Rev. bras. farmacogn. vol.26 número1

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

Penicillosides

A

and

B:

new

cerebrosides

from

the

marine-derived

fungus

Penicillium

species

Samar

S.A.

Murshid

_,

_Jihan

_M.

_Badr

_,

_Diaa

_T.A.

_Youssef

a_{DepartmentofNaturalProducts,FacultyofPharmacy,KingAbdulazizUniversity,Jeddah,SaudiArabia}

b_{DepartmentofPharmacognosy,FacultyofPharmacy,SuezCanalUniversity,Ismailia,Egypt}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received5May2015 Accepted17September2015 Availableonline10November2015

Keywords: Didemnum Penicillum Cerebrosides Penicillosides Antimicrobialactivity HeLacells

a

b

s

t

r

a

c

t

Inthecourseofourongoingefforttoidentifybioactivecompoundsfrommarine-derivedfungi,the marinefungus,PenicilliumspecieswasisolatedfromtheRedSeatunicate,Didemnumspecies.Twonew cerebrosides,penicillosidesAandBwereisolatedfromthemarine-derivedfungus,Penicilliumspecies usingdifferentchromatographicmethods.Theirstructureswereestablishedbydifferentspectroscopic dataincluding1D(1_HNMRand13_{CNMR)and2DNMR(COSY,HSQC,andHMBC)studiesaswellas}

high-resolutionmassspectraldata.PenicillosideAdisplayedantifungalactivityagainstCandidaalbicanswhile penicillosideBillustratedantibacterialactivitiesagainstStaphylococcusaureusandEscherichiacoliinthe agardiffusionassay.Additionally,bothcompoundsshowedweakactivityagainstHeLacells.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Marinemicroorganismshavereceivedagreatattentionlately; accordingly,thefungibegan toberecognizedasaliable source ofpotentially usefulnaturalproducts(Fenical,1993; Bugniand Ireland,2004).Althoughstudiesontheseorganismsbeganmuch later than theircounterparts in terrestrial environments, more thanahundrednovelcompoundshavebeenfoundannuallysince the late 1990s (Blunt et al., 2013). The high number of com-poundsreportedfromthegenusPenicilliumcouldbejustifiedby thefactthatitsdifferentspeciesaresalttolerant,fastgrowingand areobtainedeasilyfrommanysubstrates.Thispromptedmany researchers to investigate variable Penicillium species isolated fromdifferent habitats.Their extensivestudiesconcerned with biological activitiesof the isolated secondary metaboliteswere extremelyefficient.Amongthesignificantactivitiesreportedwere theantibacterial(Qietal.,2009;Devietal.,2012;Abo-Kadoum etal.,2013;Subramanietal.,2013),cytotoxicandanticancer(Wang etal.,2009a,b;Sunetal.,2012;Gaoetal.,2013;Abo-Kadoumetal., 2013;Subramanietal.,2013).

Inthecourseofourongoingsearchforbioactivecompounds fromRedSeamarine-derivedfungi,thefungusPenicilliumspecies wasisolatedfromthetunicateDidemnumspeciesandwascultured

∗ _{Correspondingauthor.}

E-mail:[email protected](D.T.A.Youssef).

inSabourauddextrosebroth.Fungalmyceliawereextractedand fractionatedusingdifferentchromatographictechniquestoafford twocompounds.Basedondifferentspectroscopicdataincluding HRESIMS,1D(1_HNMRand13_{CNMR)and2DNMR(COSY,HSQC,} andHMBC),thestructuresofthecompoundswereestablishedas cerebrosidesand namedpenicillosides A(1)and B(2).The iso-latedcompoundswereevaluatedfortheirantimicrobialactivities against different pathogens and theircytotoxic activityagainst HeLacells.PenicillosidesAandBdisplayedsignificant antimicro-bialactivitiesagainstCandidaalbicans;Staphylococcusaureusand Escherichiacolirespectivelyintheagardiffusionassay.Additionally, bothcompoundsshowedweakactivityagainstHeLacells.

Materialsandmethods

Generalexperimentalprocedures

OpticalrotationwasmeasuredonaJASCOdigitalPolarimeter. 1Dand2DNMRspectra(chemicalshiftsinppm,couplingconstants inHz)wererecordedonBrukerAvanceDRX600MHz spectrome-tersusingCD3ODassolvents.NormalandHRESIMSspectrawere recordedonaLTQOrbitrapandanAPI2000(ThermoFinnigan, Bre-men,Germany)massspectrometers.Forcolumnchromatography, silicagel(Merck,70–230meshASTM)andSephadexLH-20 (Phar-macia)wereused.Pre-coatedsilicagel60 F-254plates(Merck) were used for TLC. The HPLC separation was performed on a RP18, 250mm×10mm, 5␮m Phenomenex Luna column using

http://dx.doi.org/10.1016/j.bjp.2015.09.007

CH3CN/H2Ogradientasmobilephaseat220nmandataflowrate of2.0ml/min.

Collectionofthehosttunicateandpreparationofthefungalisolate

Themarinetunicate Didemnumspecies wascollectedin the Mangrovelocatedin Sharm El-SheikhontheEgyptian RedSea coastatdepthof1–2mduringJuly2010.Inordertoensure fun-galisolatestobeendophyticwhenobtained,asurfacesterilization oftunicatewasperformed.Thetunicatesamplewasdisinfected with5% sodiumhypochlorite, followed by70% ethanol (Li and Wang,2009),toensurethatepiphyticfungiweredestroyedbythe washingwhileassociatedfungi(ifany)werenotaffected. Approxi-mately2cm3_{ofinnertissueoftunicatematerialwashomogenized} usingasterilemortarandpestlecontaining10mlofsterile artifi-cialseawaterunderasepticconditions.Theresultinghomogenate wasdilutedwithsterileseawateratthreedilutions(1:10,1:100, and 1:1000). For fungicultivation, 100␮l of each dilution was platedin quadruplicate onto fourplates of each of the follow-ingmedia;Czapek-Doxyeastagarmedium(NaNO33g,KCl0.5g, K2HPO4 0.1g,MgSO4·7H2O0.5g,FeSO4 0.01g,sucrose30g,agar 20g,pH6.7);maltagarmedium(maltextract17g,peptone3g, agar20g)andSabourauddextroseagarmedium.Allmediawere amendedwith2%NaCland0.25%chloramphenicolasantibacterial agenttopreventbacterialgrowthandtoenrichfungigrowth.Plates werewrappedinparafilm,incubatedat28◦_{Cfor1–3weeksuntil}

themorphologyoffungicouldbedistinguished.Manypurification stepsweredoneuntilpurefungalisolateswereobtained.

Identificationoffungalstrain

ExtractionofgenomeDNAfromculturedfungalisolate

Thefungalisolatewasculturedincorrespondingbrothat28◦_C

for 2–5 days. The mycelia were harvested separately by using vacuumfiltrationanddriedwithtwolayersofpapertowel.The resultingmycelialmatwasgroundintopowderwithliquid nitro-gen.ThefungalDNAwasextractedusingQIAampDNAMiniKit (Qiagen)accordingtomanufacturer’sinstructions.

AmplificationoffungalITS-rDNAfragmentsofisolate

The genomic DNA of the fungal strain was used as the template to amplify fungal ITS-rDNA fragments using the primers ITS1 (5′_{-TCCGTAGGTGAACCTGCG-3}′_{) and ITS4 (5}′

-TCCTCCGCTTATTGATATGC-3′_{) (White et al., 1990) which were}

synthesizedbytheUniversityofUtahDNA/peptidesynthesiscore facility.ThereactionmixtureforPCRamplificationcontained5␮lof 10×reactionbufferwith15mMMgCl2(Invitrogen),2␮lof2.5mM dNTPs,0.5␮lof10␮Meachprimer,4␮loffungalDNA,0.3␮lof TaqDNApolymerase(5U␮l−1,Invitrogen),and39.7␮lofH2O.PCR conditionsincludedaninitialdenaturationat94◦_Cfor4min

fol-lowedby30cyclesofdenaturationat94◦_{Cfor50s,annealingat}

51◦_{Cfor50s,andelongationat68}◦_{Cfor1min,withafinal}

elon-gationat68◦_{Cfor10min.PCRproductswerepurifiedusingthe}

AgaroseGelDNAPurificationKit(Qiagen)andsequencedinatthe UniversityofUtahDNAsequencingfacility.

SequencefungalITS-rDNAregionsofisolate

For preliminaryidentification,sequences offungal ITS-rDNA regionsobtainedfromthemarinetunicateDidemnumspecieswere comparedwithrelatedsequencesinNationalCenterfor Biotech-nologyInformation(NCBI)(http://www.ncbi.nlm.nih.gov).Fungal ITS-rDNAsequencesacquiredinthisstudywereeditedandaligned withthebestn-BLASThitsfromGenBankintheClustalX (ver-sion1.83)program(Thompsonetal.,1997),andfurthermanually adjustedusingBioEditsoftware(Hall,1999).TheprogramMEGA

5(Tamuraetal.,2011)wasappliedtocalculatethebase composi-tionofthefungalsequences.Thesequenceanalysisbasedon99% sequenceidentitywithPenicilliumspeciesRsf-2(NCBIaccession numberEF660439.1).

Isolationandpurificationofcompounds1and2

Large scale cultureof the marine-derived fungusPenicillium specieswascarriedout.Theculturewasincubatedatroom tem-peraturefor30days.Afterthat,250mlofEtOAcwereaddedto eachflaskleftovernighttostopcellgrowth.Culturemediaand myceliawereseparatedbyvacuumfiltrationusingBuchnerfunnel. ThemyceliawereleftinMeOHovernightforextraction.Theextract wasdriedunderreducedpressure.Theviscousextract(1200mg) wasdissolvedin250mlof70%MeOHthenextractedwith hex-ane(3ml×100ml).Themethanollayerwasconcentratedunder reducedpressuretoyieldanotherviscousbrownresidue(765mg). Aportionofthealcoholicextract(600mg)wasfractionatedover silicagel(VaccumLiquidChromatography)usingCHCl3/MeOH gra-dient(100%CHCl3then2,5,7,10,15%MeOHinCHCl3)togivesix mainfractions.Fraction2(65mg,elutedwith2%MeOHinCHCl3) waspurifiedonSephadexLH-20,elutedwithMeOHtoobtainfinally tensub-fractions.Finalpurificationofthemainsubfractionswas achievedbyHPLCusinggradientsystemstartedwith5%ACN/H2O to100%ACNin15mintoaffordcompound1(3mg).Fraction3 (145mg)wasdissolvedinMeOHandsubjectedtoSephadex LH-20columnelutedwith100%MeOH.Thefractionsshowingdistinct spotswerepurifiedonsilicagelusingpet.ether/CHCl3/MeOH gra-dient(100%pet.Ether,followedby20,40,60,80%CHCl3 inpet. Ether,followedby5,10,15,20%MeOHinCHCl3).Fractionseluted with5%MeOHinCHCl3offeredimpure2.Finalpurificationwas performedbyHPLCusinggradientsystemfrom10%ACN/H2Oto 100%ACNover20mintoobtaincompound2(23.1mg).

Biologicalevaluationofcompounds1and2

Determinationoftheantimicrobialactivityofcompounds1and2 TheprocedurewasconductedintriplicateaccordingtoValgas etal.(2007)andSinghandJain(2011).Compounds1and2were testedforantibacterialactivityagainstaGrampositivebacterium (StaphylococcusaureusATCC25923),aGramnegativebacterium (Escherichiacoli ATCC25922), and yeast(CandidaalbicansATCC 14053)usingagardiffusionmethod.Accuratelymeasured0.1ml (100␮gdissolvedinDMSO)ofeachcompoundwereinsertedin thecups then incubatedat 37◦_{C for24h. Theinhibition zones}

weremeasuredandcomparedwiththereferenceantibioticsand antifungaldrugs;ampicillin,imipenemandclotrimazole(eachof 10␮g/discgiving30,30and40mminhibitionzonerespectively).

Determinationofcytotoxicactivityofcompounds1and2

Table1

NMRspectraldataofcompounds1and2(CD3OD).

Position 1 2

ıC(mult.)a ıH(mult.,JinHz) ıC(mult.)a ıH(mult.,JinHz)

1 67.4(CH2) 3.63(m) 69.7(CH2) 4.11,m;3.7,dd(10.2,3.6)

2 54.6(CH) 3.97(m) 54.6(CH) 3.97,m

3 71.5(CH) 3.64(m) 35.9(CH2) 1.69,m;1.5m

4 33.1(CH2) 2.04(m) 26.1(CH2) 1.39,m

5 32.7(CH2) 1.28(m) 40.8(CH2) 1.97,t(7.2)

6 26.5(CH2) 2.78(brt,6.0) 136.8(qC) –

7 131.0(CH) 5.34(brd,14.1) 124.8(CH) 5.14,t(6.6)

8 130.8(CH) 5.32(m) 33.8(CH2) 2.07,m

9 28.2(CH2) 2.05(m) 33.1(CH2) 1.28,m

10 14.5(CH3) 0.89,t(6.6)

11 16.1(CH3) 1.59,s

10–14 29.1–30.8(5×CH2) 1.24–1.33(m)

15 23.7(CH2) 1.24–1.33(m)

16 14.4(CH3) 0.90(t,6.6)

1′ _{174.2(qC) – 177.2(qC) –}

2′ _{72.95(CH) 4.16(m) 72.9(CH) 4.12,d(6.6)}

3′ _35.0(CH

2) 2.33(m) 73.1(CH) 3.98,dd(7.8,4.2)

4′ _26.05(CH

2) 1.60(m) 131.1(CH) 5.47,dd(15.6,7.8)

5′ _29.2(CH

2) 2.22(m) 134.6(CH) 5.71,m

6′

130.7(CH) 5.38(m)

7′ _{130.9(CH) 5.42(brd,15.0)}

8′ _29.1(CH

2) 2.06(m)

9′ _26.5(CH

2) 2.68(m)

10′ _{129.0(CH) 5.31(m)}

6′_-10′ _{30.5–33.0(5×CH}

2) 1.25–1.35,m

11′ _{129.1(CH) 5.29(brd,13.2) 14.4(CH}

3) 0.89,t(6.6)

12′

28.1(CH2) 2.05(m)

13′

-16′ _{29.1–30.8(4}

×CH2) 1.24–1.33(m)

17′ _23.6(CH

2) 1.24–1.33(m)

18′ _14.4(CH

3) 0.90(t,6.6)

1′′ _{104.7(CH) 4.26(d,7.2) 104.7(CH) 4.27,d(7.8)}

2′′ _{75.0(CH) 3.18(m) 75.0(CH) 3.18,m}

3′′

77.9(CH) 3.34(m) 77.9(CH) 3.34,m

4′′ _{71.8(CH) 3.27(m) 71.5(CH) 3.27,m}

5′′ _{78.0(CH) 3.28(m) 78.0(CH) 3.28,m}

6′′ _62.6(CH

2) 3.86(m),3.67(m) 62.6(CH2) 3.85,dd(12.0,1.2);

3.66,dd(11.4,4.8)

a_{MultiplicitywasdetectedfromHSQC.}

Spectraldata

Penicilloside A (1). Colourless amorphous powder, positive HRESIMS m/z 712.5366 (calculated for C40H74NO9, 712.5364 [M+H]+_),[

␣]D−13◦(c1.5,MeOH).NMRdata:seeTable1. Penicilloside B (2). Colourless amorphous powder; positive HRESIMS m/z 546.3646 (calculated for C28H52NO9, 546.3642 [M+H]+_),[

␣]D−19.6◦(c1.5,MeOH).NMRdata:seeTable1.

Resultsanddiscussion

Compound 1 was isolated as a colourless amorphous pow-der. Its molecular formula was suggested as C40H73NO9 based on different spectral data including HRESIMS (m/z 712.5366, [M+H]+_{), 1Dand2DNMRdata(Table1).Differentspectraldata} declaredthepresencefor asugarmoiety,anamidelinkageand aliphatic chains, thus supporting its cerebroside nature (Wang etal.,2009a,b).Analysisof1_HNMRand13_{CNMRdataprovedthe} presenceofsignalsofglucosemoiety,where1_{H NMRspectrum} revealedsignalsresonatingintherangeof3.18–4.26ppm.HSQC correlatedtheseprotonstotheircorrespondingcarbonsdetected in the range of 62.6–104.7ppm. The coupling constant of the anomericproton(J=7.2Hz)confirmedtheˇ-configurationofthe glucose moiety (Renet al.,2009; Sheet al., 2009; Peng et al., 2011).Carefulexaminationofdifferentspectraldataallowedthe assignmentoftwosubunits;subunitA(C-1→C-16)andsubunit B(C-1′

→C18′_{).FromtheCOSYandHSQC experiments,subunit}

Awasassignedas onemethyl,oneoxygenatedmethylene, two

downfield shifted methines, two protonated sp2 _{carbons and} ten methylenes. From 2D NMR spectra, C-3 wasproved to be oxygenatedasestablishedfromthechemical shiftatıC 71.5/ıH 3.64.Theothermethinewasconfirmedtobelinkedtotheamide group,thehydroxylatedmethine(C-3)andtheoxygenated methy-lene(ıC67.4/ıH3.63).Thus,thefragementfromC-1toC-3was proved. Thelocationof thesp2 _{carbonswassuggestedtobeat} C-7/C-8separatedfromC-3bythreemethylenes.Thissuggestion was confirmed from spin–spin coupling between H-7 (ı 5.34) andH2-6(ı2.78)whichinturnwascoupledwithH2-5(ı1.28). AlsoH2-4 (ı 2.04)wascoupledwithH-3(ı 3.64).Additionally, HMBCrevealedcross peaksfromH2-9(ı2.05)toC-8(ı130.8) andtoC-7(ı131.0),confirmingtheassignmentofthefragment C-1toC-8.Thelastpartofsubunit(A)wasfoundtoconsistofa terminalmethyl(ıC14.4/ıH0.90)whichwascoupledtoC-15;the adjacent methylene. H-8 revealed spin–spincoupling with C-9 (theadjacentmethylene).Thenumberofthemethylenegroups betweenC-8andtheterminalmethylC-16wasfoundtobeseven asindicated fromMSspectrum, throughthefragmentdetected at m/z 599 corresponding to a loss of the terminal methyl in additiontosevenmethylenes.Accordingly,subunitA(C-1toC-16) structurewasconfirmed.ThechemicalshiftvaluesofH2-1,H-2, H-3together withtheircorresponding carbonsare comparable tothosepreviouslyreportedforsimilarcerebrosides(Pengetal., 2011).SubunitBwasprovedtoconsistofeighteencarbons(C-1′

toC-18′_{).HMBCdeclaredcrosspeaksfromeachofH-2}′_(ı4.16),

HO

HO

OH

OH

OH

HO HO

O O

O OH

2

1

COSY

NH NH

HMBC

HO

HO HO

HO

O O

O

Fig.1.SelectedCOSYandHMBCcorrelationsofcompounds1and2.

protonatedsp2_{carbonsweredetectedinfragmentB.Their} loca-tionwasdefinedatC-6′_/C-7′_andC-10′_/C-11′_{.Thenoninterrupted}

spin–spincoupling from H2-3′ to H-6′ and from H-7′ to H-10′ supportedthis assumption. A furtherconfirmation was gained fromHMBCwhichrevealedcrosspeaksfromH2-8′(ı2.06)toeach ofC-6′_{(ı130.7)andC-7}′_{(ı130.9)andfromH}

2-9′(ı2.68)toeach ofC-10′_{(ı129.0)andC-11}′_{(ı129.1).Theterminalmethylgroup}

(CH3-18)waslinkedtoH2-17′ andC-17′ asdeclaredfromCOSY andHMBCrespectively(Fig.1).The1_Hand13_{CNMRdataforC-1}′_,

C-2′_andH-2′_{aretypicalforthosepreviouslyreportedforsimilar}

cerebrosides(Elkhayatetal.,2012).TheconnectionofsubunitA withBwasconfirmedfromHMBCthatshowedcrosspeaksfrom H-3toC-1′_{.AdditionalcrosspeakfromH-1}′′_{toC-1confirmedthe}

attachmentofsubunitAwiththeglucosemoiety.Basedonthe pre-viousdiscussion,thestructureofcompound1wasestablished.This isthefirstreportofthiscompoundfromnaturalsource.Therefore, itisconsideredasanewnaturalproductandnamedpenicillosideA.

HO

HO HO

HO

O O

O OH

OH

OH

OH HO

HO HO

HO

O O

O NH

2

1

NH

Compound2wasisolatedasacolourlessamorphouspowder. ItsmolecularformulawassuggestedasC28H51O9Nbasedon dif-ferentspectraldataincludingHRESIMS(m/z546.3646[M+H]+_),1_H NMR,13_{CNMRandHSQC(Table1).Thecerebrosidenatureof} com-pound2wassuggested from1DNMR datawhich declaredthe presenceofasugarresidue,oneormorealiphaticchainsandan amidelinkage(Elkhayatetal.,2012).Analysisof1_H–1_HCOSY spec-trum(Fig.1)ledtotheidentificationofsubunitA(C-1→C-10)and

subunitB(C-2′_→C-11′_{).Carefulinvestigationofdifferentspectral}

datadeclaredthepresenceof2sp2carbonsincludinga trisubsti-tutedolefinicmoietyinthesubunitA.Thiswasprovedfromthe carbonsignalsresonatingatı136.8(qC)and124.8(CH).Thelatter carbonwasconnectedtotheprotonatıH5.14.Thesignal resonat-ingatı136.8wasattributedtoquaternarycarbonaspredictedfrom HSQCexperiment.HMBCexperiment(Fig.1)revealedacrosspeak fromH3-11(ı1.59)tothequaternarysp2carbon(ı136.8)beside theothersp2_{carbonandC-5(ı40.8).Thissupportedthelocationof} theH3-11(ıH1.59/ıC16.1)asbeingdirectlylinkedtotheolefinic carbon(ı136.8).BothCOSYandHMBCdataconfirmedthelocation ofthedoublebondatC-6/C-7andalsoprovedthewholestructure ofsubunitA(C-1toC-10).InvestigationofthedifferentNMRdata (1_HNMR,13_{CNMR,COSY,HSQCandHMBC)provedthe} incorpo-rationof adoublebond andtwohydroxylated methineswithin subunitB. The COSYspectrumdeclared thespin–spin coupling betweentheprotonsresonatingatı3.98(H-3′_)and4.12(H-2′₎

whichare linkedtothecarbonsatı73.1(C-3′_)and72.9(C-2′₎

respectively.Thissupportedthelocationofthetwohydroxylsat C-2′_andC-3′_{.TheHMBCdemonstratedacrosspeakfromtheproton}

resonatingatı3.98(H-3′_{)totheamidiccarbonylatı177.2(C-1}′₎

andconfirmingtheassignmentofthesubstructureC-1′_toC-3′_.The

presenceofadoublebondatC-4′_/C-5′_{wasconfirmedfromthe}

pro-tonsignalsatı5.47(H-4′_)and5.71(H-5′_{)whichcorrelatedtothe}

signalsatı131.1and134.6respectivelyintheHSQCspectrum.The HMBCshowedcrosspeaksfromH-3′_{(ı3.98)toC-4}′_(ı131.1)and

hencesupportingthepresenceofolefinicmoietyatC-4′_/C-5′_.

ThelargecouplingconstantbetweenH-4′_andH-5′_(J=15.6Hz)

wasconsistentwithEconfiguration(Yoshikawaetal.,1996;Wang etal.,2009a,b).AsdeclaredfromCOSYandHMBCdata(Fig.1), thestructureofsubunitB(C-1′_toC-11′_{)wasconfirmed.The}

pres-enceofaglucosemoietywasconfirmedfromthesignalsresonating between62.6and104.7ppmin13_{CNMRspectrumalongwiththeir} correspondingprotonsignalsdetectedbetween3.18–4.18ppmin 1_{HNMRspectrum.Thelinkbetweeneach protonand its} corre-spondingcarbonwasproved fromHSQC.Thelargevalueofthe couplingconstant(J=7.8Hz)oftheanomericproton(H-1′′_,ı4.27)

confirmeditsˇ-configuration(Pengetal.,2011;Renetal.,2009; Sheetal.,2009).Theattachmentoftheglucosemoietytothe sub-unit Awas confirmedfromtheHMBC cross-peaksof H-1/C-1′′_.

Basedonthepreviousdiscussion,thestructureofcompound2was confirmed.Itisreortedhereforthefirsttimefromnaturalsource andwasgiventhegenericnamepenicillosideB.

Theantimicrobialactivityofcompounds1and2wasevaluated bydeterminingthegrowthinhibitionzone.Compound1revealed antifungalactivitytowardsC.albicansasitshowedinhibitionzone of23mm.Additionally,compound2wasactiveagainstS.aureus with19mminhibitionzoneandE.coli(20mm).Ontheotherhand, BothcompoundsshowedweakactivitywithIC50≥50␮g/mlwhen testedagainstHeLacells.

Conclusion

Investigationofthemarine-derivedfungusPenicilliumspecies yieldedtwonewcerebrosides,penicillosidesAand B(1and2). Compound1revealedantifungalactivitytowardsC.albicans. Addi-tionally,compound2showedantibacterialactivityagainstS.aureus andE.coli.Ontheotherhand,Bothcompoundsshowedweak activ-itywithIC50≥50␮g/mlwhentestedagainstHeLacells.

Authors’contribution

manuscriptandSSAMperformedtheexperiments,analyzedthe dataandwrotethemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

ThisprojectwassupportedbytheNSTIPstrategictechnologies programintheKingdomofSaudiArabia—ProjectNo. (11-BIO1556-03). The authors also, acknowledge with thanks Science and TechnologyUnit,KingAbdulazizUniversityfortechnicalsupport.

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