Application of taxifolin in preparation of product for inhibiting expression of urate transporter

Abstract

The invention discloses application of taxifolin in preparation of a product for inhibiting expression of a urate transporter, and relates to the technical field of biological medicines. The invention also provides an application of taxifolin in preparation of a product for promoting uric acid excretion and an application of taxifolin in preparation of a product for protecting human renal epithelial cells. The research finds that taxifolin has the effect of protecting human renal epithelial cells and can play a role in promoting uric acid excretion by regulating URAT1. The taxifolin not only has a good URAT1 regulating effect, but also has good safety, and compared with other similar URAT1 inhibitors, the taxifolin has good safety, protects human kidney epithelial cells, and has good application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of taxifolin in preparing a product for inhibiting the expression of uric acid transporters. Background Art

[0002] Hyperuricemia (HUA) refers to a state of the body in which the uric acid concentration in the blood exceeds the normal range. It is often associated with traditional metabolic cardiovascular risk factors such as hypertension, hyperlipidemia, type 2 diabetes, obesity, and insulin resistance. At present, basic research and epidemiological studies have suggested that uric acid is an independent risk factor for vascular diseases. 90% of hyperuricemia is caused by uric acid excretion disorders. The clearance of uric acid is mainly controlled by renal tubular reabsorption and secretion, among which reabsorption is the main factor in maintaining the concentration of uric acid in the blood. OAT1 is the main protein for uric acid secretion, which mainly excretes uric acid into the renal tubules; URAT1 is the most important uric acid reabsorption transporter in the human body. It is a membrane transporter protein encoded by the SLC22A12 gene of the organic anion encoding family SLC22A, which is mainly involved in the reabsorption of uric acid in the proximal tubules; therefore, promoting OAT1 expression while inhibiting URAT1 expression will be more conducive to the excretion of uric acid; studies have shown that due to the powerful uric acid reabsorption effect of URAT1, it has become a hot spot for the development of new uric acid-lowering drugs. At present, the main drugs for lowering uric acid in clinical practice include chemical drugs such as benzbromarone, probenecid, allopurinol, and febuxostat; among them, allopurinol and febuxostat are drugs that inhibit the production of uric acid; benzbromarone and probenecid are drugs that promote the excretion of uric acid; clinical observations have shown that existing clinical drugs have many and strong side effects; therefore, the development of new, safe and effective drugs that regulate uric acid transporters to meet the needs of patients and clinicians has good social significance.

[0003] Taxifolin (HQSS), also known as dihydroquercetin, has a structure of 3,5,7,3′,4′-pentahydroxydihydroflavone. Its molecular structure is as follows:

[0004]

[0005] Modern pharmacological studies have shown that Taxifolin has significant medicinal value in anti-oxidation, anti-inflammation, liver protection, anti-tumor, lipid regulation, anti-angiogenesis, anti-Alzheimer's disease, anti-bacterial, reducing the production of amyloid β in animal brains, anti-liver fibrosis, and treating parasites. However, there have been no reports on Taxifolin's ability to protect human renal epithelial cells, inhibit the uric acid transporter URAT1, and promote uric acid excretion. Summary of the invention

[0006] The purpose of the present invention is to provide an application of taxifolin in the preparation of a product for inhibiting the expression of uric acid transporters, so as to solve the problems existing in the above-mentioned prior art. The present invention has found that taxifolin can inhibit the expression of uric acid transporters, and further can promote uric acid excretion and protect renal epithelial cells by regulating the expression of uric acid transporters.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides application of taxifolin in preparing a product for promoting uric acid excretion.

[0009] Application of taxifolin in preparing products for inhibiting the expression of uric acid transporters.

[0010] Furthermore, the uric acid transporter is URAT1.

[0011] The present invention also provides the use of taxifolin in preparing products for promoting uric acid excretion.

[0012] Furthermore, the product promotes uric acid excretion by inhibiting the expression of uric acid transporter URAT1 in human renal epithelial cells.

[0013] The invention also provides the use of taxifolin in preparing products for protecting human renal epithelial cells.

[0014] Furthermore, the product can reduce the damaging effect of uric acid on the human renal epithelial cells.

[0015] Furthermore, the product reduces the damaging effect of uric acid on the human renal epithelial cells by inhibiting the expression of the uric acid transporter URAT1 in the human renal epithelial cells.

[0016] The present invention also provides a product for promoting uric acid excretion, protecting human renal epithelial cells or inhibiting the expression of uric acid transporters, wherein the product comprises taxifolin.

[0017] Furthermore, the product is a medicine or a health-promoting product.

[0018] Furthermore, the dosage form of the drug includes capsules, granules, tablets, oral liquids, injections or syrups; the health-promoting product includes compressed candies or solid beverages.

[0019] The present invention discloses the following technical effects:

[0020] The present invention has found that taxifolin has the effect of protecting human renal epithelial cells and can also promote uric acid excretion by regulating URAT1. Taxifolin not only has a good effect of regulating URAT1, but also has good safety. Compared with other similar URAT1 inhibitors, taxifolin has good safety and protects human renal epithelial cells, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The effect of different concentrations of uric acid on the proliferation of HK2 cells;

[0023] Figure 2 The effect of different concentrations of Taxifolin on the proliferation of HK2 cells;

[0024] Figure 3 The effect of taxifolin on the proliferation of HK2 cells damaged by uric acid;

[0025] Figure 4 The Western Blot detection results of URAT1 in each experimental group of Example 2;

[0026] Figure 5 The expression level detection results of URAT1 in each experimental group of Example 2; UA2.0 in the figure represents the model group;

[0027] Figure 6 This is a statistical analysis chart of the serum uric acid values ​​of rats in each experimental group of Example 3;

[0028] Figure 7 The Western Blot detection results of URAT1 in the kidney of rats in each experimental group of Example 3; the figure shows the results of two repeated experiments;

[0029] Figure 8 The relative expression level of URAT1 in the kidney of rats in each experimental group in Example 3. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1 Protective effect of HQSS on human renal epithelial cell line HK2 induced by uric acid

[0036] 1. Materials

[0037] Reagents: HK2 cells (from ATCC); South American fetal bovine serum (Excell); trypsin cell digestion solution (0.25% trypsin); double antibody (Sinomed); PVDF membrane 0.22μM (Millipore); CCK8 (Sinomed); uric acid (Sigma); benzbromarone (Sigma); dimethyl sulfoxide (MPbio); taxifolin (purity 99%, China National Institute for Food and Drug Control) prepared into 200mM dimethyl sulfoxide storage solution and stored at -20°C.

[0038] 2. Experimental methods and results

[0039] Resuscitate HK2 cells, and pass them when they cover 80% of the bottom of the bottle. Aspirate the medium, add 2mL of PBS, shake the culture bottle to clean the cell surface, aspirate the PBS, add 1mL of trypsin, and digestion is complete when the cells become smaller, round, and independently suspended. Add DMEM / f12 complete medium to terminate digestion, gently blow down all the cells, and transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 1000r / min for 3min. Discard the supernatant and resuspend in complete medium. Passage at a ratio of 1:3 and culture in a 37℃ 5% CO2 incubator.

[0040] 2.1 Toxicity test of uric acid and taxifolin to HK2

[0041] 2.1.1 Toxicity test of uric acid on HK2

[0042] When the cell density of HK2 cells reached 80%, 5×10 4 Cells / mL were evenly inoculated in a 96-well cell culture plate, with a cell suspension volume of 100 μL in each well. After 24 hours of cell attachment, a toxicity test was performed: experimental group, normal cell group, and blank control group were set up;

[0043] Normal cell group: HK2 cells were cultured normally without adding any test samples.

[0044] Blank control group: no cells, only the test sample, i.e. uric acid, was added to deduct the effect of the test sample on absorbance.

[0045] Experimental group: HK2 cells with a cell density of 80% were cultured at 5×10 4 Cells / mL were evenly inoculated in a 96-well cell culture plate, and 100 μL of cell suspension was inoculated in each well. Then, different concentrations of urea were added to make the final concentrations of uric acid 3mM, 2mM, 1mM, 0.5mM and 0.25mM, respectively. After the cell culture plate was placed in a 37°C, 5% CO2 cell culture incubator for 48 hours, CCK8 was used to detect cell proliferation to explore the inhibition of HK2 proliferation by different concentrations of uric acid. The absorbance (optical density, OD) at 450nm was measured with an enzyme marker to calculate the cell proliferation.

[0046] Cell growth survival rate = (OD 实验组 -OD 空白溶剂组 ) / OD 正常细胞组 ×100%.

[0047] 2.1.2 Toxicity test of taxifolin to HK2

[0048] When the cell density of HK2 cells reached 80%, 5×10 4Cells / mL were evenly inoculated in a 96-well cell culture plate, and 100 μL of cell suspension was inoculated in each well. Then, different concentrations of Taxifolin were added to make the final concentrations of Taxifolin 200 μM, 100 μM, 50 μM, 25 μM and 12.5 μM, respectively. After the cell culture plate was placed in a 37°C, 5% CO2 cell culture incubator for 48 hours, CCK8 was used to detect cell proliferation to explore the inhibitory effect of different concentrations of Taxifolin on HK2 proliferation. The absorbance at 450 nm was measured with an enzyme marker to calculate the cell proliferation.

[0049] Cell growth survival rate = (OD 实验组 -OD 空白溶剂组 ) / OD 正常细胞组 ×100%.

[0050] 2.1.3 Results

[0051] When the concentration of uric acid was 3 mM, it had a significant inhibitory effect on HK2 cells ( Figure 1 ); Taxifolin is very safe for cells and has no obvious inhibitory effect on HK2 ( Figure 2 ).

[0052] 2.2 Effect of Taxifolin on the Proliferation of Human Renal Tubular Epithelial Cells

[0053] Normal cell group: HK2 cells were cultured normally without adding any test samples.

[0054] Blank control group: no cells, only the test sample, i.e., Taxifolin, was added to deduct the effect of the test sample on absorbance.

[0055] Experimental group: 5×10 4 Cells / mL were evenly inoculated in a 96-well cell culture plate, and the volume of cell suspension inoculated in each well was 100μL. 3mM uric acid was added to each well to pretreat HK2 cells for 2h, and then different concentrations of taxifolin were added to make the final concentrations 100μM, 50μM, 25μM, and 12.5μM, respectively, with 3 replicates for each concentration. The cell culture plate was placed in a 37°C, 5% CO2 cell culture incubator for culture, and CCK8 test was performed after 48 hours. The absorbance at 450nm was measured with an enzyme reader to calculate the cell proliferation. The protective effect of taxifolin at each concentration on uric acid-damaged HK2 was determined.

[0056] Blank group:

[0057] Cell growth survival rate = (OD 实验组 -OD 空白溶剂组 ) / OD 正常细胞组 ×100%.

[0058] Results: Taxifolin in each group could counteract the growth inhibition of HK2 caused by high uric acid environment and play a role in protecting human renal epithelial cells ( Figure 3 ).

[0059] Example 2: Experimental study on the regulation of URAT1 expression in human renal epithelial cells HK2 by taxifolin

[0060] 1. Methods

[0061] Method: Take HK2 cells that are growing logarithmically and in good condition, and absorb 1 mL of trypsin to digest the cells. 5 The cells were inoculated into 6-well culture plates and adhered to the plate overnight. Then the normal group, model group (2.0 mM uric acid), positive control group (2.0 mM uric acid + 25 μM benzbromarone), HQSS-H group (2.0 mM uric acid + 50 μM HQSS), HQSS-M group (2 mM uric acid + 25 μM HQSS) and HQSS-L group (2 mM uric acid + 12.5 μM HQSS) were set up.

[0062] After 48 hours of culture, each experimental group was centrifuged at 15000r / min and 4℃ for 15min. After taking the supernatant to obtain the total protein, the following operations were performed: protein concentration was detected by BCA method; metal bath denaturation for 10min. 20μg protein was loaded in each well for gel electrophoresis, and the electrophoresis conditions were 60V 30min and 100V 80min. After transfer, the primary antibody was strictly diluted according to the antibody instructions: URAT1 antibody (1:5000), OAT1 (1:1000); internal reference β-actin (1:5000), incubated on a shaker at 4℃ overnight; the PVDF membrane was removed and washed repeatedly with TBST for 3 times, each time for 10min, and the secondary antibody (1:6000) was added and developed with ECL developer. Image Lab software was set to record the bands, and the gray value of the target protein / internal reference protein was used to represent the relative expression of the target protein; Image J was used for grayscale analysis.

[0063] 2. Results

[0064] like Figure 4 and 5 As shown, compared with the normal group, the expression of URAT1 transporter in the model group was significantly increased, with statistical differences, indicating that the model was successful; the positive drug benzbromarone can significantly downregulate the expression of URAT1 in human renal epithelial cells HK2 stimulated by uric acid; compared with the model group, the high, medium and low dose groups of taxifolin can significantly downregulate the expression of URAT1 in human renal epithelial cells stimulated by high uric acid, and restore the expression of URAT1 in human renal epithelial cells to normal levels, which can inhibit the reabsorption of uric acid; at the same time, there is no side effect of the positive control drug benzbromarone that strongly inhibits URAT1 expression, which is beneficial to the protection of the kidneys.

[0065] Example 3 Effect of Taxifolin on Serum Uric Acid (SUA) and Renal URAT1 in Rats with Hyperuricemia

[0066] 1. Methods

[0067] Thirty SD rats were randomly divided into 5 groups, with 6 rats in each group: normal group (Con), model group (Modle), high-dose HQSS group (HQSS-H, 0.3 g / kg), low-dose HQSS group (HQSS-L, 0.1 g / kg) and benzbromarone group (BM).

[0068] Except for the normal group, each group of SD rats was given adenine 100mg / kg + potassium oxonate 1.0g / kg by gavage for 21 consecutive days, and the modeling lasted until the end of the experiment; in addition to adenine and potassium oxonate, the HQSS high-dose group was given HQSS once a day at a dose of 0.3g / kg, the HQSS low-dose group was given HQSS once a day at a dose of 0.1g / kg, and the benzbromarone group was given benzbromarone once a day at a dose of 1.5g / kg. After 21 days of the experiment, the SUA levels and renal URAT1 expressions of SD rats in each group were detected.

[0069] 2. Results

[0070] As shown in Table 1 and Figure 6 As shown in the figure, the SUA level of rats in the model group increased significantly, and the SUA level of the benzbromarone group was significantly lower than that of the model group (P < 0.01); the SUA level of rats in the high-dose HQSS group and the low-dose HQSS group was also significantly reduced, with significant differences compared with the model group (P < 0.01). The results showed that HQSS has the effect of reducing serum uric acid. The results of the detection of URAT1 expression in the kidneys of SD rats in each group are shown in the figure. Figure 7-8 As shown in the figure, after grayscale analysis, it was found that HQSS could significantly downregulate the expression of URAT1 in the renal tissue of hyperuricemia model animals.

[0071] Table 1 Effect of Taxifolin on serum uric acid level in rats with hyperuricemia (μmol / L)

[0072]

[0073] Example 4 Capsules

[0074] 200 g of taxifolin, 50 g of dextrin, 5 g of low-substituted hydroxypropyl cellulose, 40 g of microcrystalline cellulose, 5 g of silica gel powder, 1 g of magnesium stearate and 10 wt % starch slurry were mixed and granulated, dried at 65° C., sieved through 45 mesh, and filled into capsules to obtain 1000 capsule preparations.

[0075] Example 5 Granules

[0076] 300 g of taxifolin, 150 g of sucrose and 50 g of maltodextrin were mixed and granulated, and 1000 packets of granules were prepared according to a conventional granule preparation method.

[0077] Example 6 Tablets

[0078] 100 g of taxifolin, 50 g of microcrystalline cellulose, 10 g of sodium carboxymethyl starch, 38 g of dextrin and 1 g of silicon dioxide were mixed evenly, and starch slurry was used as a binder to mix and granulate. The whole granules were dried according to a conventional method, 1 g of magnesium stearate was added and mixed evenly, and 1000 tablets were made according to a conventional method.

[0079] Example 7

[0080] 50 g of Taxusin, 200 g of maltodextrin, 100 g of powdered sugar and 50 g of inulin were added with 2 g of micro powdered silica gel, mixed evenly, and dry-compressed to prepare 1000 tablets.

[0081] Example 8 Oral Liquid

[0082] Add 50 g of taxifolin, 100 mg of aspartame, 1.5 g of sodium citrate, 10 g of antibacterial agent potassium sorbate and 2 g of ethylparaben to 1000 mL of purified water and fully dissolve to prepare 1000 mL of oral solution.

[0083] Example 9 Injection

[0084] Add 100 g of taxifolin, 15 g of soybean lecithin for injection, 30 g of glycerol for injection and 1 g of Tween 80 for injection to 1000 mL of water for injection, and prepare 1000 injections according to conventional methods.

[0085] Example 10 Syrup

[0086] Dissolve 100 g of Taxol in 700 mL of monosaccharide syrup and 300 mL of water, then add 1.0 g of sodium benzoate, stir evenly, and prepare 1000 mL of syrup in a conventional manner.

[0087] Example 11 Solid Beverage

[0088] 100 g of taxifolin, 150 g of sucrose and 350 g of maltodextrin were mixed and granulated, and 1000 small bags were prepared according to a conventional granule preparation method.

[0089] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of taxifolin in the preparation of products for inhibiting the expression of uric acid transporters.

2. The use according to claim 1, characterized in that: The uric acid transporter is URAT1.

3. Application of Taxifolin in the preparation of products that promote uric acid excretion.

4. The use according to claim 3, characterized in that: The product promotes uric acid excretion by inhibiting the expression of uric acid transporter URAT1 in human renal epithelial cells.

5. Application of Taxifolin in the preparation of products for protecting human renal epithelial cells.

6. The use according to claim 5, characterized in that: The product can reduce the damaging effect of uric acid on human renal epithelial cells.

7. The use according to claim 6, characterized in that: The product reduces the damaging effect of uric acid on the human renal epithelial cells by inhibiting the expression of the uric acid transporter URAT1 in the human renal epithelial cells.

8. A product for promoting uric acid excretion, protecting human renal epithelial cells or inhibiting the expression of uric acid transporters, characterized in that: The product includes taxifolin.

9. The product according to claim 8, characterized in that The product is a medicine or a health promoting product.

10. The product according to claim 9, characterized in that The dosage form of the medicine includes capsules, granules, tablets, oral liquids, injections or syrups; the health-promoting product includes compressed candies or solid beverages.

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