Glycosides and xanthine oxidase inhibitors from Conyza bonariensis

Fractionation of the xanthine oxidase inhibitory methanol extract of Conyza bonariensis afforded three glycosides, in addition to nine known compounds including amyrin, beta-sitostero1 daucosterol, syringic acid 3-hydroxy-5-methoxybenzoic acid, eugenol 4-O-glucopyranoside, and luteolin, apigenin and takakin 8-O-glucuronide. The structures of the glycosides were established by a combination of spectroscopic methods (IR, MS, 1H and 13C NMR, DEPT, COSY, HMQC and HMBC) as 4-hydroxypyridin-3-carboxylic acid 4-O-glucopyranoside, 8-hydroxy-6,7-dihydrolinalool 8-O-glucopyranoside and bonaroside [viz. 1,3,4,12-tetrahydroxy-2-(9-hexadecenoylamino)octadecane 1-O-glucopyranoside]. The in vitro enzyme assay showed that syringic acid and takakin 8-O-glucuronide displayed weak inhibitory activity against xanthine oxidase with IC50 values of 500+/-41 microM and 170+/-12 microM, respectively.     

Discovery and biological evaluation of some (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety as potent xanthine oxidase inhibitors

A series of (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety were synthesized and identified as novel xanthine oxidase inhibitors. Among them, the most promising compounds 1h and 1k were obtained with IC₅₀ values of 0.6 μM and 0.8 μM, respectively, which were more than 10-fold potent compared with allopurinol. The Lineweaver-Burk plot revealed that compound 1h acted as a mixed-type xanthine oxidase inhibitor. SAR analysis showed that the benzaldehyde moiety played a more important role than the anthraquinone moiety for inhibition potency. The basis of significant inhibition of xanthine oxidase by 1h was rationalized by molecular modeling studies.     

Consequence of the antioxidant activities and tyrosinase inhibitory effects of various extracts from the fruiting bodies of Pleurotus ferulae

This study was initiated to screen the antioxidant activities, tyrosinase inhibitory effects on the fruiting bodies of Pleurotus ferulae extracted with acetone, methanol and hot water. The antioxidant activities were performed on β-carotene–linoleic acid, reducing power, DPPH, ferrous ions chelating abilities, and xanthine oxidase. In addition to this, phenolic compounds were also analyzed. The methanolic extract showed the strongest β-carotene–linoleic acid inhibition and high reducing power as compared to other extracts. The scavenging effects on DPPH radicals, the acetonic and methanolic extracts were more effective than hot water extracts. The strongest chelating effect was obtained from the methanolic extract as compared to the tested synthetic antioxidant. Gallic acid, protocatechuic acid, caffeic acid, vanillin, ferulic acid, naringin, resveratrol, naringenin, hesperetin, formononetin and biochanin-A were detected from acetonitrile and hydrochloric acid (5:1) solvent extract. Xanthine oxidase and tyrosinase inhibitory activities of acetonic, methanolic, and hot water extracts of P. ferulae increased with increasing concentration. The results suggested that consumption of P. ferulae might be beneficial to the antioxidant, xanthine oxidase, and tyrosinase protection system of the human body against oxidative damage and others complications.     

Simultaneous determination of 16 purine derivatives in urinary calculi by gradient reversed-phase high-performance liquid chromatography with UV detection

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5-2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.     

Inhibition of mammalian xanthine oxidase by folate compounds and amethopterin

We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Bioactive ellagitannins from Cunonia macrophylla, an endemic Cunoniaceae from New Caledonia

Chemical study of Cunonia macrophylla, a New Caledonian Cunoniaceae, based on bioactive effects of a crude methanol extract of the leaves, detected bioactive tannins for the first time in this plant family. These ellagitannins have been identified as ellagic acid-4-O-beta-D-xylopyranoside (6), mallorepanin (3), mallotinic acid (1) along with corilagin (2), chebulagic acid (4), ellagic acid (5) and gallic acid (7) and have been shown to possess antimicrobial activity and to inhibit xanthine oxidase. Antimicrobial effects on bacterial human pathogens (Staphylococcus aureus, Corynebacterium accolans) and on a plant pathogen (Erwinia carotovora) as well as on a human pathogenic yeast (Candida albicans) were investigated. Activity is reported here for the first time for compounds 1, 3, 4 and 6. The inhibitory effects of all molecules against xanthine oxidase in relation to their structure was evaluated and compared. Compound 6 presented the best activity and seems to be of considerable interest for further studies.     

Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.     

3,5,2',4'-Tetrahydroxychalcone, a new non-purine xanthine oxidase inhibitor

Xanthine oxidase is a key enzyme that catalyses hypoxanthine and xanthine to uric acid and the overproduction of uric acid will lead to hyperuricemia which is an important cause of gout. In the present study, three chalcone derivatives were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, only Compound 1, 3,5,2′,4′-tetrahydroxychalcone, exhibited a significant inhibitory activity on xanthine oxidase with an IC₅₀ value of 22.5 μM. Lineweaver–Burk transformation of the inhibition kinetics data demonstrated that it was a competitive inhibitor of xanthine oxidase and Ki value was 17.4 μM. In vivo, intragastric administration of Compound 1 was able to significantly reduce serum uric acid levels and inhibited hepatic xanthine oxidase activities of hyperuricemic mice in a dose-dependent manner. Acute toxicity study in mice showed that Compound 1 was very safe at a dose of up to 5 g/kg. These results suggest that Compound 1 is a novel competitive xanthine oxidase inhibitor and is worthy of further development.     

The ability of new formamidine sugar-modified derivatives of daunorubicin to stimulate free radical formation in three enzymatic systems: NADH dehydrogenase, NADPH cytochrome P450 reductase and xanthine oxidase

Some sterically hindered N-substituted derivatives of daunorubicin are known to be poor substrates for NADH dehydrogenase, NADPH cytochrome P450 reductase and xanthine oxidase. In consequence, poor oxygen radical generation by these compounds is observed. In this study we examined a new family of sugar-N-substituted derivatives of daunorubicin bearing a bulky substituent introduced on the nitrogen atom through the amidine spacer. These compounds were found to be very active in radical formation catalyzed by all three studied enzymes. Thus, the introduction of a heterocyclic ring, even if it is bulky but flexible, on the nitrogen atom of daunosamine moiety through the one-atom spacer (amidine group), does not induce the steric hindrance effect on the interaction of daunorubicin derivatives with these flavoprotein enzymes.     

Biochemical characterization of some pyrazolopyrimidine-based inhibitors of xanthine oxidase

Inhibition of xanthine oxidase-catalyzed conversion of xanthine to uric acid by various pyrazolopyrimidine-based inhibitors (allopurinol derivatives) was evaluated and compared with the standard inhibitor allopurinol. Three compounds out of the seven compounds used in the study were found to be reasonably good inhibitors of xanthine oxidase (XO). 4-Amino-6-mercaptopyrazolo-3,4-d-pyrimidine was found to be the most potent inhibitor of XO (IC50 = 0.600 +/- 0.009 microM). 4-Mercapto-1H-pyrazolo-3,4-d-pyrimidine (IC50 = 1.326 +/- 0.013 microM) and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine (IC50 = 1.564 +/- 0.065 microM) also showed comparable inhibitory activity to that of allopurinol (IC50 = 0.776 +/- 0.012 microM). All three compounds showed competitive type of inhibition with comparable Ki values. Induction of the electron transfer reaction catalyzed by XO in the presence of these compounds monitored as reduction of 2,6-dichlorophenolindophenol (DCPIP) revealed that electron transfer by 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine is comparable to that obtained by allopurinol or xanthine. However, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine did not show DCPIP reduction. On the other hand, enzymatic reduction of cytochrome c in the presence of the three compounds was found to be insignificant and much less in comparison to allopurinol and xanthine. Therefore, both 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine and 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine displayed the inhibitory property and also did not produce XO-mediated reactive oxygen species (ROS). Since 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine was found to have some toxicity, the effect of 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine on the enzymatic formation of uric acid and ROS was investigated and it was found that this compound was inhibiting enzymatic generation of both uric acid and ROS. It can be noted that the standard inhibitor, allopurinol, inhibits uric acid formation but produces ROS.     

Characterization of the antioxidant activity of aglycone and glycosylated derivatives of hesperetin: an in vitro and in vivo study

The flavonoids are mainly present in Citrus fruits as their glycosyl derivatives. This study was conducted comparing in vitro xanthine oxidase inhibitory activity of the aglycone hesperetin and its glycosylated forms (hesperidin and G‐hesperidin) and their effects on the plasma lipid profile and the oxidative–antioxidative system (TBARS and antioxidant enzymes) in rats. The concentrations of the major conjugated metabolites in rat plasma after oral administration of these compounds were also determined. Wistar male rats were randomly assigned to three groups (n = 6) supplemented for 30 days with 1 mmol/kg body mass of hesperetin, hesperidin or G‐hesperidin. Hesperetin was a stronger xanthine oxidase inhibitor (IC₅₀ = 53 μM and K_i = 17.3 μM) than the glycosylate derivatives. Supplementation with the three compounds led to a lower (more favorable) atherogenic index, and an antioxidant preventive effect from the increase of hepatic superoxide dismutase was observed associated to HT supplementation, possibly because of the higher level of hesperetin‐glucuronide in rat plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

Kinetics and specificity of guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase towards substituted benzaldehydes

Molybdenum-containing enzymes, aldehyde oxidase and xanthine oxidase, are important in the oxidation of N-heterocyclic xenobiotics. However, the role of these enzymes in the oxidation of drug-derived aldehydes has not been established. The present investigation describes the interaction of eleven structurally related benzaldehydes with guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase, since they have similar substrate specificity to human molybdenum hydroxylases. The compounds under test included mono-hydroxy and mono-methoxy benzaldehydes as well as 3,4-dihydroxy-, 3-hydroxy-4-methoxy-, 4-hydroxy-3-methoxy-, and 3,4-dimethoxy-benzaldehydes. In addition, various amines and catechols were tested with the molybdenum hydroxylases as inhibitors of benzaldehyde oxidation. The kinetic constants have shown that hydroxy-, and methoxy-benzaldehydes are excellent substrates for aldehyde oxidase (Km values 5x10(-6) M to 1x10(-5) M) with lower affinities for xanthine oxidase (Km values around 10(-4) M). Therefore, aldehyde oxidase activity may be a significant factor in the oxidation of the aromatic aldehydes generated from amines and alkyl benzenes during drug metabolism. Compounds with a 3-methoxy group showed relatively high Vmax values with aldehyde oxidase, whereas the presence of a 3-hydroxy group resulted in minimal Vmax values or no reaction. In addition, amines acted as weak inhibitors, whereas catechols had a more pronounced inhibitory effect on the aldehyde oxidase activity. It is therefore possible that aldehyde oxidase may be critical in the oxidation of the analogous phenylacetaldehydes derived from dopamine and noradrenaline.     

Biosynthesis and cytokinin activity of 8-hydroxy and 2,8-dihydroxy derivatives of zeatin and N-6(increment-2-isopentenyl)adenine.

8-Hydroxy and 2,8-dihydroxy derivatives of the cytokinins, 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine and N-6-(increment -2-isopentenyl)adenine, have been biosynthesized by xanthine oxidase oxidation. 8-Hydroxy derivatives have been shown to be the major intermdeiates. These compounds were tested for cytokinin activity in the tobacco bioassay. The results suggest that substitution of the 8 position with a hydroxyl group causes less decrease of cytokinin activity than substitution of both the 2 and 8 positions with hydroxyl groups.     

Cis-trans isomerization of nitrofuran derivatives by xanthine oxidase

Enzymatic cis-trans isomerization of nitrofuran derivatives was 3-(5-Nitro-2-furyl)-2-(2-furyl)-demonstrated with milk xanthine oxidase. acrylamide (AF-2) and 3-(5-nitro-2-furyl)-2-(5-bromo-2-furyl)acrylamide (NFBFA) were mainly converted from the cis to the trans form by this enzyme supplemented with an electron donor. This enzymatic reaction was further characterized with respect to its cofactor requirements. Finally, a new cis-trans isomerization mechanism, which is based on transfer of a single electron by a nitroreductase such as xanthine oxidase to a nitrofuran derivative to give the anion free radical, was proposed.     

ortho-dihydroxyisoflavone derivatives from aged Doenjang (Korean fermented soypaste) and its radical scavenging activity

One new ortho-dihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone (2), and two known ortho-dihydroxyisoflavone derivatives were isolated from 5-year-old Doenjang (Korean fermented soypaste), and evaluated as potent antioxidant by comparing with other known isoflavones. 7,8,4'-Trihydroxyisoflavone (1), 7,3',4'-trihydroxyisoflavone (2), and 6,7,4'-trihydroxyisoflavone (3) inhibited DPPH (Diphenyl-1-picryl hydrazyl) formation by 50% at a concentration of 21.5+/-0.2, 28.7+/-0.4 and 32.6+/-0.6 (IC(50)), respectively, whereas three isoflavones showed weak DPPH radical scavenging activity. In xanthine oxidase (XO) system, in which both inhibition of xanthine oxidase and superoxide scavenging effect were measured in one assay. Compound 1 (IC(50)= 6.6+/-0.4 microM) and 2 (IC(50)=16.8+/-1.2 microM) show significant inhibitory activity and greater effect than allopurinol. But, compound 3 and other isoflavones showed lower inhibition activity. This study shows that the position of hydroxyl substituent at the aromatic ring of isoflavone plays an important role in radical scavenging effect.     

Quercinol, an anti-inflammatory chromene from the wood-rotting fungus Daedalea quercina (Oak Mazegill)

The fungus Daedalea quercina (oak mazegill) was examined for its capability of producing antioxidative and anti-inflammatory compounds. Bioactivity guided fractionation of the extract from a mycelial culture led to the isolation of quercinol, which was identified as (-)-(2S)-2-hydroxymethyl-2-methyl-6-hydroxychromene 1 by NMR and X-ray analyses. The cryptic hydroquinone 1 shows a broad anti-inflammatory activity against cyclooxygenase 2 (COX-2), xanthine oxidase (XO), and horseradish peroxidase (HRP) at micromolar concentrations.     

Endophytic naphthopyrone metabolites are co-inhibitors of xanthine oxidase, SW1116 cell and some microbial growths

Fractionation of the extract of Aspergillus niger. IFB-E003, an endophyte in Cyndon dactylon, gave four known compounds naphtho-gamma-pyrones rubrofusarin B, fonsecinone A, asperpyrone B and aurasperone A, which were further investigated biologically. Rubrofusarin B was shown to be cytotoxic to the colon cancer cell line SW1116 (IC50: 4.5 microgml-1), and aurasperone A inhibitory on XO (xanthine oxidase) (IC50: 10.9 micromoll-1). Moreover, the four naphtho-gamma-pyrones exhibited growth inhibitions against the five test microbes with MICs ranging in between 1.9 and 31.2 microgml(-1). The present recognition of rubrofusarin B and aurasperone A as strong co-inhibitors on XO, colon cancer cell and some microbial pathogens is of significance for the imperative discovery of new relevant therapeutic agents.     

New Guaiane-Type Sesquiterpene and Norsesquiterpene from Alisma plantago-aquatica and Their Xanthine Oxidase Inhibitory Activity

A new sesquiterpene ( 1 ) and a new norsesquiterpene ( 2 ) belonging guaiane-type skeleton together with six known compounds ( 3 – 8 ) were isolated from the rhizomes of Alisma plantago-aquatica. Their structures were determined by HR-ESI-MS, 1D and 2D NMR spectroscopic methods. Absolute configurations of new compounds were established by experimental and TD-DFT computational ECD spectra. Compounds 1 – 8 exhibited xanthine oxidase inhibitory activity with their IC₅₀ values in range of 9.4–66.7 μM. The sesquiterpenoids 1 – 5 displayed the inhibitory activity and hence they could be potential xanthine oxidase inhibitors from A. plantago-aquatica.