Design, synthesis, and biological evaluation of 5-(1H-indol-5-yl)isoxazole-3-carboxylic acids as novel xanthine oxidase inhibitors.

Xanthine oxidase (XO) is a key enzyme for the production of uric acid in the human body. XO inhibitors (XOIs) are clinically used for the treatment of hyperuricemia and gout, as they can effectively inhibit the production of uric acid. Previous studies indicated that both indole and isoxazole derivatives have good inhibitory effects against XO. Here, we designed and synthesized a novel series of N-5-(1H-indol-5-yl)isoxazole-3-carboxylic acids according to bioisosteric replacement and hybridization strategies. Among the obtained target compounds, compound 6c showed the best inhibitory activity against XO with an IC50 value of 0.13 µM, which was 22-fold higher than that of the classical antigout drug allopurinol (IC50 = 2.93 µM). Structure-activity relationship analysis indicated that the hydrophobic group on the nitrogen atom of the indole ring is essential for the inhibitory potencies of target compounds against XO. Enzyme kinetic studies proved that compound 6c acted as a mixed-type XOI. Molecular docking studies showed that the target compound 6c could not only retain the key interactions similar to febuxostat at the XO binding site but also generate some new interactions, such as two hydrogen bonds between the oxygen atom of the isoxazole ring and the amino acid residues Ser876 and Thr1010. These results indicated that 5-(1H-indol-5-yl)isoxazole-3-carboxylic acid might be an efficacious scaffold for designing novel XOIs and compound 6c has the potential to be used as a lead for further the development of novel anti-gout candidates.

A derivative of trihydroxynaphthalenone and a pyrone metabolite from the endophytic fungus Talaromyces purpurpgenus.

A newly discovered trihydroxynaphthalenone derivative, epoxynaphthalenone (1) involving the condensation of ortho-hydroxyl groups into an epoxy structure, and a novel pyrone metabolite characterized as pyroneaceacid (2), were extracted from Talaromyces purpurpgenus, an endophytic fungus residing in Rhododendron molle. The structures of these compounds were elucidated through a comprehensive analysis of their NMR and HRESIMS data. The determination of absolute configurations was accomplished using electronic circular dichroism (ECD) calculations and CD spectra. Notably, these recently identified metabolites exhibited a moderate inhibitory activity against xanthine oxidase (XOD).

Identification of Xanthine Oxidase Inhibitors from Celery Seeds Using Affinity Ultrafiltration-Liquid Chromatography-Mass Spectrometry.

Celery seeds have been used as an effective dietary supplement to manage hyperuricemia and diminish gout recurrence. Xanthine oxidase (XOD), the critical enzyme responsible for uric acid production, represents the most promising target for anti-hyperuricemia in clinical practice. In this study, we aimed to establish a method based on affinity ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) to directly and rapidly identify the bioactive compounds contributing to the XOD-inhibitory effects of celery seed crude extracts. Chemical profiling of celery seed extracts was performed using UPLC-TOF/MS. The structure was elucidated by matching the multistage fragment ion data to the database and publications of high-resolution natural product mass spectrometry. Thirty-two compounds, including fourteen flavonoids and six phenylpeptides, were identified from celery seed extracts. UF-LC-MS showed that luteolin-7-O-apinosyl glucoside, luteolin-7-O-glucoside, luteolin-7-O-malonyl apinoside, luteolin-7-O-6'-malonyl glucoside, luteolin, apigenin, and chrysoeriol were potential binding compounds of XOD. A further enzyme activity assay demonstrated that celery seed extract (IC50 = 1.98 mg/mL), luteolin-7-O-apinosyl glucoside (IC50 = 3140.51 µmol/L), luteolin-7-O-glucoside (IC50 = 975.83 µmol/L), luteolin-7-O-6'-malonyl glucoside (IC50 = 2018.37 µmol/L), luteolin (IC50 = 69.23 µmol/L), apigenin (IC50 = 92.56 µmol/L), and chrysoeriol (IC50 = 40.52 µmol/L) could dose-dependently inhibit XOD activities. This study highlighted UF-LC-MS as a useful platform for screening novel XOD inhibitors and revealed the chemical basis of celery seed as an anti-gout dietary supplement.

In Vitro Inhibition of Xanthine Oxidase Purified from Arthritis Serum Patients by Nanocurcumin and Artemisinin Active Compounds.

Curcumin and artemisinin are commonly used in traditional East Asian medicine. In this study, we investigated the inhibitory effects of these active compounds on xanthine oxidase (XO) using allopurinol as a control. XO was purified from the serum of arthritis patients through ammonium sulfate precipitation (65%) and ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose. The specific activity of the purified enzyme was 32.5 U/mg protein, resulting in a 7-fold purification with a yield of 66.8%. Molecular docking analysis revealed that curcumin had the strongest interaction energy with XO, with a binding energy of -9.28 kcal/mol. The amino acid residues Thr1077, Gln762, Phe914, Ala1078, Val1011, Glu1194, and Ala1079 were located closer to the binding site of curcumin than artemisinin, which had a binding energy of -7.2 kcal/mol. In vitro inhibition assays were performed using nanocurcumin and artemisinin at concentrations of 5, 10, 15, 20, and 25 µg/mL. Curcumin inhibited enzyme activity by 67-91%, while artemisinin had a lower inhibition ratio, which ranged from 40-70% compared to allopurinol as a control.

Design, synthesis and structure-activity relationship of N-phenyl aromatic amide derivatives as novel xanthine oxidase inhibitors.

Our previous studies suggested that N-phenyl aromatic amides are a class of promising xanthine oxidase (XO) inhibitor chemotypes. In this effort, several series of N-phenyl aromatic amide derivatives (4a-h, 5-9, 12i-w, 13n, 13o, 13r, 13s, 13t and 13u) were designed and synthesized to carry out an extensive structure-activity relationship (SAR). The investigation provided some valuable SAR information and identified N-(3-(1H-imidazol-1-yl)-4-((2-methylbenzyl)oxy)phenyl)-1H-imidazole-4-carboxamide (12r, IC50 = 0.028 µM) as the most potent XO inhibitor with close in vitro potency to that of topiroxostat (IC50 = 0.017 µM). Molecular docking and molecular dynamics simulation rationalized the binding affinity through a series of strong interactions with the residues Glu1261, Asn768, Thr1010, Arg880, Glu802, etc. In vivo hypouricemic studies also suggested that the uric acid lowering effect of compound 12r was improved compared with the lead g25 (30.61 % vs 22.4 % reduction in uric acid levels at 1 h; 25.91 % vs 21.7 % reduction in AUC of uric acid) . Pharmacokinetic studies revealed that compound 12r presented a short t1/2 of 0.25 h after oral administration. In addition, 12r has non-cytotoxicity against normal cell HK-2. This work may provide some insights for further development of novel amide-based XO inhibitors.

Pharmacological evaluation of a novel skeleton compound isobavachin (4',7-dihydroxy-8-prenylflavanone) as a hypouricemic agent: Dual actions of URAT1/GLUT9 and xanthine oxidase inhibitory activity.

Previously we discovered a novel natural scaffold compound, isobavachin (4', 7-dihydroxy-8-prenylflavanone), as a potent URAT1 inhibitor by shape and structure based on a virtue screening approach. In this study, further urate-lowering mechanism, pharmacokinetics and toxicities of isobavachin were conducted. Isobavachin inhibited URAT1 with an IC50 value of 0.24 ± 0.06 µM, and residues S35, F365, I481 and R477 of URAT1 contributed to high affinity for isobavachin. Isobavachin also inhibited glucose transporter 9 (GLUT9), another pivotal urate reabsorption transporter, with an IC50 value of 1.12 ± 0.26 µM. Molecular docking and MMGBSA results indicated that isobavachin might compete residues R171, L75 and N333 with uric acid, which leads to inhibition of uric acid transport of GLUT9. Isobavachin weakly inhibited urate secretion transporters OAT1 with an IC50 value of 4.38 ± 1.27 µM, OAT3 with an IC50 of 3.64 ± 0.62 µM, and ABCG2 with an IC50 of 10.45 ± 2.17 µM. Isobavachin also inhibited xanthine oxidase (XOD) activity in vitro with an IC50 value of 14.43 ± 3.56 µM, and inhibited the hepatic XOD activities at 5-20 mg/kg in vivo. Docking and MMGBSA analysis indicated that isobavachin might bind to the Mo-Pt catalyze center of XOD, which leads to inhibition of uric acid production. In vivo, isobavachin exhibited powerful urate-lowering and uricosuric effects at 5-20 mg/kg compared with the positive drugs morin (20 mg/kg) and RDEA3170 (10 mg/kg). Safety assessments revealed that isobavachin was safe and had no obvious toxicities. Isobavachin has little cell toxicity in HK2 cells as indicated by the MTT assay. In vivo, after treatment with 50 mg/kg isobavachin for 14 days, isobavachin had little renal toxicity, as revealed by serum CR/BUN levels, and no hepatotoxicity as revealed by ALT/AST levels. Further HE examination also suggests that isobavachin has no obvious kidney/liver damage. A pharmacokinetic study in SD rats indicated isobavachin had lower bioavailability (12.84 ± 5.13 %) but long half-time (7.04 ± 2.68 h) to maintain a continuous plasma concentration. Collectively, these results indicate that isobavachin deserves further investigation as a candidate anti-hyperuricemic drug with a novel mechanism of action: selective urate reabsorption inhibitor (URAT1/GLUT9) with a moderate inhibitory effect on XOD.

Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway.

Xanthine oxidase (XO) is an important source of reactive oxygen species. This study investigated whether XO inhibition exerts renoprotective effects by inhibiting vascular endothelial growth factor (VEGF) and NADPH oxidase (NOX) in diabetic kidney disease (DKD). Febuxostat (5 mg/kg) was administered to streptozotocin (STZ)-treated 8-week-old male C57BL/6 mice via intraperitoneal injection for 8 weeks. The cytoprotective effects, its mechanism of XO inhibition, and usage of high-glucose (HG)-treated cultured human glomerular endothelial cells (GECs) were also investigated. Serum cystatin C, urine albumin/creatinine ratio, and mesangial area expansion were significantly improved in febuxostat-treated DKD mice. Febuxostat reduced serum uric acid, kidney XO levels, and xanthine dehydrogenase levels. Febuxostat suppressed the expression of VEGF mRNA, VEGF receptor (VEGFR)1 and VEGFR3, NOX1, NOX2, and NOX4, and mRNA levels of their catalytic subunits. Febuxostat caused downregulation of Akt phosphorylation, followed by the enhancement of dephosphorylation of transcription factor forkhead box O3a (FoxO3a) and the activation of endothelial nitric oxide synthase (eNOS). In an in vitro study, the antioxidant effects of febuxostat were abolished by a blockade of VEGFR1 or VEGFR3 via NOX-FoxO3a-eNOS signaling in HG-treated cultured human GECs. XO inhibition attenuated DKD by ameliorating oxidative stress through the inhibition of the VEGF/VEGFR axis. This was associated with NOX-FoxO3a-eNOS signaling.

Xanthine oxidase mediates chronic stress-induced cerebrovascular dysfunction and cognitive impairment.

Xanthine oxidase (XO) mediates vascular function. Chronic stress impairs cerebrovascular function and increases the risk of stroke and cognitive decline. Our study determined the role of XO on stress-induced cerebrovascular dysfunction and cognitive decline. We measured middle cerebral artery (MCA) function, free radical formation, and working memory in 6-month-old C57BL/6 mice who underwent 8 weeks of control conditions or unpredictable chronic mild stress (UCMS) with or without febuxostat (50 mg/L), a XO inhibitor. UCMS mice had an impaired MCA dilation to acetylcholine vs. controls (p < 0.0001), and increased total free radical formation, XOR protein levels, and hydrogen peroxide production in the liver compared to controls. UCMS increased hydrogen peroxide production in the brain and cerebrovasculature compared to controls. Working memory, using the y-maze test, was impaired (p < 0.05) in UCMS mice compared to control mice. However, blocking XO using febuxostat prevented the UCMS-induced impaired MCA response, while free radical production and hydrogen peroxide levels were similar to controls in the liver and brain of UCMS mice treated with febuxostat. Further, UCMS + Feb mice did not have a significant reduction in working memory. These data suggest that the cerebrovascular dysfunction associated with chronic stress may be driven by XO, which leads to a reduction in working memory.

Syntheses, Crystal Structures and Xanthine Oxidase Inhibitory Activity of Aroylhydrazones.

A series of hydrazones, (E)-N'-(4-hydroxy-3-methoxybenzylidene)-4-nitrobenzohydrazide (1), (E)-4-(dimethylamino)-N'-(4-hydroxy-3-methoxybenzylidene)benzohydrazide (2), N'-(2-hydroxy-5-methylbenzylidene)-4-nitrobenzohydrazide (3) and 2-fluoro-N'-(2-hydroxy-5-methylbenzylidene)benzohydrazide (4), were prepared and structurally characterized by elemental analysis, IR and 1H NMR spectra, and X-ray single crystal determination. The xanthine oxidase inhibitory activities of the compounds were investigated. Among the compounds, N'-(3-methoxybenzylidene)-4-nitrobenzohydrazide (1) showed the strongest activity. Docking simulations were performed to insert the compounds into the crystal structure of xanthine oxidase at the active site and to investigate the probable binding modes.

Flavonoid extract of saffron by-product alleviates hyperuricemia via inhibiting xanthine oxidase and modulating gut microbiota.

Hyperuricemia was associated with the overproduction or inadequate excretion of uric acid, while its association with gut microbiota has emerged although few studies were focused on it. Previously, we have reported a flavonoid extract from saffron floral bio-residues lowered uric acid in potassium oxonate-induced hyperuricemic mice. In this study, the impacts of the flavonoid extract on potassium oxonate-induced hyperuricemic rats were evaluated through its effects on serum, renal, intestinal uric acid, and xanthine oxidase activity. At the same time, the microbial and metabolic features of the flavonoid extract against hyperuricemia were explored using 16S rRNA sequencing techniques and serum metabolomics, respectively. According to the results, the flavonoid extract lowered serum and intestinal uric acid levels in hyperuricemic rats without kidney damage. On the one hand, it inhibited serum and liver xanthine oxidase activities and down-regulated the expression of hepatic xanthine oxidase. On the other hand, it ameliorated the hyperuricemia-associated gut microbiota dysbiosis and alleviated the disturbance of serum metabolome, especially of lipid and amino acid metabolites. The results suggested that the flavonoid extract of saffron floral bio-residues exerts a potent antihyperuricemia effect by inhibiting xanthine oxidase to decrease uric acid production and modulating gut microbiota related to host metabolism.

Protective effects of corni fructus extract in mice with potassium oxonate-induced hyperuricemia.

Corni fructus is consumed as food and herbal medicine in Chinese culture. Studies have revealed that corni fructus exhibits potent antioxidant activity; however, few studies have investigated the ability of corni fructus to lower uric acid concentrations. In this study, the xanthine oxidase (XO) inhibition and uric acid-lowering effect of corni fructus extract (CFE) were evaluated in mice with potassium oxonate-induced hyperuricemia. Hyperuricemia is a chronic disease prevalent worldwide and is associated with high recurrence rates. In addition, drugs used to treat hyperuricemia induce side effects that discourage patient compliance. Hyperuricemia induces metabolic imbalances resulting in accumulative uric acid deposition in the joints and soft tissues. Hyperuricemia not only induces gout but also interrupts hepatic and renal function, thereby trigging severe inflammation and various complications, including obesity, nonalcoholic fatty liver disease, diabetes, and metabolic diseases. In this study, the ethyl acetate fraction (EAF) of CFE resulted in yields of antioxidant photochemical components significantly higher than those of CFEs formed using other substances. The EAF of CFE exhibited high free radical scavenging activity and XO inhibition and effectively lowered uric acid concentrations in the animal model of chemically induced hyperuricemia. The results of this study can serve as a reference for the prevention of preclinical gout as well as for functional food research.

Identification of novel xanthine oxidase inhibitors via virtual screening with enhanced characterization of molybdopterin binding groups.

Xanthine oxidase (XO) is an important therapeutic target for the treatment of hyperuricemia and gout. A virtual screening strategy with enhanced characterization of the molybdopterin binding group (MBG) was applied for the identification of novel XO inhibitors. Briefly, a 3D QSAR pharmacophore with fragment recognition capability was constructed by setting the MBG as a customized-pharmacophore feature. In addition, 2D QSAR was established with descriptors based on density functional theory (DFT), physical and chemical properties as well as topological properties. Descriptors related to metal ion recognition were emphasized to enhance the characterization of the MBG and to improve the screening efficiency. The 3D and 2D QSAR models were combined with the pharmacophore derived from XO-inhibitor complexes and docking with hydrogen bond constraints to screen the compound library of Specs. After two rounds of screening, six compounds with significant inhibition against XO were identified and the most active one XO-33 showed an IC50 of 23.3 nM. These compounds are structurally distinct from the known XO inhibitors, and provide new chemical prototypes for further discovery of potent and novel XO inhibitors.

Discovery of 4-(phenoxymethyl)-1H-1,2,3-triazole derivatives as novel xanthine oxidase inhibitors.

A series of 4-(phenoxymethyl)-1H-1,2,3-triazole derivatives were designed, synthesized, and evaluated for their xanthine oxidase (XO) inhibitory activities. Among these compounds, 9m emerged as the most effective XO inhibitor with an IC50 value of 0.70 µM, which was approximately 14-fold more potent than allopurinol. Additionally, compound 9m displayed favorable drug-like properties with ligand efficiency (LE) and lipophilic ligand efficiency (LLE) values of 0.33 and 3.41, respectively. We further explored the binding mode of 9m in complex with XO by molecular docking and molecular dynamics studies. In vivo hypouricemic studies also suggested that 9m could effectively lower the serum uric acid levels of rat. In summary, compound 9m could be a promising lead for further development of XO inhibitors.

Intramolecular hydrogen bond interruption and scaffold hopping of TMC-5 led to 2-(4-alkoxy-3-cyanophenyl)pyrimidine-4/5-carboxylic acids and 6-(4-alkoxy-3-cyanophenyl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-ones as potent pyrimidine-based xanthine oxidase inhibitors.

Many pyrimidine-based xanthine oxidase (XO) inhibitors with diverse chemotypes have been reported recently. Our previous study revealed that 2-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acid derivatives exhibited remarkable XO inhibitory potency. Notably, an intramolecular hydrogen bond (IMHB) formed between amino and carboxylic groups could be observed. With the hope to expand the structure-activity relationships (SARs) and obtain potential pyrimidine-based XO inhibitors, IMHB interruption and scaffold hopping were carried out on these compounds to design 2-(4-alkoxy-3-cyanophenyl)pyrimidine-4/5-carboxylic acids (11a-11n and 15a-15j) and 6-(4-alkoxy-3-cyanophenyl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-ones (19a-19j). Among them, compound 19a (IC50 = 0.039 µM) was identified as the most promising compound with substantially higher in vitro inhibitory potency than allopurinol (IC50 = 7.590 µM) and comparable to febuxostat (IC50 = 0.028 µM). The SAR analysis revealed that interrupting the IMHB through the removal of the amino group could damage the XO inhibitory potency; pyrimidine-4-carboxylic acid moiety was more beneficial for the XO inhibitory potency than the pyrimidine-5-carboxylic acid moiety. Additionally, enzyme kinetics studies suggested that compounds 11a, 15a and 19a acted as mixed-type inhibitors for XO and the removal of 6-position amino group resulted in a weakened affinity to the free enzyme, but an enhanced binding to the enzyme-substrate complex. Molecular modeling provided a reasonable explanation for the SARs observed in this study. Furthermore, in vivo hypouricemic effects demonstrated that compounds 15a and 19a could effectively reduce serum uric acid levels at an oral dose of 10 mg/kg, with 19a demonstrating a stronger effect than 15a. Therefore, our study proved that 6-(4-alkoxy-3-cyanophenyl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-ones were potent pyrimidine-based XO inhibitors and compound 19a required further structural optimization as a potential and efficacious agents for the treatment of hyperuricemia and gout.

Rapid characterisation of xanthine oxidase inhibitors from the flowers of Chrysanthemum morifolium Ramat. Using metabolomics approach.

INTRODUCTION: Hyperuricemia is the key risk factor for gout, in which the elevated uric acid is attributed to the oxidation of hypoxanthine and xanthine to uric acid by xanthine oxidase (XO). Adverse effects of the current treatments lead to an urgent need for safer and more effective alternative from natural resources. OBJECTIVE: To compare the metabolite profile of Chrysanthemum morifolium flower fraction with that of its detannified fraction in relation to XO inhibitory activity using a rapid and effective metabolomics approach. METHODS: Proton nuclear magnetic resonance (1 H-NMR)-based metabolomics approach coupled with multivariate data analysis was utilised to characterise the XO inhibitors related to the antioxidant properties, total phenolic, and total flavonoid contents of the C. morifolium dried flowers. RESULTS: The highest XO inhibitory activity, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, total phenolic and flavonoid content with strong positive correlation between them were observed in the ethyl acetate (EtOAc) fraction. Detannified EtOAc showed higher XO inhibitory activity than non-detannified EtOAc fraction. A total of 17 metabolites were tentatively identified, of which three namely kaempferol, 4-hydroxybenzoic acid and apigenin, could be suggested to be responsible for the strong XO inhibitory activity. Additive interaction between 4-hydroxybenzoic acid and apigenin (or kaempferol) in XO inhibition was demonstrated in the interaction assay conducted. CONCLUSION: Chrysanthemum morifolium dried flower-part could be further explored as a natural XO inhibitor for its anti-hyperuricemic potential. Metabolomics approach served as an effective classification of plant metabolites responsible for XO inhibitory activity, and demonstrated that multiple active compounds can work additively in giving combined inhibitory effects.

Methyl and Isopropyl N-Methylanthranilates Affect Primary Macrophage Function - An Insight into the Possible Immunomodulatory Mode of Action.

To complement the knowledge on the anti-inflammatory activity of methyl and isopropyl N-methylanthranilates, two natural products with panacea-like properties, we investigated their effects on thioglycolate-elicited macrophages by evaluating macrophage ability to metabolize MTT, macrophage membrane function, and macrophage myeloperoxidase and phagocytic activities. Moreover, two additional aspects of the inflammatory response of these compounds, their inhibitory activity on xanthine oxidase and catalase, were studied. It was found that these two compounds regulate elicited macrophage functions, most probably by interfering with the function of cell membranes and changing the reducing cellular capacity or enzyme activity of macrophages. Nonetheless, no significant inhibitory action either towards xanthine oxidase or catalase was found, suggesting that the inhibition of these enzymes is not involved in the anti-inflammatory mode of action of these two esters.

Design, synthesis, and biological evaluation of N-(3-cyano-1H-indol-5/6-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamides and 5-(6-oxo-1,6-dihydropyrimidin-2-yl)-1H-indole-3-carbonitriles as novel xanthine oxidase inhibitors.

Xanthine oxidase (XO) has been an important target for the treatment of hyperuricemia and gout. The analysis of potential interactions of pyrimidinone and 3-cyano indole pharmacophores present in the corresponding reported XO inhibitors with parts of the XO active pocket indicated that they both can be used as effective fragments for the fragment-based design of nonpurine XO inhibitors. In this paper, we adopted the fragment-based drug design strategy to link the two fragments with an amide bond to design the type 1 compounds 13a-13w,14c, 14d, 14f, 14g, 14j, 14k, and 15g. Compound 13g displayed an evident XO inhibitory potency (IC50 = 0.16 µM), which was 52.3-fold higher than that of allopurinol (IC50 = 8.37 µM). For comparison, type 2 compounds 5-(6-oxo-1,6-dihydropyrimidin-2-yl)-1H-indole-3-carbonitriles (25c-25g) were also designed by linking the two fragments with a single bond directly. The results showed that compound 25c from the latter series displayed the best inhibitory potency (IC50 = 0.085 µM), and it was 98.5-fold stronger than that of allopurinol (IC50 = 8.37 µM). These results suggested that amide and single bonds were applicable for linking the two fragments together to obtain potent nonpurine XO inhibitors. The structure-activity relationship results revealed that hydrophobic groups at N-atom of the indole moiety were indispensable for the improvement of the inhibitory potency in vitro against XO. In addition, enzyme kinetics studies suggested that compounds 13g and 25c, as the most promising XO inhibitors for the two types of target compounds, acted as mixed-type inhibitors for XO. Moreover, molecular modeling studies suggested that the pyrimidinone and indole moieties of the target compounds could interact well with key amino acid residues in the active pocket of XO. Furthermore, in vivo hypouricemic effect demonstrated that compounds 13g and 25c could effectively reduce serum uric acid levels at an oral dose of 10 mg/kg. Therefore, compounds 13g and 25c could be potential and efficacious agents for the treatment of hyperuricemia and gout.

Development of a method to screen and isolate xanthine oxidase inhibitors from black bean in a single step: Hyphenation of semipreparative liquid chromatography and stepwise flow rate countercurrent chromatography.

Black bean, in which isoflavones are the main active constituent, also contains saponins and monoterpenes. Soybean isoflavone is a secondary metabolite that is formed during the growth of soybean; it exhibits antioxidant and cardiovascular activities and traces estrogen-like effects. In this study, black bean isoflavones were extracted with n-butanol, and ultrafiltration-liquid chromatography-mass spectrometry was used to screen their activity. Subsequently, the inhibitors were isolated and purified using semipreparative liquid chromatography and stepwise flow rate countercurrent chromatography. Thereafter, five active compounds were identified using mass spectrometry and nuclear magnetic resonance experiments. Finally, the inhibition types of the xanthine oxidase inhibitors were determined using enzymatic kinetic studies. The IC50 values of daidzin, glycitein-7-O-glucoside, genistin, daidzein, and genistein were determined to be 35.08, 56.22, 30.76, 68.79, and 95.37 µg/mL, respectively. Daidzin, genistin, and daidzein exhibited reversible inhibition, whereas glycitein-7-O-glucoside and genistein presented irreversible inhibition. This novel approach, which was based on ultrafiltration-liquid chromatography-mass spectrometry and stepwise flow rate countercurrent chromatography, is a powerful method for screening and isolating xanthine oxidase inhibitors from complex matrices. The study of enzyme inhibition types is helpful for understanding the underlying inhibition mechanism. Therefore, a beneficial platform was developed for the large-scale production of bioactive and nutraceutical ingredients.

α-Amyrin and ß-Amyrin Isolated from Celastrus hindsii Leaves and Their Antioxidant, Anti-Xanthine Oxidase, and Anti-Tyrosinase Potentials.

Celastrus hindsii is a popular medicinal plant in Vietnam and Southeast Asian countries as well as in South America. In this study, an amount of 12.05 g of an α-amyrin and ß-amyrin mixture was isolated from C. hindsii (10.75 g/kg dry weight) by column chromatography applying different solvent systems to obtain maximum efficiency. α-Amyrin and ß-amyrin were then confirmed by gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR). The antioxidant activities of the α-amyrin and ß-amyrin mixture were determined via 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays with IC50 of 125.55 and 155.28 µg/mL, respectively. The mixture exhibited a high potential for preventing gout by inhibiting a relevant key enzyme, xanthine oxidase (XO) (IC50 = 258.22 µg/mL). Additionally, an important enzyme in skin hyperpigmentation, tyrosinase, was suppressed by the α-amyrin and ß-amyrin mixture (IC50 = 178.85 µg/mL). This study showed that C. hindsii is an abundant source for the isolation of α-amyrin and ß-amyrin. Furthermore, this was the first study indicating that α-amyrin and ß-amyrin mixture are promising in future therapies for gout and skin hyperpigmentation.

Natural Xanthine Oxidase Inhibitor 5-O-Caffeoylshikimic Acid Ameliorates Kidney Injury Caused by Hyperuricemia in Mice.

Xanthine oxidase (XOD) inhibition has long been considered an effective anti-hyperuricemia strategy. To identify effective natural XOD inhibitors with little side effects, we performed a XOD inhibitory assay-coupled isolation of compounds from Smilacis Glabrae Rhizoma (SGR), a traditional Chinese medicine frequently prescribed as anti-hyperuricemia agent for centuries. Through the in vitro XOD inhibitory assay, we obtained a novel XOD inhibitor, 5-O-caffeoylshikimic acid (#1, 5OCSA) with IC50 of 13.96 µM, as well as two known XOD inhibitors, quercetin (#3) and astilbin (#6). Meanwhile, we performed in silico molecular docking and found 5OCSA could interact with the active sites of XOD (PDB ID: 3NVY) with a binding energy of -8.6 kcal/mol, suggesting 5OCSA inhibits XOD by binding with its active site. To evaluate the in vivo effects on XOD, we generated a hyperuricemia mice model by intraperitoneal injection of potassium oxonate (300 mg/kg) and oral gavage of hypoxanthine (500 mg/kg) for 7 days. 5OCSA could inhibit both hepatic and serum XOD in vivo, together with an improvement of histological and multiple serological parameters in kidney injury and HUA. Collectively, our results suggested that 5OCSA may be developed into a safe and effective XOD inhibitor based on in vitro, in silico and in vivo evidence.