Application of delphinidin-3-glucoside in preparation of uric acid lowering drugs or functional products

Abstract

The invention provides application of delphinidin-3-glucoside in preparation of uric acid reducing drugs or functional products, and belongs to the technical field of medical application. According to the invention, a novel component-target-metabolic pathway (C-T-M) network is constructed, and the network provides a comprehensive method for analyzing a specific component target and the effect of the specific component target in influencing the metabolic pathway of the HUA. Molecular dynamics (MD) simulation and surface plasmon resonance (SPR) technologies are adopted, and in-vitro and in-vivo metabonomics researches are carried out. The invention discloses a uric acid reducing effect of Dp-3G in red kidney beans, and the uric acid reducing effect is realized by combining with a flavin adenine dinucleotide (FAD) site of xanthine oxidase (XO) enzyme. Therefore, the research provides a new insight for preventing and treating the HUA by specific beans and key natural medicines.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical applications, and more particularly to the application of delphinidin-3-glucoside in the preparation of uric acid-lowering drugs or functional products. Background Technology

[0002] The incidence of hyperuricemia (HUA) has increased significantly in recent years. HUA is associated with a variety of adverse health consequences, including gout, chronic kidney disease, arteriosclerosis, and hypertension. Numerous studies have documented its incidence worldwide, ranging from 8.4% to 13.3%.

[0003] Currently, xanthine oxidase inhibitor (XOI) therapy using allopurinol (AP) is recommended as the first-line option for urate-lowering therapy (ULT). While allopurinol is generally considered safe, approximately 2% of patients may experience severe allergic reactions and side effects, sometimes even leading to death, with a mortality rate of approximately 20%. Given its safety profile and potential therapeutic benefits, natural products have become a focus of research, aiming to find effective preventative or therapeutic drugs for hyperuricemia.

[0004] Hyperuricemia is the result of both genetic and environmental factors. Foods rich in purines can raise uric acid levels. Among dietary factors, the potential impact of legumes on uric acid levels is of interest. Legumes are members of the legume family, encompassing a variety of edible seeds derived from different stages of legume maturity: dried beans, peas, lentils, chickpeas, cowpeas, broad beans, and pigeon beans are considered mature legumes. Immature legumes include green beans and peas, while sprouted forms are called sprouted beans. Legumes are popular among vegetarians and health-conscious individuals. However, purine metabolism in legumes can lead to uric acid production. Conversely, certain compounds in legumes, such as fiber and XOIs, can inhibit uric acid formation. Due to the diversity of legume composition, their specific roles in hyperuricemia remain unclear. Exploring new natural compounds in legumes that may effectively lower uric acid offers hope for future therapeutic developments.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of delphinidin-3-glucoside in the preparation of uric acid-lowering drugs.

[0008] This invention also provides the application of delphinidin-3-glucoside in combination with Lesinurad in the preparation of uric acid-lowering drugs.

[0009] The present invention also provides a functional product comprising delphinidin-3-glucoside and a food-grade carrier or excipient, for helping to maintain normal uric acid homeostasis, promote uric acid excretion, and assist in lowering serum uric acid levels.

[0010] This invention integrates relevant human evidence to investigate the relationship between the consumption of 13 specific legumes and the prevalence of hyperuricemia (HUA) in a representative US population. Based on the principles of complex target pathway (CTP) networks, we constructed a novel component-target-metabolism pathway (CTM) network, providing a comprehensive approach to analyzing specific component targets and their roles in metabolic pathways influencing HUA. We then employed molecular dynamics (MD) simulations, surface plasmon resonance (SPR) techniques, and conducted in vitro and in vivo metabolomics studies. These methods revealed the uric acid-lowering effect of Dp-3G in red kidney beans, which is achieved through binding to the flavin adenine dinucleotide (FAD) site of xanthine oxidase (XO) enzyme. Therefore, our research provides new insights into the prevention and treatment of HUA using specific legumes and key natural remedies. Attached Figure Description

[0011] Figure 1 The association between legume intake and hyperuricemia in a representative US population;

[0012] Figure 2 The results of identification of key targets of key components (Cy-3G, Dp-3G, folic acid) in red kidney beans;

[0013] Figure 3 Results of molecular docking analysis;

[0014] Figure 4 To verify the binding of Dp-3G with XO and explore its impact on the XO catalytic mechanism;

[0015] Figure 5 Effects of Dp-3G on in vivo and in vitro uric acid production: Biochemical and in vivo experiments, in B, D~H, blue represents the control group, red represents the AP treatment group, and green represents the Dp-3G treatment group;

[0016] Figure 6 Design of experiments for mouse serum metabolomics;

[0017] Figure 7 The OPLS-DA metabolite was compared between the control group and the Dp-3G treatment group.

[0018] Figure 8 To depict the z-score plots of differential metabolites between the control group and the Dp-3G treatment group;

[0019] Figure 9 A heatmap showing the differential metabolites between the control group and the Dp-3G treatment group;

[0020] Figure 10 Pathway analysis and differential metabolite enrichment analysis were performed for the control group and the Dp-3G treatment group.

[0021] Figure 11 A comprehensive metabolic network analysis was performed to identify differentially expressed metabolites between the control group and the Dp-3G treatment group.

[0022] Figure 12 Serum uric acid levels under different treatments. Detailed Implementation

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] Research subject group

[0026] NHANES is a cross-sectional survey of the U.S. population designed to conduct an ongoing series of measurements on health and nutrition to meet evolving needs. The survey is conducted by the National Center for Health Statistics.

[0027] This study utilized data from the 2015-2016 National Health and Nutrition Examination Survey (NHANES) in the United States. These data included information from participants' 24-hour dietary recollection questionnaires, demographic data, physical examinations, blood biochemistry analyses, smoking and alcohol consumption surveys, and medical history questionnaires. Exclusion criteria included: (1) incomplete data from two 24-hour dietary recollections; (2) age less than 18 years or greater than 79 years; (3) lack of information on PIR, education level, BMI, serum uric acid levels, smoking and alcohol consumption habits, and the prevalence of diabetes and hypertension; (4) use of urate-lowering drugs such as allopurinol, febuxostat, or probenecid within the past 30 days; and (5) consumption of unlabeled legumes.

[0028] Initially, 9,971 participants met the inclusion criteria. After excluding 2,944 individuals with incomplete dietary recall data, further exclusion was made for those without serum uric acid data or unclear legume intake, leaving 3,957 participants. Further exclusions were made based on age, ULT treatment status, and missing covariates (BMI, PIR, education level, smoking, alcohol consumption, hypertension, and diabetes), ultimately including 2,676 participants.

[0029] Diagnosis of hyperuricemia

[0030] The 2015-2016 survey data were collected by trained technicians using Beckman Coulter UniCel DxC800Synchron and Beckman Coulter UniCel DxC 660iSynchron analyzers, and the timed endpoint method was used to measure the participants' serum uric acid levels.

[0031] HUA was defined as a serum uric acid level above 7.0 mg / dL for men and above 6.0 mg / dL for women. The entire study population was divided into a HUA group and a non-HUA group based on their serum uric acid levels.

[0032] Logistic regression analysis was performed to investigate the relationship between the intake of various specific legumes and the risk of hyperuricemia, as shown in Table 1.

[0033] Table 1. Results of regression analysis

[0034]

[0035] We conducted various analyses to examine the association between legume intake and hyperuricemia. Figure 1 A). Fisher's Precision Inspection ( Figure 1 (B) showed significant differences in the intake of red kidney beans, immature lima beans, peas, cowpeas, wild peas, or black-eyed peas between the two groups (P<0.05). The red kidney bean intake rate was significantly higher in the non-HUA group than in the HUA group (P=0.024), suggesting a potential protective effect of red kidney beans against HUA. In Model 1, the intake of immature lima beans significantly increased the risk of HUA, while the intake of peas, cowpeas, wild peas, or black-eyed peas also significantly increased the risk of HUA. Importantly, consuming red kidney beans significantly reduced the risk of HUA. Figure 1 C) confirmed the potential impact of these legume varieties on HUA risk. To further adjust for confounding factors, partially adjusted model 2 and fully adjusted model 3 were constructed. Importantly, the significant association between the intake of immature lima beans and red kidney beans and HUA risk persisted, or showed slight changes. Figure 1 C), which highlights the robustness of the relationship between the intake of immature lima beans and red kidney beans and the risk of human allergic reactions (HUA). In the general population, consumption of three specific legumes (immature lima beans; peas, cowpeas, wild peas, or black-eyed peas; red kidney beans) resulted in a VIP value higher than 1.00 in the mixed exposure effect PLS-DA model. Figure 1 C) indicates that, under mixed legume intake, there is a robust and significant association between consuming immature lima beans and red kidney beans and the risk of HUA (human genital atrophy). Figure 1 D).

[0036] We aim to identify potential targets for folic acid 5-MTHF and anthocyanins (Cy-3G and Dp-3G). Figure 2 A). Processing structured .sdf files and smiles using PharmMapper and SwissTargetPrediction. Figure 2 B). Next, we performed a KEGG global metabolic network analysis using Metaboanalyst 6.0 (https: / / genap.metaboanalyst.ca / ) to identify cross-targets. Given that HUA is caused by abnormal uric acid levels in the purine metabolism pathway, our focus was on the overlap between potential targets and genes found in this pathway. Furthermore, we obtained 66 potential HUA targets using GeneCards and CTD. KEGG enrichment analysis of these HUA targets broadened the range of HUA-related metabolic pathways, where purine metabolism pathways were also significantly characterized in the pathways enriched for HUA targets. Our analysis highlighted three genes: inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and phosphodiesterase 4D (PDE4D) associated with 5-MTHF, and XDH(XO) (Cy-3G and Dp-3G) associated with anthocyanins. These genes were significantly enriched in the purine metabolism pathway ( Figure 2 C, D).

[0037] To gain a deeper understanding of the potential mechanisms by which legumes treat hyperuricemia, we selected key genes and performed molecular docking to evaluate their binding affinity. Figure 3 A). We first focus on 5-MTHF, docking it with PDE4D (PDB ID: 1xom) and IMPDH2 (PDB ID: 1b3o) respectively. Figure 3 (B, C). Simultaneously, we evaluated the interactions between the key enzyme XO (PDBID: 2ckj) and Cy-3G and Dp-3G. The docking score between Cy-3G and XO was -7.563. Dp-3G also exhibits affinity for XO, forming hydrogen bonds with GLU267, ASN261, and VAL259 of XO, and forming a salt bridge with GLU267. The docking score between Dp-3G and XO was -11.453. Figure 3 (D, E). Notably, the docking of Dp-3G with XO achieved the lowest score (-11.453), highlighting XO as a promising target for Dp-3G in the treatment of hyperuricemia from a comprehensive perspective.

[0038] A 200 ns MD simulation was performed on the complex XO-Dp-3G structure obtained by docking, demonstrating the stability of the Dp-3G and XO combination in a simulated aquatic environment. Figure 4 A). Root mean square deviation (RMSD) of protein-ligand complexes from 200 ns MD simulations ( Figure 4(B) indicates that the RMSD remained at approximately 0.1 nm, suggesting that the XO enzyme-ligand complex acquired a stable conformation throughout the MD simulation. Furthermore, the root mean square fluctuation (RMSF) of the XO enzyme throughout the 200 ns MD simulation demonstrates that the residues surrounding the protein active site are stable after the Dp-3G structure binds to the enzyme interior. Figure 4 (C) This observation suggests that the binding of Dp-3G may affect the active site of XO enzymes, thereby potentially influencing their catalytic function. To further verify and determine the binding affinity of Dp-3G to XO, SPR assays were performed in this study. Figure 4 The diagram shows the dynamic process of Dp-3G binding with XO at different concentrations, indicating that the binding is in a stable state. With increasing concentration, the maximum binding reaction gradually increases until saturation is reached. Around 60 s, the small molecule is removed, and the response unit decreases rapidly, indicating that Dp-3G gradually dissociates from the XO surface. Figure 4 E represents the steady-state analysis result, illustrating the relationship between RU equilibrium and Dp-3G concentration. By fitting this curve, we determined the dissociation constant (KD) of the interaction between Dp-3G and XO to be 0.25 μM (2.5 × 10⁻⁶). -7 M). KD value at 10 -7 Within the M range, the results indicate that Dp-3G and XO have a moderately strong binding affinity, ensuring effective interaction without leading to irreversible binding and potential toxicity. The SPR experimental results provide strong evidence for the direct interaction between Dp-3G and XO.

[0039] Based on molecular docking and MD simulation results, and validation through SPR experiments, we further investigated the precise role of Dp-3G in XO activity. Notably, we observed that Dp-3G occupies the FAD binding site in the XO protein, forming hydrogen bonds with ASN261 and VAL259 residues. In the normal catalytic cycle of the XO enzyme, electrons are transferred from the substrate (hypoxanthine, xanthine) to FAD via the molybdenum pterin cofactor (Moco) and [2Fe-2S], ultimately completing the oxidation reaction to produce uric acid. However, upon binding to the FAD site, Dp-3G disrupts this electron transfer process. Specifically, Dp-3G interferes with the final step of electron transfer from [2Fe-2S] to FAD, thereby inhibiting the ability of XO to catalyze uric acid production. This provides a molecular mechanism explanation for Dp-3G as an XOI. Figure 4 F).

[0040] Example 1 employed a comprehensive research approach, from computer simulation analysis to experimental verification, yielding mutually supporting and validating results. The consistency among multiple methods enhances the credibility of our conclusions and strongly supports the hypothesis that Dp-3G is an XOI. These findings lay a solid foundation for further research into the bioactivity and potential therapeutic applications of Dp-3G.

[0041] Example 2

[0042] To further investigate the inhibitory effect of Dp-3G on XO in lowering uric acid levels, we performed biochemical reaction experiments using the XO reaction-UHPLC / MS coupling method. Figure 5 A).

[0043] The XOI activity of Dp-3G was demonstrated by determining uric acid production using a combined XO reaction-UHPLC method. Ultrapure water served as a negative control, while AP (100 μM) served as a positive control. Test solutions containing the desired concentration (100 μM) of Dp-3G were freshly prepared immediately before the experiment. Briefly, 50 μL of the test sample, negative control, or positive control solution was mixed with 75 μL of xanthine (0.84 mM) and incubated at 25 °C for 5 minutes. After adding 100 μL of XO (0.025 U / mL, Sigma-Aldrich, X4376), incubation continued at 25 °C for 10 minutes, followed by the addition of 80 μL of 1 M hydrochloric acid to terminate the reaction, thus obtaining the test sample.

[0044] We observed a significant decrease in uric acid levels in the AP treatment group. Allopurinol (AP) is an effective purine-like XOI and a classic drug for treating hyperuricemia (HUA). Similarly, a decrease in uric acid levels was also observed in the Dp-3G treatment group. Figure 5 BC).

[0045] Subsequently, we investigated the inhibitory effect of Dp-3G on XO in vitro. HepG2 human liver cancer cells were purchased from Wuhan Pronosei Biotechnology Co., Ltd. These cells were cultured in DMEM medium containing 10% fetal bovine serum, 1% streptomycin (0.17 mM), and 1% penicillin (0.3 mM). Cells were cultured at 37°C under suitable humidity conditions containing 5% carbon dioxide.

[0046] HepG2 cells were cultured in DMEM medium containing complete culture medium until the cells reached 85-95% confluence. Subsequently, the cells were cultured at a rate of 1×10⁶ cells / year. 5Cells were seeded at a density of [number] cells / mL into 24-well plates. The cells were then cultured at 37°C for 18 hours to ensure cell adhesion. Cells were washed with phosphate-buffered saline (PBS, 0.01M). Next, the medium was replaced with fresh medium containing 1.25 mM adenosine, and the cells were cultured for another 24 hours. Afterward, the medium was aspirated, and the cells were washed 2-3 times with PBS. For experimental treatments, cells were cultured in complete medium containing 0.025 U / mL XO (Sigma-Aldrich, X4376), with or without the addition of Dp-3G (100 μM) or AP (100 μM), for 24 hours. Finally, the uric acid content in the supernatant of each experimental group was analyzed using the UHPLC-Q-Exactive platform (UHPLC Ultimate 3000 system, Dionex, Germering, GER; Q-Exactive Orbitrap, Thermo Fisher Scientific, Bremen, GER).

[0047] We observed a significant decrease in uric acid levels in the AP treatment group and a significant decrease in uric acid levels in the Dp-3G treatment group. Figure 5 D).

[0048] Subsequently, we established a HUA mouse model and treated it with AP and Dp-3G ( Figure 5 A). Eight-week-old male ICR mice meeting SPF standards were purchased from the Experimental Animal Center of Nanjing Medical University (Nanjing, China). These mice had free access to food and water and were housed in a controlled environment with a temperature of 20 to 26 degrees Celsius and a light cycle of 12 hours / 24 hours.

[0049] These mice were randomly assigned to three groups: (1) potassium oxyketoate (PO) group (n=8 mice per group); (2) PO+AP group (n=6 mice per group); and (3) PO+Dp-3G group (n=3 mice per group). The number of animals used in our study was determined using the “power analysis” method described on the 3Rs-Reduction.co.uk website, which was based on our pilot study to ensure sufficient statistical power (α=0.05, two-sided). The sample size was also determined with reference to previous similar studies. A hyperuricemia (HUA) model was established using the uricase inhibitor PO according to standard protocols, and experiments were conducted according to previous studies using this HUA model to examine the effect of this treatment on serum uric acid levels. One hour before treatment, mice were intraperitoneally injected with PO (200 mg / kg) to increase serum uric acid levels. Given that Dp-3G exhibited predominantly parent compound characteristics in long-term oral intake studies, for comparison, Dp-3G (100 mg / kg) was administered intraperitoneally 1 hour after PO administration, while AP (5 mg / kg) served as a control group. The intraperitoneal dosage was selected based on previous research reports.

[0050] In addition, oral gavage experiments were conducted on Dp-3G and AP. Dp-3G was administered orally at a dose of 400 mg / kg (4 mice per group) 1 hour after oral PO administration, while AP (10 mg / kg) (5 mice per group) served as a positive control. All subsequent studies were conducted in a blinded manner. Mice were randomly assigned to the control group, AP treatment group, and Dp-3G treatment group, and grouping information was sealed and stored independently. AP and Dp-3G solutions were prepared by a third party to ensure consistency in appearance, volume, and administration method. Dosing procedures and sample preparation were performed by researchers unaware of the group assignments. Blinding was maintained throughout the data analysis process.

[0051] Following administration of Dp-3G, a significant increase in Dp-3G levels was observed compared to the HUA model group and the AP treatment group, indicating successful Dp-3G treatment and the presence of the parent compound for XO binding in the liver. Figure 5 E). HUA is characterized by elevated uric acid levels. For example... Figure 5 As shown in Figure F, serum uric acid levels were significantly lower in the AP treatment group and the Dp-3G treatment group compared to the HUA model group. The conclusion remained valid even after excluding the high values ​​in the control group. Furthermore, we assessed XO activity in liver extracts from each group. XO activity was significantly lower in both the Dp-3G treatment group and the AP treatment group (…). Figure 5 G). Furthermore, oral administration of Dp-3G and AP significantly inhibited the activity of hepatic XO enzymes (G). Figure 5H) highlights the inhibitory effect of Dp-3G on XO activity in vivo. Furthermore, serum uric acid levels decreased in a dose-dependent manner with increasing Dp-3G concentration. Figure 5 I). These findings collectively indicate that Dp-3G can reduce uric acid levels under biochemical, in vitro, and in vivo conditions. Furthermore, hematoxylin-eosin (H&E) staining showed no significant changes in the organ coefficients (%) of the liver and kidneys, nor in the morphology of the liver and kidneys. Figure 5 To systematically elucidate the endogenous metabolic changes in a mouse model of hyperuricemia treated with Dp-3G, we analyzed the metabolomics changes between the hyperuricemia model group and the Dp-3G treatment group. To assess the systemic metabolomics changes in Dp-3G-treated hyperuricemic mice, we used the OPLS-DA model to explore the differences in metabolomic profiles between the two groups. The resulting R2Y(cum) and Q2(cum) values ​​were 0.990 and 0.452, respectively, indicating that the OPLS-DA model has good fit and predictability. Figure 7 These results indicate that there are different metabolic phenotypes between the HUA model group and the Dp-3G treatment group. Compared with the HUA model group, 28 metabolites were altered in the Dp-3G treatment group ( Figure 8 We then investigated the biological connections between these differentially metabolites and classified them into metabolic pathways. Figure 9 The simultaneous decrease in xanthine and uric acid, while hypoxanthine levels remained unchanged, collectively indicate that Dp-3G lowers uric acid through XO. Pathway enrichment analysis showed that the differentially metabolites between the HUA model group and the Dp-3G treatment group were mainly enriched in "cysteine ​​and methionine metabolism," "purine metabolism," and "tryptophan metabolism." Figure 10 In addition, metabolite network integration analysis ( Figure 11 By integrating all differentially metabolites between the two groups into a metabolic network, the potential link between Dp-3G treatment-induced changes in overall serum metabolism was elucidated. These findings suggest that Dp-3G has additional metabolic benefits due to its effects on metabolites involved in the pathogenesis of HUA-related diseases.

[0052] The above embodiments are based on the CTM network, and Dp-3G was identified as a key XO inhibitor in red kidney beans. Dp-3G persistently reduces uric acid production through integrated molecular docking, MD simulation, SPR experiments, and biochemical, in vitro, and in vivo models, while also reversing HUA-related metabolic abnormalities in mice, including methionine, proline, and folic acid. Our study found that Dp-3G is a novel natural compound enriched in red kidney beans that can interfere with normal electron transport processes by binding to the FAD site on XO with moderate strength. This interference inhibits uric acid synthesis, reduces the risk of HUA, and also provides potentially beneficial metabolic effects. The CTM network framework introduced in this study shows promising potential in the study of environmental and metabolic diseases. Furthermore, our results significantly improve our understanding of consuming red kidney beans and its natural component Dp-3G as a novel and effective inhibitor of XO enzymes in the treatment of HUA. The mechanism by which uric acid is reduced through red kidney bean intake: Dp-3G is abundant in red kidney beans and acts as a xanthine oxidase inhibitor. It binds to XO enzymes and, by occupying the FAD binding site within the XO enzyme, interferes with the electron transport chain (Moco,[2Fe-2S], FAD) in the purine metabolic pathway, thereby blocking uric acid production. This mechanism reduces the risk of HUA and helps reverse other HUA-related metabolic changes, such as alterations in methionine, proline, and folate. These changes are associated with complications of HUA, including atherosclerosis, hypertension, and chronic kidney disease.

[0053] Example 3

[0054] Lesinurad works primarily by inhibiting the function of transport proteins in the kidneys involved in uric acid reabsorption, thus helping the kidneys excrete uric acid. Its efficacy is usually poor when used alone, therefore it is often used in combination with allopurinol to lower uric acid. This example investigates the combined effect of Dp-3G and lesinurad in mice.

[0055] Experimental animals: SPF grade mice, 18-22g, male.

[0056] After being grouped, the mice were acclimatized for one week in an environment with a temperature of 20℃ to 25℃ and a humidity of 40% to 70%, with free access to water and food. They were divided into 4 groups of 10 mice each: the model group, the Dp-3G group, the Lesinurad group, and the Dp-3G and Lesinurad combined (DL) group.

[0057] After treatment, blood samples were taken to measure serum uric acid concentration. The results were as follows: Figure 12 As shown, the combination of Dp-3G and rasinard is more effective than either drug alone.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of delphinidin-3-glucoside in the preparation of uric acid-lowering drugs.

2. Application of delphinidin-3-glucoside in combination with Lesinurad in the preparation of uric acid-lowering drugs.

3. A functional product, characterized in that, It includes delphinidin-3-glucoside and food-grade carriers or excipients, used to help maintain normal uric acid homeostasis, promote uric acid excretion, and help reduce serum uric acid levels.

Images