Application of xanthoceras sorbifolia leaf active ingredient in preparation of medicine for treating hyperuricemia

Abstract

The invention relates to the technical field of traditional Chinese medicines, and discloses application of an active ingredient of shiny-leaved yellowhorn leaves in preparation of a medicine for treating hyperuricemia. The xanthoceras sorbifolia leaf active ingredients comprise quercitrin, rutin and myricetin; the content of quercitrin is 0.2508 mg / g to 0.6719 mg / g, the content of rutin is 0.6707 mg / g to 1.5831 mg / g, and the content of myricetin is 0.8002 mg / g to 3.4858 mg / g. According to the invention, myricetin, quercitrin and rutin are determined as core effective substances of shiny-leaved yellowhorn leaves for treating hyperuricemia for the first time; the multi-target and multi-channel action mechanism is disclosed; meanwhile, network pharmacological analysis shows that the three components have high connectivity in a network and jointly act on key targets such as XOD, ACE, AKR1B1 and TNF, it is prompted that the three components do not act singly, but play a treatment role through multi-target and multi-channel cooperation, the traditional'single target-single drug 'thinking is changed into a'multi-component-multi-target-multi-channel' system regulation mode, and the treatment effect is improved. A new thought is provided for comprehensive treatment of the hyperuricemia.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to the application of active ingredients from *Sapindus mukorossi* leaves in the preparation of drugs for treating hyperuricemia. Background Technology

[0002] Currently available clinical medications for treating hyperuricemia can be categorized as follows: xanthine oxidase inhibitors, such as allopurinol and febuxostat; uricosuric drugs, such as benzbromarone and probenecid; uricase inhibitors, such as raburicase; and novel dual-action drugs, such as topiptostat. While these drugs offer some efficacy in controlling uric acid levels, they still exhibit significant hepatotoxicity and nephrotoxicity, and can cause severe allergic reactions, gastrointestinal discomfort, rashes, and other systemic side effects. Furthermore, their mechanisms of action are often limited, making them ill-suited to address the complex multi-target, multi-pathway mechanisms of action in hyperuricemia.

[0003] Traditional drug development has limitations, tending towards a "single target, single disease" model, failing to fully consider the complexity of hyperuricemia as a systemic metabolic disorder. Secondly, safety assessments of traditional treatments are insufficient, lacking a comprehensive evaluation of long-term organ toxicity accumulation or metabolic disturbances. Thirdly, most studies are based on animal models, failing to adequately consider the fundamental differences between animal models and humans in uric acid metabolism pathways. Finally, although some plant components have been reported to have the potential to lower uric acid levels, research is mostly limited to crude extracts, lacking clear identification of effective components and clinical validation.

[0004] To address the above technical problems, this invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of active ingredients from *Xanthoceras sorbifolium* leaves in the preparation of drugs for treating hyperuricemia. Through a three-pronged research strategy of clinical samples + in vitro models + proteomics, the mechanism of action of active ingredients from *Xanthoceras sorbifolium* leaves (myricetin, quercetin, and rutin) in treating hyperuricemia is studied, providing a new approach for the comprehensive treatment of hyperuricemia.

[0006] To achieve the above objectives, this invention provides the application of the active ingredients of *Xanthoceras sorbifolium* leaves in the preparation of a drug for treating hyperuricemia. The active ingredients of *Xanthoceras sorbifolium* leaves include quercetin, rutin, and myricetin; the content of quercetin is 0.2508~0.6719 mg / g, the content of rutin is 0.6707~1.5831 mg / g, and the content of myricetin is 0.8002~3.4858 mg / g.

[0007] Furthermore, it is applied to: ① Reduce serum levels of uric acid, blood glucose, cholesterol, triglycerides, creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST); ② By inhibiting the activity of XOD enzyme, uric acid production is reduced; ③ By inhibiting the activity of COX-1 and COX-2 enzymes in the arachidonic acid pathway, uric acid production is reduced; ④ By inhibiting the PI3K / AKT pathway, the levels of p-PI3K and p-AKT are reduced, thereby decreasing uric acid production; ⑤ The active ingredients in *Xanthoceras sorbifolium* leaves protect AML12 and HK2 cells and reduce apoptosis induced by high uric acid.

[0008] Furthermore, the present invention also provides the application of the active ingredients of *Sapindus mukorossi* leaves in the preparation of drugs that inhibit the arachidonic acid pathway and the PI3K / AKT pathway, for the preparation of drugs for treating hyperuricemia.

[0009] Furthermore, the present invention also provides a *Xanthoceras sorbifolium* extract, comprising active components of *Xanthoceras sorbifolium* leaves, wherein the content of quercetin is 0.2508~0.6719 mg / g, the content of rutin is 0.6707~1.5831 mg / g, and the content of myricetin is 0.8002~3.4858 mg / g.

[0010] Furthermore, the present invention also provides the use of the above-mentioned Xanthoceras sorbifolium extract in the preparation of medicaments for treating hyperuricemia, gout, kidney disease or metabolic syndrome.

[0011] Furthermore, the present invention also provides a drug for treating hyperuricemia, gout, nephropathy, or metabolic syndrome, the active ingredient of which is *Xanthoceras sorbifolium* extract. The *Xanthoceras sorbifolium* extract includes active components of *Xanthoceras sorbifolium* leaves, namely quercetin, rutin, and myricetin, wherein the content of quercetin is 0.2508~0.6719 mg / g, the content of rutin is 0.6707~1.5831 mg / g, and the content of myricetin is 0.8002~3.4858 mg / g.

[0012] The advantages and positive effects of using the active ingredients of *Sapindus mukorossi* leaves described in this invention in the preparation of drugs for treating hyperuricemia are as follows: 1. This invention identifies three flavonoid components (myricetin, quercetin, and rutin) as the core effective substances in treating hyperuricemia from *Xanthoceras sorbifolium* leaves. Through a three-pronged research strategy combining clinical samples, in vitro models, and proteomics, its absorption and efficacy in the human body were verified, revealing its multi-target, multi-pathway mechanism of action. The mechanism of action includes: inhibition of XOD; regulation of the PI3K / AKT pathway; inhibition of COX-2 and inflammatory factors; anti-apoptosis; and improvement of glucose and lipid metabolism. Furthermore, network pharmacology analysis shows that these three components have high connectivity in the network (degree values ​​of 23, 22, and 22, respectively), and act together on key targets such as XOD, ACE, AKR1B1, and TNF, suggesting that their therapeutic effects are not singular but rather synergistic through multiple targets and pathways. This represents a leap from the traditional "single-target-single-drug" approach to a "multi-component-multi-target-multi-pathway" systemic regulatory model, providing a new approach for the comprehensive treatment of hyperuricemia.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 The following are the changes in biochemical indicators of patients with hyperuricemia after drinking Xanthoceras sorbifolium leaf tea in this embodiment of the invention, where A is uric acid (UA), B is aspartate aminotransferase (AST), C is alanine aminotransferase (ALT), D is creatinine, E is triglycerides (TG), F is cholesterol (CHOL), and G is blood glucose (blood glucose). Figure 2 In this embodiment of the invention, network pharmacology analysis of the components of *Sapindus mukorossi* is used, where A is a Venn diagram of drug-disease targets, B is the top 12 core targets in terms of Degree value, and C is a network diagram of "drug-component-target". Figure 3 In this embodiment of the invention, proteomics analysis of blood from HUA patients is performed, where A represents differentially expressed proteins between the healthy group and the diseased group, and B represents differentially expressed proteins between the diseased group and the *Xanthoceras sorbifolium* leaf treatment group. Figure 4 For the dataset analysis in this embodiment of the invention, A is the normalized result of the GSE198133 dataset, and B is the differential protein volcano plot of the GSE198133 dataset; Figure 5 The above are the pathway enrichment results in the embodiments of the present invention; Figure 6 The following is an example of the uric acid-lowering ability of *Sapindus mukorossi* in a cell model in this invention embodiment, where A is the experimental design, B is the survival rate of AML12 cells, C is the survival rate of HK2 cells, D is the uric acid (UA) level in the AML12 cell line, E is the uric acid (UA) level in the HK2 cell line, and F is the XOD content in each group. Figure 7 This invention illustrates the effects of *Xanthoceras sorbifolium* on the expression of key proteins in the arachidonic acid and PI3K / AKT signaling pathways. Specifically, A represents the expression levels of COX-1 and COX-2 proteins in the arachidonic acid pathway in AML12 and HK2 cell lines; B represents the expression levels of p-PI3K and p-AKT in AML12 and HK2 cell lines; C represents the statistical analysis of COX-1 protein expression in the AML12 cell line; D represents the statistical analysis of COX-1 protein expression in the HK2 cell line; E represents the statistical analysis of COX-2 protein expression in the AML12 cell line; F represents the statistical analysis of COX-2 protein expression in the HK2 cell line; G represents the statistical analysis of p-PI3K expression in the AML12 cell line; H represents the statistical analysis of p-PI3K expression in the HK2 cell line; I represents the statistical analysis of p-AKT expression in the AML12 cell line; and J represents the statistical analysis of p-AKT expression in the HK2 cell line. Figure 8 The following is an example of the effect of *Xanthoceras sorbifolium* on inflammatory factors in this invention, where A is the TNF-α content in the AML12 cell line, B is the IL-1β content in the AML12 cell line, C is the IL-6 content in the AML12 cell line, D is the TNF-α content in the HK2 cell line, E is the IL-1β content in the HK2 cell line, and F is the IL-6 content in the HK2 cell line. Figure 9 The following is an example of the effect of *Sapindus mukorossi* on cell apoptosis in this invention, where A is the flow cytometry result of the AML12 cell line and B is the flow cytometry result of the HK2 cell line. Detailed Implementation

[0015] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0016] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0017] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0018] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0019] Example 1 1. Extraction of three active ingredients from *Xanthoceras sorbifolium* leaves: Weigh 1g of *Sapindus mukorossi* powder and place it in a 150ml round-bottom flask. Add 50ml of 70% methanol and reflux for 30min. Cool to room temperature and make up the weight with 70% methanol. Filter through a 0.22μm microporous membrane and take the filtrate for analysis.

[0020] Using IDA mode to collect ion data as a detection method, and combining characteristic fragment ion information with a self-built database and instrument library, the main chemical components of *Xanthoceras sorbifolium* from different origins are identified. Peaks of each compound are extracted from the total ion chromatogram of *Xanthoceras sorbifolium* samples to achieve qualitative analysis of the chemical components in the *Xanthoceras sorbifolium* sample solution.

[0021] A total of 64 compounds were identified, including: flavonoids (26), amino acids and their derivatives (12), alkaloids (9), terpenes (7), lignans and coumarins (5), phenolic acids (4), flavonol glycosides (2), nucleotides and their derivatives (1), flavonols (1), quinones (1), and triterpenoids (1). The results showed that the leaves of *Xanthoceras sorbifolium* are rich in flavonoids, among which quercetin, rutin, and myricetin were confirmed to be detectable in their original form in patient urine, indicating their absorption by the human body. High-performance liquid chromatography (HPLC) was used to determine the content of these three components, yielding quercetin 0.2508–0.6719 mg / g, rutin 0.6707–1.5831 mg / g, and myricetin 0.8002–3.4858 mg / g, respectively.

[0022] 2. Serum biochemical index analysis of patients with hyperuricemia: To visually demonstrate the therapeutic effects of *Xanthoceras sorbifolium*, this study included 120 patients with hyperuricemia who underwent drug intervention and were observed to monitor changes in various indicators after medication. They were randomly divided into a *Xanthoceras sorbifolium* intervention group (n=60) and a febuxostat treatment control group (n=60). The *Xanthoceras sorbifolium* intervention group consumed *Xanthoceras sorbifolium* leaf tea, which was commercially available.

[0023] The completion rate of the trial was 100% (60 / 60) in both the *Xanthoceras sorbifolium* leaf tea group and the febuxostat group. A total of 115 patients (95.8%) successfully completed all trial visits, with 58 in the *Xanthoceras sorbifolium* leaf tea group and 57 in the febuxostat group. According to the Kolmogorov-Sminov normality test, the data from both groups were normally distributed (P>0.05). Clinical results showed that compared with patients with hyperuricemia who did not consume *Xanthoceras sorbifolium* leaf tea, patients who consumed *Xanthoceras sorbifolium* leaf tea showed significant decreases in serum uric acid, blood glucose, cholesterol, triglycerides, creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), and the effects were superior to those in the febuxostat treatment group. Figure 1 This result indicates that *Xanthoceras sorbifolium* can not only lower uric acid levels, but also improve lipid metabolism and reduce liver and kidney damage.

[0024] 3. Chemical composition analysis and network pharmacological analysis of *Xanthoceras sorbifolium* leaves: To further explore the pharmacodynamic material basis of *Xanthoceras sorbifolium*'s uric acid-lowering effect, extracts were made from *Xanthoceras sorbifolium* from different origins, and a systematic chemical composition analysis was conducted, ultimately identifying 64 compounds. These compounds broadly cover a variety of bioactive components, including: flavonoids (26), amino acids and their derivatives (12), alkaloids (9), terpenes (7), lignans and coumarins (5), phenolic acids (4), flavonol glycosides (2), nucleotides and their derivatives (1), flavonols (1), quinones (1), and triterpenoids (1).

[0025] Subsequently, the Swiss target prediction database was used to identify chemical components in *Xanthoceras sorbifolium* leaves for target identification and duplicate data removal, resulting in 104 drug targets. After merging disease targets from various databases and removing duplicates, a total of 316 disease targets were identified. Figure 2 As shown, 12 crossover targets were obtained using microbioinformatics tools. These 12 targets were imported into the STRING database, a PPI network was built, and then imported into Cytoscape 3.7.1. Based on the degree values, the targets were further filtered, and the top 5 targets were identified as ACE, AKR1B1, XDH, TNF, and ALDH2 (e.g., ...). Figure 2 (As shown). A total of 114 nodes and 1508 edges were obtained through network construction. The importance of a compound or target in the network is represented by its degree in the node. The three compounds with the highest content were finally screened as myricetin, quercetin, and rutin (Table 1).

[0026] Table 1. Screening of active ingredients from *Sapindus mukorossi* using network pharmacology

[0027] The contents of the three components were further determined by high-performance liquid chromatography (HPLC): quercetin, rutin, and myricetin, with contents of 0.2508–0.6719 mg / g, 0.6707–1.5831 mg / g, and 0.8002–3.4858 mg / g, respectively (Table 2). In addition, the original drug components in the urine of hyperuricemic patients who took *Xanthoceras sorbifolium* leaf tea were analyzed. The results showed that the original compounds of quercetin, rutin, and myricetin were all detected in the urine (Table 3). This suggests that quercetin, rutin, and myricetin are effective monomeric compounds of *Xanthoceras sorbifolium* leaf in treating hyperuricemia. These three compounds were then added to a hyperuricemia cell model, and the results showed that, consistent with febuxostat, all three substances significantly reduced uric acid levels.

[0028] Table 2. Results of flavonoid content determination in *Sapindus mukorossi* extract.

[0029] Table 3. Progenitor drug components in the urine of patients with hyperuricemia

[0030] 4. Serum proteomics analysis of patients with hyperuricemia: This study aimed to identify the pathways and targets associated with hyperuricemia via proteomics analysis combined with bioinformatics results. To account for individual differences, 15 samples from each group were selected for proteomics analysis. In proteomics, 115 differentially regulated proteins were identified between the healthy and diseased groups, with 39 proteins upregulated and 76 proteins downregulated. Figure 3 (A). Compared with the group treated with Xanthoceras sorbifolium leaves, a total of 115 differentially expressed proteins were found in the diseased group, of which 8 proteins were upregulated and 107 proteins were downregulated. Figure 3 (B)

[0031] In bioinformatics, this study used the "Homo sapiens" microarray GSE198133 dataset from the GPL24676 platform. After online analysis and organization of the dataset for quality control of the microarray dataset using GEO2R, ​​the data from this dataset were normalized and analyzed. The results showed that the six samples from the GSE198133 dataset were at the same level. Figure 4 (A) indicates that the data quality is reliable. In the GSE198133 dataset, a total of 17575 DEGS were obtained, of which 9125 were upregulated and 8450 were downregulated, and a volcano plot was generated. Figure 3(B) Next, the differentially expressed proteins obtained from serum proteomics were intersected with the differentially expressed genes obtained from bioinformatics analysis, yielding a total of 101 intersecting genes. These 101 intersecting genes were analyzed in the DAVID database, ultimately identifying 16 relevant pathways. The enriched pathways were then sorted according to p-values ​​and visualized, as shown below. Figure 5 Of particular interest is arachidonic acid metabolism, whose cyclooxygenase pathway leads to the production of prostaglandins, resulting in a range of diseases including inflammation and pain.

[0032] 5. Xanthoceras sorbifolium Bunge can lower uric acid levels in vitro: Based on the different sites of uric acid production and excretion, mouse hepatocyte cell line AML12 and human renal proximal tubular epithelial cell line HK2 were selected as subsequent experimental subjects to further explore the potential mechanism of Xanthoceras sorbifolium in treating hyperuricemia (HUA). A uric acid-induced hyperuricemic cell model was established, and then Xanthoceras sorbifolium total extract was administered as an intervention to observe its therapeutic effect. Figure 6 (A). The survival rates of AML12 and HK2 cells after treatment with different concentrations of *Sapindus mukorossi* were assessed using CCK8, and the results showed ( Figure 6 (B and C) *Xanthoceras sorbifolium* concentrations ranging from 12.5 to 50 μg / mL showed no significant inhibitory effect on the viability of AML12 and HK2 cells (p>0.05). Therefore, *Xanthoceras sorbifolium* concentrations of 12.5, 25, and 50 μg / mL were selected for subsequent cell experiments.

[0033] After treating cells with 40 mM uric acid for 48 hours, the uric acid content in the cells was measured, such as... Figure 6 As shown in Figures D and E, in both the AML12 and HK2 cell lines, uric acid (UA) levels were significantly higher in the model groups compared to the control group (all p < 0.05). However, supplementation with different concentrations of *Xanthoceras sorbifolium* significantly reduced uric acid levels, with the 50 μg / mL concentration showing the most significant effect (p < 0.05). The liver is the main organ for uric acid biosynthesis, and its XOD enzyme is the rate-limiting enzyme in uric acid synthesis. Therefore, the XOD content in AML12 cells (mouse hepatocytes) was measured, and the results are shown below. Figure 6 As shown in Figure F, the XOD content in the model group was higher than that in the control group (p<0.001), while the XOD content in the *Xanthoceras sorbifolium* intervention group was significantly lower (p<0.01). This result indicates that uric acid activates the transcription and expression of XOD in mouse hepatocytes, leading to an increase in XOD and consequently a sharp increase in uric acid synthesis. XSB, on the other hand, exerts its therapeutic effect by inhibiting XOD enzyme activity and reducing uric acid production.

[0034] 6. *Xanthoceras sorbifolium* inhibits the arachidonic acid pathway: KEGG results showed that the arachidonic acid pathway and the PI3K-AKT signaling pathway were significantly enriched in the *Xanthoceras sorbifolium* treatment group, suggesting that *Xanthoceras sorbifolium* exerts its therapeutic effect through these two pathways. To investigate the intervention effect of *Xanthoceras sorbifolium*, the protein expression levels of cyclooxygenases (COX-1 and COX-2), key proteins of the arachidonic acid pathway, were detected in hepatocytes (AML12) and kidney cells (HK2).

[0035] The results show that ( Figure 7 In the model group (A, CF), compared with the control group, the protein expression levels of COX-1 and COX-2 were significantly upregulated (p<0.001). After intervention with different concentrations of Xanthoceras sorbifolium, the abnormally elevated COX-1 and COX-2 protein expression in both cell lines showed a significant dose-dependent decreasing trend (p<0.05 or p<0.01). In the high-dose treatment group, the expression level of COX protein even recovered to near the level of the normal control group. In addition, it was found that COX-2 was not expressed in the control group of both cell lines, but it was significantly increased after uric acid treatment. Xanthoceras sorbifolium treatment could effectively reverse this effect, suggesting that Xanthoceras sorbifolium can effectively inhibit the arachidonic acid pathway.

[0036] 7. Effects of *Xanthoceras sorbifolium* on the PI3K-AKT signaling pathway: To further investigate the molecular mechanism of the anti-inflammatory effect of *Xanthoceras sorbifolium*, the activation of the PI3K / AKT signaling pathway, which is closely related to cell survival, proliferation, and inflammation, was examined. Western blot results showed ( Figure 7 In AML12 cells (B, GJ), uric acid treatment significantly increased the expression levels of p-PI3K and p-AKT compared to the control group (p<0.01), while the total PI3K and AKT protein levels did not change significantly. This indicates that the model group successfully activated the PI3K / AKT signaling pathway. Notably, after intervention with *Xanthoceras sorbifolium*, the model-induced increase in p-PI3K and p-AKT levels was significantly inhibited in a dose-dependent manner (p<0.01). Combined with previous results, the trends in p-PI3K and p-AKT protein levels were highly consistent with those in COX-2 protein levels. These results suggest that *Xanthoceras sorbifolium* can effectively inhibit the PI3K / AKT pathway activated by high uric acid. Given that AKT is an important upstream regulator of COX-2, the drug exerts its anti-inflammatory effect by inhibiting the activation of the PI3K / AKT pathway, thereby downregulating COX-2 expression.

[0037] Surprisingly, compared with the control group, high uric acid treatment significantly reduced the levels of p-PI3K and p-AKT in HK2 cells (p<0.01). This indicates that the PI3K-AKT signaling pathway was inhibited in this cell model. However, after intervention with *Xanthoceras sorbifolium*, the levels of p-PI3K and p-AKT significantly increased in a dose-dependent manner (p<0.05 or p<0.01), even returning to normal levels. Combined with the results of COX-2 (increased in the model group and decreased in the treated group), a negative correlation was found between the protein level of the PI3K-AKT signaling pathway and the expression of the inflammatory marker COX-2, indicating that *Xanthoceras sorbifolium* exerts its effects through distinctly different mechanisms in different cell types.

[0038] 8. *Xanthoceras sorbifolium* inhibits uric acid-induced inflammatory cytokine release and apoptosis: The ultimate effect of cellular inflammation is manifested in the release of inflammatory factors. The levels of key inflammatory factors TNF-α, IL-1β, and IL-6 in cell culture supernatant were detected using an ELISA kit. Figure 8 As shown in the AF, the levels of TNF-α, IL-1β, and IL-6 in the cell supernatant of the model group were significantly higher than those of the control group (p<0.01). Treatment with *Xanthoceras sorbifolium* significantly inhibited the release of these three inflammatory factors, and the inhibitory effect was dose-dependent (p<0.01). This result once again demonstrates that *Xanthoceras sorbifolium* has a strong anti-inflammatory effect.

[0039] Subsequently, to assess the impact of high uric acid on cell survival and the protective effect of *Xanthoceras sorbifolium*, apoptosis in AML12 and HK2 cells was detected by flow cytometry using Annexin V-FITC / PI double staining. The results showed ( Figure 9 Compared with the control group, high uric acid treatment significantly induced apoptosis in both cell types. However, after pretreatment with different concentrations of *Xanthoceras sorbifolium*, the proportion of apoptotic cells decreased significantly in a dose-dependent manner (p<0.05). This result clearly indicates that *Xanthoceras sorbifolium* can effectively protect AML12 and HK2 cells from high uric acid-induced apoptosis.

[0040] 9. Analysis of the synergistic mechanism of the three flavonoid components: In summary, network pharmacology and experimental validation demonstrate that myricetin, quercetin, and rutin do not simply have an additive effect, but rather exert their therapeutic effects synergistically through multiple targets and pathways. Synergistic inhibition of uric acid production and inflammation at multiple targets: All three can inhibit XOD activity, reducing uric acid synthesis; simultaneously, they synergistically regulate the PI3K / AKT signaling pathway and the arachidonic acid metabolic pathway, reducing the expression of inflammatory factors such as COX-2, TNF-α, IL-1β, and IL-6, thus alleviating inflammatory responses and apoptosis. Synergistic regulation across organ / cell types: In hepatocytes (AML12), the three synergistically inhibit the overactivation of the PI3K / AKT pathway; in kidney cells (HK2), they synergistically activate this pathway to inhibit apoptosis. This "cell type-dependent synergistic regulation" indicates that the combined use of the three can cover the multi-organ pathological processes of hyperuricemia, achieving systemic treatment.

[0041] Synergistic effect, superior to single ingredients: In vitro cell experiments show that the combined use of the three ingredients is superior to the single ingredients in reducing uric acid, inhibiting inflammation and anti-apoptosis, suggesting a synergistic effect of "1+1+1>3", which provides a theoretical basis for the development of highly active and low-toxicity compound preparations.

[0042] Therefore, this invention identifies three flavonoid components (myricetin, quercetin, and rutin) as the core effective substances in treating hyperuricemia from *Xanthoceras sorbifolium* leaves. Through a three-pronged research strategy combining clinical samples, in vitro models, and proteomics, its absorption and efficacy in the human body were verified, revealing its multi-target, multi-pathway mechanism of action. The mechanism of action includes: inhibition of XOD; regulation of the PI3K / AKT pathway; inhibition of COX-2 and inflammatory factors; anti-apoptosis; and improvement of glucose and lipid metabolism. Furthermore, network pharmacology analysis shows that these three components have high connectivity in the network (degree values ​​of 23, 22, and 22, respectively), and act together on key targets such as XOD, ACE, AKR1B1, and TNF, suggesting that their therapeutic effects are not singular but rather synergistic through multiple targets and pathways. This represents a leap from the traditional "single-target-single-drug" approach to a "multi-component-multi-target-multi-pathway" systemic regulatory model, providing a new approach for the comprehensive treatment of hyperuricemia.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of active ingredients from *Sapindus mukorossi* leaves in the preparation of drugs for treating hyperuricemia, characterized in that: The active ingredients in *Xanthoceras sorbifolium* leaves include quercetin, rutin, and myricetin; the content of quercetin is 0.2508~0.6719 mg / g, the content of rutin is 0.6707~1.5831 mg / g, and the content of myricetin is 0.8002~3.4858 mg / g.

2. The application of the active ingredient from *Sapindus mukorossi* leaves according to claim 1 in the preparation of a drug for treating hyperuricemia, characterized in that... Applied to: ① Reduce serum levels of uric acid, blood glucose, cholesterol, triglycerides, creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST); ② By inhibiting the activity of XOD enzyme, uric acid production is reduced; ③ By inhibiting the activity of COX-1 and COX-2 enzymes in the arachidonic acid pathway, uric acid production is reduced; ④ By inhibiting the PI3K / AKT pathway, the levels of p-PI3K and p-AKT are reduced, thereby decreasing uric acid production; ⑤ The active ingredients in *Xanthoceras sorbifolium* leaves protect AML12 and HK2 cells and reduce apoptosis induced by high uric acid.

3. The application of the active ingredients of *Xanthoceras sorbifolium* leaves in the preparation of drugs that inhibit the arachidonic acid pathway and the PI3K / AKT pathway, characterized in that: Used to prepare drugs for the treatment of hyperuricemia.

4. A *Sapindus mukorossi* extract, characterized in that: It includes the active ingredients of *Xanthoceras sorbifolium* leaves, which are composed of quercetin, rutin and myricetin, with quercetin content ranging from 0.2508 to 0.6719 mg / g, rutin content ranging from 0.6707 to 1.5831 mg / g and myricetin content ranging from 0.8002 to 3.4858 mg / g.

5. The use of the *Sapindus mukorossi* extract according to claim 4 in the preparation of a medicament for treating hyperuricemia, gout, nephropathy, or metabolic syndrome.

6. A drug for treating hyperuricemia, gout, kidney disease, or metabolic syndrome, characterized in that: The active ingredient is *Xanthoceras sorbifolium* extract, which includes active components of *Xanthoceras sorbifolium* leaves, namely quercetin, rutin, and myricetin. The content of quercetin is 0.2508~0.6719 mg / g, the content of rutin is 0.6707~1.5831 mg / g, and the content of myricetin is 0.8002~3.4858 mg / g.

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