Application of pigeonpea extract and active ingredients in relieving hyperuricemia

Abstract

The invention provides application of pigeon pea extract and active ingredients in preparation of drugs or functional food for relieving hyperuricemia. Animal experiment research finds that intervention of pigeon pea extract and pinostrobin significantly reduces serum uric acid content of a hyperuricemia mouse, prevents serum creatinine and / or serum urea nitrogen in vivo from increasing, and can increase urine urea nitrogen and / or urine creatinine excretion; meanwhile, the pigeonpea extract and the pinostrobin can restore the kidney and liver coefficients of a hyperuricemia mouse to a normal level, remarkably relieve kidney injury caused by hyperuricemia, inhibit kidney inflammation / fibrosis reaction and restore the physiological state of the kidney to the level of a normal mouse.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine ingredients, and particularly relates to application of extract of Cajanus cajan (L.) Millsp and active ingredients in relieving hyperuricemia. BACKGROUND

[0002] The excretion of blood uric acid is blocked or increased, which will lead to hyperuricemia. Hyperuricemia can cause gout, organ damage, cardiovascular disease, hypertension, diabetes and other complications. At present, the drugs for treating hyperuricemia mainly include allopurinol, febuxostat and benzbromarone. However, these three drugs all have side effects, which can cause allergic reactions, gastrointestinal diseases (diarrhea, nausea, vomiting, etc.), abnormal liver function, blood system disorders and the like. Therefore, it is an urgent problem to be solved to find safer and more effective drugs for reducing uric acid and daily health care products.

[0003] Plant medicines are an important source for finding and developing drugs or health care products for reducing uric acid. Cajanus cajan (L.) Millsp is an important legume food crop, which is planted in more than 80 countries around the world. Its roots, stems, leaves, flowers and pods can be used as medicine, which has the effects of promoting diuresis, relieving swelling, removing blood stasis, clearing heat and detoxifying. The seeds of Cajanus cajan (L.) Millsp are rich in protein and are often used to make bean products (such as tofu, tempeh, bean paste, soy flour, soy sauce, etc.), and the tender pods and leaves in some areas can be used as vegetables, and dry and fresh leaves are used as feed. Studies have shown that the leaves of Cajanus cajan (L.) Millsp have low toxicity (LD50>5g / kg) and no accumulation toxicity after long-term feeding. Although Cajanus cajan (L.) Millsp has not been included in the national list of food and medicine, its multifunctionality has been verified by folk practice and scientific research, and it has significant potential in the field of functional food and drug development. At present, there is no clinical trial or animal model study on the direct reduction of uric acid by extract of Cajanus cajan (L.) Millsp and its component pinocembrin, and its mechanism is mostly speculated based on the commonness of legume plants and component analysis. SUMMARY

[0004] A first object of the present application is to provide the use of extract of Cajanus cajan (L.) Millsp or pinocembrin in the preparation of a medicine or functional food for relieving hyperuricemia and its complications, and the structure of the pinocembrin is as follows:

[0005]

[0006] Preferably, the complications are kidney damage caused by hyperuricemia, including kidney inflammation or fibrosis caused by hyperuricemia. Further preferably, the medicine or functional food can reduce kidney inflammation and fibrosis.

[0007] Preferably, the medicine can reduce serum uric acid, prevent the increase of serum creatinine and / or serum urea nitrogen, and increase the excretion of urinary creatinine and / or urinary urea nitrogen.

[0008] Preferably, the medicine is a combination of the extract of Caesalpinia diapenata or pinoresinol with pharmaceutically acceptable adjuvants into various dosage forms, preferably oral or injection.

[0009] Preferably, the extract of Caesalpinia diapenata is obtained by crushing Caesalpinia diapenata, then soaking in ethanol aqueous solution, filtering, and distilling under reduced pressure to obtain an ethanol extract, and then suspending the ethanol extract in water, extracting with ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure.

[0010] Further preferably, the ratio of Caesalpinia diapenata to ethanol aqueous solution is 3g / 10mL, and the volume percentage concentration of the ethanol aqueous solution is 95%.

[0011] Further preferably, the soaking extraction is performed for 3 times, the first time for 72h, the filter liquor is collected after the first soaking, the filter residue is soaked for 48h again, the filter liquor is collected after the second soaking, the filter residue is soaked for 24h for the third time, and the filter liquor after the three times of soaking is combined and distilled under reduced pressure.

[0012] Preferably, the pinoresinol is derived from Caesalpinia diapenata or other plants, or is chemically synthesized.

[0013] Beneficial effects:

[0014] 1) The present application finds that the extract of Caesalpinia diapenata and its active ingredient pinoresinol can reduce the serum uric acid level of hyperuricemic mice, prevent the increase of serum creatinine and / or serum urea nitrogen, and increase the excretion of urine urea nitrogen and / or urine creatinine; the extract of Caesalpinia diapenata and pinoresinol are highly safe, and the body weight of the mice after intervention is similar to that of the normal group, showing a steady growth trend. At the same time, the extract of Caesalpinia diapenata and pinoresinol can also restore the kidney and liver coefficients of hyperuricemic mice to normal levels, significantly alleviate the kidney damage caused by high uric acid, inhibit the inflammatory / fibrotic response of the kidney, and restore the physiological state of the kidney to normal levels.

[0015] 2) The extract of Caesalpinia diapenata and pinoresinol used in the present application are derived from natural plants and have high safety. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the effect of the extract of Caesalpinia diapenata and PSB on the kidney function and uric acid metabolism indicators of hyperuricemic mice in Example 1, wherein (A) is serum uric acid; (B) is urine uric acid; (C) is serum urea nitrogen; (D) is urine urea nitrogen; (E) is serum creatinine; (F) is urine creatinine; the data is represented as mean ± SD (n=8), ns is no significant difference, *P<0.05, **P<0.01, ***P<0.001.

[0017] Figure 2Figure 2 is the effect of Caesalpinia bonduc extract and PSB on body weight and organ coefficients of hyperuricemic mice in Example 2, wherein (A) body weight change on days 2-7 of administration; (B) kidney coefficient; (C) liver coefficient; (D) spleen coefficient; data are expressed as mean ± SD (n = 10), ns is no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.

[0018] Figure 3 Figure 3 is the effect of Caesalpinia bonduc extract and PSB on kidney pathology of hyperuricemic mice in Example 3, wherein (A) H&E staining of mouse kidney; (B) Masson staining of mouse kidney.

[0019] Figure 4 Figure 4 is the results of transcriptome data quality analysis in Example 4, wherein (A) analysis of the proportion of high-quality data; (B) correlation of gene expression levels between samples (gene expression level correlation is measured by Pearson correlation coefficient, and the closer the correlation coefficient is to 1, the higher the similarity of expression patterns between samples); (C) principal component analysis (PCA).

[0020] Figure 5 Figure 5 is the differential gene enrichment analysis of transcriptome data in Example 4, wherein (A) number of up-regulated and down-regulated genes in each comparison group; (B) gene clustering and expression trend analysis (the numbers between the trend module and the heat map in the gene expression trend heat map represent the number of differentially expressed genes in each cluster); (C) KEGG analysis of differential genes in CON group compared with HUA group; (D) KEGG analysis of differential genes in HUA group and PSB group.

[0021] Figure 6 Figure 6 is the correlation and data validation analysis of metabolomics samples in Example 5, wherein (A) QC sample correlation analysis; (B) QC sample relative standard deviation (RSD) distribution graph; (C) Pearson correlation analysis of experimental samples; (D) principal component analysis of experimental samples and QC samples.

[0022] Figure 7 Figure 7 is the kidney metabolomics analysis of CON, HUA and PSB groups of mice in Example 5, wherein (A) CON vs HUA volcano plot; (B) HUA vs PSB volcano plot; (C) cumulative trend analysis of differential metabolites in CON, HUA and PSB groups; (D) KEGG enrichment analysis of differential metabolites in CON and HUA groups; (E) KEGG enrichment analysis of differential metabolites in HUA and PSB groups; (F) changes in metabolite content in uric acid synthesis pathway compared with HUA group in PSB group (red represents down-regulation in PSB group, and yellow represents up-regulation); (G) content of hyperuricemia-related biomarkers.

[0023] Figure 8Figure 6 is the results of kidney inflammation and fibrosis-related gene expression in CON, HUA and PSB group mice in Example 6. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0025] Example 1 Effect of Caesalpinia bonduc extract and pinoresinol on uric acid, creatinine and urea nitrogen levels in hyperuricemic mice

[0026] 1.1 Experimental materials:

[0027] Experimental reagents: Caesalpinia bonduc extract, the preparation method is disclosed in patent document CN119350153A; pinoresinol, the extraction method is disclosed in patent document CN118924734A.

[0028] Experimental animals: 80 male Kunming mice (body weight 20-22g) were purchased from Guangdong Medical Experimental Animal Center. The mice were adaptively fed under the conditions of temperature 23±2℃, humidity 50-60%, 12h light and dark alternation for 1 week, and free drinking water. All animal experiments were in accordance with the guidelines of the Experimental Animal Use and Ethics Committee of Guangdong Institute of Biomedical Engineering (License No.: SYXK(YUE)2023-0183).

[0029] 1.2 Experimental method

[0030] Modeling method: hyperuricemic mouse models were induced by intraperitoneal injection of potassium oxonate (PO) and gavage of hypoxanthine (HX).

[0031] Grouping and administration: the mice were randomly divided into 8 groups (n=10): normal control group (CON), hyperuricemia group (HUA), positive control group (ALL, allopurinol, 5mg / kg / d), Caesalpinia bonduc extract treatment group (EEP, 30 and 120mg / kg / d, respectively marked as EEP-L and EEP-H) and pinoresinol treatment group (PSB, 30, 60, 120mg / kg / d, respectively marked as PSB-L, PSB-M, PSB-H). Except for the CON group, the rest of the groups were continuously injected with PO (100mg / kg / d) intraperitoneally and HX (500mg / kg / d) by gavage for 7 days. The CON group was injected with the same volume of normal saline. The ALL, EEP and PSB groups were gavaged with the corresponding drugs 1 hour after modeling, and the CON and HUA groups were gavaged with the same volume of normal saline.

[0032] Index testing: Urine samples were collected on the sixth day of dosing, and blood samples were collected one hour after dosing on the seventh day. Serum and urine uric acid levels were determined using a uric acid assay kit (Regan, phosphotungstic acid microplate method). Serum and urine urea nitrogen and creatinine levels were measured according to the instructions of the urea nitrogen (Nanjing Jiancheng, diacetyl oxime colorimetric method) and creatinine (Nanjing Jiancheng, sarcosine oxidase microplate method) assay kits.

[0033] 1.3 Experimental Results

[0034] The uric acid-lowering effects of pigeonpea extract and pinostrobin were evaluated by measuring serum uric acid levels. Figure 1 As shown in Figure A, serum uric acid levels in the hyperuricemia (HUA) group were significantly higher than those in the normal control group (CON), confirming the successful establishment of a hyperuricemia model. Allopurinol (ALL) treatment significantly reduced blood uric acid levels in hyperuricemic mice, even to levels lower than those in the CON group. Blood uric acid levels in the pigeon pea extract (EEP)-treated group were also lower than those in the CON group. Pinus sphaeroides (PSB)-treated group also reduced uric acid, but the dose-dependent effect did not reach statistical significance. These data collectively demonstrate that both pigeon pea extract and pinus sphaeroides have significant uric acid-lowering effects.

[0035] The diuretic effect of pigeonpea extract and pinostrobin was further evaluated by measuring urinary uric acid levels. Figure 1 As shown in Figure (B), urinary uric acid levels decreased to 276.22±31.24 μmol / L in the HUA group (427.39±61.37 μmol / L in the CON group), while those in the ALL group further decreased to 142.10±16.30 μmol / L. Compared with the ALL group, urinary uric acid levels in the EEP-L, EEP-H, PSB-L, PSB-M, and PSB-H groups rebounded to 234.27±39.48, 255.30±27.94, 205.46±37.66, 218.89±23.31, and 262.05±42.11 μmol / L, respectively. These results suggest that pigeonpea extract and pinospermum parkii extract are superior to allopurinol in promoting uric acid excretion.

[0036] The effects of EEP and PSB on renal function were assessed by measuring serum and urine urea nitrogen and creatinine levels. Figure 1 As shown in C, the serum urea nitrogen (SBUN) level in the HUA group was significantly higher than that in the CON group, while the SBUN level in the ALL group was even higher than that in the HUA group. In contrast, there was no significant difference between the EEP and PSB treatment groups and the HUA group. Figure 1 As shown in D, the urinary urea nitrogen (UUN) levels in the HUA and ALL groups were significantly decreased compared with the CON group; however, the UUN levels in the EEP and PSB treatment groups at all doses almost returned to normal levels. Figure 1E and 1F, serum creatinine (SCRE) and urinary creatinine (UCRE) showed similar trends in each group. These results indicated that hyperuricemia and allopurinol had adverse effects on renal function, while Caesalpinia extract and pinoresinol did not show nephrotoxicity.

[0037] Example 2 Effects of Caesalpinia extract and pinoresinol on body weight and organ coefficients of hyperuricemic mice

[0038] 2.1 Experimental method

[0039] The experimental mice were grouped and administered as in Example 1. After the last administration, the mice were sacrificed, and the kidneys, liver, and spleen were weighed to calculate the internal organ coefficients (organ coefficient = organ weight / mouse body weight).

[0040] 2.2 Experimental results

[0041] Figure 2 A is the change in mouse body weight on days 2-7 of administration. As shown in the figure, the mice in the CON group maintained daily weight gain, indicating that their health was good. The mice in the HUA group initially had normal daily weight gain, but later experienced a significant decrease. The mice in the ALL group showed progressive weight loss from day 5. In contrast, all EEP and PSB treatment groups maintained stable daily weight gain, indicating that their health was closer to that of the CON group.

[0042] As shown in Figure 2 B, the kidney coefficient of the HUA group was significantly higher than that of the CON group, suggesting hyperuricemia-induced kidney damage. ALL treatment caused the kidney coefficient to further abnormally increase, suggesting severe nephrotoxicity. However, the kidney coefficients of all EEP and PSB dose groups were not significantly different from those of the CON group, indicating that both can alleviate hyperuricemia-related kidney damage.

[0043] As shown in Figure 2 C, the liver coefficient of the HUA group was not significantly different from that of the CON group, while the liver coefficient of the ALL group was significantly higher, indicating that allopurinol has hepatotoxicity. The liver coefficients of all EEP and PSB dose groups were not significantly different from those of the CON group, indicating that they are good in liver safety.

[0044] As shown in Figure 2 D, the spleen coefficients of all treatment groups were not significantly different from those of the CON group, indicating that hyperuricemia had no significant effect on the spleen, and allopurinol, Caesalpinia extract, and pinoresinol were not toxic to the spleen.

[0045] Example 3 Effects of Caesalpinia extract and pinoresinol on kidney pathology in hyperuricemic mice

[0046] 3.1 Experimental method

[0047] The experimental mice were grouped and treated with the same medications as in Example 1. After the last dose, the mice were sacrificed and their kidney tissues were collected. Four kidneys were randomly selected from each group, rinsed with PBS buffer, and then immersed in formaldehyde solution for fixation overnight. Gradient dehydration and clearing were then performed, and the dehydrated tissues were embedded in paraffin. 5 μm thick tissue sections were prepared using a microtome. After baking, dewaxing, and hydration, they were stained with Masson staining and H&E staining, respectively. Finally, images were collected using an optical microscope.

[0048] 3.2 Experimental Results

[0049] like Figure 3 As shown in Figure A, light microscopic observation showed that the glomerular boundaries of mice in the CON group were clear, the renal tubule structure was intact, and no inflammatory cell infiltration was observed. In contrast, mice in the HUA group showed significant tubular dilation, glomerular atrophy and degeneration, and inflammatory cell infiltration within the glomeruli.

[0050] like Figure 3 As shown in Figure B, compared with the CON group, the blue-stained area in the kidneys of the HUA group increased, indicating severe tissue fibrosis. Notably, the pathological changes were more pronounced in the ALL group. These results suggest that hyperuricemia and allopurinol treatment can lead to renal impairment. However, treatment with pigeonpea extract and pinospermum parkii effectively ameliorated these pathological changes.

[0051] Example 4 Effect of Pinus glomerulone on the renal transcriptome of hyperuricemia mice

[0052] 4.1 Experimental methods

[0053] The experimental mice were grouped and treated with the same medication as in Example 1. After the last dose, the mice were killed, and transcriptome analysis was performed on the right kidney tissue of the CON group, HUA group, and PSB-H (PSB high-dose treatment) group of mice, with 3 biological replicates in each group. After extracting total RNA, sequencing was performed using the Illumina sequencing platform. The 3' end adapter sequence was removed by the Fastp tool and reads with an average quality value lower than Q20 were filtered. RSeQC was used to assess the saturation of sequencing data, and HISAT2 was used to align the filtered reads to the reference genome. The read counts of each gene were counted by HTSeq and standardized with FPKM (number of aligned reads per million per kilobase transcript). DESeq2 was used to screen differentially expressed genes (DEGs), and the screening criteria were log2-fold change value>1 and P value<0.05. Hierarchical cluster analysis of DEGs and samples was performed using the R language Pheatmap and ComplexHeatmap packages. Finally, ClusterProfiler was used to perform enrichment analysis based on GO and KEGG, and the significance threshold was set at P < 0.05.

[0054] 4.2 Experimental Results

[0055] As Figure 4 As shown in FIG. 4A, the Q30 value of the original reads was between 95.38% and 95.99%, indicating that the quality of the reads was sufficient for data analysis. Fastp was used to remove sequences with adapters at the 3' end and to remove reads with an average quality score below Q20. The average Clean Reads (%) (percentage of high-quality sequence reads to sequencing reads) of the CON group, HUA group, and PSB group samples were 98.88%, 98.87%, and 98.78%, respectively; the average Clean Data (%) (percentage of high-quality sequence bases to sequencing bases) were 98.59%, 98.60%, and 98.50%, respectively. The close proportions between the different groups of samples indicated that the data between the samples were comparable. Figure 4 As shown in FIGs. 4B and 4C, by Pearson correlation coefficient and principal component analysis, it was found that the three biological repeat samples in each group showed extremely strong correlation, indicating that the experimental repeatability was good.

[0056] To evaluate the effect of PSB on reducing uric acid at the gene level, we compared the transcriptomes of the CON, HUA, and PSB groups. As shown in FIG. 5A, 172 genes were up-regulated and 73 genes were down-regulated in the CON group compared with the HUA group; 174 genes were up-regulated and 70 genes were down-regulated in the PSB group compared with the HUA group. In terms of the number of differential genes, the gene expression pattern of the PSB group was consistent with that of the CON group. Figure 5 As shown in FIG. 5B, cluster analysis could divide the differential genes into 9 groups. Among the 473 differential genes, 316 genes (accounting for 66.81%) had consistent expression trends in the CON vs HUA and PSB vs HUA comparisons and were distributed in Cluster 3, 5, 6, 7, 8, and 9, indicating that the expression pattern of the kidney transcriptome of the CON and PSB treatment groups of mice had certain consistency, and also reflected the uric acid-lowering effect of PSB at the gene expression level. Figure 5 As shown in FIG. 5B, cluster analysis could divide the differential genes into 9 groups. Among the 473 differential genes, 316 genes (accounting for 66.81%) had consistent expression trends in the CON vs HUA and PSB vs HUA comparisons and were distributed in Cluster 3, 5, 6, 7, 8, and 9, indicating that the expression pattern of the kidney transcriptome of the CON and PSB treatment groups of mice had certain consistency, and also reflected the uric acid-lowering effect of PSB at the gene expression level.

[0057] As Figure 5KEGG analysis showed that compared with CON group, HUA group was significantly enriched in Pancreatic secretion, Steroid hormone biosynthesis, Protein digestion and absorption, Rheumatoid arthritis, TNF signaling pathway, Complement and coagulation cascades, Chemical carcinogenesis-DNA adducts, Cortisol synthesis and secretion, IL-17 signaling pathway, Aldosterone synthesis and secretion, Cushing syndrome, Cytokine-cytokine receptor interaction, etc. Among them, Steroid hormone biosynthesis / Cortisol synthesis and secretion, Rheumatoid arthritis, TNF signaling pathway, IL-17 signaling pathway and Cytokine-cytokine receptor interaction were closely related to inflammatory response. In the comparison between PSB and HUA, Cytokine-cytokine receptor interaction, TNF signaling pathway, IL-17 signaling pathway, Complement and coagulation cascades, Rheumatoid arthritis, NF-kappa B signaling pathway, PI3K-Akt signaling pathway, Chemokine signaling pathway, Viral protein interaction with cytokine and cytokine receptor, etc. were also significantly enriched Figure 5D), suggesting the potential role of PSB in preventing and treating the kidney inflammation caused by high uric acid. Compared with HUA, the cAMP signaling pathway, Relaxin signaling pathway and Serotonergic synapse signaling pathway were significantly up-regulated in PSB and CON groups, while the Complement and coagulation cascades, Hematopoietic cell lineage, Tuberculosis, TNF signaling pathway, cytokine-cytokine receptor interaction, retinol metabolism, linoleic acid metabolism, IL-17 signaling pathway and Steroid biosynthesis were significantly down-regulated Figure 5 B), indicating that HUA caused kidney inflammation and kidney fibrosis in mice, and PSB could alleviate the kidney inflammation and fibrosis caused by high uric acid in mice. At the same time, the chemical carcinogenesis-DNA adduct was also down-regulated, suggesting that PSB might have potential anti-cancer efficacy.

[0058] Example 5 Effect of pinocembrin on the kidney metabolome of hyperuricemic mice

[0059] 5.1 Experimental method

[0060] The experimental mice were grouped and administered according to Example 1. After the last administration, the mice were sacrificed, and the right kidney tissues of the CON, HUA and PSB-H (PSB high dose treatment) groups were subjected to metabolomic analysis, with 5 biological replicates in each group. A Thermo Vanquish UHPLC system (Thermo Fisher Scientific, USA) was used in combination with ACQUITY UPLC® BEH C18 columns (1.7 μm, 2.1 mm x 50 mm) to separate the samples, and a Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, USA) was used for mass spectrometry analysis. The data were analyzed by MetaboAnalyst 5.0 software, and the results are shown in Table 2. Separation was performed on an HSS T3 column (2.1 × 100 mm, 1.8 μm; Waters, Milford, MA, USA) under the following conditions: flow rate 0.3 mL / min; column temperature 40°C; injection volume 2 μL. Mass spectrometry parameters for positive ion mode: the mobile phases consisted of 0.1% formic acid in acetonitrile (B2) and 0.1% formic acid in water (A2). Gradient elution program: 0–1 min, 8% B2; 1–8 min, 8%–98% B2; 8–10 min, 98% B2; 10–10.1 min, 98%–8% B2; 10.1–12 min, 8% B2. In negative ion mode, the mobile phases consisted of acetonitrile (B3) and 5 mM ammonium formate in water (A3). The gradient elution program was as follows: 0–1 min, 8% B3; 1–8 min, 8%–98% B3; 8–10 min, 98% B3; 10–10.1 min, 98%–8% B3; 10.1–12 min, 8% B3. The mass spectrometer was a ThermoOrbitrap Exploris 120 (Thermo Fisher Scientific, USA).

[0061] Raw data were converted to .mzXML format using ProteoWizard, and peak alignment, retention time correction, and peak area extraction were performed using XCMS software. After structural identification of the metabolites, the data were preprocessed and quality controlled. OPLS-DA analysis was performed, screening for significantly differentially accumulated metabolites (DAMs) using criteria of VIP > 1 and P < 0.05. Finally, hierarchical cluster analysis of the differentially accumulated metabolites was performed using Pheatmap in the R language.

[0062] 5.2 Experimental Results

[0063] Non-targeted metabolomics analysis was performed on the kidneys of mice in the CON, HUA, and PSB-H groups. Pearson correlation analysis of the quality control samples QC showed that the metabolite expression patterns between samples were highly similar ( Figure 6 A). In the QC samples, the relative standard deviation RSD>0.8, indicating that the data is accurate ( Figure 6 B). Pearson correlation analysis of experimental samples showed that the metabolite expression patterns between samples were highly correlated ( Figure 6 C). PCA analysis of experimental samples and QC samples showed that the differences between QC samples were small, indicating that the method was stable and the data quality was good ( Figure 6 D) The experimental samples were significantly clustered within the group and significantly separated between groups, indicating high reproducibility between biological replicates.

[0064] A total of 1675 annotated metabolites were identified after screening. The HUA group upregulated 876 metabolites and downregulated 794 metabolites compared with the CON group ( Figure 7A). PSB-H group up-regulated 661 metabolites and down-regulated 636 metabolites compared with HUA group Figure 7 B). These differential metabolites were divided into 9 clusters Figure 7 C), in which cluster 3, 5, 6 group metabolites had consistent expression trends in CON group and PSB-H group, and these metabolites in these groups might be biomarkers of high uric acid kidney injury, and might be key regulatory metabolites of PSB alleviating kidney injury.

[0065] KEGG classification of the differential metabolites between CON and HUA groups showed that 44 differential metabolites were related to human diseases, 374 to metabolism, 33 to environmental information processing, 97 to organismal systems, and 7 to genetic information processing. KEGG classification of the differential metabolites between HUA and PSB-H groups showed that 58 were related to human diseases, 400 to metabolism, 40 to environmental information processing, 108 to organismal systems, and 11 to genetic information processing. KEGG enrichment analysis showed that among the top 20 significantly enriched pathways (total of 133 enriched pathways) between CON and HUA groups, the pathways closely related to inflammation and organ fibrosis after tissue injury included regulation of lipolysis in adipocytes, linoleic acid metabolism, taurine and hypotaurine metabolism, glyoxylate and dicarboxylate metabolism, arachidonic acid metabolism, cAMP signaling pathway, and aldosterone synthesis and secretion.Hyperuricemia caused metabolic disorder in mouse kidney, significantly enriched in pathways related to amino acid metabolism (aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, biosynthesis of amino acids, protein digestion and absorption, glycine, serine and threonine metabolism), sugar metabolism (TCA cycle, pyruvate metabolism and central carbon metabolismin cancer), lipid metabolism (arachidonic acid metabolism and linoleic acid metabolism) and neurotransmitter transmission (neuroactive ligand-receptor interaction, serotonergic synapse). KEGG analysis of HUA vs PSB-H group showed that 20 of the 148 significantly enriched pathways were still mainly related to inflammation and fibrosis, while also involved in amino acid, sugar and lipid metabolism. Uric acid synthesis pathway was strongly inhibited by PSB, while adenosine, inosine, guanosine and other purine metabolites were significantly up-regulated, and glycine, glutamine, uric acid and other uric acid synthesis related metabolites were down-regulated, suggesting that PSB reduced uric acid levels by inhibiting purine metabolism. The levels of high uric acid biomarkers glutamine and linoleic acid were significantly decreased after PSB treatment. In addition, ABC transporters, GABAergic synapse, biotin metabolism and mineral absorption pathways were significantly enriched, indicating that PSB might improve metabolic disorders by regulating the gut-kidney axis. In summary, PSB effectively alleviated hyperuricemia-related renal molecular injury by regulating purine metabolism and inhibiting inflammation-driven fibrosis, and its mechanism involved the synergistic effect of multiple pathways.

[0066] Example 6 Pinocembrin inhibits the expression of mouse kidney inflammation and fibrosis-related genes

[0067] 6.1 Experimental method

[0068] The experimental mice were grouped and administered in the same manner as in Example 1. After the last administration, the mice were sacrificed, and the kidney tissues of the CON group, the HUA group, and the PSB-H (high-dose PSB treatment) group were taken. Total RNA was extracted using TRIzol reagent (Invitrogen), and reverse transcription was performed using ReverTra Ace qPCR Reverse Transcription Kit (FSQ-301, TOYOHA CO., LTD.). qPCR Reverse Transcription Kit (FSQ-301, TOYOHA CO., LTD.). The real-time fluorescence quantitative PCR amplification system used ChamQ SYBR qPCR Master Mix (Q311-02, Norgen Biotek Corporation). The β-actin gene was used as an internal reference gene, and the relative expression of the target gene was calculated by 2 ^-ΔΔCt -actin gene was used as an internal reference gene, and the relative expression of the target gene was calculated by 2

[0069] Table 1 qRT-PCR Primers

[0070]

[0071] 6.2 Experimental Results

[0072] Kidney tissue sections and omics data confirmed that PSB can alleviate kidney inflammation and fibrosis induced by hyperuricemia. To further study this mechanism, we analyzed the expression of related genes by real-time fluorescence quantitative PCR (qRT-PCR). As shown in FIG. 6B, PSB treatment significantly down-regulated the expression levels of pro-inflammatory markers (TNF-α and IL-6) and fibrosis-related genes (TGF-β1, Col1α1, and FN1). Figure 8 TNF-α and IL-6) and fibrosis-related genes (TGF-β1, Col1α1, and FN1).

Claims

1. Application of pigeon pea extract or pinostrobin in the preparation of medicines or functional foods for alleviating hyperuricemia and its complications.

2. The use according to claim 1, characterized in that The complication is renal damage caused by hyperuricemia.

3. The use according to claim 2, characterized in that The renal injury is renal inflammation or fibrosis.

4. The use according to claim 3, characterized in that The medicine or functional food can alleviate kidney inflammation and fibrosis.

5. The use according to claim 1, characterized in that The drug can reduce serum uric acid, prevent the increase of serum creatinine and / or serum urea nitrogen, and increase the excretion of urine creatinine and / or urine urea nitrogen.

6. The use according to claim 1, characterized in that The medicine is prepared by combining pigeon pea extract or pinostrobin with pharmaceutically acceptable auxiliary materials to form various dosage forms.

7. The use according to claim 1, characterized in that The pigeon pea extract is obtained by crushing the pigeon pea, soaking and extracting the pigeon pea in an ethanol aqueous solution, filtering, and distilling under reduced pressure to obtain an ethanol extract, then suspending the ethanol extract in water, extracting with ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure.

8. The use according to claim 7, characterized in that The material-liquid ratio of pigeon pea to ethanol aqueous solution is 3 g / 10 mL, and the volume percentage concentration of the ethanol aqueous solution is 95%.

9. The use according to claim 7, characterized in that The soaking extraction times were 3 times, the first soaking was 72 hours, after the soaking was completed, the filtrate was filtered and collected, the filter residue was soaked again for 48 hours, the filtrate was collected after filtration, and the filter residue was soaked for the third time for 24 hours. The filtrates after the three soakings were combined and distilled under reduced pressure.

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