w w w.jo u r n als . e l s e vie r . c o m / r e v i s t a - b r a s i l e i r a - d e - f a r m a c o g n o s i a
Original
Article
Screening
of
ferulic
acid
related
compounds
as
inhibitors
of
xanthine
oxidase
and
cyclooxygenase-2
with
anti-inflammatory
activity
Shivraj
Hariram
Nile,
Eun
Young
Ko,
Doo
Hwan
Kim,
Young-Soo
Keum
∗ DepartmentofBioresourcesandFoodScience,CollegeofLifeandEnvironmentalSciences,KonkukUniversity,Seoul,SouthKoreaa
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received6May2015 Accepted23August2015 Availableonline29November2015
Keywords: Ferulicacid Gallicacid Xanthineoxidase Cyclooxygenase SAR
Gout
a
b
s
t
r
a
c
t
Theferulicandgallicacidrelatedcompoundsfromnaturaloriginwerestudiedagainstxanthineoxidase andcyclooxygenase-2alongwiththeiranti-inflammatoryactivity.Thecompoundsgallicacid,ferulic acid,caffeicacidandp-coumaricacidrevealedpromisinganti-inflammatoryactivity(30–40%TNF-␣
and60–75%IL-6inhibitoryactivityat10M).Bioavailabilityofcompoundswerecheckedbyinvitro cytotoxicityusingCCK-8celllinesandconfirmedtobenontoxic,butfoundtoxicathigherconcentration (50M).Gallic,ferulic,caffeicacidwasdemonstratedpotentialdualinhibitiontowardxanthineoxidase andcyclooxygenase-2ascalculatedbyIC50:68,70.2,and65g/ml(xanthineoxidase)and68.5,65.2,
and62.5g/ml(cyclooxygenase-2),respectively.Thestructureactivityrelationshipandinsilicodrug relevantproperties(HBD,HBA,PSA,cLogP,ionizationpotential,molecularweight,EHOMOandELUMO)
furtherconfirmedthatthecompoundswerepotentialcandidatesforfuturedrugdiscoverystudy,which wasexpectedforfurtherrationaldrugdesignagainstxanthineoxidaseandcyclooxygenase.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Goutyarthritisisawellknowndiseasewithanabruptattack causing extreme pain in and around the joints due to activi-tiesoccurredbyxanthineoxidase(Mohapatraetal.,2015).Gout isnearlyalwaysassociatedwithchronichyperuricemia,a long-lastingabnormallyhighconcentrationofuricacid(hyperuricemia) intheblood.Thehigherlevelsofuricacidcanreachapointwhere uricacidcrystals(monosodiumurate)areputdifferentlyandthis hyperuricemiaresultsinthedepositionofcrystalsofsodiumurate intissues,especiallyinkidneysandjoints(Bardin,2004;Choiand Curhan,2005).Thisprocessgeneratesoxygenmetabolites,which damagetissue,resultinginthereleaseoflysosomalenzymesthat induceaninflammatoryresponse.Thiswillleadtolocaldecrease ofpHwhichfurthercausesmoredepositionofuratecrystals(Nuki andSimkin,2006).Thediagnosisofgoutisbasedonthepresence ofmonosodiumuratecrystalsinthesynovialfluid(Martinonand Glimcher,2006).Thisdepositionwillexacerbateleadingto recur-rentepisodesofacutearthritis,theclassicmanifestationofgout. Meanwhile,controllingtheuricacidlevelinthebloodisstillthe maintargetparticularlyinthemanagementofthechronicattacks (Mandell,2002; Dinceretal.,2002).Xanthineoxidase(XO)has
∗ Correspondingauthor.
E-mail:rational@konkuk.ac.kr(Y.-S.Keum).
amajorroleintheuricacidproductionasXOisresponsiblefor catalyzing theoxidation ofhypoxanthinetoform xanthineand finally touric acid. Thus, this enzyme coordinates thereaction andproduces uric acidfromitsprecursors(Nileet al.,2013;Li etal.,2013).Similarly,certainenzymessuchas cyclooxygenase-2(COX-2)contributesitsroleinthegoutyinflammation,thoughit heightenedexpressionsinthepresenceoftheaccumulatedMSU crystals,whichinturnenhancestheproductionofinflammatory prostaglandinsleadingtotheincreasedproductionofIL-1.Thus, COX-2playsamajorroleinarousingtheinflammatoryresponses andthustakingpartintheadvancementoftheacute inflamma-tioninthegoutyarthritispatients(Pouliotetal.,1998;Mohapatra etal.,2015).Therearecertainmeasurestakeninordertotreatthe diseasebyusingnon-steroidalanti-inflammatorydrugs(NSAID), colchicineorglucocorticoids.NSAIDaretheclassofdrugswhich causesCOX-2inhibitionwhereascolchicineisanantimycotic alka-loidthatapartfromdisturbingthemicrotubulepolymerization,it alsoinhibitstheinflammationbypreventingtheIL-1processing whichwasstimulatedbytheMSUcrystals(Martinonetal.,2006; RicciottiandFitzGerald,2011).Examplesofsuchdrugsare allo-purinol,probenecidandsulfinpyrazone(Mandell,2002;Aggarwal etal.,2011).Modernmedicinesfromnaturalproductshavelittle toofferforalleviationofgout,oxidativeandinflammatory activ-ity.Thereisanurgentneedtodiscovercompoundswithxanthine oxidaseinhibitoryactivitiesbutdevoidoftheundesirableeffectsof allopurinol.Onepotentialsourceofsuchcompoundsismedicinal http://dx.doi.org/10.1016/j.bjp.2015.08.013
materialsofplantoriginwhichisusedtotreatconditionssimilar togoutyarthritis(NileandPark,2014a,b).Phenolicsandflavonoids areconsideredimportantcomponentsinthehumandietbecause of their beneficial effects on humanhealth. The phenolics and flavonoidsarenaturallyoccurringcompoundsthatareconstantly distributedinvariousfoods,fruitjuicesandbeveragesfromplant originanddisplaymanybioactiveandtherapeuticproperties,such asantioxidant,anticancer,antiviral,anti-inflammatory,and car-diacprotectiveeffects(Proestosetal.,2005;NileandPark,2014c). Mostofthetherapeuticpropertiesofphenolicsandflavonoidshave beendemonstratedtohavepotentantioxidant,anti-inflammatory andenzymeinhibitionproperties.Severalphenolicsandflavonoids havebeen describedas inhibitorsofthe xanthineoxidase(XO) enzyme,similartoallopurinolinthetreatmentofgout(Bandgar etal.,2009; Nileand Park,2013), forthis reason,researchinto ferulicacid seemspromising. Thus, the aimof this studyis to investigatethepotencyofnaturallyoccurringferulicacidrelated compoundsasxanthineoxidaseandcyclooxygenase-2inhibitors alongwiththepropertieslikeanti-inflammatoryactivity, struc-tureactivityrelationship(SAR)andinsilicodrugrelevantproperties werestudied.
Materialsandmethods
Chemicals
Xanthine oxidase, xanthine, allopurinol, myricetin, and iso-quercetinwere purchased fromSigma Chemical(Seoul,Korea). Ferulicacid(2),gallicacid(6),andtheirestersweresuppliedby Hi-MediaLaboratories,Mumbai,India.Caffeicacid(3),sinapicacid (4),p-coumaric acid(1)procuredfromSigma–Aldrich,Mumbai, India.Cyclooxygenasefluorescentinhibitorscreeningassaykitwas obtainedfromCaymanChemicalCompany(AnnArbor,MI,USA). Bovinemilkxanthineoxidaseprocuredfrom(grade1,ammonium sulfatesuspension)Sigma–Aldrich,Mumbai,India.
CO2H R1
HO R2
1 R1=R2=H
2 R1=OCH3; R2=H
3 R1=OH; R2=H
4 R1=R2=OCH3
CO2CH3
CO2R H3CO
HO 5
HO
HO OH
6R=H
7 R=CH3
8 R=(CH2)2CH3
9 R=(CH2)11CH3
10 R=(CH2)17CH3
Anti-inflammatoryandcytotoxicityassay
Pro-inflammatorycytokine production by lipopolysaccharide (LPS) in THP-1 cells was measured according to the method
describedbyBandgaretal.(2009).Duringassay,THP-1cellswere culturedwithpenicillinandstreptomycin(100U/ml)and inocu-latedwith10%fetalbovineserum(FBS,JRH)inRPMI1640culture medium (GibcoBRL, Pasley,UK). Cells weredifferentiated with phorbol myristateacetate (PMA, Sigma). Following cellplating, thetestcompounds(10M) in0.5%DMSOwaspouredtoeach wellandtheplatewereincubatedfor30minat37◦C.Finally,LPS
(Escherichiacoli0127:B8,SigmaChemicalCo.,St.Louis,MO)was added,atafinalconcentrationof1g/mlineachwell.Plateswere furtherincubated at37◦C for24hin 5%CO
2.Afterincubation, supernatantswereharvested,andassayedforTNF-␣andIL-6by ELISAasdescribedbythemanufacturer(BDBiosciences,India).The cellsweresimultaneouslyevaluatedforcytotoxicityusingCCK-8 fromDojindoLaboratories.Percentinhibitionofcytokinerelease comparedtothecontrolwascalculated.Inthisthecytotoxicitywas checkedatlower,optimumandhigherconcentration(10,25and 50M),50%inhibitoryconcentration(IC50)valueswerecalculated byanonlinearregressionmethod(BandgarandGawande,2010; NileandKhobragade,2011).
InsilicopharmacologicalpropertyandSARstudy
The pharmacological properties of the compounds, such as molecularweight,cLogPandquantumchemicaldescriptorssuch asEHOMO(Energyofhighestoccupiedmolecularorbital)andELUMO (energyoflowestunoccupiedmolecularorbital)ofthesynthesized compoundswerecalculatedusing aBioMedCaChe 6.1(FujiSuit Ltd),acomputeraidedmoleculardesignmodelingtoolforwindows 98/20000/XP operating system.Other parameters suchas HBA (hydrogenbondacceptor),HBD(hydrogenbonddonor),molecular PSA(polarsurfacearea),drugscoreanddruglikenessofthe com-poundswerealsostudiedusingonlineOsirispropertyexplorerfor drugbioavailabilityofchemicalcompounds.Sincecompoundsare consideredfororaldelivery,theywerealsoassessedfortoxicity usinginsilicoADMEpredictionmethods(BandgarandGawande, 2010;NileandKhobragade,2011).
XOinhibitoryactivity
Xanthineoxidase(XO)activitywasassayed spectrophotomet-rically by measuring the uric acid formation at 290nm using a UV-visible spectrophotometer at 25◦C. The reaction mixture
contained50mMpotassiumphosphatebuffer(pH7.6),75M xan-thineand0.08unitsofXO.InhibitionofXOactivityofindividual isolatedphenolicsfrommaize(1.5ml,2mM),wasmeasuredby fol-lowingthedecreaseintheuricacidformationat293nMat25◦C.
The enzymewaspre incubatedfor 5min,withtest compound, dissolvedinDMSO(1%,v/v),andthereactionwasstartedbythe additionof xanthine.Finalconcentrationof DMSO(1%,v/v) did notinterferewiththeenzymeactivity.TheXOkineticstudywas carriedoutusingscreeningoftenferulicacidrelatedcompounds
(10,25,50and100g/ml)comparingwithallopurinol(10,25,50
and100g/ml)aspositivecontrol.Alltheexperimentswere
per-formedintriplicatesandIC50valueswereexpressedasmeansof threeexperiments(Nileetal.,2013;NileandPark,2014a).
COX-2inhibitoryactivity
Table1
Anti-inflammatoryactivityagainstTNF-␣andIL-6.
Samples %Inhibitionat10M
TNF-␣ IL-6 Toxicity
10M 25M 50M
p-Coumaricacid 30 60 0 1.3 3.2
Ferulicacid 40 72 0 1.5 4
Caffeicacid 35 68 0 1.6 3.5
Sinapicacid 23 56 0 2.1 4.5
Alkylferulates 20 40 0 2.4 5
Gallicacid 48 75 0 1.6 3.5
Methylgallate 16 30 0.4 3 8
Propylgallate 18 35 0 2 5
Laurylgallate 20 25 0.5 4 8
Stearylgallate 15 20 0.4 2 7
Dexamethasone 60 92 0 0 3
595nm.TheCOX-2assayconsistedofa200-lreactionmixture containing150lassaybuffer,10lheme,10lCOX-2,10l fluo-rometricsubstrate,and10loftenferulicacidrelatedcompounds (25,50,100g/ml)Thereactionswereinitiatedbyquicklyadding 10lAA,thenincubatingfor5minat37◦Ctemperature.Dup-697
wasusedasapositivecontrol.Alltheexperimentswereperformed intriplicatesand IC50 valueswereexpressedasmeansof three
experiments(Lietal.,2013;Mohapatraetal.,2015).
Statisticalanalysis
Alldataareexpressedasmean±SDoftriplicateexperiments.
Resultsanddiscussion
Anti-inflammatoryandcytotoxicityassay
Alltenferulicacidrelatedcompoundswereevaluatedfor anti-inflammatoryactivitybyTNF-␣andIL-6inhibitionassays.Notall ofthe compoundsshowed a promising TNF-␣ inhibitory activ-ity except gallic acid, ferulic acid, caffeic acid and p-coumaric acidupto30–40%at10Mconcentrations,alsoapromisingIL-6 inhibitoryactivitywasshownbysamecompoundsthatisby gal-licacid,ferulicacid,caffeicacidandp-coumaricacidupto60–75% inhibitionat10Mconcentration.Ascomparedtothestandard dexamethasone,theactivityresultsarerevealedtobe compara-ble assummarized in Table1. Mostof thecompounds didnot showsignificantcytotoxicityat10Mconcentrationexceptlauryl gallate,methylgallateandstearylgallateatverynegligiblelevel butathigherconcentrationsmanyofthecompoundsrevealedcell toxicity.Theresultspresentedforferulicacidrelatedcompounds revealed excellent anti-inflammatory activities as compared to dexamethasone,a knownanti-inflammatory agent.It is known that duringinflammation and associated processes, there is an increasedproductionofsuperoxideions.Itmaybepossiblethat theinhibitionofsuperoxidegenerationinperitonealmacrophages isrelatedtotheanti-inflammatoryactivityofferulicacidrelated compounds(Nileand Khobragade,2011;Nile andPark,2014d). Pro-inflammatorycytokinessuchasTNF-␣,andIL-6areproduced andplaycriticalrolesintheinflammationprocesses.Amongthese pro-inflammatorycytokines,TNF-␣hasbeenhighlightedrecently as a main mediator in the inflammatory diseases mechanism (Maxiaadetal.,2011).Highlevelsofpro-inflammatorycytokines, includingTNF,have beendetectedin psoriaticskinlesionsand jointsofpatientswiththeinflammatorydisease(Mease,2002). TNF-␣mayhaveasignificantroleinpathogenesisofseveral inflam-matorydiseases,suchaspsoriaticarthritis, juvenilerheumatoid arthritis,andCrohn’sdisease.DecreaseintheTNF-␣andanIL-6
levelbythesecompoundssuggeststhatitmaybeusefulina vari-etyofinflammatory conditions(Ellerinet al.,2003;Kueketal., 2007).
InsilicopharmacologicalpropertyandSARstudy
Toqualifythecompound asa drugcandidate,it isanalyzed bytheparameterssetbyLipinski’sruleoffiveusingOsiris prop-ertyexplorer.ThecLogPistheimportantphysiochemicalproperty indicating thelipophilicity and the ability ofmolecule tocross thevariousbiologicalmembranes.AccordingtoLipinski’sruleof fivethecLogPvaluebelow5isfeasibleforacompoundtobea futuredrug.Thealltenferulicacidrelatedcompoundsshoweda marginallipophilicitywithintherangeof4.0–5.0.Themolecular weightpropertyofthecompoundisrelatedtoitsinvivo adminis-tration.Alltheferulicacidrelatedcompoundshavethemolecular weightwithintheacceptablerange, thatis,400–500.The com-pounds showedtheHBA below10 and HBDbelow 5,which is alsowithinthelimitsetbyLipinski’srule.Thepolarsurfacearea (PSA)>140A2is thoughttohavelow oralbioavailability,which isalsorevealedwithintherangeforthesecompounds(Table2). Interestinglythecompoundsalsopresentedabetterdruglikeness values.Overall,thecompoundsferulicacid,gallicacid,caffeicacid andsinapicacidshowedagooddrugscore,calculatedby combin-ingallparameters.Drugtoxicityisafactorofgreatimportancefor apotentialcommercialdrug,sinceasignificantnumberofdrugs aredisapprovedinclinicaltrialsbasedontheirhightoxicityprofile (Bandgaretal.,2009;BandgarandGawande,2010).Thetoxicityof thecompoundsiscalculatedintermsofmutagenicity,tumorigenic, reproductivelyeffectiveandirritant.Allthecompoundswere con-firmedasnon-mutagenicandthereforewerebiologicallysafefor intake.
XOinhibitionbyferulicacidrelatedcompounds
Table2
Insilicopharmacologicalparametersforbioavailabilityofferulicacidrelatedcompounds.
Sample Molecularformula Molecular weight (g/mol)
Meltingpoint (◦C)
EHOMO ELUMO HBD HBA Mol.PSA cLogP Solubility Drug likeness
Drug score
p-Coumaricacid C9H8O3 164 212 −4.28 −0.65 0 4 56.20 4.45 −4.20 0.53 0.32 Ferulicacid C10H10O4 194 170 −5.52 −0.61 0 5 56.20 4.12 −4.50 0.52 0.40
Caffeicacid C9H8O4 180 224 −4.25 −0.58 0 4 56.20 4.60 −4.20 0.41 0.38
Sinapicacid C11H12O5 224 204 −5.34 −0.70 0 4 65.5 5.40 −5.34 −0.22 0.35 Alkylferulates C40H70O4 614 96 −9.55 −0.85 0 6 85.5 6.86 −8.50 0.85 0.28
Gallicacid C7H6O5 170 260 −4.10 −0.60 0 4 55.40 4.11 −4.10 0.55 0.38
Methylgallate C8H8O5 184 202 −4.61 −0.56 0 4 55.20 4.20 −4.22 −0.32 0.30 Propylgallate C10H12O5 212 150 −5.45 −0.55 0 5 60.5 5.10 −4.40 −0.23 0.25 Laurylgallate C19H30O5 338 98 −6.28 −0.68 0 5 72.5 6.85 −5.30 −0.42 0.22 Stearylgallate C25H42O5 423 105 −6.85 −0.75 0 6 75.5 6.20 −6.10 −0.65 0.20
InhibitionofCOX-2byferulicacidrelatedcompounds
Five ferulic acid related compounds demonstrated COX-2-inhibitingactivity(Fig.2), among whichgallic acid, ferulicacid andcaffeicacidshowedaninhibitor>50%at100g/ml.The mech-anism of anti-inflammatory action is assumed to be mediated throughtheinhibitionofCOX-1andCOX-2.Theseenzymes cat-alyzethebiosynthesisofprostaglandinH2 fromthearachidonic
acidsubstrate.TheinhibitionofCOX-1resultsinsomeundesirable side-effects,whereasCOX-2inhibitionprovidestherapeuticeffects inpain,inflammation,cancer,glaucoma,Alzheimer’sand Parkin-sondisease(BlobaumandMarnett,2007).Therefore,weintended toexaminetheCOX-1andCOX-2inhibitoryactivityofferulicacid relatedcompoundsdemonstratedonpurifiedenzymesasa mecha-nismofitstopicalanti-inflammatoryaction.Alsoitwasfoundthat manyphenolic compoundssuchaschlorogenicacid, gallicacid,
Concentration (μg/ml)
XO inhibitors
Inhibition r
atio (%)
IC
50
(
μ
g/mL)
20 0 20 40 60 80
0 20 30
10 40 50 60 70 80 100 120
A
B
40
p-Coumar ic acid
Fer ulic acid
Caff eic acid
Sinapic acid Alkyl f
erulates Gallic acid
Meth yl gallate
Prop yl gallate
Laur yl gallate
Stear
yl gallateAllopur inol
60 80 100
p-Coumaric acid
Ferulic acid
Caffeic acid
Alkyl ferulates
Gallic acid
Methyl gallate
Allopurinol
Concentration (μg/ml)
COX-2 inhibitors
Inhibition r
atio (%)
IC
50
(
μ
g/mL)
20 0
10 20 30 50
40
0 20 30
10 40 50 60 70 60 70
A
B
40
p-Coumar ic acid
Fer ulic acid
Caff eic acid
Sinapic acid Alkyl f
erulates Gallic acid
Meth yl gallate
Prop yl gallate
Laur yl gallate
Stear
yl gallate DUP-697
60 80 100
Ferulic acid
Caffeic acid
Gallic acid
DUP-697
Fig.2.(A)Inhibitionratio(%)and(B)thecalculatedIC50offerulicandgallicacidrelatedcompoundsforinhibitionofCOX-2.Thefivecompoundsdemonstrated COX-2-inhibitingactivityamongwhichferulicacid,caffeicacidandgallicacidshowedaninhibition>50%at100g/ml.DUP-697isapositivecontrol.
rosmarinicacid,catechin,hesperidin,hesperetin,lutenolin, narin-genin,naringinhavebeendemonstratedpossessingtheinhibitory effectforCOX-2expression(Rasoetal.,2001;Shenetal.,2008). Thustheresultindicatesthattheferulicacidrelatedcompounds mayreducetheinflammatoryactivitybyvirtue ofinhibition of COX-2enzymewhichprovidespro-inflammatoryactivity.
Theresultindicatesthattheferulicacidrelatedcompoundsmay reducetheinflammatoryactivitybyvirtueofinhibitionofxanthine oxidaseandCOX-2enzymewhichprovidepro-inflammatory activ-ity.Thesecompoundsmightbeactsasgood sourceofxanthine oxidaseandCOX-2inhibitoryagents,butfurtherinvivoandclinical studyshouldberequiredfortheircharacterizationasadrugagainst variousdiseasesanddisordersrelatedtoactivityofxanthine oxi-daseandCOX-2enzymes.Furthermoretheindividualtheferulic acidrelatedcompoundscanbesubjectedtooptimizationsoasto designand developalead anti-inflammatoryandantigoutdrug withanimprovedpotency.Fordevelopmentofrationaldrug devel-opmentagainstXOandcyclooxygenase-2itisimportanttocheck thestructure–activityrelationshipsandotherfactorsofrelatedto ferulicacidcompoundssuchasbindingaffinity,kineticparameters andchangesatactivesiteofthesestudiedenzymeswerediscussed, whichisexpectedforfurtherdrugdevelopmentanddesign.
Authors’contributions
SHNperformedthelaboratoryworkincludinginvitroxanthine oxidaseandcyclooxygenase-2activity,alsoinsilicostudyanddata analysis.VBassistedinanti-inflammatoryassays.DHKprovided idea andproject planwithsubsequent editing and checkingof manuscriptdataandSHNcontributedtocriticalreadingand writ-ingofthemanuscript.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare
thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.
Righttoprivacyandinformedconsent. Theauthorsdeclarethat
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest. Acknowledgement
ThisstudywassupportedbyKU-Researchprofessor program-2015,KonkukUniversity,Seoul,SouthKorea.
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