Chemical composition, lethality and antifungal activities of the extracts of leaf of Thaumatococcus

13 Pages·2016·0.56 MB·English

by  Oluwagbenga Adeogun

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Full Length Article
Chemical composition, lethality and antifungal
activities of the extracts of leaf of
Thaumatococcus daniellii against foodborne fungi
Oluwagbenga Adeogun a,*, Adedotun Adekunle a, Anofi Ashafa b
aFacultyofScience,UniversityofLagos,Akoka,Lagos,Nigeria
bFacultyofNaturalandAgriculturalSciences,UniversityoftheFreeStates,QwaQwaCampus,SouthAfrica
A R T I C L E I N F O A B S T R A C T
Articlehistory: Theconsumers’interestinnaturalpreservativesseemedtohaveincreasedbecauseofthe
Received20June2016 apathy and negative effect of synthetic preservatives.This study explored the use of
Receivedinrevisedform1 Thaumatococcusdanielliileavesaspotentialpreservativesagainstfungiresponsibleforthe
November2016 spoilageoforangejuiceandcornjell-o.Theleavesoftheplantwereextractedwithdiffer-
Accepted4November2016 entsolvents–acetone,aqueous,ethanolandhexane–andassayedagainsttestfungiusing
Availableonline17November2016 discdiffusionmethod.Theminimuminhibitoryandfungicidalconcentrationsweredeter-
mined.Thequalitativeandquantitativephytochemicalanalysesofleavesoftheplantswere
Keywords: carriedout,themostactiveextractofthefreshanddriedleaveswasassayedviaGC–MS
CornJell-O fortheessentialoilsandHPLCfingerprintingwasusedtodeterminethephytochemicalcon-
Extracts stituentsofthemostactiveextracts.ThetoxicitytestoftheextractsagainstBrineshrimp
Foodspoilage wascarriedoutafter24hoursofexposure.Theethanolextractsoftheplantswerethemost
Sweetorangejuice activebasedontheantifungalassay.Thetoxicitytestrevealedthattheextractsoftheplants
Thaumatococcusdaniellii werenon-toxicontheBrineshrimpsatLD50(592.95µg/mland281.12µg/mlforaqueousand
ethanolextracts).Thequalitativephytochemicalscreeningrevealedthepresenceofalka-
loid,tannin,saponins,flavonoids,steroids and terpenoids in the leaf of the plant.The
quantitativephytochemicalanalysisofthemostactiveextractintheplantsrevealedalka-
loidswiththehighestcontentof107.48mg/100g.TheGC–MSanalysesofthefreshleaves
oftheplantsrevealedthepresenceofisodecaneat15.16%.TheGC–MSanalysesofthedried
leavesrevealed1,2,3-trimethylbenzeneinT.danielliwiththehighestpercentageof15.48%.
TheHPLCanalysisrevealedthepresenceofvariousphytochemicalconstituentsinthedried
leavesoftheplant.ThisstudyhasbeenabletoascertainthepotencyofT.danielliileaves
onfungiresponsibleforspoilageofsweetCitrussinensis(orange)juiceandCornJell-O(‘Eko’)
which contribute to continuous drive towards the use and total acceptance of natural
antimicrobials.
©2016Beni-SuefUniversity.ProductionandhostingbyElsevierB.V.Thisisanopen
accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-
nc-nd/4.0/).
* Correspondingauthor.FacultyofScience,UniversityofLagos,Akoka,Lagos,Nigeria.Fax:[phone].
E-mailaddress:[email protected](O.Adeogun).
http://dx.doi.org/10.1016/j.bjbas.2016.11.006
2314-8535/©2016Beni-SuefUniversity.ProductionandhostingbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND
license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
beni-suef university journal of basic and applied sciences 5 (2016) 356–368 357
tion, maturing and ripening from January until mid-April
1. Introduction
(Onwuemeetal.,1979).
The leaf of T.daniellii is used extensively in cooking and
Foodproductsareknowntobesusceptibletospoilageduring wrappingfoodbothfordomesticuseandcommercialenter-
storage.Thespoilageoffoodproductscanbeattributedtothe prise.Itsusestranscendtheconfinesoftheruraldwellersas
effectandactionofmicro-organismsandextracellularactivi- evidencedbyitsusesforspecialpackagingofricebyapromi-
ties of enzymes on them (Barth et al., 2009; Ledenbach and nentandwellspreadurban-basedfastfoodrestaurant(Aiyeloja
Marshall, 2009).The action of microorganisms changes the andAjewole,2005).
sensorypropertiesandreducestheeconomicvaluesofthefood Thisstudywasaimedtodeterminein-vitroantifungalas-
products.There are documented evidence in several litera- sessmentofessentialoilsandorganicextractsonfoodborne
turethatfilamentousfungiandyeastarenoticeableorganisms fungi isolated from Corn Jell-O and fruit juices and also de-
associatedwithspoilageoffoodproducts(Adebayoetal.,2014; terminethein-vivolethalitystatusonbrineshrimpsfortoxicity
Aoudouetal.,2010).Themalaisearisenduetomicrobialcon- appraisaloftheleafextracts.
taminations of food products signals the need to explore
available control methods to reduce the impact of microbes
onfoodandenhancetheshelf-lifeoffoodproducts(Aponiene
etal.,2015;Jorgen,2005). 2. Materialandmethods
Theappreciableinterestinsearchoffoodpreservativeas
2.1. Glasswares,consumables,equipmentandsolvents
control method from natural products has since been gath-
eringconsiderableattentionbasicallybecauseofthenegative
TheglasswareswereproductsofThermoFisherScientificand
health implications of synthetic preservatives and the safe
theconsumableswereobtainedfromlocalscientificstoresin
healthstatusofmostnaturalproductsfromplants.Plantshave
Nigeria.Therotaryevaporatorusedwasfromlaboratoryand
beenusedforagesbymanyculturestoenhancetheflavour
analyticalsupplies(PTY),Durban,SouthAfrica,platformshaker
andaromaoffoods.Earlycultureshaveassessedthevalueof
fromLabconlaboratoryconsumables(PTY),Durban,SouthAfrica
these plants since time immemorial as preservative agents
and the solvents were of high analytical grade from Merck
(Erturk,2006).
chemicals(PTY),Wadeville,SouthAfrica.TheGC–MSsystem
In sub-SaharanAfrica with less technologies and lack of
used was Hewlett Packard and HPLC system used was
accesstorefrigeratingequipment,thenativesemploytheuse
SchimadzuProducts,Japan.
ofleavestowrapfoodtoenhanceitsshelf-life.Musasapientum,
Musaparadisiaca,Colanitida,Colaacuminata,Piliostigmareticulatum,
Theobromacacao,Colocasiaesculenta,Cannaindica,Megaphrynium 2.2. Sourceofspecimens
macrostachyumandThaumatococcusdanielliileaves(Adegunloye
et al., 2006) are used to package and present foods to clien- 2.2.1. Plantmaterial
tele.Theuseoftheseleavesisaveryancienttraditionofthe T.danielliileaveswerecollectedfromIlogboinOttalocalgov-
people;thebasisofwhichcannotbeeasilyascertained,buta ernment(Nigeria,N06°43.296′,E003.09.403)inMay,2014.After
cursory look at these leaves reveals that they all have large thetaxonomicalidentificationoftheplantspecies,avoucher
surfaceareas,i.e.canandareusedtohold/package/wraplarge specimen(LUH6087)wasdepositedatLagosUniversityHer-
volumesoffood(Ojekaleetal.,2007).Theleafextractsofthese bariumforrepositorypurposes.
plantshavebeendocumentedtopossessantimicrobialprop-
erties(Adegunloyeetal.,2006;Erturk,2006;Ojekaleetal.,2007;
2.2.2. Sourcesoffungi
Yazdani et al., 2011) and essential oils from plants have
Thefungiusedforthisstudywereisolatedfromorangejuice
beengenerallyrecognizedbeingsafeandthetoxicityonfood
andCornJell-O(‘Eko’);theywereidentifiedbycomparingtheir
mightbequiteinconsequential(Barbosaetal.,2014;Mithetal.,
morphology with fungi descriptions of Bryce (1992) and
2014).
Beveridge(2001).
Thaumatococcusdaniellii(Benn.)Benth.,commonlyknownas
ThefungiweresenttoCABI,Surrey,Egham,formolecular
Katempe,belongs to family Marantaceae.It grows through-
identificationandconfirmationofthefungi.
outthehot,humidtropicalrainforestandseawardzoneofWest
Africa(Burkill,1997).Thenaturalhabitatistheundergrowth
offoresttrees.T.danielliiisparticularlyfoundinsomeparts 2.3. Preparationofinocula
ofGhana,Coted’Ivoire,Nigeria,SierraLeone,CentralAfrican
Republic,UgandaandIndonesia(Burkill,1997). Thefungiforanti-fungalassayswereobtainedviapureculture
Katemfeisarhizomatous,perennialandmonocotyledon- platesoffungidevoidofcontaminations.Thesporesuspen-
ousherb,propagatingitselfbyrhizomes(Onwuemeetal.,1979). sions of the fungi were made using sterilized normal saline
TheinflorescenceofT.danielliiusuallyarisesfromthelowest waterof10ml.Thetopofthefungalcoloniesof3–7daysold
nodeandmaybesimpleorforkedwithspikesabout8–10cm weretouchedwithaloopandseedintosterilizednormalsaline
inlengthandbracts,usuallyumbricate,about3–4cminlength water of 10ml in the test tube.Drops of the spore suspen-
(Onwuemeetal.,1979).Theflowersthatmaybeaslongasthe sionswerelaterdroppedonasterileglassslideusingasterile
bractsforminshortspikesclosetothegroundatthebaseof dropper.The suspensions were observed under the micro-
aswollenpetiole.Theplantflowersmostoftheyearbutismost scopetoascertainthepresenceofthefungalspore;thespores
prolificfromJulyuntillateOctober,followedbyfruitforma- were later counted using haemocytometer (Adekunle and
358 beni-suef university journal of basic and applied sciences 5 (2016) 356–368
Ikumapayi, 2006) and then further diluted to approximately placedonanaluminumfoilandwrapped,latersterilizedin
106CFU/ml(Kosanicetal.,2012). anautoclaveandthediscsweredriedinanovenat60°Cfor
30min.Thediscswerelatersoakedintheextractsprepared
2.4. Preparationofplantextractsforantifungalassay for24hourspriortouse.
2.4.1. Preparationofplantorganicextracts 2.4.6. Preparationofextractconcentrationsforanti-fungal
The leaves of Thaumatococcus daniellii were shade-dried for assay
twentytwodaysandweregroundusinganindustrialmiller. The concentrations of plant extracts were determined after
Two hundred grams of leaves was soaked with solvents: dryingtheplantextractsusingthemethodadaptedbyZakia
aqueous,acetone,ethanolandhexaneindifferentconicalflasks etal.(2015).Theconcentratedextractswerereconstitutedwith
pluggedwithcottonplugsrespectivelyandobservedonashaker 100mlofdimethylsulfoxide(DMSO)andtheconcentratedex-
for seventy two hours.The extracts were filtered through a tracts were later varied into different concentrations: 0.1g
WhatmanNo.1filterpaperandMuslinclothforseveraltimes (10mg/ml),0.5g(50mg/ml),1g(100mg/ml),5g(500mg/ml)
andconcentratedtodrynesswiththeaidofarotaryevapo- and10g(1000mg/ml).
rator.Thestockswerekeptat4°Cinarefrigeratoruntilfurther
use(Silvaetal.,2014). 2.5. AntifungalactivityofT.danielliileaf’sextracts
2.4.2. Essentialoilextraction 2.5.1. Determinationofanti-fungalassayofplantextracts
Theoilextractionwasobtainedwiththeadoptionofmethods againstisolatedfungi
describedbySilvaetal.(2014).Theoilwasobtainedviahydro- ThediscdiffusionmethodbyAdekunleandIkumapayi(2006)
distillationof0.5kgoffreshanddriedleavesseparatelyfor3 andZakiaetal.(2015)wasadoptedfortheanti-fungalassay.
hours using Clevenger-type apparatus.The aqueous emul- Thetestswereconductedwithauthenticatedpureculturesof
sion was concentrated and submitted to extraction with the fungi to determine their respective tolerance of the ex-
dichloromethaneandthesolventwasevaporatedusingrotary tracts.0.5ml of the stocked suspension of each fungus was
evaporator.Theresultingessentialoilwasstoredat4°Cprior droppedasepticallyusingadropperovertheentireagarsurface
tofurtheranalyses.Theessentialoilwassolubilizedinn-hexane ofafreshlypreparedpotatodextroseagar(PDA)plateandevenly
forgaschromatographyandmassspectrometryanalysis. spreadusingaspreader.Thiswasdonetoensureanevendis-
tributionofthesporesandlaterdriedatroomtemperaturefor
2.4.3. Determinationofpercentageyieldoftheextracts 15minpriortotheapplicationofthediscsontheagarplates.
Theyieldsoftheextractsweredeterminedusingthemethod The discs (4) which have been soaked in reconstituted sol-
ofDellavalleetal.(2011)withslightmodification.Theweights ventswereplacedatthefouredgesonthesurfaceoftheagar
oftheMcCartneybottlesof28mlwerenotedafterrinsingand platewiththeaidofasterilizedforceps.Theinoculatedplates
oven-drying.TheextractswerelaterputintotheMcCartney werelaterplacedinanincubatorat28°Ctoascertainifthere
bottlesafterevaporatingviarotaryevaporatorandleftdevoid would be inhibition or not around the extract-impregnated
ofanysolventontheworkingbenchbeforetheweightsofthe discs.Theplateswereobservedafter48hours.Theextentof
bottlesweretaken.Thepercentageyieldsofextractswerecal- inhibitionwasdeterminedbymeasuringthediameterofthe
culated,thus inhibitionzoneusingatransparenthalf(1/2)metrerule(Booths,
1971;Zakiaetal.,2015).Themeanzoneofinhibitionofthethree
Percentage(%)yield:W1−W2 ×100 replicated tests of the plants extracts on the test organisms
X 1 was expressed in millimetres.The discs were also impreg-
g
natedinequivalentvolumeofdimethylsulfoxide(DMSO)as
whereX istheinitialweightofdriedplantsamples;W isthe negative control and Nystatin (anti-fungal drug) as positive
g 1
weightofMcCartneybottles+Pastyextract;andW istheweight control.
2
ofemptyMcCartneybottles.
2.5.2. Determinationofminimuminhibitoryconcentration
2.4.4. Testforpurityoftheorganicextractsandessentialoils (MIC)
Eachoftheextractsobtainedwastestedtoensureitspurity Minimuminhibitoryconcentration(MIC)wasdeterminedfor
viadroppingonasterileplatecontainingpotatodextroseagar each plant extract showing antimicrobial activity.Modified
andwellspreadontheagar.Theplateswereincubatedat27°C methodsofNgondaetal.(2012)andMishraetal.(2015)were
based on the method described by Okigbo et al. (2009).The adopted.Theconcentrationofextractwithnoobservablefungal
plateswereexaminedforpossiblegrowthofcontaminants;the growthwastakenastheminimuminhibitoryconcentration.
absenceofwhichconfirmedthepurityofthetestextracts.
2.5.3. Determinationofminimumfungicidalconcentration
2.4.5. Preparationofdiscforanti-fungalassay (MFC)
Thediscpreparationforanti-fungalactivityinvolvestheuse Minimumfungicidalconcentration(MFC)wasdeterminedfor
offilterpapers(WhatmanNo.1filterpaper).Theywerepre- each plant extract showing antimicrobial activity.Modified
paredasadaptedbyAdekunleandIkumapayi(2006)andValle methods of Ngonda et al. (2012), Oro et al. (2015) and Mihai
et al. (2016); the filter papers were perforated using a perfo- and Popa (2015) were adopted; the Petri dishes of the ex-
rator.Theperforatedfilterpapers,whichwerethediscs,were tractsconcentrationsthatshowednovisiblefungalgrowthwere
beni-suef university journal of basic and applied sciences 5 (2016) 356–368 359
seededintofreshlypreparedpotatodextroseagarplatetoassay 2.7.1.3. Testforphlobatannins. Inbrief,aqueousextractwas
forthefungicidaleffectoftheextracts.Theplatescontaining boiledwith1%aqueoushydrochloricacidandobservedforde-
thetestorganismswereincubatedat28°C.Theminimumfun- positionofredprecipitateasindicationofphlobatannins.
gicidalconcentrationwasregardedasthelowestconcentration
thatdidnotyieldanyfungalgrowthonthesolidmediumused. 2.7.1.4. Testforsaponins. Approximately2gofpowderedma-
terialwasboiledin20mlofdistilledwaterinawaterbathand
filtered.Next,10mlofthefiltratewasmixedwith5mlofdis-
2.6. Lethalitytestusingbrineshrimp
tilledwaterandshakenvigorouslyandobservedforastable
persistentfroth.Thefrothingwasmixedwiththreedropsof
Thebrineshrimpslethalitytestusingthelarvaeofbrineshrimp
oliveoilandshakenvigorouslyagainandthenobservedfor
nauplii,ArtemiasalinaL.,wascarriedoutusingthemethodde-
theformationofemulsionasindicationofsaponin.
scribedbyKayodeandAfolayan(2015),withslightmodification.
1mgml−1concentrationoftheextractwaspreparedfromthe
2.7.1.5. Testforflavonoids. Aportionofthepowderedmate-
originalstocksolutionofT.danielliithatwasusedforantifun-
rialwasheatedwith10mlofethylacetateoverasteambath
gal assays. The extracts were further diluted to final
forthreeminutes.Themixturewasfilteredand4mlofthefil-
concentrationsof20,40,60,80and100µgml−1indifferentvials
trate was shaken with 1ml of dilute ammonia solution.
usingDMSO.Tennaupliiweretransferredintoeachvialusing
Developmentofyellowcolourationisanindicationofthepres-
Pasteurpipettesandwerenotgivenfoodbecausehatchedbrine
enceofflavonoids.
shrimpcansurviveupto48hourswithoutfoodastheystill
feedontheiryolkduringthisperiod.Thecontrolvialscontain
2.7.1.6. Testforsteroids. Inthistest,2mlofaceticanhydride
normalsalinesolutionanddimethylsulfoxide(DMSO)sepa-
was added to 0.5g of ethanolic extract with 2ml concen-
rately.Theexperimentswerereplicatedthreetimes.After24
tratedHSO.Thecolourchangefromviolettoblueorgreen
2 4
hoursofincubation,thecontentofeachvialwastransferred
isindicationofsteroids.
into 65mm Petri dish and examined; the number of surviv-
inglarvaewascountedandthepercentageofmortalitywas
2.7.1.7. Testforterpenoids(Salkowski’stest). Inbrief,5mlof
evaluated.Larvaewereconsidereddeadiftheydidnotexhibit
extractwasmixedwith2mlchloroformand3mlHSO was
2 4
any form of movement during several seconds of observa-
carefullyaddedtoformalayer.Areddishbrowncolouration
tion.Extractsareregardedasnon-toxicifitsLC isgreaterthan
50 oftheinterfacewasindicationofterpenoids.
100µgml−1inbrineshrimplethalityassay.
2.7.1.8. Testforcardiacglycosides(Keller–Kilianitest). Inthis
test,5mloftheextractwastreatedwith2mlofglacialacetic
2.7. Qualitativeandquantitativephytochemical
acidcontainingonedropofferricchloridesolution.Thiswas
screening
underplayedwith1mlofconcentratedHSO.Abrownringof
2 4
the interface indicates a deoxysugar characteristic of
Theacetone,aqueous,hexaneandethanolextractsofT.daniellii
cardenolides.Avioletringmayappearbelowthebrownring,
weresubjectedtoqualitativepreliminaryphytochemicalscreen-
whileintheaceticacidlayer,agreenishringmayformjust
ingbyadoptingthestandardmethodsasdescribedbyHarbone
graduallythroughoutthethinlayer.
(1973),TreaseandEvans(2009),Sofowora(1993),Edeogaetal.
(2005),andAshafaandUmebese(2012).Theywerelatersub-
2.7.2. Quantitativephytochemicaldetermination
jectedtoquantitativeanalysisofthephytochemicalconstituents
basedonthemethodsofEdeogaetal.(2005)andProhpand
2.7.2.1. Determinationofalkaloids. Fivegramsofthesample
Onoagbe(2012).
wasweighedintoa250mlbeakerand200mlof10%aceticacid
inethanolwasadded.Thebeakerwascoveredandallowedto
2.7.1. Qualitativephytochemicalscreeningoftheplant standfor4hours.Itwasthenfilteredandtheextractwascon-
extracts centratedonawater-bathtoone-quarteroftheoriginalvolume.
Concentratedammoniumhydroxidewasaddeddropwiseto
2.7.1.1. Test for alkaloids. Briefly,0.5g of the powdered ma- theextractuntiltheprecipitationwascomplete.Thewholeso-
terial was stirred in 5ml of 1% aqueous hydrochloric acid, lutionwasallowedtosettleandtheprecipitatewascollected
heatedonawaterbathandfiltered.Then,1mlofthefiltrate andwashedwithdiluteammoniumhydroxide(2M)andthen
wastreatedwithafewdropsofMayer’sreagentandasecond filtered.Theresidueifavailableisthealkaloidwhichisthen
portion was treated the same way only with Dragendroff’s driedandweighed.
reagent.Turbidity of precipitation with either of those re-
agentswastakenaspreliminaryevidenceforthepresenceof 2.7.2.2. Determination of tannin. Five hundred milligrams of
alkaloidsintheextract. the sample was weighed into a 50ml plastic bottle. Fifty
millilitresofdistilledwaterwasaddedandshakenfor1hour
2.7.1.2. Testfortannins. Inthetestfortannins,0.5gofdried in a mechanical shaker.This was filtered into a 50ml volu-
powderedsamplewasboiledin20mlofwaterinatesttube metricflaskandmadeuptothemark.Then5mlofthefiltrate
andfiltered.Fewdropsof0.1%ferricchloridewereaddedand waspipettedoutintoatesttubeandmixedwith2mlof0.1M
observedforbrownishgreenorablueblackcolourationasin- FeCl in0.1NHCland0.008Mpotassiumferrocyanide.Theab-
3
dicationoftannins. sorbancewasmeasuredat120nmwithin10min.
360 beni-suef university journal of basic and applied sciences 5 (2016) 356–368
2.7.2.3. Determinationofsaponin. Thesampleswereground matographicseparation.Theinjectionvolumeof20µlwasused.
and20gofeachwasputintoaconicalflaskfollowedbythe Theisocraticmobilephasecomprisedmethanol:acetonitrile
addition of 100ml of 20% aqueous ethanol.They were then (60:40).Analysiswasperformedataflowrateof0.6mL/min.
heatedoverahotwaterbathfor4hourswithcontinuousstir- Ten(10)milligramsoftheplantextractswasdissolvedinap-
ringatabout55°C.Themixturewasfilteredandtheresidue propriatesolventsfilteredthroughWhatmanpaperNo.1into
wasre-extractedwithanother200mlof20%ethanol.Thecom- volumetricflaskandmadeupto25mL.
binedextractswerereducedto40mloverwaterbathatabout
90°C.Theconcentratewastransferredintoa250mlseparatory 2.10. Statisticalanalysis
funneland20mlofdiethyletherwasaddedandshakenvig-
orously.Theaqueouslayerwasrecoveredwhiletheetherlayer The data obtained were analysed using analysis of variance
was discarded.The purification process was repeated.60ml (ANOVA) and compare using Duncan’s multiple range test
ofn-butanolwasadded.Thecombinedn-butanolextractswere (DMRT)(Leechetal.,2008).
washedtwicewith10mlof5%aqueoussodiumchloride.The
remainingsolutionwasheatedinawaterbath.Afterevapo-
rationthesamplesweredriedintheoventoaconstantweight. 3. Results
Saponincontentwascalculatedaspercentage.
3.1. Plantidentity
2.7.2.4. Determinationofflavonoids. Tengramsofthestembark
extractswasextractedrepeatedlywith100mlof80%aqueous TheT.danielliiplantwasidentifiedwithvoucherno.LUH6087
methanol at room temperature.The whole solution was fil- attheLagosUniversityHerbarium.
teredthroughWhatmanfilterpaperNo.42(125mm).Thefiltrate
waslatertransferredintoacrucibleandevaporatedintodryness 3.2. Testisolates
overawaterbathandweighedtoaconstantweight.
TheisolatedtestfungiareAspergillusaculeatus,Aspergillusniger,
2.8. Analysisofessentialoils Aspergillusflavusstrain,Rhizopusstolonifer,Issatchenkiaorientalis,
Meyerozyma guilliermondii, Fusarium oxysporum, Paecilomyces
The essential oils of the extracted plant samples (fresh and variotii, Penicillium crustosum, Trichoderma harzianum and
dried)wereanalysedbasedonthemethoddescribedbyAshafa Meyerozymacaribbica.
etal.(2010);onehundredandfiftygramsoffreshanddried
leavesofT.danielliiwassubjectedtohydrodistillationforthree
3.3. PercentageyieldofT.danielliiextracts
hoursusingaClevenger-typeapparatus.
TheoilwasanalysedusingHewlettPackard6890GasChro-
The yield of the extracts obtained from 200g of leaves of T.
matographlinkedwithHewlettPackard5973massspectrometer
danielliiwasdeducedasmentionedinSections2.4.2.and2.4.3;
systemwhichwasequippedwithaHP5-MScapillarycolumn
itwasobservedthattheaqueousextracthadthehighestyield
(30m×0.25mm,filmthickness0.25µm,AgilentTechnologies
amongthesolventsusedforextractionasdepictedinTable1.
Wilmington,DE,USA).Theoventemperaturewasprogrammed
fromseventytotwohundredandfortydegreeCelsiusatthe
3.4. Purityoftheextract
rateof5°Cmin−1.Theionsourcewassetattwohundredforty
degreeCelsiuswithionizationvoltageof70eV.Heliumwasused
The observable activity of the extract on medium as shown
as a carrier gas. Spectra were analysed using the Hewlett
in Plate 1 indicated that the extract has not been contami-
PackardEnhancedChemStationG1701programforwindows.
natedprior.
Thecomponentsoftheoilswereidentifiedbymatchingtheir
spectraandretentionindiceswiththoseoftheWiley275(Wiley,
3.5. AntifungalassaysofT.danielliionfungiisolated
NewYork)inthecomputerlibraryandliterature.Percentage
fromorangejuiceandCornJell-o(‘Eko’)
compositionwascalculatedusingthesummationofthepeak
areasofthetotaloilcomposition.
Theconcentrationofacetone,ethanol,hexaneandaqueous
extractsfromT.danielliiincreasedwithanincreaseindiam-
2.9. Determinationofphytoconstituentsbyhigh
eter of zone of inhibition as depicted in Table 2.Analysis of
performanceliquidchromatography
variance revealed that the rate of inhibition differs signifi-
cantly (p<0.05)alongthevariedconcentrationsoftheextracts
Theconstituentsofthecrudeextractsofethanolwhichwere
against each test isolate.The concentrations of some of the
quite inhibitory on all organisms tested against were deter-
mined using a method employed by Ngonda et al. (2012);
Schimadzu LC-10AD HPLC system (Japan),equipped with a
Table1–Yieldofplantsafterextraction.
SchimadzuSPD-10AVUV–VISspectrophotometerdetectorwith
athermostatedflowcellandaselectabletwowavelengthsof Solvent Yieldofextract(g) Yieldofextract(%)
190–370nmor371–600nmwithSCL-10Asystemcontrol.The Acetone 9.6 4.7
detectorsignalwasoperatedatultravioletwavelengthdetec- Ethanol 24.1 12.1
tionat254nm.AnAgilentZORBEXSB-C18(AgilentTechnologies, Hexane 21.6 10.8
USA)column(3.5µm,4.6mm×150mm,i.d.)wasusedforchro- Aqueous 19.8 39
beni-suef university journal of basic and applied sciences 5 (2016) 356–368 361
poninsinallthetestextractsofT.danielliileaves;steroidswere
presentinacetoneandaqueousextractsandflavonoidswere
presentinethanolandaqueousextracts.
3.7.2. Quantitativephytochemicaldetermination
Theresultofquantitativephytochemicalconstituentsofethanol
extractofT.danielliileaveswhichwasthemostactiveresult
wasdepictedinTable5b.
3.8. EssentialoilanalysisoffreshanddriedleavesofT.
daniellii
Table6aandbshowsthepercentageofessentialoilcompo-
sition of the dried and fresh leaf samples of T. daniellii
respectively.
Plate1–Purityoftheextractonpotatodextroseagar.
3.9. HPLCdeterminationofthephytoconstituents
testfungisuchasM.guilliermondiiincreasedfrom10±0mm
at0.1gto14±0.6mmat10gwhileM.caribbicaincreasedfrom 3.9.1. HPLCdeterminationoftheethanolextractofT.
9.7±0mmat0.1gto13.7±0.3mmat10gandthisappliesto daniellii
othertestisolateswithdifferentextractsfromT.danielliileaves. TheanalyticalHPLCusedtoanalysethephytochemicalcom-
Thecomparativeactivitiesofantifungaldrug(Nystatin)ontest poundsofT.daniellirevealedthepresenceof19phytochemical
isolates seemed to be less potent compared to the test ex- compoundsasshowninTable7.
tractsfromT.danielliileaves.Thealphabets(a,b,c,d,e,fand
g)wereusedasdistinguishingcharacterfortheirsignificant
differences.Table2alsoshowsthattheactivitiesofethanol 4. Discussion
extractweremorepronouncedwithhigheractivitiesacrossall
testedisolatescomparedwiththeactivitiesofaqueous,acetone
Theconsumerinteresttowardsnaturallyderivedantimicro-
andhexaneextracts.Table3showstheminimuminhibitory
bials for the enhancement of food products has created
concentrationoftheextractsfromT.danielliileaves.Itwasob-
attention because of the documented side-effects of syn-
servedbasedonTable3thattheethanolextracthadthelowest
theticpreservatives.Severalauthorshaveexaminedthepotency
minimuminhibitoryconcentrationamongthetestextractson
ofessentialoilsandotherplants’antimicrobialsforfooden-
testisolates;therangeoftheMICofethanolextractisbetween
hancement(Hintzetal.,2015;Hyldagaardetal.,2012;Regnier
0.5gand1.0g.Theminimumfungicidalconcentrationexhib-
etal.,2012;Tareketal.,2014;Welietal.,2014).Thisstudyhas
itedthepotencyoftheextractstokillthetestfungiatarange
been able to ascertain the potential of T.daniellii as an anti-
of0.1g–10.0gasseeninTable2anditwasalsomanifested
microbialagentagainstfoodspoilagefungiisolatedorangejuice
thattherateofthefungicidalactivitiesofethanolwashigher
andCornJello.
thanothertestextractsofT.danielliileaveswithvaluesthat
TheextractsofT.daniellii’sleavesobtainedfromacetone,
rangebetween0.1gand5.0gonmostofthetestisolates.
aqueous,ethanolandhexanewereassayedforthisstudy.The
percentageyieldoftheaqueousextractoftheextractwasthe
3.6. Brineshrimpslethalityassayafter24hoursof
highestamongthefoursolventsusedwhichagreeswiththe
exposure
workofDhananietal.(2013)andcorroborateearlierworkby
Adekunle and Ikumapayi (2006) in which the yield of the
Thelethalitydoseofextractsrequiredtokillfiftypercentof
aqueousextractwashigherthantheyieldofethanolextract
thepopulationofBrineshrimpsisdepictedinTable4.Itshows
of Funtumia elastica and Mallotus oppositifolius.Francois et al.
thattheethanolandaqueousextractsofT.danielliiof281.12µg/
(2015)positedthathigheryieldoftheaqueousextractscom-
ml and 592.95µg/ml are non-toxic because they were above
pared with the ethanol, benzene and chloroform extracts
thegeneraltoxicitytestagreementofLD beingabove100µg/
50 suggest higher proportion of water-soluble plant compo-
mlfortoxic-freesubstances.
nents.Theyieldofthehexaneextractsshowsthepresenceof
somenon-polarcompoundsinthethreeplantswhichagrees
3.7. Phytochemicalscreening withtheworksofOkworietal.(2006),PatelandKumar(2008),
Adeshinaetal.(2012),Ekwealoretal.(2012),Souryadeepetal.
The results of screened qualitative and quantitative phyto- (2012),andSardashtiandAdhami(2013).Thesubstantialyield
chemicalconstituentsofdifferentextractsofT.danielliileaves oftheethanolextractcanbeattributedtothefactthatethanol
areshowninTable5aandb. is an organic solvent and will dissolve organic compounds
better, hence liberate the active compounds in the plants
3.7.1. Qualitativephytochemicalscreening (Anukworjietal.,2012;Onifade,2002).Theyieldofacetoneex-
The qualitative phytochemical constituents of T.daniellii as tracts was momentous because of the polarity and non-
shown in Table 5a signify the presence of alkaloids and sa- polaritystatusofthesolventwhichencouragesabsorptionof
3
6
2
b
e
n
i
-
s
u
Table2–ConcentrationandzonesofinhibitionofThaumatococcusdanielliileafextractsagainstthetestfungi. ef
ACETONE 0.1 5±0a 5±0a 5±0a 5±0a 9±0b,c 10±0c 5±0a 6±0a 5±0a 5±0a 9.7±0.3c un
0.5 6.3±0.3b 6±0b 5±0a 5±0a 9.3±0.3c 10.3±0.3c 5±0a 9±0b,c 9.7±0.3c 9.3±0.3c 10.7±0.3c iv
1.0 10±0.6d 9±0.3d 8.7±0.3b 9±0c 11.3±0.9d 12.7±0.3d 8.3±0.3b 9.7±0.3c 11±0.6d 11.3±0.3d 12±0.6d er
5.0 10±0.6d 9.3±0.3e 10±0c 10±0.6d 13.3±0.7e 12.7±0.3d 9±0c 11±0.6d 12.7±0.3e 11.3±0.7d 13±0d,e si
t
10.0 12.7±0e 13±0.3f 12±0.6d 14.3±0.3e 14.7±0.9e 14±0.6e 11±0d 13±0.6e 13.3±0.3e 13±0.6e 13.7±0.3e y
NYSTATIN PO 8±0c 7.3±0.3c 8±0b 8±0b 7.3±0.3b 8±0b 8±0b 8.3±0.3b 8±0b 7.7±0.3b 7.7±0.3b j
o
HEXANE 0.1 5±0a 5±0a 5±0a 5±0a 7.6±0.7b 10±0c 5±0a 5±0a 5±0a 5±0a 10.3±0.3c u
r
0.5 8±0b 8±0b 6.7±0.3a 9.7±0.7b,c 9±0.3c 11.3±0.3d 8.3±0.3b 5±0a 5±0a 5±0a 11.3±0.3d n
1.0 9.3±0.3d 9±0b 8.7±0.3c,d 9.7±0.3b,c 9.7±0.3c 12±0e 9.3±0.3d 5±0a 5±0a 8.3±0.7b,c 12±0d al
5.0 10.7±0.3d 9±0.6b 9.3±0.3d,e 9.7±0.7b,c 9.7±0.3c 12.3±0.3e 9±0.3c,d 9±0.3c 9.3±0.3c 9±0c 12±0d o
f
10.0 12±0.6e 6.3±0a,b 10±0.6e 10±1c 12±0.6d 13±0f 9±0.6c,d 9.3±0.3c 9.3±0.3c 9±0c 11.3±0.3d
b
NYSTATIN PO 8±0c 7.3±0.3a,b 8±0c 8±0b 7.3±0.3b 8±0b 8±0b 8.3±0.3b 8±0b 7.7±0.3b 7.7±0.3b a
s
ETHANOL 0.1 8±0b 7±0.6c 7±0b 10±0c 9±0c 9.3±0.3c 8±0.6b 10±0.3c 8.7±0.3b,c 9±0c 10±0.5c ic
0.5 9±0c 10±0c 7.7±0.3b 10±0.3c 9.3±0.3c 9.3±0.3d 8.7±0.3b 10±0.6c 9.7±0.3b,c,d 9.7±0.3c 10.7±0.3c a
1.0 10.3±0.7d 10.3±0.3c 10±0.6c 11±1c,d 10.7±0.6d 11.3±0.3d 10±0c 10.3±0.3c 10.3±0.8c,d 9.7±0.7c 11.3±0.3d nd
5.0 10.7±0.3d 10.7±0.3c 11±0d,e 11.7±0.7c,d 11.7±0.7d,e 11.7±0.3d,e 10.7±0.3c 11.3±0.7d 11.3±0.9d 11.7±0.7c 11.7±0.3d,e a
10.0 11.7±0.33e 10.7±0.3c 12±0.6e 12.3±0.3d 12.3±0.3e 12.7±0.6e 10.3±0c 13.7±0.3e 12.3±0.7e 12.3±0.3d 12±0e pp
NYSTATIN PO 8±0b 7.3±0.3b 8±0b 8±0b 7.3±0.3b 8±0b 8±0b 8±0b 8±0b 7.7±0.3b 7.7±0.3b li
e
WATER 0.1 5±0a 5±0a 7±0c 6±0b 5±0a 5±0a 5±0a 5±0a 5±0a 7.7±0.9b 6±0b d
0.5 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a 11.3±0.3c 7±0c s
c
1.0 5±0a 5±0a 6±0b 5±0a 5±0a 5±0b 5±0a 5±0a 5±0a 14.7±0.3e 7.3±0.3c ie
5.0 5±0a 5±0a 6.7±0.3c 5±0a 5±0a 8±0b 5±0a 5±0a 5±0a 13±0.6c 8.7±0.3d nc
10.0 9.7±0.7c 10±0c 7±0c 10±0.6d 10±0.7c 11.3±0.7c 10±0c 10±0.6c 10±0c 11.3±0.9c 10.7±0.3e es
NYSTATIN PO 8±0b 7.3±0.3b 8±0c 8±0c 7.3±0.3b 8±0b 8±0b 8.3±0.3b 8±0b 7.6±0.3b 7.7±0.3c 5
NO DMSO 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a 5±0a (
2
0
ThelettersdenotesignificancedifferencebasedonDuncanMultipleRangeTest.Theletterswhentheyarethesame,itindicatesnosignificanceandwhenthelettersaredifferent,itconnotes 1
6
significance. )
3
5
6
–
3
6
8
beni-suef university journal of basic and applied sciences 5 (2016) 356–368 363
Table3–Minimuminhibitoryconcentrationandminimumfungicidalconcentrationoftestextractsonisolatedfungi.
Isolates Acetone Hexane Ethanol Aqueous
MIC MFC MIC MFC MIC MFC MIC MFC
ANS 1.0 10 1.0 10 0.5 10 10 –
AN 1.0 10 5.0 – 0.5 0.5 10 10
AFS 1.0 10 5 – 0.5 10 10 –
AF 5 10 5 – 0.5 5 10 –
MCR 5 10 0.5 – 0.1 5 10 –
YST 0.1 1.0 0.5 10 0.1 1.0 10 –
YSTA 0.1 1.0 0.1 1.0 0.1 1.0 10 –
YSTB 1 1.0 0.1 1.0 0.5 1.0 10 –
FSR 5 – 1.0 – 1.0 1.0 10 –
YB 0.5 5.0 5.0 – 0.1 5.0 10 –
PENU 0.5 5.0 5.0 – 0.5 5.0 10 –
TRI 0.5 1.0 5.0 – 0.1 5 0.5 1.0
ANS:Aspergillusaculeatus;AN:Aspergillusniger;AFS:AspergillusflavusStrainA;AF:Aspergillusflavusstrain;MCR:Rhizopusstolonifer;YST:Issatchenkia
orientalis;YSTA:Meyerozymaguilliermondii;YSTB:Meyerozymacaribbica;FSR:Fusariumstolonifer;YB:Paecilomycesvariotii;PENU:Penicilliumcrustosum;
TRI:Trichodermaharzianum.
polarcompoundsandnon-polarcompoundsandthisasser- vativeandfungicidalcompoundswerepresentintheleaves
tionupholdearlierreportsofEllof(1998),Sheelaetal.(2012), ofT.daniellii,sincetheywereabletoinhibitthegrowthofthe
Sivasankaridevietal.(2013),andDzoyemetal.(2014). testfungi;thisrevelationisinconsonancewithearlierreports
Theascertainedpureextractdevoidofcontaminationagrees onseveralstudiesbutondifferentfungalorganisms(Ajayiand
withtheworkofCheesbrough(2000),Okigboetal.(2009),and Ojelere,2013,2014;Awosanetal.,2014;GrilloandLawal,2010).
Anukworjietal.(2012).Thisstudyalsorevealedthatpreser- Hencethefourextractsusedhavethepotentialapplicationin
Table4–TheeffectofThaumatococcusdanielliileafextractsonbrineshrimps.
Conc.(µg/ml) TotalNo. Control Ethanol Hexane Acetone Aqueous
DMSO Salinewater
20 10 0 0 1 5 4 0
40 10 0 0 3 6 5 0
60 10 0 0 2 8 7 2
80 10 0 0 2 9 8 0
100 10 0 0 4 9 8 1
LD50 281.12µg/ml 22.77µg/ml 31.27µg/ml 592.95µg/ml
Table5–(a)Qualitativephytochemicalscreeningoftheplant’leaf.(b)Quantitativephytochemicalscreeningofthe
plants’leaf.
(a)
S/No. Compound Thaumatococcusdaniellii
Acetoneextract Ethanolextract Hexaneextract Aqueousextract
1 Alkaloid + + + +
2 Tannins − + − +
3 Phlobatannins − − − −
4 Saponins + + + +
5 Flavonoids − + − +
6 Steroids + − − +
7 Terpenoids − − − +
8 Cardiacglycosides − − − −
(b)
Plants Phytoconstituents(mg/100g)
Alkaloids Tannin Saponins Flavonoids
Thaumatococcusdaniellii 98.96 81.77 22.92 35.03
+,Presence;−,Negative.
364 beni-suef university journal of basic and applied sciences 5 (2016) 356–368
Table6–(a)EssentialoilanalysisofdriedleavesofThaumatococcusdaniellii.(b)EssentialoilanalysisoffreshleavesofT.
daniellii.
(a)
PeakNo. Driedleaves(Thaumatococcusdaniellii)
Plantconstituents(essentialoils) RT RI Composition(%)
1 2,2,5,5-tetramethylhexane 8.20 0 1.78
2 2-Fluoro-3,3-dimethylbutanol 8.30 0 0.99
3 1-Aza-thiapentane 8.64 813 0.68
4 Isodecane 8.70 0 1.66
5 Ethylbenzoylpyruvate 8.83 0 1.63
6 3,3-dimethylbutyrophenone 8.92 0 2.69
7 6-Iodo-1-heptene 9.21 0 0.53
8 1,3,5-Trimethylbenzene 9.40 0 11.80
9 1,2-Dineopentyldisulfane 9.83 1824 0.98
10 2,3-Dimethyl-3-phenybutan-2-ol 9.91 0 3.58
11 8-(Phenylsilyl)-3-phenyoct-1-ene 10.18 0 0.55
12 (E)-Ethyl5-N-benzamido-2-pentenoate 10.41 0 1.33
13 5,9,9-Trimethylspiro(3–5)nona-5,7-dien-1-one 10.51 0 3.11
14 1-Methylbutylnitrite 10.58 0 0.55
15 1,4,5,8-tetrathiadelin 10.67 0 1.07
16 o-Diethylbenzene 10.84 0 0.98
17 (z)-Lyratylacetate 10.90 0 1.01
18 1-methyl-3-isoprophylbenzene 11.61 0 2.17
19 Hendecane 11.18 0 4.73
20 Perillaacetate 11.55 0 1.41
21 n-Nonane 12.78 0 2.98
22 Dodecane,1,1-difluoro 14.26 0 1.19
23 Bibenzene 15.60 0 1.33
24 1-methylbutylnitrite 16.95 0 0.57
25 Octyldisulfide 19.34 0 0.52
26 4-Hydroxycarbonyl-2-oxobutyltriphenylphosphoniumbromide 24.39 0 2.41
27 1,3-Diphenyl-1-(trimethylsilyl)oxy)-1(E)heptene 31.63 0 0.56
28 :(E)-1-[(1′,1’′dimethylethyl)diphenylsilyl]-2-(trimethylsilyl)ethylene 33.99 0 0.61
29 2-(N-methyl-N-acetylamino)-4,4-diphenyl-4-ethylcarbonylbutane 34.35 0 0.52
30 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene)tyramine 34.35 0 0.54
31 4,4-Dimethyl-2-oxo-tetrahydrofur-3-yl2-[Tris(trimethylsilyl)silyl]-4-phenyl-but-3-enoate 34.66 0 0.50
(b)
PeakNo. Freshleaves(Thaumatococcusdaniellii)
Plantconstituents(essentialoils) RT RI Composition(%)
1 2,6-Dimethyloctane 8.18 887 1.58
2 5,6-dimethylundecane 8.57 1185 0.68
3 2,3,4-Trimethylheptane 8.70 0 1.70
4 1,3,5-Trimethylbenzene 8.77 0 4.61
5 3-Ethyltoluene 8.83 0 1.47
6 3-Methylnonane 8.90 0 2.26
7 1-Ethyl-2-methylbenzene 9.10 1006 1.83
8 1-Isobutyl-3-methylcyclopentane 9.20 956 1.19
9 Isodecane 9.43 0 15.27
10 2-Phenylbutane 9.68 0 0.86
11 2-Butyloctanol 9.76 1393 1.05
12 4-Methyldecane 9.83 0 3.91
13 (1,3-dimethylbutyl)cyclohexane 10.006 0 0.71
14 3,7-Dimethylnonane 10.01 0 0.79
15 (1-Propyldecyl)cyclohexane 10.12 1909 2.13
16 Octadecylhexanoate 10.19 2574 0.75
17 p-Ethyethylbenzene 10.35 0 1.06
18 1,3-Hexanedione,1-phenyl-2,5-dimethyl- 10.42 1633 2.70
19 1,4-Diethylbenzene 10.51 0 3.77
20 2-Methyl-decane 10.57 0 2.21
21 (1,3,3-Trimethylnonyl)benzene 10.68 1738 3.84
22 1,2-Dimethyl-3-ethylbenzene 10.85 0 1.37
23 Thymene 10.90 0 1.80
24 p-isopropyl-Benzaldehyde 11.01 0 1.80
25 1-Methylindan 11.06 0 0.52
26 2-Propyl-2-heptenal 11.10 0 0.58
27 Hendecane 11.18 0 8.94
28 p-Cumicaldehyde 11.27 1230 0.83
29 (1,1,4,6,6-pentamethylheptyl)Benzene 12.35 0 0.65
30 Nonan 11.44 0 0.60
31 p-Cymol 11.61 1042 1.12
32 (+)-(R)-p-Mentha-1,8-dien-4-ol 12.14 0 0.99
33 Hexadecylchloride 12.22 0 0.65
34 n-Tridecane 15.65 1413 1.49
35 Isododecane 12.78 0 0.57
36 n-Tetradecane 15.65 1413 1.49
37 n-Octadecane 18.18 0 1.12
38 2-Keto-butyric-acid 20.44 0 0.80
39 n-Hexadecoicacid 22.12 0 0.60
40 9,12,15-Octadecatrienal 23.93 0 1.36
41 Di-(2-ethylhexyl)phthalate 30.12 0 1.81
beni-suef university journal of basic and applied sciences 5 (2016) 356–368 365
Table7–HPLCdeterminationoftheethanolextractofThaumatococcusdaniellii.
SpectrumMaxPlot
Pk#name Retentiontime Starttime Stoptime Area Areapercent
1 11.774 11.11 12.07 25,716,659 8.68
2 12.363 12.09 12.85 38,686,413 13.06
3 13.152 12.85 13.74 11,859,356 4.01
4 14.283 13.74 14.61 38,402,254 12.97
5 14.709 14.61 15.11 16,543,520 5.59
6 15.456 15.11 15.58 5,930,881 2.00
7 15.947 15.58 16.32 11,253,413 3.80
8 16.597 16.32 16.85 2,555,085 0.86
9 17.291 16.85 17.64 12,976,393 4.38
10 18.080 17.64 18.33 10,591,257 3.58
11 18.720 18.33 19.25 4,658,620 1.57
12 19.723 19.25 20.47 8,264,670 2.79
13 20.800 20.47 21.12 2,565,391 0.87
14 21.611 21.12 22.55 29,746,553 10.05
15 24.651 22.83 25.69 29,364,214 9.92
16 26.219 25.69 26.74 7,885,506 2.66
17 27.339 26.74 28.74 36,272,676 12.25
18 28.907 28.74 29.34 1,872,750 0.63
19 29.781 29.42 29.98 965,989 0.33
theinhibitoryactivityagainstthetestfungiisolatedfromorange extractsasagainstothersolventsinexhibitingantifungalac-
juiceandspoiltCornJell-O(‘Eko’).Howevertheefficacyofthe tivitiesagainstthetestfungimightnotbeunconnectedwith
extracts differed based on the concentration of the extract, thesolubilityoftheplantinthedifferentextractingsolvents;
solventofextractionandwitheachtestfungus. moreinhibitingeffectobservedwithethanolextractsthanthe
The observation of the activities of the extracts against otherextractscouldbeexplainedbythefactthatethanolis
testfungishowedthattheactivityoftheaqueousandhexane acidic and in solution donates a proton which makes the
extractsagainstthefungiwerequiteineffectiveatanycon- mediumacidic(NwankwoandAmaechi,2013),anyorganism
centrationexceptinfringesamongtestorganismswhichagrees inthesolutionwillaccepttheprotonasabase(Nwankwoand
with Alo et al. (2012) who reported that aqueous extract of Amaechi, 2013).The increase in concentration of the hydro-
Ocimumgratissimumdidnotinhibitthegrowthoftestorgan- genionsinhibitstheactivitiesofthemicroorganismthereby
isms.TheworkofOluremietal.(2010)corroboratesAloetal. resulting in their death (Uruquiaga and Leighton, 2000).The
(2012) that some organisms are not susceptible to the ex- apparent reduction in the spectrum of activity in other ex-
tract’sactivityandtheresistancetoantimicrobialagentscannot tracts may signal a possible loss of potency in the event of
beeliminatedbutcurtailedsincesomeorganismsareintrin- extractionandfurtherpurificationoftheplantcomponents.
sicallyresistant.Theinactivityofhexaneandaqueousextracts The activities of the various extracts were gauged by
also agrees with earlier works by El-Mahmood (2010).The determining their minimum inhibitory and fungicidal con-
absenceofactivityinthehexaneandaqueousextractsofT. centrations. The minimum inhibitory and fungicidal
daniellii’sleavesmighteitherbeduetothemoresolubilityof concentrations are tests of in-vitro susceptibility that is pre-
theactiveprinciplesinothersolventsthanhexaneandaqueous dictiveofin-vivotherapeuticefficacy.Theminimuminhibitory
solvents(Francoisetal.,2015;ParekhandChanda,2007)orthe concentration,whichisthelowestconcentrationofpotency
presenceofactivecomponentsininsufficientquantitiesinthe oftheextractthatinhibitedthevisiblegrowthoffungiafter
crudeextractstoshowtheactivitywiththedoselevelsem- incubationasshowninTable3,signalledacetoneandethanol
ployed (Taylor et al., 2001).The activity of the acetone and extracts at 0.1mg/ml against I. orientalis, M. caribbica and
ethanolextractswasquiteinhibitoryonalltestfungiandthese Meyerozyma guilliermondii among the test fungi with lowest
occur as the concentration increases which agrees with minimuminhibitoryconcentrationneededfortheinhibition.
EkwenyeandElegalem(2005).Athigherconcentrationwider Theminimuminhibitoryconcentrationsinhexaneandwater
zone of inhibition was produced and it reduced as the con- werequiteinfinitesimallylow,observedonlywithM.caribbica
centrationreduces.Cowan(1999)reportedthattheethanolic and M.guilliermondii when their growth was hindered with
extractsshowedbetterresultsascomparedtoaqueousbecause hexaneextractofT.daniellii.Theminimuminhibitoryconcen-
oftheabilitytoextractorganiccompoundsandthisencour- trationwasalsolowinT.harzianumwheninhibitedwithwater
ages the release of greater amount of active antimicrobial extract of T.daniellii.The minimum inhibitory concentration
components.Thehighactivityoftheacetoneextractagainst activityoftheextractsagainstthetestfungiagreeswithseveral
thetestfungiagreeswithVaghasiyaandChanda(2010)and worksbyOkigboetal.(2009),GrilloandLawal(2010),Aloetal.
Abraham and Thomas (2012).Minhas et al. (2013) acknowl- (2012),AshafaandUmebese(2012),andHarunaetal.(2013).
edgedtheineffectivenessofacetoneextractagainstAspergillus Theminimumfungicidalconcentration,whichisthecon-
flavus but recognized the activeness of the acetone extract centrationoftheextractswithnoobservablegrowthoffungi
againstsomebacteriaspecies.Thepredominanceofethanol after incubation as shown in Table 5,indicated T.daniellii at