Review Article Xanthine Oxidase: Isolation, Assays of Activity, …downloads.hindawi.com/journals/jchem/2015/294858.pdf · Review Article Xanthine Oxidase: Isolation, Assays of Activity,

Review ArticleXanthine Oxidase Isolation Assays of Activity and Inhibition

Danijela A KostiT Danica S DimitrijeviT Gordana S StojanoviT Ivan R PaliTAleksandra S YorZeviT and Jovana D Ickovski

Department of Chemistry Faculty of Science and Mathematics University of Nis Visegradska 33 18 000 Nis Serbia

Correspondence should be addressed to Danica S Dimitrijevic danicadimitrijevic7gmailcom

Received 18 November 2014 Accepted 21 January 2015

Academic Editor Patricia Valentao

Copyright copy 2015 Danijela A Kostic et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Xanthine oxidase (XO) is an important enzyme catalyzing the hydroxylation of hypoxanthine to xanthine and xanthine to uricacid which is excreted by kidneys Excessive production andor inadequate excretion of uric acid results in hyperuricemia Thispaper presents a detailed review of methods of isolation determination of xanthine oxidase activity and the effect of plant extractsand their constituents on it Determining the content and activities of XO can be used for diagnostic purposes Testing inhibitionof XO is important for detection of potentially effective compounds or extracts that can be used to treat diseases that are causedby increased activity of XO In vitro bioassays are used to examine test material for XO inhibition as inhibitors of XO may bepotentially useful for the treatment of gout or other XO induced diseases Several authors reported on the XO inhibitory potentialof traditionally used medicinal plants

1 Introduction

Reactive oxygen species (ROS) such as hydrogen peroxidesuperoxide radical anion hydroxyl radical alkylperoxyl rad-ical nitric oxide and singlet oxygen are often associated withsome physiopathological states in human Oxidative stresscaused by an imbalance between antioxidant systems andthe production of oxidants including ROS is consideredto contribute to a wide variety of degenerative processesand diseases such as atherosclerosis Parkinsonrsquos diseaseAlzheimerrsquos dementia and reperfusion injury of brain orheart [1 2] and also can be associated with the pathogenesisof various conditions such as aging arthritis cancer andinflammation [2 3] ROS are generated inside the humanbody as a consequence of the exposure to a multitudeof exogenous chemicals in our ambient environment likeduring UV light irradiation and by X-rays and gamma raysor produced during metal catalyzed reactions [4] andor anumber of endogenous metabolic processes involving redoxenzymes and bioenergetics electron transfer [5] Endogenousfactors leading to formation of ROS can be neutrophilsand macrophages during inflammation or byproducts ofmitochondrial catalyzed electron transport reactions andvarious other mechanisms [6] Endogenous sources of ROS

include mitochondria cytochrome P450 metabolism per-oxisomes and inflammatory cell activation [7] so as othercellular sources of superoxide radicals present such as theenzyme xanthine oxidase which catalyzes the reaction ofhypoxanthine to xanthine and xanthine to uric acid In bothsteps molecular oxygen is reduced forming the superoxideanion followed by the generation of hydrogen peroxide [8]

Besides these many studies have now confirmed thatexogenic antioxidants especially supplied by foods are essen-tial for counteracting oxidative stress These antioxidantsmainly come from plants in the form of phenolic com-pounds (flavonoids phenolic acids and alcohols stilbenestocopherols and tocotrienols) [9] Many studies have sug-gested that flavonoids exhibit biological activities includingantiallergenic antiviral anti-inflammatory and vasodilatingactions These pharmacological effects are linked to theantioxidant properties of flavonoids Protective effects offlavonoids are ascribed to their capacity to suppress ROSformation by inhibiting some enzymes or chelating traceelements involved in free radical production scavenge radicalspecies and more specially the ROS and improve regulationantioxidant defense [10ndash13]

The aim of this study is to give an overview of methodsfor isolation and determination of XO activity

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 294858 8 pageshttpdxdoiorg1011552015294858

2 Journal of Chemistry

S

OH

OGlu1261

N

NN

N

O

H

HO

H

Xanthine

HO

SH

N

NN

N

O

H

HO

H

N

NN

N

O

H

HO

H

O

S

S

OH

N

NN

N

O

H

HO

H

H

O

Uric acid

O O

O

O

Ominus

H2

O

H+

eminus

MoVI

MoVI

MoIV

MoV

Scheme 1 Transformation of xanthine to uric acid by XO

N

N N

N

O

H

HHypoxanthine

N

N N

N

O

H

HXanthine

Xanthineoxidase

O

H

N

N NN

OH

H

Allopurinol

N

N N

N

O

H

HO

HXanthine

Xantineoxidase

N

N N

N

O

H

HO

H

O

H

Uric acid

Figure 1 Inhibition of xanthine oxidase by allopurinol to prevent conversion of hypoxanthine to xanthine andor uric acid

2 Xanthine Oxidase Mechanism of Action

In 1902 Schardinger [14] showed that milk contains anenzyme capable of oxidizing aldehydes to acids accompaniedby the reduction of methylene blue this enzyme was thencommonly called the ldquoSchardinger enzymerdquo In 1922 Morganet al [15] showed that milk contains an enzyme capable ofoxidizing xanthine and hypoxanthine with the concomitantreduction of O

2to H2O2 and this enzyme was called XO

Hass and Hill [16] and Hass and Lee [17] reported that milkcontains a substance which they called ldquoitaterdquo capable of

oxidizing nitrite to nitrate in the presence of an aldehyde andO2under other conditions and thatmilk could reduce nitrate

to nitrite In 7 1938 Booth [18] presented strong evidence thatthe Schardinger enzyme was in fact XO

XO is a homodimer with molecule mass of 290 kDaXanthine oxidase belongs to themolybdenum-protein familycontaining one molybdenum one of the flavin adeninedinucleotides (FAD) and two iron-sulfur (2Fe-2S) centers ofthe ferredoxin type in each of its two independent subunitsThe enzyme contains two separated substrate-binding sitesXO catalysed the oxidation of hypoxanthine to xanthine and

Journal of Chemistry 3

Table 1 Assay procedure of effect of plant extract on xanthine oxidase activity

Reference Tested sample Assay procedures

[19] Erythrinastricta Roxb fractions

Sample fraction (1mL 5ndash100 120583gmL) phosphate buffer (pH 75) (29mL) xanthineoxidase (01mL 01 unitsmL in phosphate buffer) preincubation 15min (25∘C)substrate solution (2mL 150120583M xanthine in buffer) incubation 30min (25∘C) HCl(1mL 1M)

[20] Lanostanoids from Ganodermatsugae

Test solution 70mM phosphate buffer (pH 75) enzyme solution (01 unitsmL in70mM phosphate buffer (pH 75)) preincubation 25∘C for 15min substrate(150120583M xanthine in the same buffer)

[21] Food extracts and componentsTest sample xanthine (08mL 150 120583M) hydroxylamine (02mM) and EDTA(01mM) all in sodium phosphate buffer (02M pH 75) XO (02mL 2342mili02M phosphate buffer) incubation 30min (37∘C) HCl (01mL 5M)

[22] Malaysian medicinal plant

Sodium phosphate buffer (pH 75) (300120583L 50mM) 100 120583L of sample solution indistilled water or DMSO enzyme solution (100 120583L 02 unitsmL of xanthineoxidase in phosphate buffer) 100120583L of distilled water preincubation 15min (37∘C)xanthine (200120583L 015mM) incubation 30min (37∘C) 200 120583L of 05MHCl

[23] Edible plants of the TurkmenSahra region

Plant extract (0250mL in 50mM potassium phosphate buffer pH = 74) xanthine(0330mL 015mM) potassium phosphate buffer (pH 74) (0385mL 50mM) XO(0035mL in 50mM potassium phosphate buffer)

[24] Anacardic acidSodium carbonate buffer containing 01mM EDTA (pH 100) (276mL 40mM)xanthine (006mL 10mM) sample (006mL in DMSO) xanthine oxidase (012mL004 units)

[25] Czech medicinal plantsPhosphate buffer (pH 75) (400120583L 120mmolL) xanthine (pH 75) (330 120583L150120583molL) extract stock solution (250 120583L) enzyme solution (20120583L 05 unitsmLin buffer)

[26] Centaurium erythraea infusion Xanthine (44120583M in 1 120583MNaOH) XO (029 unitsmL in 01M EDTA) lyophilizedinfusion (52 104 208 417 833 and 1667 120583gmL)

[27] Dried S anacardiumseeds 01 UmL XO 02mL xanthine (026M) in 50mM phosphate buffer (pH = 74)

subsequently to uric acid [32ndash34] During the reoxidation ofXO molecular oxygen acts as electron acceptor producingsuperoxide radical and hydrogen peroxide [35] During thesereactions superoxide anion radicals (O

2

∙minus) and H2O2are

formed [35] Superoxide anion radicals spontaneously orunder the influence of enzyme superoxide dismutase (SOD)transformed into hydrogen peroxide and oxygen Thesereactions can be written as follows [36] and on Scheme 1

hypoxanthine +O2+H2O 997888rarr xanthine +H

2O2

xanthine + 2O2+H2O 997888rarr uric acid + 2O

2

∙minus+ 2H+

xanthine +O2+H2O 997888rarr uric acid +H

2O2

2O2

∙minus+ 2H+ 997888rarr H

2O2+O2

(1)

Uric acid is breakdown product of ingested and endoge-nously synthesized purines DNA andRNA are degraded intopurine nucleotides and bases which are then metabolizedvia the action of xanthine oxidase to xanthine and uricacid These later steps are irreversible and generate superox-ide anions Uric acid undergoes no further metabolism inhumans and is excreted by the kidneys and intestinal tract[37]

According to this higher concentrations of uric acid maybe response to the higher levels of xanthine oxidase activityand to the oxidative stress which is characteristic for manyvascular disease states [38] The overactivity of XO results ina condition known as gout [39] a common rheumatic disease

and an acute inflammatory arthritis [40] The treatment forhyperuricemia and gout is either increasing the excretion ofuric acid or reducing the uric acid production

Xanthine oxidase inhibitors (XOI) are very useful for this[19] The inhibition of XO reduces both vascular oxidativestress and circulating levels of uric acidThe inhibition of XOby allopurinol is showed in Figure 1

Thus XO inhibitors may be useful for treatment ofmany other diseases [41 42] Among the many known XOinhibitors allopurinol oxypurinol and febuxostat have beenused widely for the treatment of hyperuricemia and gout[43] XO inhibitors can act either at the purine binding sitesuch as allopurinol [44 45] or at the FAD cofactor site suchas benzimidazole [46] XO inhibitors act by blocking thebiosynthesis of uric acid from purine in the body [47] andit is believed that either increasing the excretion of uric acidor reducing the uric acid production helps to reduce the riskof gout [48]

3 Isolation and Purification of XO

Isolation of XO as the widespread enzymes among differentspecies involves the extraction of the enzyme from a widerange ofmaterials (bacteria milk organs of different animalsetc) and its purification from crude extract XO is con-centrated in the milk fatlipid globule membrane (MFGM)in which it is the second most abundant protein afterbutyrophilin Therefore all isolation methods use cream as


Table 2 Plant extract and pure compounds as potent XOI

Reference Plant Extract or compound Inhibition effect IC50 or ofinhibition

[28] Semecarpus anacardiumFractionated methanolic extract by hexane ethyl acetate andbutanolIsolated compound tetrahydroamentoflavone (THA)

THAIC50 100 nM

[29] Ajuga iva L Crude 85 methanol extract fractionated by chloroform(CE) ethyl acetate (EAE) and water (AE)

IC50 3878ndash5835120583Mquercetin equivalent (QE)

[30] Teucrium polium Methanol (ME) chloroform (CE) and ethyl acetate (EAE)crude extracts

CE 079 ME 1059 EAE1175 120583MQE

[21] Food extracts Various extracts and compounds

Hesperetin 35 120583MQETheaflavin-331015840-digallate49 120583MQECranberry juice purple grapejuice and black teaIC50 24ndash58 of extractsSage cinnamon thyme androsemary infusions IC50129ndash204 of infusion

[31] Flavonoids Genistein apigenin quercetin rutin and astilbin

In vitro no significant inhibitoryeffectIn vivo quercetin rutin andastilbin-potent XOI

[20] Ganoderma tsugaeMurr(Polyporaceae)

CE isolated compounds new lanostanoids sugaric acids AB and C tsugarioside A and four known compounds3120573-hydroxy-5120572-lanosta-824-dien-21-oic acid3-oxo-5120572-lanosta-824-dien-21-oic acidergosta-722-dien-3120573-ol and212057331205729120572-trihydroxy-5120572-ergosta-722-dieneME two new lanostanoids sugariosides B and C and amixture of the already known compounds51205728120572-epidioxyergosta-622-dien-3120573-ol and51205728120572-epidioxyergosta-69(11)22-trien-3120573-ol

Potent XOI

[25] Plant species from CzechRepublic Different crude plant extracts

Methylene chloride ME ofPopulus nigra and Betulapendula with IC50 of 83 and259 120583gmL80 EE of Caryophyllusaromaticus and Hypericumperforatum 50 120583gmL

[19]

Erythrina stricta(Papilionaceae) distributedin India China Thailand

and Vietnam

Fractions of the hydromethanolic extract of leaves

Chloroform fraction (IC50 212 plusmn16 120583gmL)pet ether (IC50 302 plusmn22 120583gmL)ethyl acetate (IC50 449 plusmn14 120583gmL)and residual fraction (IC50 100 plusmn33 120583gmL)

[26] Centaurium erythraea(Gentianaceae)

C erythraea flowering tops infusionIdentified compound several esters of hydroxycinnamicacids namely p-coumaric ferulic and sinapic acids

Noncompetitively inhibitingxanthine oxidase

the starting material the cream is washed and churned toyield a crude MFGM preparation dissociating and reducingagents are used to liberate XO from membrane lipoproteinsand some form of chromatography is used for purification[49]

Since the discovery of XO many authors tried to purifyand characterize this enzyme Schardinger [14] is a partially

purified XO and found that reduced activity of the enzyme isnot only a consequence of the removal of fat from milk butalso to reduce the concentration of the enzyme on the surfacedue to the absorption of fat molecules XO from milk washighly purified by Ball in 1939 [50] He used continuous stepsof centrifugation on milk in order to separate cream layerrich in XO Ball [50] separated XO from cream Since then


XO has been isolated and purified by several authors [51 52]Hart and coworkers [53] prepared XO from milk accordingto the procedure described earlier by Palmer et al [54] Theyseparated cream from milk and then added salicylate andEDTA as concentrated solutions to the cream

The published purification procedures for xanthine oxi-dase include proteolytic cleavage calcium chloride treatmentseveral ammonium sulfate fractionations dialysis and sev-eral chromatographic steps [55 56] Ozer et al [57] isolatedXO from fresh bovine milk modifying previous purificationprocedures to achieve high-yield purification procedureThey added EDTA and toluene in fresh milk and after themilk was churned and cooled the (NH

4)2SO4was addedThe

suspension was centrifuged and the precipitate formed wasdiscarded The supernatant was brought to 50 saturationwith solid ammonium sulfate

Baghiani et al [29] purified XO from mammalian milk(bovine) in the presence of 10mM of dithiothreitol byammonium sulphate fractionation followed by affinity chro-matography on heparin agarose

Zhang et al [58] isolated and purified XO fromArthrobacter M3 The Arthrobacter M3 culture was trans-ferred into induction medium The cells were harvested bycentrifugation and were resuspended in sodium phosphate

4 XO Activity Assay Medicinal Importance

Determining the content and activities of XO can be used fordiagnostic purposes The most frequently used method forthe determination of XO activity is described by Marcocciet al [59] and Cos et al [34] The measurement is carriedout in buffer pH 74 which is the most common carbonateor phosphate puffer at 25 or 37∘C The incubation periodis made by various authors from 15 to 30min EDTA isoften added to complexation of metals present in the testsample Spectrophotometric determination of XO activity isbased on measuring uric acid production from xanthine orhypoxanthine substrate at around 295 nmThe assay mixturealways contains xanthine as a substrate and sample Reactionis initiated by adding the XO Higher values indicate apathological condition

Some studies support the hypothesis that uric acid isconnected with elevated vascular events in patients withhypertension diabetes and known cardiovascular disease[60ndash63] The treatment for hyperuricemia and gout is eitherincreasing the excretion of uric acid or reducing the uricacid production Xanthine oxidase inhibitors (XOI) are veryuseful for this [19] The inhibition of XO reduces bothvascular oxidative stress and circulating levels of uric acidAllopurinol is XOI with high potential Inhibitory activitiesof plant extracts and their constituents are compared withthe activity of allopurinol as standard In Table 1 is given theassay procedure of effect of plant extract on xanthine oxidaseactivity

Xanthine oxidase inhibitors (XOI) are typically usedin the treatment of nephropathy and renal stone diseaseslinked to hyperuricemia There has been recent interest inthe potential benefit of XOI in the prevention of vascular

Table 3 IC50 values of flavonoids and allopurinol for inhibition ofXO

Compounds XO IC50 (120583M) plusmn SD(plusmn)-Taxifolin gt100(+)-Catechin gt100(minus)-Epicatechin gt100(minus)-Epigallocatechin gt10041015840-Hydroxyflavanone gt30Naringenin gt507-Hydroxyflavanone 380 plusmn 70Chrysin 084 plusmn 013Apigenin 070 plusmn 023Luteolin 055 plusmn 004Baicalein 279 plusmn 0013-Hydroxyflavone gt100Galangin 180 plusmn 007Kaempferol 106 plusmn 003Quercetin 262 plusmn 013Fisetin 433 plusmn 019Morin 101 plusmn 070Myricetin 238 plusmn 013Allopurinol 024 plusmn 001Luteolin + epigallocatechin (1 1) 076 plusmn 008

disease because of emerging evidence suggesting a rolefor serum uric acid in the development of cardiovasculardisease the enzyme is an important source of oxidativestress in the vasculature [64] XOI are agents that directlyinhibit the synthesis of uric acid in vivo Certain activeconstituents present in crude plant extracts like flavonoidsand polyphenolic compounds have been reported to possessXOI [65 66] These findings have opened the possibility ofisolation of new natural compounds which can be possibleinhibitors of XO and led to the growing interest in theinvestigation of medicinal plants The activity of flavonoidsas inhibitors of xanthine oxidase in vitro has been reportedThe absence of a hydroxyl group at C-3 enhances slightly theinhibition effect on XO [34 67 68]

In traditional medicine are used many herbs and theirextracts in the treatment of various diseases that are the resultof increased XO activity Scientists have studied why someplants and their extracts have an inhibitory effect on theactivity of XO (Table 3) [69]

In Table 2 are given plant extract and pure compounds aspotent XOI

The structure-activity relationship of flavonoids asinhibitors of xanthine oxidase and as scavengers of thesuperoxide radical produced by the action of the enzymexanthine oxidase was investigated The hydroxyl groups atC-5 and C-7 and the double bond between C-2 and C-3 wereessential for a high inhibitory activity on xanthine oxidaseFlavones showed slightly higher inhibitory activity thanflavonols [34]


5 Conclusions

This review is an overview of methods for the isolationand determination of XO activity in vivo and in vitro andinhibition by plant extracts and their constituents For iso-lation the most used methods are extraction centrifugationand chromatographic separation Plant extracts and theirconstituents show good inhibitor activity and therefore mayhave a positive impact on the prevention of disease causedby increased activity of XO Elevated concentrations of uricacid in the blood stream of human body lead to formation ofgout characterized by hyperuricemia and recurrent attacksof arthritis so xanthine oxidase (XO) inhibitors may serve astherapeutic agents for hyperuricemia andor gout Xanthineoxidase inhibitors are agents that directly inhibit the synthesisof uric acid in vivo Certain active constituents present incrude plant extracts like flavonoids and polyphenolic com-pounds have been reported to possess XOI These findingshave opened the possibility of isolation of new naturalcompounds which can be potent inhibitors of XO and led tothe growing interest in the investigation of medicinal plants

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Financial support of this work is provided by the SerbianMinistry of Education and Science Project no ON 172047

References

[1] K Sachidanandam S C Fagan and A Ergul ldquoOxidative stressand cardiovascular disease antioxidants and unresolved issuesrdquoCardiovascular Drug Reviews vol 23 no 2 pp 115ndash132 2005

[2] B Halliwell and J M C Gutteridge ldquoFree radicals ageing anddiseaserdquo in Free Radicals in Biology and Medicine pp 422ndash437Clarendron Press Oxford UK 1989

[3] U Gawlik-Dziki M Swieca D Sugier and J Cichocka ldquoCom-parison of in vitro lipoxygenase xanthine oxidase inhibitoryand antioxidant activity of Arnica montana and Arnica chamis-sonis tincturesrdquoActa Scientiarum Polonorum HortorumCultusvol 10 no 3 pp 15ndash27 2011

[4] E Cadenas ldquoBiochemistry of oxygen toxicityrdquo Annual Reviewof Biochemistry vol 58 pp 79ndash110 1989

[5] G-R Zhao Z-J Xiang T-X Ye Y-J Yuan and Z-X GuoldquoAntioxidant activities of Salviamiltiorrhiza and Panax notogin-sengrdquo Food Chemistry vol 99 no 4 pp 767ndash774 2006

[6] E Cadenas and K J A Davies ldquoMitochondrial free radicalgeneration oxidative stress and agingrdquoFree Radical Biology andMedicine vol 29 no 3-4 pp 222ndash230 2000

[7] M Inoue E F Sato M Nishikawa et al ldquoMitochondrialgeneration of reactive oxygen species and its role in aerobicliferdquoCurrentMedicinal Chemistry vol 10 no 23 pp 2495ndash25052003

[8] M Valko M Izakovic M Mazur C J Rhodes and J TelserldquoRole of oxygen radicals inDNAdamage and cancer incidencerdquo

Molecular and Cellular Biochemistry vol 266 no 1-2 pp 37ndash562004

[9] M Laguerre J Lecomte and P Villeneuve ldquoEvaluation of theability of antioxidants to counteract lipid oxidation existingmethods new trends and challengesrdquoProgress in Lipid Researchvol 46 no 5 pp 244ndash282 2007

[10] M Ferrali C Signorini B Caciotti et al ldquoProtection againstoxidative damage of erythrocyte membrane by the flavonoidquercetin and its relation to iron chelating activityrdquo FEBSLetters vol 416 no 2 pp 123ndash129 1997

[11] A J Elliott S A Scheiber C Thomas and R S PardinildquoInhibition of glutathione reductase by flavonoids A structure-activity studyrdquo Biochemical Pharmacology vol 44 no 8 pp1603ndash1608 1992

[12] R Hirano W Sasamoto A Matsumoto H Itakura O Igarashiand K Kondo ldquoAntioxidant ability of various flavonoids againstDPPH radicals and LDL oxidationrdquo Journal of NutritionalScience and Vitaminology vol 47 no 5 pp 357ndash362 2001

[13] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology and Medicine vol 20 no 1 pp35ndash43 1996

[14] F Schardinger ldquoUeber das Verhalten der KuhmilchgegenMethylenblau und seine Verwendungzur Unterscheidung vonungekochterund gekochter Milchrdquo Zeitschrift fur Untersuchungder Nahrungs- und Genussmittel vol 5 no 22 pp 1113ndash11211902

[15] E JMorgan C P Stewart and F GHopkins ldquoOn the anaerobicand aerobic oxidation of xanthin and hypoxanthin by tissuesand by milkrdquo Proceedings of the Royal Society of London SeriesB vol 94 no 657 pp 109ndash131 1922

[16] P Hass and T G Hill ldquoObservations on certain reducing andoxidizing reactions in milkrdquo Biochemical Journal vol 17 pp671ndash682 1923

[17] P Hass and B Lee ldquoFurther observations on certain reducingand oxidizing reactions in milkrdquo Biochemical Journal vol 18pp 614ndash620 1924

[18] V H Booth ldquoThe specificity of xanthine oxidaserdquo BiochemicalJournal vol 32 pp 494ndash502 1938

[19] M Umamaheswari K AsokKumar A Somasundaram TSivashanmugam V Subhadradevi and T K Ravi ldquoXanthineoxidase inhibitory activity of some Indian medical plantsrdquoJournal of Ethnopharmacology vol 109 no 3 pp 547ndash551 2007

[20] K-W Lin Y-T Chen S-C Yang B-L Wei C-F Hung andC-N Lin ldquoXanthine oxidase inhibitory lanostanoids fromGanoderma tsugaerdquo Fitoterapia vol 89 no 1 pp 231ndash238 2013

[21] T P Dew A J Day and M R A Morgan ldquoXanthine oxidaseactivity in vitro effects of food extracts and componentsrdquoJournal of Agricultural and Food Chemistry vol 53 no 16 pp6510ndash6515 2005

[22] S M N Azmi P Jamal and A Amid ldquoXanthine oxidaseinhibitory activity from potential Malaysian medicinal plant asremedies for goutrdquo International Food Research Journal vol 19no 1 pp 159ndash165 2012

[23] S M Motamed and F Naghibi ldquoAntioxidant activity of someedible plants of the Turkmen Sahra region in northern IranrdquoFood Chemistry vol 119 no 4 pp 1637ndash1642 2010

[24] N Masuoka and I Kubo ldquoCharacterization of xanthine oxidaseinhibition by anacardic acidsrdquo Biochimica et Biophysica ActamdashMolecular Basis of Disease vol 1688 no 3 pp 245ndash249 2004


[25] J Havlik R G de la Huebra K Hejtmankova et al ldquoXanthineoxidase inhibitory properties of Czech medicinal plantsrdquo Jour-nal of Ethnopharmacology vol 132 no 2 pp 461ndash465 2010

[26] P Valentao E Fernandas F Carvalho P B Andrade R MSeabra and M L Bastos ldquoAntioxidant activity of Centauriumerythraea infusion evidenced by its superoxide radical scav-enging and xanthine oxidase inhibitory activityrdquo Journal ofAgricultural and Food Chemistry vol 49 no 7 pp 3476ndash34792001

[27] A Nagao M Seki and H Kobayashi ldquoInhibition of xanthineoxidase by flavonoidsrdquo Bioscience Biotechnology and Biochem-istry vol 63 no 10 pp 1787ndash1790 1999

[28] S Boumerfeg A Baghiani M Djarmouni et al ldquoInhibitoryActivity on xanthine oxidase and antioxidant properties ofTeucrium polium L extractsrdquo Chinese Medicine vol 3 pp 30ndash41 2012

[29] A Baghiani S Boumerfeg M Adjadj et al ldquoAntioxidants freeradicals scavenging and xanthine oxidase inhibitory potentialsof Ajuga iva L extractsrdquo Free Radicals and Antioxidants vol 1no 4 pp 21ndash30 2011

[30] J Huang S Wang M Zhu J Chen and X Zhu ldquoEffectsof genistein apigenin quercetin rutin and astilbin on serumuric acid levels and xanthine oxidase activities in normal andhyperuricemicmicerdquo Food and Chemical Toxicology vol 49 no9 pp 1943ndash1947 2011

[31] Y-T Tung and S-T Chang ldquoInhibition of xanthine oxidase byAcacia confusa extracts and their phytochemicalsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 2 pp 781ndash7862010

[32] F Candan ldquoEffect of Rhus coriaria L (Anacardiaceae) onsuperoxide radical scavenging and xanthine oxidase activityrdquoJournal of Enzyme Inhibition and Medicinal Chemistry vol 18no 1 pp 59ndash62 2003

[33] A Mittal A R J Phillips B Loveday and J A Windsor ldquoThepotential role for xanthine oxidase inhibition in major intra-abdominal surgeryrdquoWorld Journal of Surgery vol 32 no 2 pp288ndash295 2008

[34] P Cos L Ying M Calomme et al ldquoStructure-activity relation-ship and classification of flavonoids as inhibitors of xanthineoxidase and superoxide scavengersrdquo Journal of Natural Prod-ucts vol 61 no 1 pp 71ndash76 1998

[35] E E Kelley N K H Khoo N J Hundley U Z Malik B AFreeman and M M Tarpey ldquoHydrogen peroxide is the majoroxidant product of xanthine oxidaserdquo Free Radical Biology andMedicine vol 48 no 4 pp 493ndash498 2010

[36] J Flemmig K Kuchta J Arnhold and H W Rauwald ldquoOleaeuropaea leaf (PhEur) extract as well as several of its isolatedphenolics inhibit the gout-related enzyme xanthine oxidaserdquoPhytomedicine vol 18 no 7 pp 561ndash566 2011

[37] J Dawson and M Walters ldquoUric acid and xanthine oxidasefuture therapeutic targets in the prevention of cardiovasculardiseaserdquo British Journal of Clinical Pharmacology vol 62 no6 pp 633ndash644 2006

[38] F J Nieto C Iribarren M D Gross G W Comstock and RG Cutler ldquoUric acid and serum antioxidant capacity a reactionto atherosclerosisrdquo Atherosclerosis vol 148 no 1 pp 131ndash1392000

[39] A Burke E Smyth and G A FitzGerald ldquoAnalgesicndashantipyretic agents pharmacotherapy of goutrdquo in The Pharma-cological Basis of Therapeutics L L Brunton J S Lazo and KL Parker Eds pp 706ndash710 McGraw-Hill Medical PublishingDivision New York NY USA 11th edition 2006

[40] H K Choi D B Mount and A M Reginato ldquoPathogenesis ofgoutrdquo Annals of Internal Medicine vol 143 no 7 pp 499ndash5162005

[41] M T T Nguyen S Awale Y Tezuka Q Le Tran and SKadota ldquoXanthine oxidase inhibitors from the heartwood ofVietnamese Caesalpinia sappanrdquo Chemical and PharmaceuticalBulletin vol 53 no 8 pp 984ndash988 2005

[42] PHiggins LD Ferguson andMRWalters ldquoXanthine oxidaseinhibition for the treatment of stroke disease a novel therapeu-tic approachrdquo Expert Review of Cardiovascular Therapy vol 9no 4 pp 399ndash401 2011

[43] T M Ngoc N M Khoi D T Ha et al ldquoXanthine oxi-dase inhibitory activity of constituents of Cinnamomum cassiatwigsrdquo Bioorganic and Medicinal Chemistry Letters vol 22 no14 pp 4625ndash4628 2012

[44] R Hille and V Massey ldquoTight binding inhibitors of xanthineoxidaserdquo Pharmacology andTherapeutics vol 14 no 2 pp 249ndash263 1981

[45] T R Hawkes G N George and R C Bray ldquoThe structureof the inhibitory complex of alloxanthine (1H-pyrazolo[34-d]pyrimidine-46-diol) with the molybdenum centre of xan-thine oxidase from electron-paramagnetic-resonance spec-troscopyrdquo Biochemical Journal vol 218 no 3 pp 961ndash968 1984

[46] E B Skibo ldquoNoncompetitive and irreversible inhibition ofxanthine oxidase by benzimidazole analogues acting at thefunctional flavin adenine dinucleotide cofactorrdquo Biochemistryvol 25 no 15 pp 4189ndash4194 1986

[47] T Unno A Sugimoto and T Kakuda ldquoXanthine oxidaseinhibitors from the leaves of Lagerstroemia speciosa (L) PersrdquoJournal of Ethnopharmacology vol 93 no 2-3 pp 391ndash3952004

[48] M Umamaheswari K Asokkumar A T Sivashanmugam ARemyaraju V Subhadradevi and T K Ravi ldquoIn vitro xanthineoxidase inhibitory activity of the fractions of Erythrina strictaRoxbrdquo Journal of Ethnopharmacology vol 124 no 3 pp 646ndash648 2009

[49] P F Fox and A L Kelly ldquoIndigenous enzymes inmilk overviewand historical aspectsmdashpart 1rdquo International Dairy Journal vol16 no 6 pp 500ndash516 2006

[50] E G Ball ldquoXanthine oxidase purification and propertiesrdquoTheJournal of Biological Chemistry vol 128 pp 51ndash67 1939

[51] N Y Farkye ldquoIndigenous enzymes in milk other enzymesrdquo inAdvanced Dairy Chemistry Volume 1 Proteins P F Fox Ed pp339ndash367 Elsevier Applied Science London UK 1992

[52] N Y Farkye ldquoIndigenous enzymes in milk other enzymesrdquo inAdvanced Dairy Chemistry P F Fox and P L H McSweeneyEds Elsevier 2003

[53] L IHartMAMcGartoll H R Chapman andRC Bray ldquoThecomposition ofmilk xanthine oxidaserdquoBiochemical Journal vol116 no 5 pp 851ndash864 1970

[54] G Palmer R C Bray and H Beinert ldquoDirect studies on theelectron transfer sequence in xanthine oxidase by electron para-magnetic resonance spectroscopy I Techniques and descriptionof spectrardquo The Journal of Biological Chemistry vol 239 pp2657ndash2666 1964

[55] C A Nelson and P Handler ldquoPreparation of bovine xanthineoxidase and the subunit structures of some iron flavoproteinsrdquoThe Journal of Biological Chemistry vol 243 no 20 pp 5368ndash5373 1968

[56] W R Waud F O Brady R D Wiley and K V Rajagopalan ldquoAnew purification procedure for bovine milk xanthine oxidase


effect of proteolysis on the subunit structurerdquo Archives ofBiochemistry and Biophysics vol 169 no 2 pp 695ndash701 1975

[57] N Ozer M Muftuoglu D Ataman A Ercan and I HOgus ldquoSimple high-yield purification of xanthine oxidase frombovine milkrdquo Journal of Biochemical and Biophysical Methodsvol 39 no 3 pp 153ndash159 1999

[58] Y Zhang Y Xin H Yang et al ldquoNovel affinity purificationof xanthine oxidase from Arthrobacter M3rdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 906 pp 19ndash24 2012

[59] L Marcocci L Packer M-T Droy-Lefaix A Sekaki and MGardes-Albert ldquoAntioxidant action of Ginkgo biloba extractEGb 761rdquoMethods in Enzymology vol 234 pp 462ndash475 1994

[60] J F Baker E Krishnan L Chen andH R Schumacher ldquoSerumuric acid and cardiovascular disease recent developments andwhere do they leave usrdquoTheAmerican Journal of Medicine vol118 no 8 pp 816ndash826 2005

[61] S Kivity E Kopel E Maor et al ldquoAssociation of serum uricacid and cardiovascular disease in healthy adultsrdquoTheAmericanJournal of Cardiology vol 111 no 8 pp 1146ndash1151 2013

[62] S G Wannamethee ldquoSerum uric acid and risk of coronaryheart diseaserdquoCurrent Pharmaceutical Design vol 11 no 32 pp4125ndash4132 2005

[63] E J Newman F S Rahman K R Lees C J Weir and MRWalters ldquoElevated serum urate concentration independentlypredicts poor outcome following stroke in patients with dia-betesrdquo DiabetesMetabolism Research and Reviews vol 22 no1 pp 79ndash82 2006

[64] P Higgins J Dawson and M Walters ldquoThe potential forxanthine oxidase inhibition in the prevention and treatmentof cardiovascular and cerebrovascular diseaserdquo CardiovascularPsychiatry and Neurology vol 2009 Article ID 282059 9 pages2009

[65] W S Chang Y J Lee F J Lu and H C Chiang ldquoInhibitoryeffects of flavonoids on xanthine oxidaserdquo Anticancer Researchvol 13 no 6 pp 2165ndash2170 1993

[66] L Costantino A Albasini G Rastelli and S Benvenuti ldquoActiv-ity of polyphenolic crude extracts as scavengers of superoxideradicals and inhibitors of xanthine oxidaserdquo Planta Medica vol58 no 4 pp 342ndash344 1992

[67] D E C van Hoorn R J Nijveldt P A M van Leeuwen et alldquoAccurate prediction of xanthine oxidase inhibition based onthe structure of flavonoidsrdquo European Journal of Pharmacologyvol 451 no 2 pp 111ndash118 2002

[68] R Arimboor M Rangan S G Aravind and C Aru-mughan ldquoTetrahydroamentoflavone (THA) from Semecarpusanacardium as a potent inhibitor of xanthine oxidaserdquo Journalof Ethnopharmacology vol 133 no 3 pp 1117ndash1120 2011

[69] M T Nguyen S Awale Y Tezuka J Y Ueda Q Tran andS Kadota ldquoXanthine oxidase inhibitors from the flowers ofChrysanthemum sinenserdquo Planta Medica vol 72 no 1 pp 46ndash51 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


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Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

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Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


S

OH

OGlu1261

N

NN

N

O

H

HO

H

Xanthine

HO

SH

N

NN

N

O

H

HO

H

N

NN

N

O

H

HO

H

O

S

S

OH

N

NN

N

O

H

HO

H

H

O

Uric acid

O O

O

O

Ominus

H2

O

H+

eminus

MoVI

MoVI

MoIV

MoV

Scheme 1 Transformation of xanthine to uric acid by XO

N

N N

N

O

H

HHypoxanthine

N

N N

N

O

H

HXanthine

Xanthineoxidase

O

H

N

N NN

OH

H

Allopurinol

N

N N

N

O

H

HO

HXanthine

Xantineoxidase

N

N N

N

O

H

HO

H

O

H

Uric acid

Figure 1 Inhibition of xanthine oxidase by allopurinol to prevent conversion of hypoxanthine to xanthine andor uric acid

2 Xanthine Oxidase Mechanism of Action

In 1902 Schardinger [14] showed that milk contains anenzyme capable of oxidizing aldehydes to acids accompaniedby the reduction of methylene blue this enzyme was thencommonly called the ldquoSchardinger enzymerdquo In 1922 Morganet al [15] showed that milk contains an enzyme capable ofoxidizing xanthine and hypoxanthine with the concomitantreduction of O

2to H2O2 and this enzyme was called XO

Hass and Hill [16] and Hass and Lee [17] reported that milkcontains a substance which they called ldquoitaterdquo capable of

oxidizing nitrite to nitrate in the presence of an aldehyde andO2under other conditions and thatmilk could reduce nitrate

to nitrite In 7 1938 Booth [18] presented strong evidence thatthe Schardinger enzyme was in fact XO

XO is a homodimer with molecule mass of 290 kDaXanthine oxidase belongs to themolybdenum-protein familycontaining one molybdenum one of the flavin adeninedinucleotides (FAD) and two iron-sulfur (2Fe-2S) centers ofthe ferredoxin type in each of its two independent subunitsThe enzyme contains two separated substrate-binding sitesXO catalysed the oxidation of hypoxanthine to xanthine and


















2




2O2


2

∙minus+ 2H+


2O2

2O2


2O2+O2

(1)












THAIC50 100 nM




CE 079 ME 1059 EAE1175 120583MQE







Potent XOI



[19]


and Vietnam












4)2SO4was addedThe















5 Conclusions




Acknowledgment


References



















































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


















2




2O2


2

∙minus+ 2H+


2O2

2O2


2O2+O2

(1)












THAIC50 100 nM




CE 079 ME 1059 EAE1175 120583MQE







Potent XOI



[19]


and Vietnam












4)2SO4was addedThe















5 Conclusions




Acknowledgment


References



















































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





THAIC50 100 nM




CE 079 ME 1059 EAE1175 120583MQE







Potent XOI



[19]


and Vietnam












4)2SO4was addedThe















5 Conclusions




Acknowledgment


References



















































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




4)2SO4was addedThe















5 Conclusions




Acknowledgment


References



















































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


5 Conclusions




Acknowledgment


References



















































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Review Article Xanthine Oxidase: Isolation, Assays of Activity, …downloads.hindawi.com/journals/jchem/2015/294858.pdf · Review Article Xanthine Oxidase: Isolation, Assays of Activity,