Inhibitory effects of flavonoids on aldehyde oxidase activity

Flavonoids are an important group of natural compounds that can interfere with the activity of some enzymes. In this study, effects of various flavonoids on aldehyde oxidase (AO) activity were evaluated in vitro. AO was partially purified from guinea pig liver. The effects of 12 flavonoids from three subclasses of flavon-3-ol, flavan-3-ol and flavanone on the oxidation of vanillin and phenanthridine as substrates of AO and xanthine as a substrate of xanthine oxidase (XO) were investigated spectrophotometrically. Among the 12 flavonoids, myricetin and quercetin were the most potent inhibitors of both AO and XO. In general, the oxidation of vanillin was more inhibited by flavonoids than that of phenanthridine. Almost all of the flavonoids inhibited AO activity more potently than XO, which was more evident with non-planner flavanols. A planner structure seems to be essential for a potent inhibitory effect and any substitution by sugar moieties reduces the inhibitory effects. This study could provide a new insight into AO natural inhibitors with potential to lead to some food-drug interactions.     

Flavonoid glycosides isolated from unique legume plant extracts as novel inhibitors of xanthine oxidase

Legumes and the polyphenolic compounds present in them have gained a lot of interest due to their beneficial health implications. Dietary polyphenolic compounds, especially flavonoids, exert antioxidant properties and are potent inhibitors of xanthine oxidase (XO) activity. XO is the main contributor of free radicals during exercise but it is also involved in pathogenesis of several diseases such as vascular disorders, cancer and gout. In order to discover new natural, dietary XO inhibitors, some polyphenolic fractions and pure compounds isolated from two legume plant extracts were tested for their effects on XO activity. The fractions isolated from both Vicia faba and Lotus edulis plant extracts were potent inhibitors of XO with IC(50) values range from 40-135 μg/mL and 55-260 μg/mL, respectively. All the pure polyphenolic compounds inhibited XO and their K(i) values ranged from 13-767 μM. Ten of the compounds followed the non competitive inhibitory model whereas one of them was a competitive inhibitor. These findings indicate that flavonoid isolates from legume plant extracts are novel, natural XO inhibitors. Their mode of action is under investigation in order to examine their potential in drug design for diseases related to overwhelming XO action.     

Molecular modeling of flavonoids that inhibits xanthine oxidase

The inhibition of xanthine oxidase activity by various flavonoids was assessed. All of the tested flavonoids were competitive inhibitors, and from the kinetic analysis suggested that flavonoids bind to the reactive site. To further understand the stereochemistry between these flavonoids and xanthine oxidase, structure-based molecular modeling was performed. Apigenin was the most potent inhibitor which showed the most favorable interaction in the reactive site. The bicyclic benzopyranone ring of apigenin stacked with phenyl of Phe 914, and the phenolic group stretched to the space surrounding with several hydrophobic residues. Quercetin and myricetin composed a 3-hydroxyl group on benzopyranone which resulting in reduction of binding affinity. The phenolic group of genistein positioned in opposite orientation comparison with apigenin, and resulted in a weaker interaction with xanthine oxidase. Isovitexin showed the weakest inhibitory effect among the compounds tested. The bulky group of sugar in isovitexin may hamper its interaction with xanthine oxidase.     

Synthesis,structure-activity relationships,and mechanistic studies of 5-arylazo-tropolone derivatives as novel xanthine oxidase (XO) inhibitors

Xanthine oxidase (XO) is an enzyme that contains molybdenum at the active site and catalyzes the oxidation of purine bases to uric acid. Even though XO inhibitors are widely used for the treatment of hyperuricemia and gout, only very few such compounds are clinically used as drugs for the treatment of these diseases. Given the unique physicochemical properties of tropolone, i.e., its chelating effect and the pKa value that is similar to that of carboxylic acid, we have synthesized 22 5-arylazotropolone derivatives as potential XO inhibitors. In vitro enzyme-inhibitory assays for XO revealed that 3-nitro derivative 1j showed the most potent XO inhibitory activity, which is by one order of magnitude more potent than allopurinol. An enzyme-kinetic study revealed that 1j inhibited the production of uric acid by XO both competitively and non-competitively. A docking-simulation study of 1j with XO suggested that the carbonyl and hydroxyl groups of the tropolone ring interact with the hydroxy group that acts as a ligand for molybdenum and the amino acid residues around the active site of XO.     

Inhibition of Xanthine Oxidase by Flavonoids

Various dietary flavonoids were evaluated in vitro for their inhibitory effect on xanthine oxidase, which has been implicated in oxidative injury to tissue by ischemia-reperfusion. Xanthine oxidase activity was determined by directly measuring uric acid formation by HPLC. The structure-activity relationship revealed that the planar flavones and flavonols with a 7-hydroxyl group such as chrysin, luteolin, kaempferol, quercetin, myricetin, and isorhamnetin inhibited xanthine oxidase activity at low concentrations (IC₅₀ values from 0.40 to 5.02 μM) in a mixed-type mode, while the nonplanar flavonoids, isoflavones and anthocyanidins were less inhibitory. These results suggest that certain flavonoids might suppress in vivo the formation of active oxygen species and urate by xanthine oxidase.     

Renierol from marine sponge Haliclona.SP.: A natural inhibitor of xanthine oxidase with hypouricemic effects

The purpose of this study was to evaluate the inhibitory effect of renierol, extracted from marine sponge Halicdona.SP., on xanthine oxidase (XO) and its hypouricemic effect in vivo. Renierol and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid and superoxide radical from xanthine. Renierol inhibited XO in a concentration-dependent and competitive manner. IC₅₀ value was 1.85 μg·ml^{? 1} through the measuring of uric acid and was 1.36 μg.ml^{? 1} through the measuring of superoxide radical. Renierol was found to have an in vivo hypouricemic activity against potassium oxonate-induced hyperuricaemia in mice. After oral administration of renierol at doses of 10, 20 and 30 mg.kg^{? 1}, there was a significant decrease in the serum urate level (4.08 ± 0.09 mg.dl^{? 1}, P < 0.01), (3.47 ± 0.11 mg.dl^{? 1}, P < 0.01) and (3.12 ± 0.08 mg.dl^{? 1}, P < 0.01), when compared to the hyperuricaemic control (6.74 ± 0.23 mg.dl^{? 1}). Renierol was a potent XO inhibitor with hypouricemic activity in mice.     

2-Styrylchromones As Novel Inhibitors of Xanthine Oxidase. A Structure-activity Study

The purpose of this study was the evaluation of the xanthine oxidase (XO) inhibition produced by some synthetic 2-styrylchromones. Ten polyhydroxylated derivatives with several substitution patterns were synthesised, and these and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid from xanthine. The synthesised 2-styrylchromones inhibited xanthine oxidase in a concentration-dependent and non-competitive manner. Some IC 50 values found were as low as 0.55 μM, which, by comparison with the IC 50 found for allopurinol (5.43 μM), indicates promising new inhibitors. Those 2-styrylchromones found to be potent XO inhibitors should be further evaluated as potential agents for the treatment of pathologies related to the enzyme's activity, as is the case of gout, ischaemia/reperfusion damage, hypertension, hepatitis and cancer.     

2-styrylchromones as novel inhibitors of xanthine oxidase. A structure-activity study

The purpose of this study was the evaluation of the xanthine oxidase (XO) inhibition produced by some synthetic 2-styrylchromones. Ten polyhydroxylated derivatives with several substitution patterns were synthesised, and these and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid from xanthine. The synthesised 2-styrylchromones inhibited xanthine oxidase in a concentration-dependent and non-competitive manner. Some IC50 values found were as low as 0.55 microM, which, by comparison with the IC50 found for allopurinol (5.43 microM), indicates promising new inhibitors. Those 2-styrylchromones found to be potent XO inhibitors should be further evaluated as potential agents for the treatment of pathologies related to the enzyme's activity, as is the case of gout, ischaemia/reperfusion damage, hypertension, hepatitis and cancer.     

Screening and Inhibition Mechanism of Xanthine Oxidase Inhibitors in Ethanolic Extracts of Chimonanthus salicifolius Hu Leaves

This study aimed to evaluate the inhibition of the ethanol elutions of Chimonanthus salicifolius Hu leaves (CsHL) against xanthine oxidase (XO). The results of XO inhibition assay and enzymatic superoxide free radical scavenging assay in vitro showed that 70 % ethanol eluate (EE) had the best inhibitory effect and followed by 40 % EE. High performance liquid chromatograph analysis showed that quercetin and kaempferol were the potential active components of XO inhibition. The inhibition mechanism of quercetin and kaempferol on XO was investigated by kinetic analysis and fluorescence quenching titration assay. The molecular simulation further revealed that quercetin and kaempferol bind to XO mainly by hydrogen bonding and van der Waals, blocking the entry of substrates and leading to the inhibition of XO. In conclusion, the CsHL have inhibitory effects on XO activity, which provides a theoretical basis for relieving or preventing hyperuricemia and gout as a natural food or medicinal plant in the future.     

Xanthine Oxidase Catalyzes the Synthesis of Retinoic Acid

Milk xanthine oxidase (xanthine: oxygen oxidore-ductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min^?1, determined at pH 7.0 with 1nM XO and all trans-retinaldehyde varying between 0.05 to 2μM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4μM and 2μM allopurinol respectively and inhibited 48% by 10 μM xanthine in enzyme assays performed at 2μM all trans-retinaldehyde. The K_i value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 μM.     

Kinetic study on the inhibition of xanthine oxidase by acylated derivatives of flavonoids synthesised enzymatically

Studies have reported that flavonoids inhibit xanthine oxidase (XO) activity; however, poor solubility and stability in lipophilic media limit their bioavailability and applications. This study evaluated the kinetic parameters of XO inhibition and partition coefficients of flavonoid esters biosynthesised from hesperidin, naringin, and rutin via enzymatic acylation with hexanoic, octanoic, decanoic, lauric, and oleic acids catalysed by Candida antarctica lipase B (CALB). Quantitative determination by ultra-high performance liquid chromatography–mass spectrometry (UHPLC–MS) showed higher conversion yields (%) for naringin and rutin esters using acyl donors with 8C and 10C. Rutin decanoate had higher partition coefficients (0.95), and naringin octanoate and naringin decanoate showed greater inhibitory effects on XO (IC₅₀ of 110.35 and 117.51?μM, respectively). Kinetic analysis showed significant differences (p?K_m values, whereas the values for V_max were the same, implying the competitive nature of XO inhibition.     

New xanthine oxidase inhibitors from the fruiting bodies of Tyromyces fissilis

Excessive uric acid production, which causes gout and hyperuricemia, can be blocked by inhibiting xanthine oxidase (XO). However, some agents to block on XO often cause side effects, thereby necessitating the identification of new inhibitors. During the screening of XO inhibitors from various mushroom extracts, we found that a methanolic extract of the fruiting bodies of Tyromyces fissilis, an inedible and non-toxic fungus, showed inhibitory activity. Both n-hexane and ethyl acetate layers, obtained by partitioning this extract exhibited XO inhibitory activity. Subsequently, using an activity-guided separation method, eight active compounds (1–8) were isolated. The structures of five of the new compounds, 2–4, 6, and 7, were elucidated by spectral analysis and chemical derivatization. All compounds had a salicylic acid moiety with an aliphatic group at the C-6 position. Notably, 2-hydroxy-6-pentadecylbenzoic acid (1) showed the highest level of XO noncompetitive inhibition (58.9 ± 2.2% at 25 µM).     

Synthesis, biological evaluation, and molecular docking of N-{3-[3-(9-methyl-9H-carbazol-3-yl)-acryloyl]-phenyl}-benzamide/amide derivatives as xanthine oxidase and tyrosinase inhibitors

Claisen-Schmidt condensation of 3-formyl-9-methylcarbazole with various amides of 3-aminoacetophenone afforded N-{3-[3-(9-methyl-9H-carbazol-3-yl)-acryloyl]-phenyl}-benzamide/amide derivatives. All compounds were investigated for their in vitro xanthine oxidase (XO), tyrosinase and melanin production inhibitory activity. Most of the target compounds had more potent XO inhibitory activity than the standard drug (IC(50)=4.3-5.6μM). Interestingly, compound 7q bearing cyclopropyl ring was found to be the most potent inhibitor of XO (IC(50)=4.3μM). Molecular modelling study gave an insight into its binding modes with XO. Compounds 7a, 7d, 7e, 7g, and 7k were found to be potent inhibitors of tyrosinase (IC(50)=14.01-17.52μM). These results suggest the possible use of these compounds for the design and development of novel XO and tyrosinase inhibitors.     

拐枣中总黄酮提取工艺优化及其抗黄嘌呤氧化酶活性研究

为了研究拐枣总黄酮(TF)最佳提取工艺及其体外抗黄嘌呤氧化酶(xanthine oxidase,XO)活性。在单因素试验基础上,实验选择料液比、提取时间和乙醇浓度为自变量,应用Box-Benhnken中心组合法进行3因素3水平试验设计,以拐枣总黄酮得率为响应值,进行响应面分析,并采用D101大孔树脂对总黄酮进行进一步富集纯化,在此基础上研究拐枣总黄酮体外抗XO活性。结果表明,拐枣总黄酮的最佳提取条件为料液比15.0 mL/g、提取时间1.85 h、乙醇浓度53.0%,在此条件下,拐枣总黄酮的平均提取率可达到2.56%。体外活性结果表明,我们首次发现拐枣总黄酮具有显著的抗XO活性,其IC_(50 )为18.63±1.67μg/mL。本研究可为从拐枣中寻找和发现天然抗XO活性分子提供参考,同时可为拐枣的开发利用提供思路。     

Inhibition of xanthine oxidase by theaflavin: Possible mechanism for anti-hyperuricaemia effect in mice

Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. Excessive production of uric acid leads to hyperuricaemia. Due to the serious side effects of allopurinol, it is an urgent need to explore new XO inhibitors. Herein, the effects of theaflavin (TF1) on XO and anti-hyperuricaemia effect in hyperuricemic mice were investigated. Kinetic analysis indicate that TF1 is a reversible competitive inhibitor and has a significant inhibitory effect on XO with an IC₅₀ value of 63.17 ± 0.13 μmol/L. Analysis of fluorescence spectra suggests that TF1 causes the obvious fluorescence quenching of XO, which is mainly driven by hydrophobic interactions and hydrogen bonds. Docking studies demonstrate that TF1 interacts with dozens of amino acid residues surrounded in the active cavity of XO, including Glu-879, Pro-1012, Thr-1010, Val-1011, Lys-771, Glu-802, Pro-1076, Leu-873, Leu-1014, Asn-768, Leu-648 and Phe-649. The inhibitory mechanism may be the insertion of TF1 into the active site of XO, which hinders the substrate xanthine to enter into the site. Furthermore, the results from animal experiments demonstrate that TF1 is effective in reducing serum uric acid in mice. These findings suggest that TF1 may be a potential drug candidate for the treatment of hyperuricaemia.     

Inhibition of xanthine oxidase — xanthine — iron mediated lipid peroxidation by eugenol in liposomes

The effect of eugenol on xanthine oxidase (XO) xanthine(X)-Fe⁺³-ADP mediated lipid peroxidation was studied in liver microsomal lipid liposomes. Eugenol inhibited the lipid peroxidation in a dose dependent manner as assessed by formation of thiobarbituric acid reactive substances. When tested for its effect on XO activity per se, (by measuring uric acid formation) eugenol inhibited the enzyme to an extent of 85% at 10 µm concentration and hence formation of O₂ also However, the concentration of eugenol required for XO inhibition was more in presence of metal chelators such as EDTA, EGTA and DETAPAC, but not in presence of deferoxamine, ADP and citrate. The antiperoxidative effect of eugenol was about 35 times more and inhibition of XO was about 5 times higher as compared to the effect of allopurinol. Eugenol did not scavenge O₂ generated by phenazine methosulfate and NAD but inhibited propagation of peroxidation catalyzed by Fe²⁺ EDTA and lipid hydroperoxide containing liposomes. Eugenol inhibits XO-X-Fe⁺³ ADP mediated peroxidation by inhibiting the XO activity per se in addition to quenching various radical species. (Mol Cell Biochem 166: 65-71, 1997)     

Antioxidant,anti‐inflammatory,and enzyme inhibitory activity of natural plant flavonoids and their synthesized derivatives

The synthesized flavonoid derivatives were examined for their antioxidant, anti‐inflammatory, xanthine oxidase (XO), urease inhibitory activity, and cytotoxicity. Except few, all the flavonoids under this study showed significant antioxidant activity (45.6%–85.5%, 32.6%–70.6%, and 24.9%–65.5% inhibition by DPPH, ferric reducing/antioxidant power, and oxygen radical absorption capacity assays) with promising TNF‐α inhibitory activity (42%–73% at 10 μM) and IL‐6 inhibitory activity (54%–81% at 10 μM) compared with that of control dexamethasone. The flavonoids luteolin, apigenin, diosmetin, chrysin, O^3?, O⁷‐dihexyl diosmetin, O^4?, O⁷‐dihexyl apigenin, and O⁷‐hexyl chrysin, showed an inhibition with IC₅₀ values (4.5‐8.1 μg/mL), more than allopurinol (8.5 μg/mL) at 5 μM against XO and showing more than 50% inhibition at a final concentration (5 mM) with an IC₅₀ value of ranging from 4.8 to 7.2 (μg/mL) in comparison with the positive control thiourea (5.8 μg/mL) for urease inhibition. Thus, the flavonoid derivatives may be considered as potential antioxidant and antigout agents.     

Xanthine oxidase-dependent ROS production mediates vitamin A pro-oxidant effects in cultured Sertoli cells

Several studies have suggested that vitamin A (retinol, ROH) presents pro-oxidant properties in biological systems. Recent studies point out that xantine oxidase, a ROS-generating enzyme, catalyses ROH oxidation to RA in vitro. These works stimulated the authors to investigate whether xanthine oxidase could be involved on the ROH pro-oxidative effects reported in cultured Sertoli cells. In vitro, it was demonstrate that xanthine oxidase generates superoxide in the presence of ROH as assessed by superoxide mediated-NBT reduction. Superoxide production is potentiated in the presence of NADH and inhibited by allopurinol. In Sertoli cells, ROH treatment increased xanthine oxidase activity and inhibition of the enzyme with allopurinol attenuated ROH-induced ROS production, protein damage and cytotoxicity. Moreover, inhibition of ROH oxidation to RA by retinaldehyde dehydrogenase inhibitor potentiated both xanthine oxidase-dependent ROS production and cell damage in ROH-treated cells. The data show that xanthine oxidase may play a role on vitamin A pro-oxidant effects.     

Novel Xanthine Oxidase (XO) inhibitory phenylindanes produced by thermal reaction of caffeic acid

The products from the thermal reaction of chlorogenic and caffeic acids, which is a model process of roasting coffee beans, exhibited xanthine oxidase (XO) inhibitory activity. From caffeic acid, six inhibitory phenylindanes were identified, and a new phenylindane displayed the highest inhibitory activity among them. The activity of these phenylindanes may contribute to XO inhibition-related functions of roasted coffee beverages.     

Effects of Greek legume plant extracts on xanthine oxidase,catalase and superoxide dismutase activities

Legumes are considered to have beneficial health implications, which have been attributed to their phytochemical content. Polyphenols are considered the most important phytochemical compounds extensively studied for their antioxidant properties. The aim of the present study was to examine the effects of potent antioxidant legume plant extracts on xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD) activities. XO exerts a dual role, as it is the major contributor of free radicals during exercise while it generates uric acid, the most potent antioxidant molecule in plasma. CAT and SOD are two of the main enzymes of the antioxidant defence of tissues. We demonstrate that the majority of the extracts inhibited XO activity, but they had no effect on CAT inhibition and SOD induction when used at low concentrations. These results imply that the tested extracts may be considered as possible source of novel XO inhibitors. However, we have shown that allopurinol administration, a known XO inhibitor, before exercise reduces performance and induces oxidative stress in rats. Considering the fact that the extracts examined had an inhibitory effect on XO activity, possibly posing a restriction in their characterization as antioxidants, phytochemical antioxidant administration before exercise should probably be reconsidered.