Chemical Composition, Antibacterial and Antifungal Activities of Crude Dittrichia viscosa

13 Pages·2017·0.4 MB·English

molecules
Article
Chemical Composition, Antibacterial and Antifungal
Activities of Crude Dittrichia viscosa (L.) Greuter
Leaf Extracts
WafaRhimi 1,2,IssamBenSalem2,*,DavideImmediato3,MouldiSaidi2,AbdennacerBoulila4
andClaudiaCafarchia3
1 FacultédesSciencesdeBizerte,UniversitédeCarthage,7021Zarzouna,Tunisia;[email protected]
2 LaboratoryofBiotechnologyandNuclearTechnolog,NationalCentreofNuclearScienceand
Technology(CNSTN),SidiThabetTechnopark,2020Ariana,Tunisia;offi[email protected]
3 DipartimentodiMedicinaVeterinaria,UniversitàdegliStudidiBari,70010Valenzano,Italy;
[email protected](D.V.);[email protected](C.C.)
4 LaboratoryofNaturalSubstancesLR10INRAP02,NationalInstituteofResearchandPhysico-chemical
Analyses,BiotechpoleofSidiThabet,2020Ariana,Tunisia;[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditors:HideyukiIto,TsutomuHatanoandTakashiYoshida
Received:12May2017;Accepted:3June2017;Published:30June2017
Abstract: The small amount of data regarding the antifungal activity of Dittrichia viscosa (L.)
Greuter against dermatophytes, Malassezia spp. and Aspergillus spp., associated with the few
comparativestudiesontheantimicrobialactivityofmethanolic,ethanolic,andbutanolicextracts
underpinsthestudyhereinpresented. Thetotalcondensedtannin(TCT),phenol(TPC),flavonoid
(TFC),andcaffeoylquinicacid(CQC)contentofmethanol, butanol, andethanol(80%and100%)
extractsofD.viscosawereassessedandtheirbactericidalandfungicidalactivitieswereevaluated.
The antibacterial, anti-Candida and anti-Malassezia activities were evaluated by using the disk
diffusionmethod,whereastheanti-Microsporumcanisandanti-Aspergillusfumigatusactivitieswere
assessed by studying the toxicity effect of the extracts on vegetative growth, sporulation and
germination. ThemethanolicextractcontainedthehighestTPCandCQCcontent. Itcontainsseveral
phytochemicals mainly caffeoylquinic acid derivatives as determined by liquid chromatography
withphotodiodearrayandelectrosprayionisationmassspectrometricdetection(LC/PDA/ESI-MS)
analysis. AllextractsshowedanexcellentinhibitoryeffectagainstbacteriaandCandidaspp.,whereas
methanolicextractexhibitedthehighestantifungalactivitiesagainstMalasseziaspp.,M.canisand
A.fumigatusstrains. Theresultsclearlyshowedthatallextracts,inparticularthemethanolicextract,
mightbeexcellentantimicrobialdrugsfortreatinginfectionsthatarelifethreatening(i.e.,Malassezia)
orinfectionsthatrequiremandatorytreatments(i.e.,M.canisorA.fumigatus).
Keywords: Dittrichiaviscosa;antifungalactivities;Candidaspp.;Malasseziaspp.;Microsporumcanis;
Aspergillusfumigates
1. Introduction
Thegrowingworldwideconcernaboutthealarmingincreaseintherateofhumanandanimal
infections caused by antibiotic-resistant microorganisms have spurred the interest of the scientific
communityindevelopingalternativemethodsfortheircontrol[1].Manykindsofnaturalextractsfrom
medicinalplantscontainingphenolicandflavonoidcompoundshaveexcellentbiologicalproperties
and are used as alternative therapies. Among the large variety of Mediterranean folkloric herbs,
DittrichiaviscosabelongingtotheAsteraceaefamily,hasproventobeasourceofnaturalproducts
formingthebasisforalternativemedicineandnaturaltherapies[2–4]. Dittrichiaviscosawasstudied
Molecules2017,22,942;doi:10.3390/molecules22070942 www.mdpi.com/journal/molecules
Molecules2017,22,942 2of13
against antibiotic-resistant microorganisms, antibacterial activity and anti-fungal activity against
Candida albicans and Fusarium species [5,6]. To the best of our knowledge, reports on antifungal
activityofD.viscosaagainstdermatophytes,Malasseziaspp. andAspergillusspp. arescantorlimited
to Microsporum canis. In particular, dermatophytes are a group of fungi which have the ability to
invadethekeratinizedtissues(skin,hair,nails)causingcutaneousinfectionsinhumansandanimals
commonlyknownasdermatophytosis[7]. Theyaredistributedworldwideandsomeofthemare
considered zoonotic, being transmitted from animals to humans [8]. The treatment of infections
is mandatory due to the contagious and the zoonotic nature and usually requires long antifungal
therapywithazoles[9]. Inaddition,thesetreatmentsarenotusuallyperformedinfoodproducing
animalssincetheyaremoreexpensive,andtreatedanimalsneedlongwithholdingbeforeusingin
foodprocessingindustry[10].
ThefungalgenusMalasseziaispartofthenormalskinmicrobiota. Theseyeastscausehumanand
animalskindisordersinimmune-competenthostsandsystemicinfectionsinimmune-compromised
patientswhichusuallyrequireprolongedtreatmentwithand/orhighdosesofantifungalagents[11,12].
Inaddition,recentstudiesclearlyshowthatthesamespecieswithinthegenusofMalasseziafurfur
andMalasseziapachydermatisarecharacterizedbyhighminimalinhibitoryconcentration(MIC)values
againstallazoledrugscommonlyemployedinthetreatmentoftheinfections.
Finally,Aspergillusspeciesarefoundworldwideinhumansandinalmostalldomesticanimals
andbirdsaswellasinmanywildspecies,causingawiderangeofdiseasesfromlocalizedinfections
tofataldisseminateddiseases,aswellasallergicresponsestoinhaledconidia[13]. Someprevalent
forms of animal aspergillosis are invasive fatal infections and are difficult to treat. In addition, the
environmentaldiffusionofA.fumigatusstrainspresentingazoleresistantphenomenaisworldwide
reported[13].
Thus,thisstudyaimedto:(i)quantifythephenolicandflavonoidscontentofD.viscosaleafextract
withdifferentsolvents;(ii)evaluatetheiractivitiesagainstgrampositiveandnegativebacteria,and
againstCandidaspp. (i.e.,Candidaalbicans,Candidakrusei,Candidaprapsilos);and(iii)toassesstheir
activitiesagainstMalasseziaspp. (MalasseziapachydermatisandMalasseziafurfur),Aspergillusfumigatus
andMicrosporumcanis.
2. ResultsandDiscussion
2.1. PhytochemicalScreening
Thetotalcondensedtannin(CTC),phenol(TPC),flavonoid(TFC),andcaffeoylquinicacid(CQC)
content of different D. viscosa extracts are reported in Table 1. They are expressed as mg catechin
equivalent(CE),mggallicacidequivalent,mgquercetinequivalent(QE)andmgofchlorogenicacid
equivalent(ChlAE)pergdryextract,respectively.
The CTC amounts varied from 7.05 ± 1.6 to 27.15 ± 2.21 mg CE/g, being the highest in the
methanolic extract (Table 1). The TPC ranged from 75.34 ± 1.30 to 123.39 ± 1.22 mg GAE/g, the
highestcontentretrievedinmethanolicand80%ethanolicextracts(Table1).
TheCQCamountsofD.viscosaextractsrangedfrom57.11±0.98to87.61±1.06mgChlAE/g
(Table1)andthehighestamountofCQCwasregisteredinmethanolicextract. TheTFCvariedfrom
30.86 ± 1.28 to 58.03 ± 1.85 mg QE/g and the highest content was registered in butanolic extract
(Table1). ThemethanolicextractcontainsthehighestCTC,TPCandCQCvalueswhilethebutanolic
extractcontainedthehighestamountofTFC.
TheresultsofthisstudyclearlyindicatethatphenolicandflavonoidscontentofD.viscosacrude
extractsvaryaccordingtothesolventextractionprocedure. Inparticular,thisstudyreportsforthe
firsttimethepresenceofcondensedtanninsinthisplantspecies. Indeed,nopreviousstudieshave
evaluated CTC in D. viscosa leaves, but results herein indicate that the amount within methanolic
extractsareinthesamerangeasthoseofsomeAsteraceaespeciessuchasArtemisiagenus[14]. Onthe
contrary, the TPC values of Tunisian D. viscosa extracts were in same range or slightly lower than
Molecules 2017, 22, 942 3 of 13
the solvent, and it was highest when the solvent polarity increased. Similar results were reported by
Negi and Jayaprakasha when studying methanol extracts of Punica granatum peel [16]. However, a
high value of TFC in our extracts was detected in butanolic extract, suggesting that the flavonoid
Molecules2017,22,942 3of13
composition of D. viscosa might comprise of substances with a high solubility in butanol, like luteolin
derivatives [17,18].
thosereportedfromTurkishorMoroccansamples[4,15],thussuggestingthatTPCvaluesofD.viscosa
doesTnoabtlvea 1r.y Caocncdoerndsiendg tlayntnoinpsl,a tnottaol rpigoliynp.hAenccoolsr,d tointagll yfl,atvhoenoTiPdCs, aanmdo cuanfftesoydleqpueinnidc aocnidt hcoenpteonlta roift yof
thesodlivffeenretn,ta Dnd. viitscwosaa slehafi gehxtersatctws. henthesolventpolarityincreased. Similarresultswerereported
byNegiandJayaprakashawhenstudyingmethanolextractsofPunicagranatumpeel[16]. However,
Polyphenols and Flavonoids Content Ethanolic Ethanolic 80% Butanolic Methanolic
ahighvalueofTFCinourextractswasdetectedinbutanolicextract,suggestingthattheflavonoid
CTC(mgCAE/g extract) 14.29 ± 1.30 a 7.05 ± 1.6 b 16.86 ± 1.62 c 27.15 ± 2.21 d
compositioTnPCof (mD.gvGiAscEos/ga emxtirgahctt)c omprise1o1f7.s5u8b ±s 1ta.2n9c ae sw12it3h.3a9 ±h i1g.2h2 sbo lu7b5il.3it4y ±i n1.3b0u ct an1o2l3,.l0i7k e± l1u.6t9e ob lin
derivativesT[F1C7, 1(m8]g.QE/g extract) 57.79 ± 1.76 a 49.23 ± 1.039 b 58.03 ± 1.85 a 30.86 ± 50 c
CQC (mgCGAE/g extract) 71.85 ± 0.35 a 73.13 ± 1.06 a 57.11 ± 0.98 b 87.61 ± 1.06 c
Table1V.aCluoensd feonllsoewdetda nbnyi nthse, tsoatmalep loetlyteprh aelnonogls ,thtoet raolwfla avroen nooitd ssi,gannifdiccaanftfleyo ydliqffueirnenict a(pc i<d 0c.o0n5)t.e ntof
differentD.viscosaleafextracts.
2.2. Phenolic Profile of D. viscosa Extracts
PolyphenolsandFlavonoidsContent Ethanolic Ethanolic80% Butanolic Methanolic
The HPLC-PDA/ESI-MS analysis allowed us to tentatively identify 18 phenolic compounds in
CTC(mgCAE/gextract) 14.29±1.30a 7.05±1.6b 16.86±1.62c 27.15±2.21d
the methaTnPoCli(cm DgG. vAiEsc/goseax terxatcrt)act (Figure1 117).. 5T8h±e 1p.2h9eanoli1c2 3fr.3a9c±tio1n.2 2ofb me7t5h.a34no±li1c. 3D0.c visc1o2s3a.0 e7x±tr1a.c6t9 wb as
dominatedT FbCy (cmagffQeEo/yglqexutrinacitc) acid deriva5t7iv.7e9s± su1c.7h6 aas ch4l9o.2r3o±ge1n.0ic39 abcid, 5d8i.c0a3f±fe1o.y85lqauinic3 0a.c8i6d± is5o0mcers,
and caffCeoQyCl (gmlugcCoGsAe Ea/sg ietx strhaoctw) n in Tabl7e1 .28.5 O±t0h.e35r ahyhy7d3.r1o3x±yc1i.0n6naamic5 7a.1c1id±s 0li.9k8eb coum87a.6r1ic± a1c.i0d6 cand
caffeic acid deVriavluaetsivfoellso wweedreby atlhseos admeteelcettteedra. longtherowarenotsignificantlydifferent(p<0.05).
Some flavonoid compounds were also detected. They were represented exclusively by quercetin
2d.2e.riPvhaetnivoelisc P(er.ogfi.,l eqoufeDrc.evtiisnco-Osa-hEexxtroascitdse, quercetin glucuronide, quercetin dimethyl ether isomers),
and the flavonol catechin glucoside. The identification of phenolic compounds by HPLC-PDA-ESI-
TheHPLC-PDA/ESI-MSanalysisallowedustotentativelyidentify18phenoliccompoundsin
MS as shown in Table 2 confirmed our photochemical screening findings about the richness of
themethanolicD.viscosaextract(Figure1). ThephenolicfractionofmethanolicD.viscosaextractwas
D. viscosa extract in caffeoylquinic acid derivatives, and in agreement with previous reports from
dominatedbycaffeoylquinicacidderivativessuchaschlorogenicacid,dicaffeoylquinicacidisomers,
Israel, Turkey and Tunisia [4,15,19]. From these results, it emerges that D. viscosa might be advised
andcaffeoylglucoseasitshowninTable2. Otherhyhydroxycinnamicacidslikecoumaricacidand
as potential source of bioactive components especially caffeoylquinic acid derivatives.
caffeicacidderivativeswerealsodetected.

FFiigguurree 11.. CChheemmiiccaall cchhaarraacctteerriizzaattiioonn ooff mmeetthhaannoolliicc eexxttrraacctt ooff DD.. vviissccoossaa lleeaavveess bbyy HHPPLLCC--PPDDAA--EESSII--SS..
TThheep peeaakkssa arreen nuummbbeerreedda anndda assssigignnmmeenntstsa arereg giviveenni ninT Tabablele2 .2.
Someflavonoidcompoundswerealsodetected. Theywererepresentedexclusivelybyquercetin
derivatives(e.g.,quercetin-O-hexoside,quercetinglucuronide,quercetindimethyletherisomers),and

theflavonolcatechinglucoside. TheidentificationofphenoliccompoundsbyHPLC-PDA-ESI-MS
asshowninTable2confirmedourphotochemicalscreeningfindingsabouttherichnessofD.viscosa
Molecules2017,22,942 4of13
extractincaffeoylquinicacidderivatives,andinagreementwithpreviousreportsfromIsrael,Turkey
andTunisia[4,15,19]. Fromtheseresults,itemergesthatD.viscosamightbeadvisedaspotentialsource
ofbioactivecomponentsespeciallycaffeoylquinicacidderivatives.
Table2.Retentiontime(RT),wavelengthsofmaximumabsorption(λmax),massspectraldata,relative
occurrence,andtentativeidentificationofphenoliccompoundsinmethanolicextractofD.viscosaleaves.
Compound RT(min) λmax [M−H]− FragmentIons ProposedStructure Occurrence
341(100),
1 4.856 292sh–322 377 Caffeicacidhexoside ++
179,119
191(100),
2 5.346 292sh–322 341 3-Caffeoylquinicacid ++
137,128
3 11.876 292sh–322 353 191(100),161 Chlorogenicacid +++
191, 4-Caffeoylquinicacid
4 13.856 292sh–322 353 +
161(100) isomer
5 17.222 293–310 467 163(100) Coumaricacidderivative +
267(100),
6 22.365 - 429 Feruloylcaffeolglycerol +
173,161
301(100),
7 27.982 360,262 463 Quercetinhexoside +
331,255
8 28.007 293sh–321 463 301(100) Hydroxyluteolinhexoside +
9 30.452 293sh–354 477 301(100) Quercetinglucuronide +
10 30.714 294sh–354 477 301(100),161 Quercetinglucuronide +
191(100),
11 32.888 292sh–322 353 5-Caffeoylquinicacid +
179,161
191(100), Dicaffeoylquinicacid
12 33.463 292sh–322 353 ++++
179(32) isomer
191(78),
Caffeoylquinicacid
13 37.98 292–322 353 179(100), +++
isomer
161(80)
14 44.561 290,320 339 135(100) Caffeoylglucose +++
314(100),
299(80),
Quercetin-dimethylether
15 47.234 253–349 329 285(70), +++
isomer
271(53),
243(50)
314(100),
299(85) Quercetin-dimethylether
16 47.395 253–349 329 +++
271(75), isomer
241(40)
314(100),
Quercetin-dimethylether
17 49.315 253–349 329 299(85), +
isomer
285,243
289(40),
18 55.566 278 493 165(100), Catechinglucoside +++
139(80)
+:lowinabundance;++:moderateinabundance;+++:highinabundance);++++:veryhighinabundance.
2.3. Antibacterial,Anti-CandidaandAntifungalActivityofD.viscosaExtracts
Table3showstheinhibitoryeffectsofD.viscosaextractsagainstGrampositive(i.e.,Staphylococcus
aureus,Enterococcusfeacium,Streptococcusagalactiae)andGramnegativebacteria(i.e.,Escherichiacoli
andSalmonellatyphimurium)withtheinhibitionhalorangingfrom9.5to34.5mm. Nostatistically
significantdifferenceswererecordedbetweendifferentextracts. Thehighestantimicrobialactivity
wasobservedagainstEnterococcusfeacium(G+)andStreptococcusagalactiae(G+)withinhibitionzones
of34.5±0.7mmand29±1.41mm,respectively.
Molecules2017,22,942 5of13
Table3. Antibacterialpropertiesofextractsunderstudy,expressedasdiameterofinhibitionhalo
(inmm)versusseveralstrains.
Concentration
Bacterialspps. Ethanol Ethanol80% Butanol Methanol
(mg/mL)
50 12±1.41a 11.5±0.70a 12.5±0.70a 12±0.70a
Eshershiacoli 10 11±1.41a 10±0.0a 10.5±0.0a 10±0.0a
50 10.5±0.70a 9.5±0.70a 10.5±0.70a 10±0.0a
SalSalmonellatyphimurium
10 9.5±0.70a 0±0.0b 9.5±0.70a 9.5±0.0a
50 34±1.41a 28.5±0.0b 34.5±0.70a 34.5±0.7a
Enterococcusfeacium
10 30±0.0a 25±0.0b 28±0.0c 29±0.0d
50 28±1.41a 28±1.41a 29±1.41a 29±1.41a
Streptococcusagalactiae
10 18.5±0.70a 17±0.0a 21.5±1.41b 18±1.14a
50 25±0.0a 25±0.0a 22.5±0.70b 20±0.0c
Staphylococusaureus
10 13.5±0.70a 10±0.0b 13±1.41a 11±0.0c
Valuesfollowedbythesamesuperscriptalongtherowarenotsignificantlydifferent(p<0.05).
Theresultsofanti-Candidaandanti-MalasseziaactivitiesarereportedinTable4. Thediameter
halorangedfrom7to14.5mmaccordingtoextractconcentration. Nosignificantdifferenceswere
recordedamongtheactivityofdifferentextractsagainstCandidaspecies.
Table4.Anti-CandidaandAnti-Malasseziapropertiesofextractsunderstudy,expressedasdiameterof
inhibitionhalo(inmm)versusseveralstrains.
Concentration
CandidaandMalasseziaspp. Ethanol Ethanol80% Butanol Methanol
(mg/mL)
50 10.25±0.58a 9.66±1.52a 8.75±1.73a 10.75±0.95a
CandidaprapsilosisATCC22019
10 8.66±1.73a 8.5±1.73a 8.66±1.73a 10±0.95a
50 10±1.41a 10.5±0.57a 10±1a 10±0.0a
CandidakruseiATCC6258 10 9.5±0.7a 10±0.0a 9±0.82a 10±0.0a
50 13.5±0.70a 13.5±0.70a 14.5±0.70a 14.5±0.70a
CandidaalbicansATCC10231 10 12±0.0a 11.5±0.70a 13±0.00a 12±1.41a
50 10.5±0.57a 11±0.00a 10.25±0.5a 10±2.0a
CandidaalbicansCD1358 10 10.25±0.5a 10±0.57a 9.5±0.57a 9.5±2.0a
50 10.25±0.5a 11.0±0b 10±0.0a 10±0.81a
CandidaalbicansCD1378 10 10±0.5a 10.33±0.0a 10±0.5a 10±0.0a
50 10.66±0a 10.33±1.89a 10.75±0.5a 11±0.81a
Candidaalbicans CD140 10 10±0.5a 8.25±1.89a 10.33±0.57a 9.66±0.57a
50 9.5±1.91a 10.5±0.57a 10.75±0.5a 11±0.81a
CandidaalbicansCD1408 10 7±1.15a 10±0.57a 6.66±0.57a 9.66±0.57a
50 10±0.0a 10±0.0a 10.33±0.57a 11±00a
MalasseziapachydermatisCBS1879
10 9.33±1.15a 9.33±0.57a 9.66±1.52a 9.33±0.57a
50 10.33±0.57a 10.66±0.57a 9.33±1.15a 10.66±0.57a
MalasseziapachydermatisCD112
10 7.66±0.57a 7.66±0.57a 7±0.0a 10.33±0.57b
50 10.33±1.55a 10.33±0.57a 9.66±0.57a 9.66±0.57a
MalasseziapachydermatisCD90
10 0±0.0a 7.66±0.57b 7.33±0.57b 9.33±0.57c
50 10.66±1.54a 10.33±1.52a 8.33±0.57a 9.66±1.15a
MalasseziafurfurCBS1978
10 0±0.0a 7±0.0b 0±0.0a 8±1.0b
50 9.33±0.57a 9.66±1.52a 8.33±1.52a 9±1.73a
MalasseziafurfurCD1006
10 0±0.0a 0±0.0a 0±0.0a 8±1.0b
50 8±1.0a 9±0.0a 0±0.0b 9±1.0a
MalasseziafurfurCD1029
10 0±0.0 0±0.0 0±0.0 0±0.0
Regardingthebiologicalactivity,theresultshereinarenotonlyconfirmedexistingdataabout
the antibacterial activities of crude extracts of D. viscosa, but are extended our knowledge on the
Molecules2017,22,942 6of13
antifungalactivitiesagainstdifferentCandidaspp. (i.e.,C.parapsilosisandC.krusei),Malasseziaand
A.fumigatusstrains.
All the extracts investigated exhibited antibacterial and anti-Candida activities which are
independentoftheextractionsolvent,butdependentontheextractconcentrations,suggestingthat
bothflavonoidandphenoliccompoundsmightactasantibacterialandanti-Candidadrugs[20]. Itis
wellknown,thatluteolinderivatives,isorhamnetinandinparticular3(cid:48)-di-O-methylquercetinand
3-O-methyquercetin from Jordanian D. viscosa have an excellent inhibitory efects against B. cereus,
S. typhimurium and S. aureus. Phenolic compounds such as hydroxycinnamic acids derivatives
(caffeoylquinic acid and chlorogenic acid) or p-coumaric acid are also potent inhibitors of E. coli,
K. pneumoniae, B. cereus and C. albicans [20,21]. Both phenolic and flavonoid compounds provoke
damage in bacterial or yeast cell walls and cytoplasmic membranes [21,22]. Interestingly, the
gram-positivebacteriatestedweresignificantlymoresensitivetoD.viscosaextractsthangram-negative
bacteria,mostlikelyduetothepresenceofalipopolysaccharide(LPS)membraneinGram-negative
bacteria,beingmoreresistanttotheforeignagents[23]. TheabsenceoftheseLPSinmembranecellof
Candidaspp. makesthemvulnerableagainstforeignagents.
Theanti-Malasseziainhibitionzonerangedfrom0to11mm.Amongtheyeastpopulationstestedin
thisstudy,Malasseziaspeciespresentasusceptibilityprofilevaryingaccordingtothespeciesandstrain
(Table4). Inparticular,allextractsshowedgoodbroad-spectrumactionagainstM.pachydermatisfrom
dogotitis/dermatitiswhereasthelowesteffectivenessagainstMalasseziafurfurisolatedfromhuman
bloodstreaminfections. Theseresultsarenotsurprisingsincesimilartrendswereobservedwhenthe
susceptibilityofM.pachydermatisandM.furfurtoazoleswascomparedduetothevariabilityofthe
cellwallchemicalcompositionofMalasseziayeasts[24]. Theanti-Malasseziaactivityofourextractsnot
onlyvariedaccordingtoMalasseziaspecies,butalsotothesolventusedforextractionwithmethanol
extractmostactiveagainstM.furfur(Table4). Indeed,theextractspreparedwiththehighpolarity
solvents (methanol) were more effective against Malassezia species including M. furfur than those
usinglowpolaritysolvents. SimilartrendshavebeenobservedusingchloroformicextractofLawsonia
inermisleavesoraqueousextractsofAlliumcepaandAlliumsativumagainstMalasseziafurfur[25]. The
anti-MalasseziaactivitiesofD.viscosaextractsmaybeexplainedbythehighTFCandCQCcontent
identifiedinmethanolextractsthusconfirmingpreviousresultswithI.paraguariensisextracts[26].
ToxicityassaysandtheeffectonfungalgerminationofextractsagainstM.canisandA.fumigatus
arereportedinTables5and6,respectively. Thegerminationandsporulationwereexpressedasmean
values(±standarddeviation)ofLog10ofcolonyformingunits(CFU)/mLandvegetativegrowthas
meanvalue(±standarddeviation)ofcolonydiameters(Ø)ofthreeindependentexperiments. All
D.viscosaextractswereabletocompletelyinhibitthegerminationofM.canisatconcentrationhigher
than1mg/mL.ThegerminationofA.fumigatuswascompletelyinhibitedatconcentrationshigher
than 10 mg/mL. D. viscosa extracts affect both M. canis vegetative growth and sporulation, being
non-toxicforM.canisCD1279andM.canisCD1447onlywhenethanolicand80%ethanolicofD.
viscosaextractswereusedat1mg/mL(Table5). AllD.viscosaextractsaretoxictoA.fumigatus,except
forthestrainsCD1435andCD1441. Inparticular,allD.viscosaextractsataconcentrationof1mg/mL
arenon-toxicforCD1435,withtheexceptionof80%ethanolicextractwhichisnottoxicfortheA.
fumigatusCD1441atthisconcentration.
Molecules2017,22,942 7of13
Table5.EffectsofD.viscosaextractsonconidiagermination,vegetativegrowthandsporulationofM.canis.Degreeoftoxicity(Tvalue)wasalsoreported.
Ethanol Ethanol80% Butanol Methanol
[C]
Germination Sporulation Germination Sporulation Germination Sporulation Germination Sporulation
mg/mL Growth T Growth T Growth T Growth T
Strains (Log10 (Øcm) (Log10 Value (Log10 (Øcm) (Log10 Value (Log10 (Øcm) (Log10 Value (Log10 (Øcm) (Log10 Value
CFU/mL) CFU/mL) CFU/mL) CFU/mL) CFU/mL) CFU/mL) CFU/mL) CFU/mL)
50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
CD 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1279 5 0 2.4±0.3 3.54±0.1 14.71 0 2.55±0.1 4.06±0.1 23.08 0 2.5±0.1 3.47±0.1 14.54 0 1.8±0.1 2.97±0.1 9.31
1 3.72±0.1 3.65±0.4 4.79±0.1 77.78 3.87±0.1 3.85±0.1 4.77±0.1 76.91 3.56±0.2 3.5±0.1 4.58±0.1 53.55 3.49±0.1 2.55±0.1 4.43±0.1 38.45
Control 4.30±0.1 5.45±0.1 4.91±0.1 100 4.30±0.2 5.45±0.1 4.91±0.1 100 4.30±0.2 5.45±0.1 4.91±0.1 100 4.3±0.2 5.45±0.1 4.91±0.1 100
50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
CD 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1243 5 0 2.1±0.14 3.13±0.1 10.62 0 2.25±0.1 3.41±0.04 13.85 0 1.85±0.1 3.06±0.1 9.52 0 1.85±0.1 2.86±0.1 8.61
1 3.73±0.1 2.55±0.1 4.22±0.2 48.97 3.81±0.1 2.85±0.1 4.22±0.02 45.41 3.24±0.1 2.85±0.1 3.79±0.1 25.71 3.24±0.1 2.35±0.1 3.43±0.1 15.11
C 4.18±0.1 5.55±0.1 4.51±0.1 100 4.18±0.1 5.55±0.1 4.51±0.1 100 4.18±0.1 5.55±0.1 4.51±0.1 100 4.18±0.1 5.55±0.1 4.51±0.1 100
50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
CD 5 0 2.5±0.1 3.36±0.2 16.58 0 2.85±0.1 3.55±0.2 21.12 0 2.45±0.1 3.81±0.1 14.77 0 1.65±0.1 2.81±0.1 9.16
1447 1 3.85±0.1 3.45±0.1 4.43±0.1 66.31 3.88±0.3 3.8±0.1 4.26±0.1 17.19 2.52±0.1 3±0.1 4.29±0.1 50.23 2.29±0.3 2.65±0.1 4.26±0.1 46.31
C 4.46±0.1 4.15±1 4.61±0.1 100 4.46±0.1 4.15±0.1 4.61±0.2 100 4.46±0.1 4.15±0.1 4.61±0.1 100 4.46±0.1 4.15±0.1 4.61±0.1 100
*Tvalue=verytoxic(0≤T≤30);toxic(31≤T≤45);moderatelytoxic(46≤T≤60);non-toxic(T>60);[C]:Concentration(mg/mL);C:ControlConcentration(mg/mL);C:Control.
Table6.EffectsofD.viscosaextractsonconidiagermination,vegetativegrowthandsporulationofA.fumigatus.Degreeoftoxicity(Tvalue)wasalsoreported.
Ethanol Ethanol80% Butanol Methanol
[C]
Germination Sporulation Germination Sporulation Germination Sporulation Germination Sporulation
mg/mL Growth T Growth T Growth T Growth T
Strains (Log10 (Øcm) (Log10 Value (Log10 (Øcm) (Log10 Value (Log10 (Øcm) (Log10 Value (Log10 (Øcm) (Log10 Value
CFU/mL) CFU/mL) CFU/mL) CFU/mL) CFU/mL) CFU/mL) CFU/mL) CFU/mL)
50 0 1.25±0.1 5.09±0.2 2.88 0 1.55±0.1 6.25±0.2 4.71 0 1.35±0.1 5.12±0.2 3.11 0 1.15±0.1 4.08±0.3 2.59
CD 10 3.66±0.1 2.35±0.1 6.45±0.2 7.13 3.51±0.1 2.85±0.1 6.81±0.1 13.62 3.47±0.3 3.35±0.2 6.18±0.1 8.56 3.46±0.3 1.95±0.1 5.08±0.6 4.75
1435 5 4.06±0.1 5.9±0.1 6.72±0.2 19.33 4.20±0.1 6.25±0.1 7.23±0.0 29.86 4.03±0.3 5.7±0.14 6.51±0.1 16.44 4.143±0.1 5.35±0.1 6.18±0.3 14.45
1 5.21±0.2 7.85±0.1 7.75±0.1 73.53 5.03±0.1 8.15±0.1 7.79±0.1 78.41 4.97±0.1 8.15±0.1 7.74±0.1 73.71 4.7±0.1 8.25±0.1 7.66±0.1 64.9
C 5.24±0.1 8.9±1.0 7.91±0.1 100 5.24±0.1 8.9±1.0 7.91±0.6 100 5.24±0.1 8.95±1.0 7.91±0.1 100 5.24±0.1 8.95±1.1 7.91±0.1 100
50 0 1.2±0.0 5.35±0.2 2.86 0 1.65±0.1 5.23±0.2 3.85 0 1.2±0 5.13±0.3 2.81 0 1.1±0 4.67±0.1 2.54
CD 10 3.55±0.1 3.4±0.3 6.59±0.1 9.93 3.68±0.1 3.4±0.0 6.56±0.1 9.8 3.19±0.1 3.3±0.5 6.17±0.2 8.33 2.67±0.1 2.95±0.1 5.64±0.1 8.25
1441 5 4.07±0.2 5.25±0.1 6.96±0.1 17.2 4.34±0.1 5.4±0.3 7.04±0.10 18.78 3.95±0.1 4.55±0.2 6.84±0.1 14.34 3.8±0.1 4.4±0.2 6.72±0.1 13.1
1 4.99±0.1 7.55±0.2 7.73±0.5 47.5 5.06±0.1 7.95±0.1 7.9±0.1 63.81 4.83±0.1 7.4±0.1 7.57±0.1 38.74 4.66±0.1 7.8±0.1 7.47±0.1 34.82
C 5.35±0.1 8.8±0.1 8.15±0.1 100 5.53±0.1 8.8±0.1 8.15±0.1 100 5.35±0.1 8.84±0.1 8.15±0.1 100 5.35±0.1 8.8±0.1 8.15±0.1 100
50 0 1.25±0.1 5.06±0.1 2.84 0 1.6±0 5.19±1.1 3.83 0 0 0 0 0 1.2±0.0 0.00 0
10 4.3±0.1 3.05±0.2 5.76±0.5 7.2 4.58±0.1 3.8±0.1 6.5±0.2 9.72 2.67±0.1 2.95±0.1 5.85±0.1 6.94 4.3±0.1 2.55±0.2 5.61±0.1 5.97
CD 5 5.08±0.1 5.55±0.2 6.69±0.1 14.37 5.19±0.1 6.6±0.1 7.78±0.1 23.54 3.96±0.1 3.95±0.1 6.84±0.1 12.23 4.68±0.1 3.6±0.1 6.18±0.2 8.84
1438 1 5.45±0.1 7.65±0.2 7.89±0.1 49.38 5.76±0.1 8.0±0.3 7.78±0.01 41.49 4.12±0.1 7.62±0.1 7.78±0.1 40.84 5.3±0 7.55±0.2 7.69±0.1 40.73
C 5.92±0.1 8.95±1.0 8.31±0.2 100 5.92±0.1 8.95±1.0 8.31±0.2 100 5.92±0.1 8.95±1.0 8.31±0.2 100 5.92±0.1 8.95±1.0 8.31±0.2 100
Molecules2017,22,942 8of13
ThepresentstudyshowsthatallD.viscosaextractssignificantlydecreasethevegetativegrowth,
germination,conidiaproductionofbothM.canisandA.fumigatus,thusconfirmingpreviousresults
against dermatophytes or other fungal species (i.e., Cladosporium cucumerinum, Botrytis cinerea,
Pseudoperonospora cubensis, Phytophthora infestans, Erysiphe graminis and Puccinia helianthi [5,22].
However,allextractsevincedaconcentration-dependentinhibitoryactivity,whichvariesaccordingly
tofungalgenus. Infact,theA.fumigatusstrainsseemstobelesssusceptiblethanM.canisaspreviously
reportedusingacetoneextractsofArctotisarctotoides[22]. Additionallythehighestantifungalactivity
was observed with methanol extracts in both fungal species, thus suggesting the efficacy of both
TPCandCQAcontentasantifungaldrugs[21,27]. Themechanismofactionofphenoliccompounds
againstfungiwaspreviouslyexplainedbyseveralstudiesandmightbeduetothemembranelipid
perturbation. SungandLee(2010)[28]demonstratedthatphenolicacidsmightcausedisruptionof
iontransport,whereasTeodoroetal. (2015)[20]indicatedthatthehydroxylgroupandcarboxylic
acidgroupsofpheonliccompoundsplaysanimportantroleindestabilizingthefungalcytoplasmic
membrane. EvenifthelowtoxicityvaluesofD.viscosamethanolicextractinonestrainofA.fumigatus
needtobeconfirmed,thehereinobtainedresults,suggestedthatconcentrationshigherthan1mg/mL
should be employed in controlling A. fumigatus strains. The antifungal activity against Malassezia
yeasts,M.canisandA.fumigatusisofinterestsincethecontroloftheseinfectionsisthesubjectofdebate
inthescientificcommunity. Inparticular,Malasseziayeastinfectionsinanimals,mainlydogs,maybe
unresponsivetoantifungaltherapyandtheanimalsusuallyhaverecurrencesthusrequiringmultiple
drug regimens [24]. The treatment of M. canis infections in animals is mandatory because of the
zoophilicnatureofthisfungus,butitisnotalwayspossibleinanimalsusedforfoodproduction[29].
Finally,thehighazolesresistancephenomenaregisteredinAspergillusspp. strainsalsosuggests
theusefulnessofstudiesonnewantifungaldrugs[30]. Allthesefindingspromotetheemploymentof
drugsofplantorigin.
3. MaterialsandMethods
3.1. ChemicalandReagents
Vanillin(C H O ),catechin(C H O ),Folin-Ciocalteureagent,sodiumcarbonate(Na CO ),
3 8 3 15 14 6 2 3
gallicacid(C H O ),aluminumchloride(AlCl ),potassiumacetate(C H KO ),rutin(C H O ),
7 6 5 3 2 3 2 27 30 16
quercetin(C H O ),sodiummolybdatedihydrate(Na MoO ),dipotassiumhydrogenphosphate
15 10 7 2 4
(K HPO ),potassiumdihydrogenphosphate(KH PO ),chlorogenicacid(C H O )werepurchased
2 4 2 4 16 18 9
fromSigma-Aldrich®(Steinheim,Germany). SolventsofanalyticalandHPLCgradewerepurchased
fromCarloErbaReactif-CDS(ValdeReuil,France).
3.2. PlantMaterial
TheleavesoftheplantswerecollectedinJune2015fromuncultivatedlandinSidiThabet,located
intheNorthEastofTunisia(latitude36◦55(cid:48)45”N,longitude10◦06(cid:48)02.10”E,altitude30m).
3.3. PreparationofExtracts
Dried and ground leaves (10 g) were macerated in four different solvents (ethanol (80% and
100%),methanolandbutanol)(10:100w/v)for48hwithshakingatroomtemperature. Theextracts
werefilteredwithWhatmanNo. 1filterpaperandthefiltrateevaporatedtodrynessusingarotary
evaporator. In order to test the antimicrobial activities, the samples were solubilized in dimethyl
sulfoxide(DMSO)toobtainconcentrationsof1,5,10and50mg/mL.
Molecules2017,22,942 9of13
3.4. PhytochemicalScreening
3.4.1. CondensedTanninsContent(CTC)
The CTC was determined as previously described [31]. In particular, 0.5 mL of extract was
condensedusing3mLofvanillinat4%inmethanoland1.5mLofconcentratedhydrochloricacid
(HCl). Themixturewaskeptinthedarkfor15minat20 ◦CandtheCTCweremeasuredusinga
Jenway6300spectrophotometer(Cole-Parmer,Staffordshire,UK)atabsorbanceof500nm. TheCTC
wascalculatedfromcalibrationcurveusingcatechin(CAE)asastandardandresultswereexpressed
asmilligramsofcatechinequivalentpergramm(g)ofdryextract(mgCAE/g).
3.4.2. TotalPhenolContent(TPC)
TheTPCwasdeterminedusingtheFolin-Ciocalteumethod[32]. Briefly,0.5mLofeachdissolved
extract was mixed with 2.5 mL of Folin-Ciocalteu reagent in each test tube. After 4 min, 2 mL of
saturatedsodiumcarbonate(Na CO )solution(7.5%)wasaddedtothemixture.Thereactionmixtures
2 3
wereincubatedfor2h. Methanolwasusedastheblank. Allassayswereconductedintriplicateand
theresultswereaveraged. TheTPCwascalculatedfromacalibrationcurveusinggallicacid(GAE)as
thestandardandtheresultswereexpressedasmilligramsofgallicacidequivalentpergramofextract
(mgGAE/g).
3.4.3. TotalFlavonoidContent(TFC)
The TFC was quantified using the aluminum chloride colorimetric assay with slight
modifications[33]. Inbrief,0.5mLofeachsolutionextractwasmixedwith1.5mLmethanol,0.1mL
of10%aluminumchloride,0.1mLof1mol/Lpotassiumacetatesolutionand2.8mLdistilledwater.
Themixturewasallowedtostandfor15min,andabsorbancewasmeasuredat415nm. Allassays
wereconductedintriplicateandtheresultswereaveraged. TheTFCwascalculatedfromacalibration
curveusingquercetin(QE)asthestandard,andtheresultwasexpressedasmgofquercetinequivalent
pergramdryextract(mgQE/g).
3.4.4. CaffeoylquinicAcid(CQC)Content
The CQC content of extracts was quantified using the molybdate colorimetric method [34].
Sodium molybdate (16.5 g), dipotassium hydrogen (8.0 g) phosphate, and potassium dihydrogen
phosphate (7.9 g) were dissolved in 1 liter of deionized water to prepare the molybdate reagent.
ForeachI.viscosaextractsolution0.3mLwasmixedwith2.7mLofmolybdatereagent. Themixture
wasincubatedatroomtemperaturefor10min. Absorbancewasmeasuredat370nm. Allassayswere
conductedintriplicateandtheresultswereaveraged. TheCQCwascalculatedfromacalibration
curve using chlorogenic acid (ChlA) as the standard and the result was expressed as mg of ChlA
equivalentpergdryextract(mgChlA/g).
3.5. CharacterizationofPhenolicCcompoundsbyHPLC-PDA-ESI-MS
The phenolic compounds present in methonolic extract were tentatively identified using the
chromatographicseparationmethodaspreviouslyreported[4].
ChromatographicseparationwasperformedonanAlliancee2695HPLCsystem(Waters,Bedford,
MA,USA)equippedwithaRP-xTerraMScolumn(150×4.6mmi.d.,3.5µmparticlesize),photodiode
arraydetector(PDA)andinterfacedwithatriplequadruplemassspectrometer(MSD3100,Waters)
fittedwithanESIionsource.Thesample(20µL)waselutedthroughthecolumnwithagradientmobile
phaseconsistingofA(0.1%formicacid)andB(acetonitrileacidifiedwithformicacid0.1%)withaflow
rateof0.5mL/min. Thefollowingmultisteplinearsolventgradientwasused: 0–40min: 14–26%B;
40–60min: 15%B;60–75: 0%B;75–80min: 14%B.TheHPLC-PDA-ESI-MSchromatogramspectral
Molecules2017,22,942 10of13
datawerestoredandprocessedwithMasslynx4.1datasystem. Eachpeakinthechromatogramwas
accomplishedinasinglechromatographicruninordertobeidentified[35].
3.6. AntibacterialandAntifungalActivities
3.6.1. BacterialStrains
Fivereferencebacterialstrains,includingGram-positive(i.e.,StaphylococcusaureusATCC6538,
Enterococcus feacium ATCC 19434, Streptococcus agalactiae ATCC 12386), and Gram-negative (i.e.,
Escherichia coli ATCC 8739, 29212 and Salmonella typhimurium ATCC 14028), were used to assess
theantibacterialpropertiesoftheextracts.
3.6.2. FungalStrains
Candidaspp.
Three references strains of Candida (i.e., Candida krusei ATCC 6258, Candida parapsilosis ATCC
22019,CandidaalbicansATCC10231),andfourCandidaalbicansstrains(i.e.,CD1358,CD1378,CD1407,
CD1408)isolatedfromcloacaoflayinghens,wereusedtoevaluatetheanti-CandidaactivityofI.viscosa
extracts. AllstrainswereobtainedfromthefungalcollectionoftheDepartmentofVeterinaryMedicine
attheUniversityofBari(AldoMoro,Italy).
Malasseziaspp. Strains
AtotalofsixMalasseziaspp. strains(threeMalasseziapachydermatisandthreeMalasseziafurfur)
weretested. Tworeferencestrains(i.e.,M.pachydermatisCBS1879andM.fufurCBS1978),twostrains
isolatedfromdogswithdermatitisand/orotitis(i.e.,M.pachydermatisCD112andCD90),twoM.furfur
strainsfromhumanskin(i.e.,M.furfurCD1029),andonefromahumanbloodstreaminfection(i.e.,
M.furfurCD1006)weretested.
Aspergillusfumigatusstrains
ThreeA.fumigatusstrains(CD1435,CD1438andCD1441)weretested. Allstrainswereisolated
fromtherespiratorytractofcriticallyillhumanpatients. Allstrainswereobtainedfromthefungal
collectionoftheDepartmentofVeterinaryMedicineattheUniversityofBari.
Microsporumcanisstrains
ThreeM.canisstrains(CD1243,CD1447,andCD1279),isolatedwithskinlesionsfromhuman,
cat,anddogweretested,respectively.AllstrainswerestoredinthefungalcollectionoftheDepartment
ofVeterinaryMedicineattheUniversityofBari.
3.7. DeterminationofAntibacterial,Anti-CandidaandAntifungalActivityofI.viscosaExtract
Theantibacterial,anti-Candidaandanti-Malasseziaactivitieswereevaluatedbythediskdiffusion
method[36],whereastheantifungalactivityofI.viscosaextractsagainstM.canisandA.fumigatuswas
assessedbystudyingthetoxicityeffectoftheextractonvegetativegrowthandsporulationaswell
theireffectonfungalgermination.
3.7.1. ToxicityAssay
TheantifungalactivityofD.viscosaextractsagainstM.canisandA.fumigatuswasassessedas
previouslyreported[37]. Inparticular,theantifungalpropertiesofextractswereassessedbyapplying
thefollowingmathematicalmodelinordertoevaluatethedegreeoftoxicity:
T=20[VG]+80[SR]/100 (1)