Application of cinchona element Ia as uric acid reducing medicine

Abstract

The invention discloses an application of cinchona element Ia as a uric acid lowering drug, and finds that the cinchona element Ia has a remarkable inhibition effect on expression of GLUT9 protein for the first time, and reabsorption of the kidney to uric acid can be effectively reduced by inhibiting expression of the GLUT9 protein, so that excretion of the uric acid from urine is promoted, the serum uric acid level is reduced, and the toxic and side effects of the kidney are reduced. And a new effective way is provided for treatment of hyperuricemia and gout.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of cinchonasin Ia as a uric acid-lowering drug. Background Technology

[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorders or reduced uric acid excretion. Long-term hyperuricemia can lead to the deposition of urate crystals in joints and surrounding tissues, causing gouty arthritis, and may also affect the kidneys, causing serious complications such as uric acid nephropathy. With changes in people's lifestyles and dietary structures, the incidence of hyperuricemia and gout is increasing year by year, and it has become one of the common diseases that endanger human health.

[0003] Uric acid is the end product of purine metabolism in humans, mainly existing in the body as urate. Urate transporters are crucial for regulating serum uric acid levels. In the human body, two-thirds of uric acid is excreted in urine, while the proximal tubules of the kidneys reabsorb most of the filtered uric acid. This process is primarily mediated by urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9). Therefore, GLUT9 is an important drug target for the treatment of hyperuricemia and gout.

[0004] Currently, many natural extracts have been disclosed to have GLUT9 inhibitory activity, such as apigenin, resveratrol, apigenin, quercetin, baicalin, and naringenin. Cinchonain Ia, as a natural extract, has been documented in studies for its inhibitory effects on amyloid β42 aggregation (Anti-Alzheimer'sflavanolignans from Ceiba pentandra aerial parts, Fitoterapia, 2020, Jun:143: 104541), antioxidant activity (Characterization of antioxidant compounds in Eriobotrya fragrans Champ leaf, Scientia Horticulturae, 2008), and antiviral and antibacterial activity (Bioactive Constituents from Iryanthera megistophyll, J. Nat. Prod. 2002,65, 1412-1416). However, no existing technology has disclosed the inhibitory effect of cinchonain Ia on GLUT9.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide an application of cinchonasal Ia as a uric acid-lowering drug. Cinchonasal Ia can significantly inhibit the expression of GLUT9 protein, thereby reducing the reabsorption of uric acid by the kidneys, promoting uric acid excretion, and effectively reducing serum uric acid levels.

[0007] To achieve the above objectives, the present invention provides the application of cinchonasin Ia as a uric acid-lowering drug.

[0008] The chemical structural formula of cinchonain Ia is as follows: Preferably, the cinchonasin Ia is used as a medication for treating hyperuricemia and / or gout.

[0009] High uric acid and gout are metabolic diseases caused by abnormal purine metabolism or impaired uric acid excretion. This invention demonstrates through experiments that cinchonasin Ia can significantly inhibit the expression of GLUT9 protein, thereby reducing the reabsorption of uric acid by the kidneys, promoting uric acid excretion, and effectively lowering serum uric acid levels.

[0010] The purpose of this invention is to provide the application of cinchonasin Ia as a drug for inhibiting GLUT9 protein expression.

[0011] The application of cinchona bark extract Ia of the present invention as a uric acid-lowering drug has the following advantages: This invention is the first to discover that cinchona bark extract Ia has a significant inhibitory effect on the expression of GLUT9 protein. By inhibiting GLUT9 protein expression, it can effectively reduce the reabsorption of uric acid by the kidneys, thereby promoting the excretion of uric acid in urine and lowering serum uric acid levels. This provides a new and effective approach for the treatment of hyperuricemia and gout. As a naturally derived compound, cinchona bark extract Ia has the potential advantage of high safety, and its development and application are expected to provide new options for the clinical treatment of hyperuricemia and gout. Attached Figure Description

[0012] Figure 1 The structure of Cinchonain Ia (A), the molecular docking of Cinchonain Ia and GLUT9 (B~C), and the binding site of Cinchonain Ia and GLUT9 (D) of this invention are shown.

[0013] Figure 2This is the binding site of Cinchonain Ia and GLUT9 in this invention.

[0014] Figure 3 The results of evaluating the binding affinity of Cinchonain Ia to GLUT9 for this invention; (A) Binding of Cinchonain Ia at different concentrations at different times; (B) Affinity analysis results.

[0015] Figure 4 The results of the drug affinity response target stability experiment of the present invention are shown in Figure 1; (A) protein immunoblotting results; (B) GLUT9 protein levels in each group at different concentrations of Pronase.

[0016] Figure 5 The results of cell thermal migration in this invention are shown in Figure 1; (A) results of protein immunoblotting; (B) GLUT9 protein levels in each group at different temperatures; n=3.

[0017] Figure 6 The effects of Cinchonain Ia on serum uric acid (A) and urinary uric acid (B) levels in a hyperuricemic mouse model and on the expression level of GLUT9 protein in mouse kidneys (C~D); n=3.

[0018] Note: In the figure, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, nsP>0.05. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] It should be noted that: Unless otherwise specified in the examples, conditions should be followed according to standard conditions or the manufacturer's recommendations. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0021] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0022] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0023] In the description of this invention, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0024] This invention provides an application of cinchonain Ia as a uric acid-lowering drug. The inventors discovered in their research that cinchonain Ia can bind to the GLUT9 protein (glucose transporter 9), inhibiting GLUT9 expression and increasing uric acid excretion, thereby lowering uric acid levels in mice. Therefore, cinchonain Ia offers a new potential drug option for the treatment of hyperuricemia and gout. This discovery not only expands the application field of cinchonain Ia but also opens up new avenues for drug development for related diseases.

[0025] The following experimental examples illustrate in detail the application of cinchonasin Ia provided by the present invention as a uric acid-lowering drug.

[0026] Experiment Example 1: Molecular docking experiment of compound CinchonainIa with GLUT9 Molecular docking analysis was used to assess the binding potential of compound Cinchonain Ia to GLUT9, as detailed below: The 3D structure of compound Cinchonain Ia was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), and the structure of GLUT9 protein (8Y66) was obtained from the RCSB PDB database (https: / / www.rcsb.org / ). Molecular docking simulations were performed using AutoDockVina software to evaluate ligand-receptor interactions.

[0027] like Figure 1As shown in Figures A through C, the results indicate that compound Cinchonain Ia may bind to GLUT9, with a binding energy of -9.7 kcal / mol. Figure 1 D and Figure 2 The diagram shows the potential binding sites of the compound Cinchonain Ia to the GLUT9 complex. These findings suggest that Cinchonain Ia in PC may exert its anti-hyperuricemic effect by binding to GLUT9.

[0028] Experimental Example 2: Determination of Affinity by Surface Plasmon Resonance To further verify the binding potential of Cinchonain Ia and GLUT9, surface plasmon resonance (SPR) was used to determine their affinity, as follows: A CM5 chip with two channels was used. 50 mM N-hydroxysuccinimide (NHS) and 200 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) were mixed in a 1:1 ratio and injected into both channels at a flow rate of 10 μL / min for 600 seconds. The protein was diluted to 100 μg / mL (pH 4.0) with 10 mM acetate buffer and immobilized on the chip surface. Subsequently, 1 M ethanolamine was injected at the same flow rate for 300 seconds to block remaining active sites. The chip was then equilibrated with flow buffer consisting of 1× PBS-P (containing 20 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, and 0.05% P20 surfactant) at a flow rate of 10 μL / min. The binding and dissociation of different concentrations of Cinchonain Ia with GLUT9 protein were measured in each cycle. Affinity (KD) was analyzed using T200 analysis software, where KD represents the equilibrium dissociation constant.

[0029] The results are as follows Figure 3 As shown in Figures A and B, the binding affinity constant between GLUT9 fixed on the CM5 chip and Cinchonain Ia is 1.54 × 10⁻⁶. -5 M.

[0030] Experiment Example 3: Drug Affinity Response Target Stability and Cell Thermal Migration Analysis Experiment This experiment validated the binding potential of compound Cinchonain Ia to GLUT9 through drug affinity response target stability assays and cell thermal migration assays. GLUT9 protein extracted from cells was divided into a Cinchonain Ia group and a DMSO control group. The drug affinity response target stability and cell thermal migration analyses are detailed below:

[0031] (1) Cell heat transfer analysis experiment The Cinchonain Ia group received treatment with 200 μM of the compound Cinchonain Ia (using DMSO as solvent), while the control group received DMSO treatment. Proteins were sequentially heated at 37–52 °C for 5 minutes, followed by assessment of target interactions using Western blotting.

[0032] (2) Drug affinity response target stability experiment The proteins were also treated with different concentrations of Pronase E, with both groups treated with 200 µM Cinchonain Ia or DMSO, followed by incubation at 40 °C for 30 minutes to induce protein denaturation. The results were analyzed by Western blotting.

[0033] like Figure 4 As shown in Figures A and B, the drug affinity response target stability assays revealed that when the concentration of Pronase E increased (the ratio of GLUT9 protein to Pronase E was 1:250–1:1000), the GLUT9 protein level in the Cinchonain Ia group using a 1:1000 Pronase ratio was significantly higher than that in the DMSO group, while complete protein degradation was observed in the DMSO group. Figure 5 As shown in Figures A and B, the cell thermal migration results indicated that the GLUT9 protein level in the Cinchonain Ia group was significantly higher than that in the DMSO group as the temperature increased (43–52 °C). These results suggest that Cinchonain Ia can directly bind to the GLUT9 protein.

[0034] Experiment Example 4: Construction of a mouse model of hyperuricemia and drug treatment Twenty-four male mice (6-7 weeks old) were purchased from Wuhan Shubeili Biotechnology Co., Ltd. (Wuhan, China). The animals were housed in a standard environment at 24°C with a 12-hour light / dark cycle and free access to food and water. After 7 days of acclimatization, the mice were randomly divided into four groups: control group, model group, 20 mg / kg group, and 40 mg / kg group, with six mice in each group. The control group was fed a standard diet; the hyperuricemia group was fed a hyperuricemic diet for 8 weeks, consisting of a standard diet supplemented with 15% yeast and 2% potassium oxophosphate. Starting from week 5, mice in the constructed model group (i.e., the hyperuricemia group) were administered Cinchonain Ia by gavage. Rats in the 20 mg / kg and 40 mg / kg groups were administered 20 mg / kg and 40 mg / kg of Cinchonain Ia by gavage, respectively, with Cinchonain Ia dissolved in 0.5% sodium carboxymethyl cellulose (CMC). Starting from week 5, the control group received an equal volume of CMC daily for 4 weeks. After the experiment, the abdominal aortic blood samples were thawed at 4°C, and the serum uric acid and urine uric acid levels were detected using a fully automated biochemical analyzer.

[0035] like Figure 6 As shown in the figure, the results of the mouse experiment showed that the serum uric acid (UA) level in the Cinchonain IA group (20 mg / kg group and 40 mg / kg group) was significantly lower than that in the hyperuricemia model group, while the urinary uric acid (U-UA) level was significantly higher than that in the model group. Figure 6 (A and B). Compared with the model group, GLUT9 protein expression was significantly reduced in the Cinchonain IA group (A and B). Figure 6 (Figures C and D, with GAPDH used as an internal control). These results indicate that Cinchonain Ia can inhibit GLUT9 expression and promote uric acid excretion in vivo, thereby reducing serum uric acid levels.

[0036] In summary, this invention reveals that cinchona bark extract Ia can effectively reduce serum uric acid levels in hyperuricemic mice by binding to and inhibiting the expression of the GLUT9 protein. This finding not only confirms the significant effect and clear mechanism of action of cinchona bark extract Ia in lowering uric acid—namely, regulating uric acid transport and excretion by targeting the GLUT9 protein—but also provides a novel and potentially valuable drug for the treatment of hyperuricemia and gout.

[0037] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Application of cinchonasin Ia as a uric acid-lowering drug.

2. The application according to claim 1, characterized in that, The cinchonasin Ia is used as a medication for treating hyperuricemia and / or gout.

3. Application of cinchonasalin Ia as a drug to inhibit GLUT9 protein expression.

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