Determination of Antimicrobial, Antioxidant and

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2018Vol.4 No.1:4

Research Article

Journal of Pharmaceutical Microbiology

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Abegunde MT1, Akinpelu DA1,2,3, Omololu-Aso J1*, Otusanya OO4 and Akinlolu JT1

1 DepartmentofMicrobiology,ObafemiAwolowoUniversity,IleIfe,OsunState,Nigeria

2 MicrobiologyandBiotechnologyUnit,AgriculturalResearchCouncil,AnimalProductionInstitute,Irene,Pretoria0062,SouthAfrica

3 PlantScienceDepartment,theFreeStateUniversity,QwaqwaCampus,SouthAfrica

4 DepartmentofBiologicalSciences,WesleyUniversity,Ondo,Nigeria

Corresponding author: JosephOmololu-Aso

[email protected]

DepartmentofMicrobiology,ObafemiAwolowoUniversity,IleIfe,OsunState,Nigeria.

Tel: +2348033770933

Citation: AbegundeMT,AkinpeluDA, Omololu-AsoJ,OtusanyaOO, AkinloluJT(2018)DeterminationofAntimicrobial,AntioxidantandPhytochemicalPropertiesofCocos nucifera linn EndocarpExtractonBacteriaAssociatedwithHumanInfection.JPharmMicrobiol.Vol4No.1:4.

Determination of Antimicrobial, Antioxidant and Phytochemical Properties of Cocos

nucifera linn Endocarp Extract on Bacteria Associated with Human Infection

AbstractThestudyinvestigatedthattheantioxidantpropertiesandmodeofactionofCocos nuciferaLinnendocarpextractsonpathogensassociatedwithhumaninfectionswithaviewofproducingnaturalproductthatserveasapotentialtemplatefornewantibacterialagainstmultipleresistantbacterialpathogens.

Cocos nuciferaendocarpwascollectedfromIle-Ife,OsunState,Nigeria,andovendriedat40oCforfourdaysandgroundintofinepowder.Thepowderedsamplewascoldextractedusingmethanolandsteriledistilledwater in ratio3:2 (v/v).Themixtureobtainedwasconcentratedin vacuousingrotaryevaporatortodriveout theorganic solvent,while theaqueous layerwas subsequently lyophilized.The crude extract obtainedwas screened for the antimicrobial activity againstpanelofbacterialstrainsimplicatedinhumaninfections.Thecrudeextractwaslaterpartitionedusing fourdifferentorganic solvents inorderof theirpolarity.Theantibacterialpotentialsofthecrudeextractalongwiththefractionsobtainedwere determined using agar-well diffusion method. The minimum inhibitoryconcentration andminimum bactericidal concentration of the extracts againstthebacterialstrainswerealsodetermined.Therateofkilling,protein,nucleotideand potassium ion leakagewere determined using Staphylococcus aureus andEscherichia coli as representativeofGrampositiveandGramnegativebacteriarespectively.Themostactivefractionwhichwasethylacetatefractionwasfurtherpartially purified by thin layer and column chromatography. The antimicrobialactivityoftheresultingsampleswastestedagainstthepreviouslyusedbacterialstrains. Themost active sample of partially purified ethyl acetate fractionwasanalyzedusingGasChromatography-MassSpectrometry.

The results showed that the endocarp extract of C. nucifera and variousfractions obtained from it exhibited varying degree of antibacterial activities.The phytochemical screening of the extract showed the presence of phenols,flavonoids,tannins,alkaloids,triterpenesandsaponins.Theminimuminhibitoryconcentrationofthecrudeextractrangedbetween0.27and8.75mg/mLwhilethemostactivefractionrangedbetween0.31and2.50mg/mL.Therateofkillassay showed that the percentage of the cells killed increasedwith increasingconcentrationofthefractionandcontacttimeintervals.Leakageofnucleotides,proteinandpotassiumionsfromtestcellsalsofollowedthesametrendobservedforkillingrate.Cocos nuciferaendocarpextractexhibited50%inhibitionoffreeradicalsat0.011mg/mL,whereastheascorbicacidusedasstandardhadIC50of0.020mg/mL.ThemajoractiveconstituentofthepurifiedsamplewasidentifiedasEthylVanillin.

Cocos nucifera endocarp extracts which possessed antioxidant propertiesexhibitedappreciableantibacterialactivitiesagainstthetestpathogens.

Keywords: Endocarp extract; Phytochemical screening; Antimicrobial activity;antioxidant;Pathogens

Received: April17,2018; Accepted: May07,2018;Published: May14,2018

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2 This article is available in: http://pharmaceutical-microbiology.imedpub.com/archive.php

IntroductionThe oldest written evidence of medicinal plant usage forpreparation of drugswas found on a Sumerian clay slab fromNagpur which comprised 12 recipes for drug preparationreferring to over 250 various plants [1].Nearly 80%of peopleliving in the developing countries especially in Africa dependon herbal medicine for their health needs including wounds,infectiousandmetabolicdiseases[2].Variousplantparts,suchasherbs,spices,fruit,vegetablesandtropicalplantshavebeenshowed to contain these natural antimicrobials which are ofintensemedical benefits [3]. Plant produces a wide variety ofsecondarymetaboliteswhicharenatural bioactive compoundsasameansofsurvivalinahostileenvironment[4]andareusedasprecursorsoras the leadcompounds in thepharmaceuticalindustry.

Africaisreputedfortheextraordinaryrichnessofitsflorawhichconstitutesthousandsofspecies[5].Theresearchonthefolkloreusesofplants,citesthat75%mayprovidenovelmedicinalplants,whichmayleadtoherbaldrugdiscoveryandformulation[6,7].It is inrecordthatthepotentialofplantsingeneralandhigherplantsinparticularasasourceofnewdrugshasnotbeenfullyexplored. The secondary metabolites (bio-active compounds)producedbytheseplantshavebeenlinkedtotheirhighmedicinalpotencyandenablethemtobeusedasasourceofrawmaterialsintheexplorationofantimicrobialagentsintheindustry[8].

These antimicrobial compounds also serve as alternatives toformally approved chemically synthesized artificial drugs towhichmany infectiousmicroorganisms have become resistant[9]. Some individual plant extract may have been subjectedto specific pharmacological test (e.g. for cardiac activity only)however,thesameextractmaybeexaminedforothertypesofactivitiessuchaspainrelieving,anti-inflammation,antidiarrhealetc.[10].

Theincidencesofresistanceofpathogenstoexistingantibioticscallforthesearchfornewantimicrobialsespeciallyfromnaturalorigin.Accumulationofoxidesinhumansystemwhichultimatelyleadstoageingcouldbecontrolledbytheuseofmedicinalplantswith promising source of antimicrobials and antioxidants. ThisstudygaveaquantitativeevaluationontheantibacterialactivitiesandmodeofactionofendocarpextractofCocos nuciferafoundintheenvironment.

Materials and MethodsMaterialsMicroorganisms: Thebacteriausedinthisstudywereobtainedfrom culture collections of Prof. DA Akinpelu, Department ofMicrobiology,ObafemiAwolowoUniversity, Ile-Ife,OsunState,Nigeria.Thebacteriastrainswere:

Gram positive: Bacillus cereus (NCIB 6349), Bacillus polymyxa (LIO), Bacillus stearothermophilus (NCIB 8222), Bacillus subtilis (NCIB 6349), Clostridium sporogenes (NCIB 532),Enterococcus faecalis(NCIB775),Micrococcus luteus(NCIB196),Shigelladysentariae (LIO), Staphylococcus aureus (NCIB 8588)andStreptococcus agalaticae (CIRv).

Gram negative:Escherichia coli (NCIB86),Klebsiella pneumonia (NCIB418),Pseudomonas aeruginosa(NCIB950),Proteus mirabilis (LIO),Proteus vulgaris(NCIB67),Pseudomonas fluorescens (NCIB3756),Vibrio fluvialis (LIO),Vibrio furnissii (LIO).

Preparation of bacteria used for the experiment: Thebacterialstrains used in the experiment were re-activated in nutrientbrothandincubatedat37°Cfor18h.Theorganismswerestoredon sterile nutrient agar slants inMcCartney bottles at 4°C andsubcultured at three months interval to maintain them forfurtheruse.

MethodsDrying and extraction of the plant sample: The endocarp ofCocos nucifera used for this study was collected within Ile-Ifeenvironment inOsunState,Nigeria inthemonthofJuly,2016.TheplantwasidentifiedintheHerbariumoftheDepartmentofBotany,ObafemiAwolowoUniversity,Ile-Ife,Nigeria.Thevouchernumber is IFE-1742.Theendocarpwasdried inhotairovenat40°Cuntilaconstantweightofthesamplewasobtained.Thiswasgroundintofinepowder.Exactly2000gofthepowderedsamplewasextractedusingmethanolandsteriledistilledwaterinratio3:2 (v/v) for four dayswith regular agitation. The supernatantcollectedwaslaterfilteredintoacleansteriledriedconicalflask.Thefiltratewasconcentratedin vacuotodriveoffthemethanolandlaterlyophilized.

Sensitivity testing of the crude extract and fractions of C. nucifera endocarp against bacterial strains: The sensitivitytestingof theextractwasdeterminedusingagar-welldiffusionmethodasdescribedbyIrobietal.andRusellandFurrwithsomemodifications [11,12]. Thebacterial strainswerefirst grown innutrientbrothfor18hbeforeuse.The18holdtestorganismswere then standardized using MacFarland (108 cfu/mL of 0.5McFarlandstandards).TheinoculumwasthenstreakedontoanalreadysterilizedMueller-Hintonagar(LabM)plate.Wellswerethen bored into themedium using a sterile 6mm cork borer.Thewellswerefilledupwith35mg/mLpreparedsolutionoftheextract.Carewas takennot toallowsolutionof theextract tospillonthesurfaceofthemedium.Theplateswerethenallowedtostandonthelaboratorybenchforabout1-2htoallowproperinflow of the solution into themedium before incubating theplate in an incubator at 37°C for 24 h. The plates were laterobservedforthezonesofinhibition.Theeffectsoftheextractson bacterial strains were compared with standard antibiotic;streptomycinandampicillin.

Determination of minimum inhibitory concentrations (MICs) of the crude extract and the fractions of C. nucifera endocarp on bacterial strains: TheMICs of the extract was determinedusingthemethoddescribedbyAkinpeluandKolawole[13].Two-folddilutionoftheextractwaspreparedand2mLofdifferentconcentrations of the solution was added to 18 mL of pre-sterilizedmoltennutrientagartogivefinalconcentrationrangeof 0.273 to 35.0 mg/mL. The medium was then poured intosterilepetridishesandallowedtoset.Thesurfacesofthemediawereallowedtodrybeforestreakingwith18holdstandardizedbacterialcultures.Theplateswerelaterincubatedat37°Cforup

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to72h after andwereexamined for thepresenceor absenceofgrowth.Minimuminhibitoryconcentrationwastakenasthelowestconcentrationthatpreventedbacterialgrowth.

Determination of minimum bactericidal concentrations (MBCs) of the crude extract of C. nucifera and the fractions on bacterial strains: Minimum bactericidal concentrations of the extractweredeterminedinaccordancewiththemethodofOlorundareetal.withsomemodifications[14].SamplesfortheMBCweretaken from lineof streakonMICplateswithoutvisiblegrowthand then streaked onto extract-free freshly prepared nutrientagarmediumplates.Theplateswerethenincubatedat37°Cfor48hrs.TheMBCwastakenas the lowestconcentrationof theextractthatdidnotallowanybacterialgrowthonthesurfaceoftheagarplatesattheendof48hincubationperiod.

Phytochemical screening of the extract: Endocarpcrudeextractof the plant was subjected to phytochemical screening usingTrease,EvansandHaborne,Baxtertotestforalkaloids,tannins,flavonoids, steroids, saponins, reducing sugars and cardiacglycoside [15,16]. Viable counts of the test organisms wereinitiallydetermined.A0.5mLvolumeofknowncelldensity(byviablecountsof108 cfu/mL)fromeachorganismsuspensionwasaddedto4.5mLofdifferentconcentrationofthefractions.Thesuspensionwasthoroughlymixedandheldatroomtemperature(28-30°C) and the killing ratewasdeterminedover aperiodof2h.Exactly0.5mLvolumeofeachsuspensionwaswithdrawnatatime intervalsand transferred to4.5mLofnutrientbrothrecoverymedium containing 3% “Tween 80” to neutralize theeffectsoftheantimicrobialcompoundscarry-overfromthetestsuspensions.Thesuspensionwasproperly shaken thenseriallydilutedinsterilephysiologicalsaline.Exactly0.5mLofthefinaldilutionofthetestorganismwastransferredintopre-sterilizednutrient agar at 45°C and plated out. The plateswere allowedto set and incubated upside down at 37°C for 72 h. Controlexperimentwassetupwithoutinclusionofantimicrobialagent.Viablecountsweremadeintriplicatesofeachsample.

Determination of potassium ions leakage from the test bacterial strains using active fractions: The method of Allwood, HugoandGalewasused for thisassay [17,18].CellsofE. coliandS. aureus from 18 hr old nutrient broth culture were washed in0.09w/vNaCl(normalsaline).WashedsuspensionofE. coliandS. aureus (approximately 108 cells) were treated with variousconcentrations of the fractions relative toMIC at varioustimeintervalsfor2h.Eachsuspensionwasthencentrifugedat10,000rpm and supernatant collectedwas assayed for potassium ionusingatomicabsorptionspectroscopy.Normalsalineinoculatedwiththesamequantityofinoculumswasusedascontrol.

Determination of nucleotides leakage from the test strains by the active fractions: The method described by Joswick et al.with somemodificationwasused todetermine the leakageofthenucleotidesfromthetestcells.CellsofE. coliandS. aureus from18hroldnutrientbrothculturewerewashedin0.9%w/vnormalsaline [19].WashedsuspensionofE. coliandS. aureus (inoculums approximately 108 cells) were treatedwith variousconcentrationsofthefractionsrelativetoMICsatvarioustimeintervalsfor2h.Eachsuspensionwasthencentrifugedat10,000

rpm and the optical density of the supernatant collected wasmeasured at 260 nm wavelength using spectrophotometer.Normal saline inoculated with the same quantity of inoculumwasusedascontrol.

Determination of protein leakage from the test strain by the active fraction: CellsofE. coliandS. aureusfrom18hrnutrientbroth culture were separately washed in 0.9% w/v normalsaline.Washed suspension of E. coli and S. aureus (inoculumssize approximately 108 cells 0.5 Mcfarland standards) weretreated with various concentration of the fraction relative toMICsatvarioustimeintervalof2h.Eachsuspensionwasthencentrifugedat7000rpmandsupernatantcollectedwasassayedforproteinusingBradfordmethod[20].Inassayingforprotein,0.4mLBradford reagentwasadded to1.6mL sample (0.2mLsupernatantplus1.4mLsteriledistilledwater)tomakeup2mLtotalvolume.Opticaldensity(OD)oftheresultingsolutionwasthereafter takingat595nmafter5min.TheopticaldensityofeachofthesampleswascalculatedfromtheequationofthebestlinearregressionlineobtainedfromthegraphofBovineSerumAlbumin(BSA)standardcurve.

Preparation of Bovine serum albumin standard curve: Bovineserum albumin stock solution of concentration 100 µg/mLwasfirstprepared.Varying concentrationof thebovine serumalbumin was thus prepared from the stock solution. Bradfordreagent(0.4mL)wasaddedtothevariousbovineserumalbuminconcentrations.Thiswasallowedtostandforfiveminuteafterwhichtheopticaldensitywasmeasuredat600nm.Thevariousopticaldensityobtainedwerethereafterplottedagainstbovineserumalbuminconcentrationstoformstandardalbumincurve.Theconcentrationsofproteininthesampleswerethencalculatedfromtheequationofbest-fitlinearregressionlineobtainedfromthegraphofthebovineserumalbuminstandardcurve.

DPPH antioxidant assay of C. nucifera: Theantioxidantactivity(AA%) of each substance was assessed by DPPH free radicalassay.ThemeasurementwasperformedasdescribedbyBrand-williamsetal.[21].EndocarpextractofC. nucifera wasreactedwithDPPHradicalinethanolsolution.The0.5mLoftheextractwasmixedwith3mLofabsoluteethanoland0.3MDPPHradicalsolution of 0.5 mM solution in ethanol. When DPPH reactswith an antioxidant compound, which can donate hydrogen,it is reduced. The colour changewhenDPPH reactedwith theextractwas read at 517nmafter30minof reactionusingUVspectrophotometer.Themixtureofethanol(3.3mL)andsample(0.5mL)servedasblank.Thecontrolsolutionwaspreparedbymixingethanol(3.5mL)andDPPHradicalsolution(0.3mL).Thescavengingactivitypercentage(AA%)wasdeterminedaccordingtoMensoretal.[22].

DPPHradicalscavengingactivity%=(A A )blank sample

100Ablank

−×

Partial purification of the fractions using thin layer and column chromatography: Thebestsolventsystemfortheelutionofethylacetatefractiononcolumnchromatographywasdeterminedbyelutingthefractionswithdifferentsolventssystemsonthinlayerchromatography(TLC)plate.Todothis,solventwaspouredintodevelopingchamber to0.5cmdepth.Partof the insideof the

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chamberwaslinedwithfilterpaper,coveredwithlid,swirlgentlyandallowedtostandtillTLCplatewasprepared.TheTLCplatewascutoutintoaconvenientsizewithoutdisturbingthecoatingontheadsorbent.Alinewhichservedastheoriginwascarefullyat0.5cmmarkabovethebottomoftheplatewithpencil.Theethylacetatefraction(1mg)wasdissolvedin1mLofmethanolandtheresultingsolutionwascarefullyspottedonthepreparedTLC plate at the origin with micro-capillary tube. The spottedplatewasafterwardsdevelopedinTLCchamber.TheTLCplatewasallowedtodevelop inthechamberuntilsolventfrontwasabout0.5cmbelowthetopoftheTLCplate.TheTLCplatewasremoved;solventfrontmarkedcarefullywithpencilandallowedtodry.TheplatewasvisualizedunderUV-lightat254and366nmtoviewanyfluorescence.Thespotsobservedwerecircledwithpencil.ThesolventsystemdeterminedforethylacetatefractionelutionbaseontheRfvalueswasinorderofn-hexane,n-hexane-ethylacetate(1:1),n-hexane-ethylacetate(2:8),n-hexane-ethylacetate(1:9),ethylacetate,ethylacetate-methanol(9:1),ethylacetate-methanol(8:2)andethylacetate-methanol(7:3).

Thereafter, exactly 5 g of the ethyl acetate fraction wasdissolved in a minimal amount of methanol adsorbed on 25g of silica gel of 60-200mesh size and then allowed to dry. Itwasthenchromatographedonsilicagelcolumn(650x40mm)and gradiently eluted with pre-determined solvent system.Column chromatography of ethyl acetate fraction yieldedETHYL-A, ETHYL-B, ETHYL-C and ETHYL-D partially purifiedsamples.Theeluteswerecollectedintesttubes,analysedusingTLC to determine fractions with similar retention factors (Rf).Fractions with similar retention factors were bulked together,concentrated to dryness in vacuo and stored in a container inrefrigeratorforfurtheruse.Antimicrobialactivityofthepartiallypurifiedfractionswascarriedoutusingthemethodasdescribedpreviously.

Gas chromatography-mass spectrometry analysis of partially purified ethyl acetate fraction of C. nucifera endocarp extract: Gas chromatography-Mass spectrometry (GC-MS) analysis ofthe partially purified ethyl acetate fraction of C. nucifera wasperformed using Agilent technologies GC system comprisingonAOC-20i auto-sampler andaGas chromatograph interfacedto a triple axismass spectrometer detector equippedwith anElite-5MS (5% diphenyl/95% dimethyl poly siloxane) fused toa capillary column (30 x0.25µmdf). ForGC-MSdetection, anelectron ionization system was operated in electron impactmodewithionizationenergyof70eV.Heliumgas(99.999%)wasusedasacarriergasataconstantflowrateof1mL/min,andaninjectionvolumeof2µLwasemployed(asplitratioof10:1).

The injector temperature was maintained at 250°C, the ion-source temperature was 200°C, the oven temperature wasprogrammedfrom110°C(isothermalfor2min)withanincreaseof10°C/minto200°C,then5°C/minto280°C,endingwitha9minisothermal at 280°C.Mass spectrawere taken at70 eV; a scaninterval of 0.5 second. The solvent delaywas 0 to 6min, andthetotalGC/MSrunningtimewas34.667seconds.Therelativepercentage amount of each component was calculated bycomparingitsaveragepeaktothetotalareas.Themass-detector

used in thisanalysiswasAgilentTechnologies-5975Cwhilegaschromatography model was Agilent Technologies-7890A. Theinjector model used was Agilent Technologies-7683B, and thesoftware adopted to handlemass spectra and chromatogramswasaNISTversion14.0L.

ResultsTheextractcollectedwasdarkbrownincolourandtheyieldwas110.50g(5.53%).

Antibacterial activities of crude extract of c. nucifera endocarp against various bacterial strainsSensitivitypatternsexhibitedbythecrudeextractofC. nucifera againstbacterialstrainswasusedforthisstudyasshowninTable 1.Nineteenoutofthetwenty-fivebacterialstrainstestedweresusceptibletotheactivityoftheextractatafinalconcentrationof 35 mg/mL. The test bacteria strains that were resistant tothe extract included Enterococcus faecalis, Escherichia coli (CIU), Staphylococcus aureus (CIU), Pseudomonas aeruginosa (CIU),Salmonella typhi (LIO),Vibrio fluvialis (LIO).Thezonesofinhibition exhibited by the extract against the test organismsranged between 8.67 mm and 18.00 mm. The highest zoneof inhibition (18.00 mm) was exhibited against Micrococcus luteus(NCIB196)whilethelowestzoneofinhibition(8.67mm)was exhibited against P. fluorescens. On the other hand, allorganisms testedagainst the standardantibiotics-streptomycinandampicillinataconcentrationof1mg/mLweresusceptibletothesecompoundsexceptClostridium sporogenes,E. coli(CIU),Plesiomonas shigelloides(LIO),P. aeruginosa(CIU),P. Fluorescens (NCIB3756),S. aureus (CIU)andStreptococcus agalatiae (CIRv)that was resistant to streptomycin. Klebsiella pneumoniae (NCIB 418),M. luteus (NCIB 196),P. aeruginosa (NCIB 950),P. aeruginosa(CIU),S. aureus(CIU),V. fluvialisandV. furnissii (LIO)wereresistanttoampicillin.

Antimicrobial activities exhibited by fractions obtained from crude extract of C. nucifera on the test bacterial strainsThree fractions obtained from the crude extract ofC. nucifera endocarpand these are ethyl acetate, butanol and aqueousfractions.Chloroformandn-hexanedidnotshowaffinityforthebioactivecomponentsof theextract.Theantimicrobial activityof various fractions fromC. nucifera crude extract is shown inTable 2.

The aqueous, butanol and ethyl acetate fractions exhibitedantimicrobialactivitiesagainstvariousbacterialstrainsusedforthis study. The zonesof inhibitionexhibitedby these fractionsagainstthetestorganismsrangedbetween8.33mmand20.33mm.TheEthylacetatefractionshowedthehighestantimicrobialactivityamongthesefractions.Thisfractioninhibitedthegrowthofnineteenoutof thetwenty-fivetestorganisms.Thezoneofinhibition exhibited by ethyl acetate fraction against variousBacillus species rangedbetween10.66mmand17.66mm.Ontheotherhand,zonesofinhibitionobservedforstrainsofE. coli

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Table 1: ThesensitivitypatternsexhibitedbycrudeextractofC. nucifera andthestandardantibioticsagainsttheteststrains.

Zone of inhibition (mm)**

Bacterial Strains Crude extract (35 mg/mL) (Mean ± SD)

Streptomycin (1 mg/mL) (Mean ± SD)

Ampicillin (1 mg/mL) (Mean ± SD)

M/W01:01

Bacillus cereus (NCIB6349) 15.00±1.63 28.00±1.73 20.70±1.10 0Bacillus polymyxa (LIO) 12.00±0.00 17.33±1.73 0 0

Bacillus stearothermophilus(NCIB8222) 14.33±0.94 27.67±2.08 16.33±1.53 0Bacillus subtilis (NCIB3610) 13.33±1.54 28.33±1.53 16.00±3.61 0

Clostridium sporogenes (NCIB532) 10.66±0.94 0 14.00±2.51 0Corynebacterium pyogenes(LIO) 10.66±0.94 20.33±2.31 21.00±2.65 0Enterococcus faecalis (NCIB775) 0 20.67±2.52 17.33±2.52 0

Escherichia coli(NCIB86) 14.00±0.94 20.33±2.31 20.67±1.15 0Escherichia coli(CIU) 0 0 18.70±1.20 0Escherichia coli (CISt) 10.66±0.94 21.00±2.65 0 0

Klebsiella pneumoniae (NCIB418) 15.33±1.88 20.67±2.52 0 0Micrococcus luteus (NCIB196) 18.00±0.94 22.33±2.52 16.33±2.89 0Plesiomonas shigelloides (LIO) 15.00±1.41 0 0 0

Proteus mirabilis(CIU) 12.00±0.00 19.00±2.65 17.67±2.52 0Proteus vulgaris(CIU) 10.00±0.67 16.67±2.08 16.33±3.21 0

Pseudomonas aeruginosa (NCIB950) 10.00±0.00 18.00±2.00 0 0Pseudomonas aeruginosa (CIU) 0 0 0 0

Pseudomonas fluorescens (NCIB3756) 8.67±0.94 0 20.70±1.10 0Salmonella typhi (LIO) 0 20.33±2.31 19.00±2.65 0

Shigella dysentariae(LIO) 16.66±0.94 25.30±1.10 24.70±1.30 0Staphylococcus aureus(NCIB8588) 16.00±1.88 22.33±2.52 27.30±1.20 0

Staphylococcus aureus(CIU) 0 0 0 0Streptococcus agalaticae (CIRv) 10.00±1.88 0 24.70±1.30 0

Vibrio fluvialis (LIO) 0 17.30±1.20 0 0Vibrio furnissii (LIO) 10.00±1.88 26.70±2.30 0 0

Abbreviations: CIRv:ClinicalIsolatesfromRectovagina,CISt:ClinicalIsolatesfromStool,CIU:ClinicalIsolatesfromUrine,NCIB:NationalCollectionofIndustrialBacteria,LIO:LocallyIsolatedOrganism,mm**:Meanofthreereplicates,M/W:Methanol/Water,0:Notsensitive.

rangedbetween9.66and18.00mm.Plesiomonas shigelloides,Pseudomonas aeruginosa (CIU), Salmonella typhi, Shigella dysentariae and Vibrio furnissii were not susceptible to theethylacetatefraction.Ontheotherhand,butanolandaqueousfractionsshowedantibacterialactivitiesagainstsixandthreetestorganismsrespectively.

Susceptibility patterns exhibited by partially purified ethyl acetate fractions against the test bacterial strainsSusceptibilitypatternsexhibitedbypartiallypurifiedfractionofethylacetateagainst theorganismsare shown inTable 3. Thepartially purified Ethyl-D was not active against the bacterialstrainswhileEthylA,EthylBandEthylCshowedvaryingdegreesof activities. Ethyl C showed the highest antimicrobial activityamongthesepartiallypurifiedsamples.

Ethyl- A exhibited antibacterial activity against one out of thetwenty-fivetestbacterialstrainsusedforthisstudywhileEthyl-Bexhibitedactivityagainstfiveofthetwenty-fivebacterialstrainsandthezonesof inhibitionagainst thebacterial strains rangedbetween8.66mmand18.00mm.TheEthyl-Cexhibitedactivityagainstnineoutofthetwenty-fivetestbacterialstrainsandthe

zonesofinhibitionsrangedbetween9.67mmand17.33mm.ThesusceptiblebacterialstrainsareshowninTable 3.Otherpartiallypurifiedsample,thatis,Ethyl-Ddidnotshowactivityonanyofthetestorganisms.

The minimum inhibitory concentrations and the minimum bactericidal concentrations exhibited by crude extract of C. nucifera against susceptible bacterial strainsThe minimum inhibitory concentrations and the minimumbactericidalconcentrationsexhibitedbythecrudeextractofC. nuciferaagainstsusceptiblebacterialstrainsareshowninTable 4.TheMICsofthecrudeextractagainstthesusceptiblebacterialstrains rangedbetween0.27mg/mLand8.75mg/mLwhile thelowest MICs of 0.27 mg/mL were observed against B. cereus (NCIB6349)andE. coli (NCIB86).ThehighestMICof8.75mg/mLwasobservedagainstP. aeruginosa(NCIB950).TheMBCsofthecrudeextractagainstthesusceptiblebacterialstrainsrangedbetween 1.09mg/mL and 17.5mg/mLwhile the lowestMBCsof1.09mg/mLwereobservedagainstB. cereus (NCIB6349)andS.Aureus (NCIB 8588). The highestMBC of 17.50mg/mLwasobservedagainstP. aeruginosa(NCIB950).

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The minimum inhibitory and the minimum bactericidal concentrations exhibited by ethyl acetate fractions against the susceptible bacterial strainsThe ethyl acetate fraction exhibited appreciable antimicrobialactivitywhencomparedtothoseshownbyaqueousandbutanolfractions.Thusethylacetatefractionwaschosenforfurthertests.VariousMICandMBCswereexhibitedbyethylacetatefractionagainst thesusceptible testorganisms (Table 5).TheMICsandMBCs were exhibited by this fraction against the organismsrangedbetween0.31mg/mLand2.50mg/mL.The lowestMICof0.31mg/mLwasobservedagainstE. coli(CISt)andthehighestMICs of 2.50 mg/mL were observed against B. cereus (NCIB6349),B. polymyxa(LIO)andMicrococcus luteus(NCIB196).TheMinimum bactericidal concentrations (MBC) exhibited by thisfractionrangedbetween0.63mg/mLand5.00mg/mL.

Phytochemical screening of the plant extractPhytochemicalscreeningoftheextractshowedthepresenceofsaponins,tannins,flavonoids,alkaloids,triterpenesandphenolsasshowninTable 6.

The extent and rate of killing of S. aureus exhibited by ethyl acetate fractionTheextentandrateofkillingofS. aureus byethylacetatefractionat1xMIC,2xMICand3xMICconcentrationsindicatedinFigure 1.Itspercentageoftheorganismkilledbythefractionat1xMICconcentrationin15minwas30.1%whilethepercentageofcellkilledat30minroseto50%.After60minofcontacttimewiththisfraction, thepercentageof theorganismskilledwas57.9%.Asthecontacttimeincreasedto90min,thepercentageofthecellskilledwas70.6%andthisroseto86.8%after120minofcontacttime.Whentheconcentrationoftheethylacetatefractionwasdoubled,thepercentageoforganismskilledwas41.7%at15minofthecontacttime.At30minofcontacttime,thepercentageofthecellskilledincreasedto73.3%;andwithanincreaseinthecontacttimeto60min,thepercentageoforganismskilledwas85%.

Eachpoint represents themean log10 survivalofbacterial cellsat a particular time interval in the presence of the fraction.After90minofthecontacttimeinterval,thepercentageoftheorganismskilledhas increasedto90.2%,while itroseto95.5%after120minofcontacttime.Theextentandrateofkillofthe


Bacterial Strains Aqueous(10 mg/mL) (Mean ± SD)

Butanol(10 mg/mL) (Mean ± SD)

Ethyl acetate(10 mg/mL) (Mean ± SD)

M/W01:01

Bacillus cereus (NCIB6349) 0 13.33±0.94 17.66±1.89 0Bacillus polymyxa (LIO) 0 14.00±0.94 15.00±2.37 0

Bacillus stearothermophlus (NCI822) 0 20.33±1.41 10.66±0.94 0Bacillus subtilis (NCIB3610) 0 0 13.00±2.00 0

Clostridium sporogenes (NCIB532) 0 0 0 0Corynebacterium pyogenes (LIO) 0 0 15.00±2.35 0Enterococcus faecalis (NCIB775) 0 0 14.33±3.77 0

Escherichia coli (NCIB86) 14.00±0.94 10 18 0Escherichia coli (CIU) 0 0 15.00±0.47 0Escherichia coli (CISt) 0 0 9.66±0.47 0

Klebsiella pneumoniae (NCIB418) 8.33±0.47 18.00±0.94 10.67±0.58 0Micrococcus luteus (NCIB196) 0 0 14.67±1.53 0Plesiomonas shigelloides(LIO) 0 0 0 0

Proteus mirabilis(CIU) 0 0 11.00±2.00 0Proteus vulgaris(CIU) 0 0 17.33±2.82 0

Pseudomonas aeruginosa(NCIB950) 0 0 13.67±1.15 0Pseudomonas aeruginosa(CIU) 0 0 0 0

Pseudomonas fluorescens(NCIB356) 0 0 12.66±3.77 0Salmonella typhi(LIO) 0 0 0 0

Shigella dysentariae(LIO) 0 0 0 0Staphylococcus aureus(NCIB8588) 13.66±1.41 10.66±0.94 14.67±1.53 0

Staphylococcus aureus(CIU) 0 0 17.33±2.08 0Streptococcus agalaticae(CIRv) 0 0 14.67±1.53 0

Vibrio fluvialis(LIO) 0 0 9.67±1.15 0Vibrio furnissii(LIO) 0 0 0 0

Table 2: ThesensitivitypatternsexhibitedbyfractionscollectedfromC. nuciferacrudeextractonthetestbacterialstrains.

Abbreviations: CIRv:bClinicalIsolatesfromRectovagina,CISt:ClinicalIsolatesfromStool,CIU:ClinicalIsolatesfromUrine,NCIB:NationalCollectionofIndustrialBacteria,LIO:LocallyIsolatedOrganism,mm**: Meanofthreereplicates,M/W:Methanol/Water,0:Notsensitive.

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Bacterial Strains Aqueous (10 mg/mL) (Mean ± SD)

Butanol (10 mg/mL) (Mean ± SD)

Ethyl acetate (10 mg/mL) (Mean ± SD)

M/W 01:01

Bacillus cereus (NCIB6349) 0 13.33±0.94 17.66±1.89 0Bacillus polymyxa (LIO) 0 14.00±0.94 15.00±2.37 0Bacillus stearothermophlus (NCI822) 0 20.33±1.41 10.66±0.94 0Bacillus subtilis (NCIB3610) 0 0 13.00±2.00 0Clostridium sporogenes (NCIB532) 0 0 0 0Corynebacterium pyogenes (LIO) 0 0 15.00±2.35 0Enterococcus faecalis (NCIB775) 0 0 14.33±3.77 0Escherichia coli (NCIB86) 14.00±0.94 10 18 0Escherichia coli (CIU) 0 0 15.00±0.47 0Escherichia coli (CISt) 0 0 9.66±0.47 0Klebsiella pneumoniae (NCIB418) 8.33±0.47 18.00±0.94 10.67±0.58 0Micrococcus luteus (NCIB196) 0 0 14.67±1.53 0Plesiomonas shigelloides(LIO) 0 0 0 0Proteus mirabilis(CIU) 0 0 11.00±2.00 0Proteus vulgaris(CIU) 0 0 17.33±2.82 0Pseudomonas aeruginosa(NCIB950) 0 0 13.67±1.15 0Pseudomonas aeruginosa(CIU) 0 0 0 0Pseudomonas fluorescens(NCIB356) 0 0 12.66±3.77 0Salmonella typhi(LIO) 0 0 0 0Shigella dysentariae(LIO) 0 0 0 0Staphylococcus aureus(NCIB8588) 13.66±1.41 10.66±0.94 14.67±1.53 0Staphylococcus aureus(CIU) 0 0 17.33±2.08 0Streptococcus agalaticae(CIRv) 0 0 14.67±1.53 0Vibrio fluvialis(LIO) 0 0 9.67±1.15 0Vibrio furnissii(LIO) 0 0 0 0

Table 3: AntimicrobialactivitiesofpartiallypurifiedfractionsofC. nucifera extractagainstbacterialstrains.

Abbreviations: CIRv:ClinicalIsolatesfromRectovagina,CISt:ClinicalIsolatesfromStool,CIU:ClinicalIsolatesfromUrine,NCIB:NationalCollectionofIndustrialBacteria,LIO:LocallyIsolatedOrganism,mm**: Meanofthreereplicates,M/W:Methanol/Water,0:Notsensitive.

Bacterial StrainsCrude extract

MIC (mg/mL) MBC (mg/mL)Bacillus cereus(NCIB6349) 0.27 1.09

Bacillus polymyxa(LIO) 2.19 8.75Bacillus sterothermophilus(NCIB822) 2.19 8.75

Clostridium sporogenes(NCIB532) 4.38 8.75Corynebacterium pyogenes(LIO) 4.38 8.75

Escherichia coli(NCIB86) 0.27 4.38Escherichia coli(CISt) 0.54 2.19

Klebsiella pneumoniae(NCIB418) 1.09 2.19Micrococcus luteus(NCIB196) 4.38 8.75Plesiomonas shigelloides(LIO) 2.19 8.75

Proteus mirabilis(LIO) 2.19 4.38Proteus vulgaris(LIO) 2.19 4.38

Pseudomonas aeruginosa(NCIB950) 8.75 17.5Pseudomonas fluorescens(NCIB3756 2.19 8.75

Shigella dysentariae(LIO) 1.09 2.19Staphylococcus aureus(NCIB8588) 0.55 1.09

Streptococcus agalaticae(CIRV) 4.38 8.75Vibrio furnissii(LIO) 4.38 8.75

Table 4: Theminimuminhibitoryconcentrationsandminimumbactericidalconcentrationsofthecrudeextractexhibitedagainstthetestbacterialstrain.

Abbreviations: CIRv:ClinicalIsolatesfromRectovagina,CISt:ClinicalIsolatesfromStool,CIU:ClinicalIsolatesfromUrine,NCIB:NationalCollectionofIndustrialBacteria,LIO:LocallyIsolatedOrganism.

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fractionat3xMICconcentrationfollowedthesametrendwiththeconcentrationsaforementioned.Astheconcentrationsofthefraction increasedwith increase intime, thepercentageof theorganismskilledalsoincreased.

The extent and rate of killing of E. coli exhibited by the ethyl acetate fractionTheextentandrateofkillingofE. coli byethylacetatefractionat1xMIC,2xMICand3xMICconcentrationindicatedinFigure 2. The percentage of the organisms killed by the fraction at 1xMICconcentrationin15minwas5.1%,thepercentageofthecellskilledat30minroseto24.1%.After60minofcontacttimewiththisfraction,thepercentageofthecellskilledwas47.6%.Whenthecontacttimewasincreasedto90min,thepercentageofthecellskilledwas65.1%andthisroseto82.5%after120minof contact time. When the concentration of the fraction wasdoubled,thepercentageoforganismskilledbythefractionwithin15minwas 15.1%.At 30minof contact time, thepercentageof cells killed increased to49.1%;andwith the increase in thecontacttimeto60min,thepercentageoforganismkilledgotto67.9%.After90minofthecontacttimeinterval,thepercentageof theorganismskilledhas increased to89.6%while it rose to94.8%after120minofcontacttime.Theextentandrateofkillingbythefractionat3xMICconcentrationfollowedthesametrendasinprevioustest.

Eachpointrepresentsthemeanlog10survivalofbacterialcellsataparticulartimeintervalinthepresenceofthefraction.

The effect of ethyl acetate fraction on potassium leakage from S. aureus Cells The potassium ion leakages from S. aureus cells due to theeffect of ethyl acetate fraction at 1 x MIC, 2 x MIC, 3 x MICconcentrationsindicatedinFigure 3,whereamountofpotassiumionleakedfromS. aureus cellsat1xMICin15minwas0.91µg/mL,whiletheamountleakedfromthecellsroseto0.98µg/mLafter30minofcontacttime.At60minofcontacttimeintervalwiththisfraction,theamountofpotassiumleakedwas1.09µg/mL.Whenthecontacttimewasincreasedto90min,thequantityofpotassiumionleakedfromthecellswas1.14µg/mLandthisincreasedto1.28µg/mLafter120minofcontacttime.

When the cells were exposed to the fraction at 2 x MICconcentration,theamountofpotassiumionsleakedoutofthecellsat15minwas0.99µg/mL.At30minofcontacttime,theamount of potassium ion leaked from the cells increased to1.01µg/mL,with increased in the contacttime to60min, thepotassiumionleakedroseto1.12µg/mL.At90mincontacttime,the quantity of potassium ion leaked increased to 1.22 µg/mLandthisincreasedto1.38µg/mLafter120minofcontacttime.Theeffectofthisfractionat3xMICconcentrationfollowedthesame trend as exhibited earlier on. The amount of potassiumionleakedfromthecellsat15minofcontacttimewas1.00µg/mL and this rose to 1.22 µg/mL after 30min of contact time.Whenthecontacttimewasincreasedto60min,thequantityofpotassiumionleakedincreasedto1.36µg/mL.At90mincontacttime,thequantityleakedoutofthecellsroseupto1.41µg/mLand1.50µg/mLafter120minofcontacttime.

Eachpointrepresentstheamountofpotassiumionleaked(µg/mL)fromthecellsataparticulartimeintervalinthepresenceofthefraction.

The effect of ethyl acetate fraction on potassium leakage from E. coli cells ThepotassiumionleakedfromE. coli cellsduetoeffectofethylacetate fraction at 1 x MIC, 2 x MIC, 3 x MIC concentrationsindicated inFigure 4.At1xMICconcentrations in15min, thequantity of potassium ion leaked was 0.72 µg/mL, while thisamountroseupto0.73µg/mLat30mininterval.Andwiththeincreaseinthecontacttimeto60min,thepotassiumionleakedwas0.76µg/mL.Therewasanincreaseinpotassiumionleakedfromthecellat60mincontacttimeto0.82.Lastly,at120minofcontacttime,thequantityofpotassiumionleakedroseto0.89µg/mL

Bacterial StrainsEthylacetate fraction

MIC (mg/mL) MBC (mg/mL)Bacillus cereus(NCIB6349) 2.50 5.00

Bacillus polymyxa(LIO) 2.50 5.00Bacillus stearothermophilus 1.25 5.00Bacillus subtilis(NCIB3610) 1.25 5.00

Corynebacterium pyogenes(LIO) 0.63 1.25Enterococcus faecalis(NCIB775) 0.63 1.25

Escherichia coli(NCIB86) 1.25 2.50Escherichia coli(CIU) 1.25 2.50Escherichia coli(CISt) 0.31 0.63

Klebsiella pneumoniae(NCIB418) 1.25 2.50Micrococcus luteus(NCIB196) 2.50 5.00Plesiomonas shigelloides(LIO) 2.50 5.00

Proteus mirabilis(CIU) 2.50 5.00Proteus vulgaris(CIU) 2.50 5.00

Pseudomonas fluorescens (NCIB3756) 1.25 2.50

Staphylococcus aureus(NCIB8588) 0.63 1.25Staphylococcus aureus(CIU) 0.63 1.25

Streptococcus agalaticae(CIRv) 1.25 2.50Vibrio fluvialis(LIO) 2.50 5.00

Abbreviations: CIRv: Clinical Isolates from Rectovagina, CISt: ClinicalIsolates from Stool, CIU: Clinical Isolates from Urine, NCIB: NationalCollectionofIndustrialBacteria,LIO:LocallyIsolatedOrganism.

Table 5: The minimum inhibitory concentrations and minimumbactericidalconcentrationsoftheethylacetatefractionagainstthetestbacterialstrains.

Chemical Test ResultAlkaloids Positive

Cardiacglycosides NegativeFlavonoids PositivePhenols Positive

Triterpenes PositiveSteroids NegativeSaponins PositiveTannins Positive

Table 6: Phytochemicalcompoundsrevealedfrom Cocos nuciferaextract.

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contacttime.Whenthecontacttimewas increasedto90min,the quantity of potassium ion leaked increased to 1.20 µg/mLand this got to1,23µg/mLafter120minof contacttime.Theeffectofthisfractionat3xMICconcentrationfollowedthesametrendaspreviouslydescribedforotherconcentrations.

Eachpointrepresentstheamountofpotassiumionleaked(µg/mL)fromthecellsataparticulartimeintervalinthepresenceofthefractions.

Theextentand rateofkillingofS.aureus cellsbyethylacetatefractionat1xMIC( ),2xMIC( ),3xMIC ( )andcontrol( ).

Figure 1

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When the cells were exposed to the fraction at 2 x MICconcentration, the amountof potassium ion leakedoutof thecellsat15mincontacttimewas0.98µg/mL.At30minofcontacttime, the amount leaked out from the cells increased to 1.03µg/mL.Thequantityleakedincreaseto1.13µg/mLat60minof

The effect of ethyl acetate fraction on potassium ionleakagefromS. aureuscellsatconcentration1xMIC( ),2xMIC( ),3xMIC( )andcontrol( ).

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The effect of ethyl acetate fraction on nucleotide leakage from S. aureus cellsThenucleotideleakagefromS. aureuscellsduetotheeffectofethylacetatefractionat1xMIC,2xMIC,3xMICconcentrationsindicatedinFigure 5.Theamountofnucleotideleakedfromthecellsat1xMICconcentrationin15minwas1.33µg/mL,whilethisamountroseupto1.40µg/mLafter30minofcontacttime.At60minofcontacttimeintervalwiththisfraction,theamountofnucleotideleakedoutwas1.47µg/mL.Whenthecontacttimeincreasedto90min,thequantityleakedoutofthecellswas1.48µg/mLandthisincreasedto1.53µg/mLafter120minofcontacttime.When the cellswere exposed to the fraction at 2 xMICconcentration,theamountofnucleotidethatleakedoutofthecellsat15minwas1.40µg/mL.At30minofcontacttime,theamountofnucleotideleakedfromthecellsincreasedto1.42µg/mL,andat60mininterval,thenucleotideleakedwas1.49µg/mL.Whenthecontacttimewasincreasedto90min,thequantityofnucleotideleakedincreasedto1.58µg/mLandthisincreasedto1.60µg/mLafter120minofcontacttime.Theeffectofthisfraction at 3 xMIC concentration followed the same trend asexhibited earlier onwithother concentrations. The amount ofnucleotideleakedfromthecellsat15minofcontacttimewas1.47µg/mLandthisroseto1.55µg/mLafter30minofcontacttime. When the contact time was increased to 60 min, thequantity of nucleotide leaked increased to 1.74 µg/mL, while itroseto1.93µg/mLat90min.Finally,thequantityofnucleotidesleaked out of the test cells at 120min contact timewas 2.00 µg/mL.

Eachpointrepresentstheamountofnucleotideleaked(µg/mL)fromthecellsataparticulartimeintervalinthepresenceofthefraction.

The effect of ethyl acetate fraction on nucleotide leakage from E. coli cells Thenucleotide leakage fromE. coli cellsdue toeffectof ethylacetate fraction at 1 x MIC, 2 x MIC, 3 x MIC concentrationsindicatedinFigure 6.TheamountofnucleotideleakedfromE. coli cells at 1 xMIC concentration in 15minwas 0.37 µg/mL,whiletheamountleakedfromthecellsat30minroseupto0.42µg/mL.At60mincontacttime,thenucleotideleakedoutofthecellswas0.69µg/mL.Whenthecontacttimewaslaterincreasedto90min, theamount leakedout fromthecellswas0.81µg/mL.Lastly,at120minofcontacttime,thequantityofnucleotideleakedroseto0.88µg/mL.

When the cells were exposed to the fraction at 2 x MICconcentration,theamountofnucleotideleakedoutofthecellsat 15min contact timewas 0.98 µg/mL. At 30min of contacttime, the amount rose up to 1.03 µg/mL. The quantity leakedout of the cells increased to 1.13 µg/mL at 60min of contacttime.When the contact time reached 90min, the quantity ofnucleotideincreasedto1.20µg/mLandthisgotupto1.23µg/mLafter120minofcontacttime.Theeffectof this fractionat3 xMIC concentration followed the same trend as previouslydescribedforotherconcentration.

The effect of ethyl acetate fraction on protein leakage from S. aureus cellsTheproteinleakagefromS. aureuscellsduetotheeffectofethylacetatefractionat1xMIC,2xMIC,3xMICconcentrationsareshowninFigure 7.TheamountofproteinleakedfromS. aureus cellsat1xMICconcentrationsin15minwas4.62µg/mL,whilethe amount leaked from the cells rose to 6.04µg/mL after 30minofcontacttime.At60minofcontacttimeintervalwiththisfraction,theamountofproteinleakedwas10.50µg/mL.When

TheeffectofethylacetatefractiononnucleotideleakagefromS. aureuscellsatconcentration1xMIC( ),2xMIC( ),3xMIC( )andcontrol( ).

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the contact time increased to 90min, the quantity of proteinleaked from the cells was 11.42 µg/mL and this increased to12.86µg/mLafter120minofcontacttime.

When the cells were exposed to the fraction at 2 x MICconcentration,theamountofproteinthatleakedoutofthecellsat15minwas9.04µg/mL.At30minofcontacttime,theamountleakedfromthecellsincreasedto9.28µg/mL,withincreasedinthecontacttimeto60min,theproteinleakedwas11.62µg/mL.When the contact timewas increased to 90min, the quantityofproteinleakedincreasedto12.85µg/mLandthisroseupto15.58 µg/mL after 120min of contact time. The effect of thisfraction at 3 xMIC concentration followed the same trend asexhibited earlier on with other concentration. The amount ofproteinleakedfromthecellsat15minofcontacttimewas11.72µg/mLandthisroseto16.79.

The effect of ethyl acetate fraction on protein leakage from E. coli cellsTheproteinleakagefromE. coli cellsduetoeffectofethylacetatefractionat1xMIC,2xMIC,3xMICconcentrationindicatedinFigure 8.Theamountofprotein leakedfromE. colicellsat1xMIC in15minwas4.28µg/mL,while theamount leaked fromthecellsat30minroseupto4.41µg/mL.Whenthecontacttimewasincreasedto60min,theproteinoutofthecellswas5.71µg/mL.Whenthecontacttimewas later increasedto90min, theamountleakedfromthecellswas6.90µg/mL.Lastly,at120minofcontacttime,thequantityofproteinleakedroseto8.60µg/mL.

When the cells were exposed to the fraction at 2 x MIC

concentration,theamountofproteinleakedoutofthecellsat15mincontacttimewas5.11µg/mL.At30minofcontacttime,thequantityofprotein leaked fromthecells increasedto6.78µg/mL.Thequantityleakedincreaseto7.44µg/mLat60minofcontacttime.Whenthecontacttimewas increasedto90min,around 8.37µg/mLof proteinwas leakedout of the cells andthisgotto11.86µg/mLafter120minofcontacttime.Theeffectofthisfractionat3xMICfollowedthesametrendaspreviouslydescribedforotherconcentrations.

The antioxidant activity of C. nucifera endocarp extractThe in vitroantioxidantassayofC. nucifera revealsappreciableantioxidantpotentialscomparedtoascorbicacidasthestandard.The results of the antioxidant activity of C. nucifera indicatedin Figure 9. Ascorbic acid used as the standard exhibited 50%inhibition (IC50) at a concentration of 0.020 mg/mL. HighestpercentageinhibitionexhibitedbyC. nuciferaendocarpextractwas 79.80% at concentration of 0.015 mg/mL. The IC50 of C. nuciferaendocarpextractwasfoundtobe0.011mg/mLwhichislessthanthatofthestandardascorbicacidby50%.

The GC-MS analysis of partially purified ethyl acetate fraction of C. nucifera extractThe Gas Chromatogram-Mass Spectrometry chromatogramanalysis of the partially purified ethyl acetate extract of C.

7: TheeffectofethylacetatefractiononproteinleakagefromS. aureus cellsatconcentration1xMIC ( ),2xMIC( ),3xMIC( )andcontrol( ).Note: Each point represents the amount of proteinleaked(µg/mL)fromthecellsataparticulartimeintervalinthepresenceofthefraction.

Figure 7

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nuciferaendocarprevealedseventeenpeakswhichindicatedthepresenceofseventeenconstituents(Figure 10).Oncomparisonof the constituents mass spectra with National InstituteStandardandTechnology(NIST)14.0library,2-methoxy-phenol,2-Methoxy-4-vinylphenol, 2,6-dimethoxy-phenol, Ethyl-vanillin,3-hydroxy-4-methoxy-benzadehyde, 2,4-di-tert-butylphenol,2-Propenoic acid, 3-(4-hydroxy-3-methoxyphenyl), Coniferylaldehyde, 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol,1-(2,5-Dimethoxyphenyl)-propanol, Hexadecanoic acid,1-octadecene, Methyl 9-cis, 11-trans-octadecadienoate,8-Octadecanoicacid,1-Octadecene,5-Eicosene,1-Tetracosanolwere identified to be present in the partially purified ethylacetatefractionofC. nuciferaendocarpextract.Oftheseventeencompounds,themostprevailingcompoundswereEthyl-vanillinwithretentiontimeof8.996,1-Octadecenewithretentiontime15.916andn-Tetracosanolwithretentiontime19.525.

DiscussionThe crude extract at a concentration of 35 mg/mL inhibitedthe growth of nineteen out of twenty-five test strains (Table 1). On the other hand, the standard antibiotics; streptomycinandampicillinusedaspositive controls inhibitedeighteenandsixteenout of twenty-fivebacterial strains tested respectively.Theethylacetatefractionwasthemostactivefractionexhibitingappreciable level of antibacterial activity against the testbacterial strains at a concentrationof 10mg/mL. This fractioninhibitednineteenbacterial strainswhilebutanolandaqueousfractionsinhibitedsixandthreebacteriaoutofthetwenty-fivetestorganismsrespectivelyatthesameconcentrationof10mg/mL(Table 2).ThisisanindicationthatethylacetatewouldbeabettersolventforextractingbioactivecomponentsofC. nucifera

powderedendocarp.Antibacterialactivitiesofpartiallypurifiedsamples obtained from ethyl acetate viz., Ethyl A, B, C and Dalso exhibited varying activities against the bacterial strainstestedthefindingswhichwereinsupportoftheworkconductedbyAdleret al. [23] inwhichC. nucifera extractwasapointertowardsdevelopmentofantimicrobialdrugofnaturalorigintotreat infections caused by some pathogens and in this case S. agalaticaeknowntocauseneonatalsepsisandProteus mirabilis,thecausativeagentofurinary tract infectionswhichwerealsosusceptibletotheextract.

Theminimuminhibitoryconcentration(MICs)andtheminimumbactericidalconcentration(MBCs)exhibitedbythecrudeextractandethylacetatefractionwerealsoinvestigated.ThelowestMICexhibitedbythecrudeextractagainstthetestbacterialstrainswas 0.27mg/mL and the lowestMBCwas 1.09mg/mL (Table 4).Ontheotherhand,thelowestMICexhibitedbythefractionwas0.31mg/mLwhilethelowestMBCwas0.63mg/mL(Table 5).AccordingtoShanmughapriyaetal., theMIC indexofplantextract that is equal or less than 2mg/mL is considered to bebactericidalwhilethosewiththeMICsabove2mg/mLbutlessthan16mg/mL,suchextractcouldbetakenasbacteriostatic[24].AchintoandMunirudinconcludedintheirstudythatalowMICvalueofmedicinalplantextract indicatesabetterantibacterialactivity[25].ThisobservationshowsthatextractsobtainedfromC. nucifera showed appreciable antibacterial activities havingexhibitedlowMICsagainstpathogensusedforthisstudy.Thusthisextractcouldserveasagoodsourceofpotentantimicrobialcompoundsorcouldbeusedsynergisticallywithothersyntheticantibiotics to combat gradual development of resistant to theexistingantibioticsbysomepathogens.

Themedicinal valuesof plant lie in phytochemical compounds

AntioxidantactivitiesofC. nucifera.Figure 9

y = 2179.2x + 23.493R² = 0.8609

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which produced definite physiological actions on the humanbody[26].Thiscorroboratedourfindingsasthephytochemicalsrevealed in the C. nucifera extract were alkaloids, flavonoids,phenols, triterpenes, saponins and tannins (Table 6). Phenoliccompounds possess medicinal properties such as antiageing,anticarcinogenic, anti-inflammatory, anti-therosclerosis,cardiovascular protection and improvement of endothelialfunctionaswell as cellproliferationactivities [27]. Inaddition,naturalantioxidantsmainlycomefromplantinformofphenoliccompounds[28].

Scalbert, reported that Phenols present in C. nucifera extractcouldpromotehumanhealthand reduce theeffectsofageingand that flavonoids, one of the phytochemicals present in theextractofC. nucifera havetheabilitytoscavengehydroxylandsuperoxide anion radicals which are important for diseasesassociated with oxidative damage of membranes, proteinsand DNA [29]. Flavonoids may reduce risk of cancers, as wellas preventing menopausal symptoms. Thus C. nucifera couldserveasamajorsourceofnaturalflavonoidsthatcouldbeusedto scavenge free radicals produced in human system. In ourfindings,existenceofthisphytochemicalsupportstheusefulnessofthisplantinfolkloremedicineforpreventingageinginhuman.Saponins found inawiderangeofplantsexisted in C. nucifera extract.Saponinsareresponsible forthemostof theobservedbiological effects in medicinal plants [30]. Interactions ofsaponinswithcellshowlysingofthemembrane[31].EvenlowconcentrationsofsomesaponinshavebeenreportedbyEidetal.toapparentlyenhancetheuptakeofpolarsecondarymetabolites,thusincreasingtheiractivityinanapparentlysynergisticfashion[32]. Saponins, along with other polar secondary metabolitesinC. nuciferaextractcouldhavecontributedtoitsantibacterialactivityofthisplant.Ukohaetal.reportedtanninstobeusefulasananti-inflammatoryagentandtreatmentofburnsandwoundsandalso tannin-rich remedies tobecytotoxicandantiparasitic[33].TanninsarenaturallyoccurringplantpolyphenolsandarefoundpresentinC. nuciferaextract.Theyareabletoformstableprotein–tannincomplexesandthusinteractwithwidevarietyofproteintargetsinmicrobesanditspresencein C. nuciferaextractconfirmedthepotencyoftheextractagainsttestbacterialstrainsusedforthisstudy[31].

Alkaloids areanotherphytochemical compoundpresent in theextractofC. nucifera.Alkaloidsexhibitawiderangeofbiologicalactivities which includes antibacterial, anti-asthmatic, anti-inflammatoryandanti-anaphylactic[34,35].Alltheseconfirmthetherapeuticpropertiesofthisextractandthereforecanbeusedforthetreatmentofseveralailmentscausedbymicroorganisms.

Triterpenoidswas also part of the phytochemical constituentsobservedtobepresentinC. nuciferaextract.Thiscompoundhasbeenreportedtobecytotoxicagainsthuman larynxcarcinomaandbreastcancer[36].Thebiologicalactivitiesofthiscompoundindicate that C. nucifera can be utilized to produce drugs fortreatment of cancer and infections associated with varioushumanpathogens.Theantimicrobialactivityofterpenoidswhicharelipophiliccompoundshasbeenassociatedwithbacterialcellmembranedisruption [37]. Thismighthave contributed to the

modeofactionsofC. nucifera extractwhichledtothedeathoftheseorganisms.

-vitrotimekillassayareexpressedastherateofkillingbyafixedconcentrationofanantimicrobialagentandareoneofthemostreliable methods for determining tolerance of microrganismsto drugs [38]. The killing rates exhibited by ethyl acetatefraction against the test organisms increased with increase inconcentrationsandtimeintervals(Figures1and2).Forinstance,thepercentage reduction in cell viability ofS.aureus after 15,30, 60, 90 and120 min contact time with this fraction at 1 xMIC concentration were 30%,50%, 57.9%, 70.6% and 86.8%respectively.ThepercentagereductionincellviabilityofE. coli atthesametimeandthesameconcentrationwere5.1%,24.1%,47.6%, 65.1% and 82.5%. Potent bacterial killing was evidentas extract concentration gets higher and time increases. ThisobservationwassimilartotheantibacterialeffectofHemidesmus indicusreportedbySarithaetal.[38].TheabilityofthisfractiontocompletelyinhibitthesepathogensatminimalcontacttimeandatlowconcentrationsisanindicationthatbioactivecompoundsinC. nucifera couldbeusedforthedevelopmentofantimicrobialcompounds for treatment of infections caused by pathogens.Such antimicrobials could be useful in combating infectionscausedbymultidrugresistantmicroorganisms.

Inadditiontokillingrateexhibitedbyethylacetatefraction,othermodes of action including leakage of potassium ions, proteinsandnucleotideswerestudied.Thefractionexhibitedappreciableleakageofprotein,potassiumionsandnucleotides(Figures3-8)which is an indication of bacterial cell membrane disruptionby the antimicrobial compounds present in this extract. Theethylacetatefractionexhibitedappreciablepotentialstocausepotassium ion efflux from the test bacterial strains (Figures 3and 4). The concentrations of potassium ions leaked from thetest bacterial strains increased with increase in concentrationof the fractionsaswell as increase in contacttimeof thecellswith the fractions.Thecytoplasmicmembranedamagecausedby antimicrobial agents could have caused the cations to befreelytransportedoutofthecellandthusledtothedeathofthecell.Potassiumionisinvolvedinthemaintenanceofaconstantinternal pH andmembranepotential [39], therefore the effluxofpotassiumoutofthemembranewillhavedetrimentaleffecton the cell functions and lead to the cell death. The resultsobtainedfromthisstudyshowedthatC. nuciferaextractexerteditscidaleffectsonthetestcellsthroughdisruptionoftheircellmembranes.AccordingtoZasloff,antibacterialagentscouldactbydisruptingthecytoplasmicmembraneofbacteriaandassuchcausedestabilizationandpermeabilization[40].Thisobservationsupport themodeofactionexhibitedbyethylacetate fractionobtained from C. nucifera crude extract. Spencer reportedthat many antimicrobial compounds that act on the bacterialcytoplasmic membrane induce the loss of 260 nm absorbingmaterialandthesewereobserved intheethylacetatefractionusedagainst the testorganisms [41].Theresultobtained fromthis assay showed that the active fraction was able to causeleakageofnucleotidesfromthecellsandthuslossofnucleotidethroughadamagedcytoplasmicmembrane(Figures 3 and 4).

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Theproteinleakagefromthetestmicroorganismsasobservedinthisstudywasproportionaltotheconcentrationofthefractionand the time of exposure of the cells to the solution of thisfraction.Fromall indications,ethylacetatefractionactsonthetestcellsandcausedtheirdeaththroughcytoplasmicdisruptionasaresultofincreaseinconcentrationandcontacttime.ThisisanindicationofmonophasiceffectasreportedbyAkinpeluetal.[42].

ThestabilityofDPPH free radicalmethod isa sensitivewayofdetermining the antioxidant activity of plant extract [43].OurfindingsshowedthattheextractofC. nuciferaendocarpexhibitedantioxidantactivitywhichwasfoundtobecomparablewiththeascorbicacidthatwasusedasstandard(Figure9).Cocosnucifera extract significantly inhibited hydroxyl radicals produced byDPPHinthisstudyandcouldserveasafreeradicalinhibitororscavengerusingitsprotondonatingability[44].ThisstudyfurthersupportstheusefulnessofthisC. nuciferainscavenginghydroxylradicals(OH-)formedinthebiologicalsystemsofhumanswhichhave been recognized as extremely damaging [45]. This plant

canserveasapointertowardsdevelopmentofantioxidantdrugof natural origin. Suchdrug could go a longway in healthcaredelivery.

In GC-MS analysis (Figure 10),major constituents of themostactive partially purified fraction of the endocarp extract of C. nucifera was found to be Ethyl- vanillin, 1- Heniecosanol and1- Tetracosanol, constituents of which greatly contributed tokillingeffectsexhibitedbyC. nuciferaextracts thereforeusefulin production of potent antimicrobial compound with broadspectrumactivity.

Author ContributionsAll contributing authors have agreed to the submission of thismanuscript forpublication.DAA,MTAconceivedanddesignedthe study, performed the experiments, interpreted the resultsJOAhelpedtoformulatethehypothesis indesigningofprojectandwrotethepaper.OOOandTJAmonitoredendocarpextractproduction experiment, analyzed the data, interpreted theresultsandeditedthemanuscripts.

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