Rev. bras. farmacogn. vol.27 número2

RevistaBrasileiradeFarmacognosia27(2017)251–253

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Short

communication

Cytotoxicity,

antioxidant

and

antibacterial

activity

of

four

compounds

produced

by

an

endophytic

fungus

Epicoccum

nigrum

associated

with

Entada

abyssinica

Jean

P.

Dzoyem

,

Raduis

Melong

,

Armelle

T.

Tsamo

,

Timoleon

Maffo

,

Deccaux

G.W.F.

Kapche

_,

_Bonaventure

_T.

_Ngadjui

_,

_Lyndy

_J.

_McGaw

_,

_Jacobus

_N.

_Eloff

a_{DepartmentofParaclinicalSciences,FacultyofVeterinaryScience,UniversityofPretoria,Pretoria,SouthAfrica} b_{DepartmentofOrganicChemistry,FacultyofScience,UniversityofYaoundéI,Yaoundé,Cameroon}

c_{DepartmentofChemistry,HigherTeachers’TrainingCollege,UniversityofYaoundéI,Yaoundé,Cameroon}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received31May2016 Accepted1August2016 Availableonline7December2016

Keywords:

Antimicrobial Freeradicalscavenging Cytotoxicity Endophyticfungi

Epicoccumnigrum

a

b

s

t

r

a

c

t

Four compounds including beauvericin, parahydroxybenzaldehyde, indole-3-carboxylic acid and quinizarinwereisolatedfromendophyticfungusEpicoccumnigrumandtheircytotoxicity,antibacterial andantioxidantactivitywereevaluated.BeauvericinhadremarkableactivityagainsttwoGram-negative strains(BacilluscereusandSalmonellatyphimurium)withrespectiveMICvaluesof3.12and6.25␮g/ml. Allthecompoundshad weakcytotoxic effecton bothnormal andtumorcells.LC50 valuesranged from40.42to86.56␮g/ml,31.87to86.57␮g/mland21.59to67.27␮g/mlonVerocells,THP-1and RAW264.7respectively.Thepresentstudyshowed thatthesecompoundscould bedeveloped for theformulation of antioxidant-richtherapeutic dietsand as atherapeutic agentagainst bacterial infections.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Overthepastfewdecades,multi-drugresistancein microorgan-ismshasemergedasaseriousprobleminhealthcareduemostly totheimproperusageofantibiotics.Inaddition,accumulated evi-denceindicatesthatoxygenradicalssuchassuperoxidearekey moleculesinthepathogenesisofvariousinfectiousdiseases.Some pathologiesarisingduringbacterialinfectionscanbeattributedto oxidativestressandgenerationofreactivespecies(Pohanka,2013). Freeradicals,especiallyreactiveoxygenspecies(ROS),havebeen identifiedtoplayacrucialroleinthepathogenicprocessesinmany humandisordersincludingcancerandmicrobialinfectiousdiseases

(Oyinloye et al., 2015). It is essential toinvestigatenovel

anti-cancerandantibioticdrugswithlesschanceofthedevelopment ofresistanceagainstthem.Fungiareremarkablyadiversegroup, whichcanpotentiallyprovideawidevarietyofmetabolitessuch asalkaloids,flavonoids,phenols,steroidsandterpenoids(Joeland

Bhimba,2013)Bioactivenaturalproductsfromendophyticfungi,

isolatedfromdifferentplantspecies,haveattractedconsiderable

∗Correspondingauthor.Permanentaddress:DepartmentofBiochemistry,Faculty ofScience,UniversityofDschang,Dschang,Cameroon.

E-mail:jean.dzoyem@univ-dschang.org(J.P.Dzoyem).

attentionfromnaturalproductchemistsandbiologistsinthelast decades(Nisaetal.,2015).EpicoccumnigrumLink,Pleosporaceae isanendophyticfungusthathasbeenassociatedwiththe biolog-icalcontrolofphytopathogens,andtheproductionofsecondary metabolites(Fávaroetal.,2012).Insearchofnewbioactive com-poundsofnaturalorigin,thisstudywasundertakentoevaluatethe antibacterialandantioxidantactivitiesandthecytotoxicityoffour compoundsisolatedfromtheendophyticfungalspeciesE.nigrum

isolatedfromEntadaabyssinicaSteud.exA.Rich.,Fabaceae.

Materialsandmethods

FreshleavesofEntadaabyssinicaSteud.exA.Rich.,Fabaceae, were collectedin May2012at Balatchi (Mbouda), inthe West regionofCameroon,andidentifiedattheNationalHerbariumof Cameroon,bycomparisontoexistingvoucherspecimennumber HNCNo.10672/SFR/CAM.Endophytefungiwereisolatedand iden-tifyaccordingtoTalontsietal.(2013).

Forthecompoundsisolation,theproducingstrainRAM10-1was culturedonPDagarat25◦_{Cforsixdays.Then,theculturewas}

extractedwithethylacetate(EtOAc)andconcentratedtodryness

invacuotoaffordcrudeextract(19.1g).Theextractwas fraction-ated bysilica gelcolumn chromatography using CH2Cl2–MeOH gradientelutiontoprovidethreemajorfractionsaccordingtoTLC.

http://dx.doi.org/10.1016/j.bjp.2016.08.011

252 J.P.Dzoyemetal./RevistaBrasileiradeFarmacognosia27(2017)251–253

Thesecondfractionwaspurified onsilicagelCCand Sephadex LH-20usingMeOHtoaffordbeauvericin(1;15mg;Mw781;m.p. 147–148◦_{C)(Guptaetal.,1995).Thethirdfractionwaspurified}

onSephadex LH-20usingCH2Cl2–MeOH(1:1),and subfractions werefurtherseparatedbypreparativeelutingwithCH2Cl2–MeOH andpurifiedwithSephadexLH-20usingMeOHtoafford parahy-droxybenzaldehyde (2,6mg;Mw 122; m.p. 112–114◦_{C), (Ayer}

andTrifonov,1993),indole-3-carboxylicacid(3,5mg;Mw161;

m.p. 232–234◦_{C) (Bano et al., 1986) and quinizarin (4, 6mg;} Mw240;m.p.200–202◦_{C)(Faridand Ahmed,2013).The}

struc-tures of these compounds were identified by analysis of their spectroscopic data and by comparison with those reported in theliteratureaspreviouslydescribed (Melong etal.,2014).The antimicrobialandantioxidantactivityaswellasthecellgrowth inhibitionwereperformedaspreviouslyreported(Supplementary

data).

All experiments were conducted in triplicate and values expressedasmean±standarddeviation.Differencesbetween val-ueswereassessedforsignificanceusinganalysisofvarianceand resultswerecomparedusingtheFisher’sleastsignificantdifference (LSD)at5%significancelevel.

Resultsanddiscussion

The antibacterial activity of compounds 1–4 against differ-ent test organisms is shown in Table 1. Results showed that theirantibacterialpotentialagainstallthepathogenstested var-iedfrom weaktosignificantwithMIC values ranging between

3.12 and 100␮g/ml (Kuete, 2010). Compounds 2–4 had

mod-erate toweakactivity. Interestingly,compound 1 (beauvericin) andcompound3(indole-3-carboxylicacid)hadremarkable activ-ity against Gram-negative strains (Staphylococcus aureus) with respectiveMICvaluesof3.12␮g/mland6.25␮g/ml.Thisfinding is consistent with literature report on the antibacterial activ-ity of indole-3-carboxylic acid and beauvericin. A novel series ofindole-3-carboxylicacidderivativeswerepreviouslyreported topossesspotentantibacterialactivityagainstEnterococcus fae-calis(Himajaetal.,2010).Usingthediskdiffusionmethod,Meca

etal.(2010)foundthatafter48hofincubationtime,thesmallest

amountofbeauvericinthatinhibitedBacilluspumiluswas0.1mg perdisk.Similarly,Xuetal.(2010)reportedthemedianeffective inhibitoryconcentration(IC50)valuesofbeauvericinagainstsixtest bacteria(Bacillussubtilis,Staphylococcushaemolyticus,Pseudomonas

Table1

AntibacterialandantioxidantactivityofcompoundsisolatedfromEpicoccumnigrum(MICin␮g/ml;antioxidantvaluesexpressasIC50,␮g/mlinDPPHandABTSand␮mol

FeSO4/ginFRAP).

Compounds MIC(␮g/ml) IC50,␮g/ml ␮molFeSO4/g

Sa Bc St Pa Ef Ec DPPH ABTS FRAP

1 3.12 12.5 12.5 100 – – – – 2.63±0.14a

2 50 25 – 50 100 25 38.43±4.85a _49.45

±6.52a _12.12

±1.25b

3 6.25 100 100 – 50 100 88.97±8.91b _{– 6.34}

±1.14c

4 50 50 50 100 – 100 11.36±2.83c _10.86

±2.73b _27.17

±1.66d

Gentamicin 0.5 0.5 2 0.25 0.25 1

Trolox nd nd nd nd nd nd 8.71±2.03d _10.38±2.4b _nd

Ascorbicacid nd nd nd nd nd nd 3.44±1.9e _4.15±1.21c _nd

–,>100␮g/ml;Sa,Staphylococcusaureus;Ef,Enterococcusfaecalis;Bc,Bacilluscereus;Ec,Escherichiacoli;Pa,Pseudomonasaeruginosa;St,Salmonellatyphimurium;nd,not determined.Datarepresentthemean±SDofthreeindependentexperiments;valueswithdifferentlettersaresignificantlydifferentatp<0.05.

Table2

Cytotoxicity(IC50in␮g/ml)offourcompoundsisolatedfromEpicoccumnigrumandtheirselectivityindex(SI)againstnormalcelllines.

Compounds Vero THP-1 RAW264.7

IC50 SI IC50 SI

1 86.56±3.94a _76.56±5.76a _{0.92 64.48±6.17}a _1.09

2 38.33±0.60b _31.87

±4.07b _{2.21 48.62}

±4.70b _1.45

3 70.41±5.69c _76.56

±4.44a _{0.92 67.27}

±4.12a,c _1.05

4 40.42±2.18b _86.56

±7.76a,c _{0.81 21.59}

±4.22d _3.26

Doxorubicin 9.35±0.66c _{– nd 0.5±0.00}e _nd

Puromycin 5.32±0.90c _0.4±0.02d _{176.03 1.15±0.17}f _61.23

J.P.Dzoyemetal./RevistaBrasileiradeFarmacognosia27(2017)251–253 253

lachrymans,Agrobacteriumtumefaciens, Escherichiacoliand Xan-thomonasvesicatoria)tobebetween18.4and70.7␮g/ml.Therefore, theantibacterialactivityofbeauvericineitherdeterminedbythe diameterofinhibitionzoneorbyIC50isinagreementwiththe activitydeterminedintermsofMICinthisstudy.

Freeradicalscavengingactivitiesofthetestedcompoundswere evaluatedusingtheDPPHandABTSassayswhiletheFRAPassay wasusedtoevaluatethereducingpower.Resultsarepresentedin

Table1.Theanthraquinonequinizarinhadthegreatestscavenging

activitywithIC50valuesof10.86and11.36␮g/mlintheABTSand DPPHassaysrespectively.Similartoourfindings,anthraquinone compoundshavebeenreportedtobeverygoodelectronand/or hydrogendonors(Zenginetal.,2015).Thecytotoxicityresultsare showninTable2.Comparedtodoxorubicinandpuromycin, com-poundshadnoconsiderablecytotoxiceffectonbothnormaland tumorcells.Compoundshad LC50 values rangingfrom40.42to 86.56␮g/ml, 31.87to86.57␮g/ml and 21.59to67.27␮g/ml on Vero cells, THP-1and RAW 264.7cells respectively. Quinizarin (compound4)hadmoderatecytotoxicityagainstRAW264.7cells withLC50valueof21.59␮g/mlandSIof3.26.Rossietal.(2010) previouslyreportedtheantiproliferativepotentialofquinizarinon B16-F10melanomamurinecells,Inaddition,severaldata demon-stratethepromisingactionofanthraquinonesasanticanceragents

(Kueteetal.,2015).Thepresentstudyshowedthatthefour

com-pounds isolated from theendophytic fungus E.nigrum possess antibacterialandantioxidantactivitywithnotoxiceffectonnormal Verocells.Quinizarinhadremarkableantioxidantactivitywhile beauvericinhad potent antibacterialactivity. Thesecompounds couldbe recommended for theformulation of antioxidant-rich therapeuticdietsandasatherapeuticagentagainstbacterial infec-tionsrespectively.

Authorscontributions

JPDcarriedouttheexperimentsandwrotethemanuscript.RM, ATTandTMcontributedtothecompoundisolationand identifica-tion.GDKWFandBTNsupervisedthechemicalpartofthestudy. JNEandLJMsupervisedtheworkandprovidedthefacilitiesfor biologicalactivitiesstudy.Allauthorsreadandapprovedthefinal manuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheUniversityofPretoriaprovidedapostdoctoralfellowship toJPD.TheNationalResearchFoundationand MedicalResearch Councilprovidedfundingtosupportthisstudy.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.08.011.

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