidney structural injury caused by hyperuricemia, and promoting the excretion of uric acid in the kidney, and can be used to prepare drugs for treating hyperuricemia or drugs for kidney injury caused by hyperuricemia. The present invention provides a new active ingredient for the treatment of hyperuricemia and the recovery of kidney injury caused by hyperuricemia, and provides a new choice for the individualized clinical medication of hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It shows the effects of 1-O-sinapoyl-β-glucose on the body weight, kidney coefficient and serum uric acid of HUA mice in Example 1 of the present invention; Figure A shows the change in body weight during drug administration, Figure B shows the kidney coefficient, and Figure C shows the blood uric acid concentration of mice; # Indicates compared with the normal group, p <0.05; ### Indicates compared with the normal group, p <0.001; ** Indicates compared with the Mod group, p <0.01; Figure 2 It shows the effects of 1-O-sinapoyl-β-glucose on the kidney function of HUA mice in Example 1 of the present invention; Figure A shows the blood creatinine content of mice; Figure B shows the serum urea nitrogen content of mice; Figure C shows the creatinine clearance rate of mice; Figure D shows the HE staining results of the kidneys and livers of mice; ### Indicates compared with the normal group, p <0.001; * Indicates compared with the Mod group, p <0.05; ** Indicates compared with the Mod group, p <0.01; Figure 3Effect of 1-O-sinapoyl-β-glucose on uric acid excretion in the kidneys of HUA mice in Example 1 of the present invention; Panel A shows the bands of uric acid transporters in the mouse kidneys; Panels B-E show the gray values of the bands of uric acid transporters in the mouse kidneys. # Indicates compared with the normal group, p <0.05; ### Indicates compared with the normal group, p <0.001; * Indicates compared with the Mod group, p <0.05; ** Indicates compared with the Mod group, p <0.01. Detailed implementation manners

[0022] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0023] Hyperuricemia is characterized by an elevated serum uric acid level, which not only causes gout but also damages the kidneys through various mechanisms. The clinical treatment of hyperuricemia usually needs to be individualized according to the specific conditions of the patients. Therefore, it is necessary to develop more new active substances that can be used to treat hyperuricemia and relieve the symptoms of patients to provide more suitable choices for clinical medication for different patients.

[0024] The present invention first discovers that 1-O-sinapoyl-β-glucose can significantly reduce the serum uric acid in hyperuricemic mice, improve the renal function injury and renal structure injury caused by hyperuricemia, and promote the excretion of uric acid in the kidneys. Based on the above research findings, the embodiments of the present invention provide the application of 1-O-sinapoyl-β-glucose in the preparation of drugs for treating hyperuricemia.

[0025] The embodiments of the present invention also provide the application of 1-O-sinapoyl-β-glucose in the preparation of drugs for improving renal injury caused by hyperuricemia.

[0026] The following will illustrate the solutions of the present invention through specific embodiments.

[0027] Unless otherwise specified, the reagents and drugs used in the following embodiments are all obtained from commercial channels. Except for the content specifically described, the experiments involved in the following embodiments all adopt the conventional experimental methods in the art.

[0028] Example 1 This example investigated the effects of 1-O-sinapoyl-β-glucose on serum uric acid and renal tissue in hyperuricemic mice.

[0029] 1. Experimental Materials and Methods 1) Experimental Animals SPF-grade male C57BL / 6J mice, 8 weeks old, weighing 20 - 25 g.

[0030] 2) Experimental Grouping and Drug Administration Male C57BL / 6J mice were randomly divided into 4 groups: normal group (Normal), model group (Mod), positive drug benzbromarone 50 mg / kg group (Ben-50), and 1-O-sinapoyl-β-glucose 50 mg / kg group (SG-50), with 10 mice in each group. Benzbromarone and 1-O-sinapoyl-β-glucose were dissolved in ultrapure water according to the dosing dose and administered by gavage. Modeling drugs: potassium oxonate (150 mg / kg) and adenine (50 mg / kg) were dissolved in ultrapure water to make a suspension solution, and the volume of 20 mL / kg was administered by gavage, freshly prepared before use. Drugs were administered at 9:00 - 11:00 am every day. Except for the normal group and the model group which were given ultrapure water, the other two groups were given the corresponding drugs. One hour later, except for the normal group which was given ultrapure water, the other three groups were given the modeling drugs. The body weights of the mice were weighed before drug administration every day. The above animal experiment operations continued for 21 days.

[0031] 3) Collection of Blood Samples, Urine, and Kidney and Liver Tissues On the 20th day of drug administration, immediately after 1 h of gavage administration of the modeling drug, the mice were placed in a metabolic cage, fasted but not water-restricted, and 24 h urine was collected. After taking them out of the metabolic cage, drugs were administered again in the same way as above. One hour after the last drug administration of each group of mice, according to the dosing order, blood samples were collected by eye socket blood collection. After standing at room temperature for 2 h, they were centrifuged at 3500 g for 20 min, and the supernatant was taken and stored at -80 °C for later use. After eye socket blood collection, the mice were sacrificed according to the dosing order, and the kidneys and livers were taken for subsequent research.

[0032] 4) Processing of Plasma Samples and Detection of Uric Acid Concentration The uric acid concentration in plasma was detected by UPLC method.

[0033] Plasma treatment method: Take 10 μL of plasma and add 9 times the volume (90 μL) of 0.3 mol / L HClO4. Vortex thoroughly and mix evenly, then place in an ice bath for 30 min. Centrifuge at 12000 g for 10 min at 4℃; Take 200 μL of the supernatant and add 40 μL of 0.8 mol / L Na2HPO4·12 H2O. Mix thoroughly and centrifuge at 12000 g for 10 min at 4℃; Pipette 20 μL of the supernatant and add 180 μL of acetonitrile. After mixing, centrifuge at 14000 g for 10 min. Take 100 μL of the supernatant and transfer it to an injection vial for testing.

[0034] The UPLC chromatographic conditions are as follows: Chromatographic column: ACQUITY UPLC BEH Amide (1.7 mm, 2.1×50 mm); Mobile phase: 0.1% acetic acid aqueous solution / acetonitrile = 10 / 90, v / v; Flow rate: 0.3 mL / min; Column temperature: 30℃; Detection wavelength: 285 nm; Injection volume: 10 μL.

[0035] 5) Determination of creatinine and urea nitrogen levels Respectively, measure the levels of serum creatinine (SCr) and serum urea nitrogen (BUN) according to the kit instructions.

[0036] 6) HE staining of mouse kidneys and livers Fix mouse kidney and liver tissues in formalin (10% paraformaldehyde solution). After trimming, place them in an embedding cassette, dehydrate with gradient ethanol, make them transparent in xylene, then infiltrate with wax and embed. Cut into 5 μm thin sections, flatten and dry them in hot water. After dewaxing, perform eosin staining. After staining, dehydrate with absolute alcohol and make them transparent in xylene. Finally, mount the slides and observe the pathological morphology of kidney tissues under a light microscope.

[0037] 7) Detection of the expression levels of proteins GLUT9, URAT1, OAT1, and ABCG2 in mouse kidney tissues by Western-blot method Weigh approximately 30 mg of kidney tissue into an EP tube, add 300 μL of RIPA lysis buffer (containing 1 mM PMSF and phosphatase inhibitors), use a homogenizer to fully disrupt the tissue, let it stand on ice for 30 min to fully lyse the tissue, centrifuge at 12000 g and 4 °C for 10 min, and take the supernatant. Quantify the total protein to 80 μg using the BCA protein quantification method, adjust the protein concentration to be consistent with physiological saline, add 4× protein loading buffer at a volume ratio of 3:1, vortex and heat in a metal bath at 100 °C for 10 min. After the protein is fully denatured, store it at -20 °C. Use SDS-polyacrylamide gel (SDS-PAGE) electrophoresis to separate the proteins and transfer them to a PVDF membrane. Cut out the target bands, incubate with the corresponding primary antibody overnight at 4 °C. The next day, wash the bands with PBST for 10 min × 3 times, then incubate with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. After the secondary antibody incubation, wash with PBST again for 10 min × 3 times. Prepare the chemiluminescence solution, immerse the bands in the developing solution, let it stand for dozens of seconds, and then use the ChemiDoc MP Imaging System to image the protein bands, and use the Image J software to perform band gray scale quantification. Use β-actin as the internal reference protein and repeat the experiment three times.

[0038] 8) Statistical analysis The experimental data are expressed as Mean ± S.E.M., and the Graph Pad Prism Version (8.1.1) statistical software is used to evaluate the significant differences between the means through T-test and one-way analysis of variance (One-way ANOVA), and set p< 0.05 as statistically significant.

[0039] 2. Experimental results 1) Effects of 1-O-sinapoyl-β-glucose on body weight, kidney index and serum uric acid in hyperuricemia (HUA) mice During the experiment, the mice in the normal group had smooth and shiny fur, good mental state, active and agile movements, and their body weight continued to increase. The mice in the Mod group had dull fur, slow movements, and their body weight decreased rapidly. The mice in the drug administration groups (Ben-50 group and SG-50 group) were in good condition, and their body weight decreased more slowly than the Mod group. Compared with the normal group, the kidney index of the mice in the Mod group was significantly increased, and the serum uric acid content of the mice was significantly increased. Compared with the Mod group, after treatment with 1-O-sinapoyl-β-glucose, the kidney index and serum uric acid content of the mice were significantly decreased. As Figure 1 shown.

[0040] 2) Effects of 1-O-sinapoyl-β-glucose on kidney and liver functions in HUA mice Compared with the normal group, the serum creatinine and urea nitrogen levels in the Mod group were significantly increased. After treatment with 1-O-sinapoyl-β-glucose compared with Mod, the serum creatinine and urea nitrogen levels in mice were significantly decreased. Compared with the normal group, the creatinine clearance rate in the Mod group of mice was significantly decreased; compared with the Mod group, the creatinine clearance rates in the benzbromarone 50 mg / kg group and the 1-O-sinapoyl-β-glucose group of mice were significantly increased, indicating that 1-O-sinapoyl-β-glucose could significantly improve the impaired renal function in mice caused by hyperuricemia. Compared with the normal group, there were phenomena such as dilatation of the renal tubular lumen, inflammatory infiltration, and necrosis of renal tubular epithelial cells in the kidney tissues of the Mod group of mice. Compared with the Mod group, the dilatation of the renal tubular lumen in the kidneys of the benzbromarone 50 mg / kg group of mice was improved, and the marginal boundary of the renal tubules was relatively complete and clear; after treatment with 1-O-sinapoyl-β-glucose, the dilatation of the renal tubular lumen was significantly improved, the inflammatory infiltration and the necrosis and exfoliation of renal tubular epithelial cells were reduced, and the marginal boundary of the renal tubules was clear and complete. The results revealed that the treatment with 1-O-sinapoyl-β-glucose could significantly improve the renal structural damage in HUA mice.

[0041] Compared with the normal group, no obvious pathological changes were observed in the hepatocytes in the liver tissues of the Mod group of mice, and the morphology, nuclear structure, and arrangement of the hepatocytes remained normal, indicating that HUA mice did not cause liver damage.

[0042] As Figure 2 shown.

[0043] 3) Effects of 1-O-sinapoyl-β-glucose on renal uric acid excretion in HUA mice Compared with the normal group, the expression levels of the renal uric acid reabsorption transporters GLUT9 and URAT1 in the Mod group of mice were up-regulated, and the expression levels of the uric acid secretion proteins OAT1 and ABCG2 were down-regulated, proving that the renal uric acid reabsorption increased, the secretion decreased, and the uric acid excretion was blocked. Compared with the Mod group, the expression levels of the renal uric acid reabsorption transporters GLUT9 and URAT1 in the 1-O-sinapoyl-β-glucose group of mice were significantly down-regulated, and the expression levels of the uric acid secretion proteins OAT1 and ABCG2 were significantly up-regulated. The results showed that 1-O-sinapoyl-β-glucose could promote renal uric acid excretion by regulating the protein expression levels of uric acid-related transporters (down-regulating GLUT9 and URAT1, up-regulating OAT1 and ABCG2). As Figure 3 shown.

[0044] Example 2 This example provides an oral liquid for treating hyperuricemia and / or improving renal injury caused by hyperuricemia, and its preparation method is as follows: Dissolve 4000 g of sucrose in 700 mL of purified water, stir until completely dissolved to obtain syrup. Premix 50 g of 1-O-sinapoyl-β-glucose with 150 mL of glycerol and 5 g of sodium carboxymethylcellulose, circulate and grind through a colloid mill until the particle size is ≤50 μm, add it to the above syrup, mix evenly, then add 1 g of sodium benzoate and 3 g of citric acid, stir for 30 minutes, add purified water to 100 mL, mix evenly, sterilize through a 0.45 μm filter membrane, and fill and seal in a brown glass bottle to obtain the product.

[0045] Example 3 This example provides a tablet for treating hyperuricemia and / or improving renal injury caused by hyperuricemia, and its preparation method is as follows: Mix 500 g of 1-O-sinapoyl-β-glucose, 1.2 kg of microcrystalline cellulose, 600 g of lactose, and 100 g of corn starch, add 50 g of sodium carboxymethyl starch, mix again, then add 20 g of colloidal silicon dioxide and 30 g of magnesium stearate, and directly press the tablets after mixing for 2 minutes. The obtained plain tablets can be film-coated as needed.

[0046] Example 4 This example provides a granule for treating hyperuricemia and / or improving renal injury caused by hyperuricemia, and its preparation method is as follows: Mix 600 g of 1-O-sinapoyl-β-glucose, 1.2 kg of dextrin, 900 g of lactose, and 200 g of mannitol, add 100 g of polyvinylpyrrolidone K30 ethanol solution (30% concentration) as a wetting agent, granulate with a swing granulator through a 16-mesh sieve, dry at 40 - 45°C until the moisture content is ≤2.0%, screen with a 14-mesh sieve, add 30 g of magnesium stearate and mix for 5 minutes, and then package into aluminum-plastic composite film bags to obtain the product.

[0047] Example 5 This example provides a hard capsule for treating hyperuricemia and / or improving renal injury caused by hyperuricemia, and its preparation method is as follows: Mix 750 g of 1-O-sinapoyl-β-glucose with 3 kg of pregelatinized starch and 1 kg of microcrystalline cellulose, add 150 g of cross-linked carboxymethylcellulose sodium, mix again, then add 50 g of colloidal silicon dioxide and 50 g of magnesium stearate, and fill into gelatin capsule shells with an automatic capsule filling machine after mixing for 2 minutes, and then package to obtain the product.

[0048] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of 1-O-sinapoyl-β-glucose in the preparation of a medicament for treating hyperuricemia, characterized in that, The active ingredient of the drug includes 1-O-sinapoyl-β-glucose.

2. The application according to claim 1, wherein The drug is a drug for promoting uric acid excretion.

3. The application according to claim 1, characterized in that, The active ingredient of the drug further includes other ingredients that can inhibit uric acid production, promote uric acid excretion, promote uric acid decomposition, and / or alkalize urine.

4. The application according to any one of claims 1 to 3, characterized in that The dosage form of the drug is an oral preparation. 5.1 - O - sinapoyl - β - glucoside in the preparation of drugs for improving renal injury caused by hyperuricemia, characterized in that, The active ingredient of the drug includes 1-O-sinapoyl-β-glucose.

6. The application according to claim 5, wherein The renal injury is renal function injury.

7. The application according to claim 5, characterized in that, The renal injury is renal structural injury.

8. The application according to claim 5, characterized in that, The active ingredient of the drug further includes other ingredients that can improve renal injury.

9. The application according to any one of claims 5 to 8, characterized in that The dosage form of the drug is an oral preparation.

Images