194

Vol. 31, 2013, No. 2: 194202 Czech J. Food Sci.

Biological Activities of Essential Oils and Methanol Extracts
of Five Ocinun Species against Pathogenic Bacteria
Sou+r:SAHA
1,2
,Tnvni^n1nDHAR
2
,Cnn:on:SE^GUPTA
2
andPnv1nnornGHOSH
1
1
Departnent of Botany, Cytogenetics and Plant Biotechnology Research Unit, University
of Kalyani, ^adia, West Bengal, India,
2
Departnent of Botany, Microbiology Research Unit,
University of Kalyani, ^adia, West Bengal, India
Abstract
SnS.,DnvT.N.,Svouv:C.,GnosnP.D.(2013):Biological activities of essential oils and methanol
extracts of five Ocinun species against pathogenic bacteria.CzechJ.FoodSci.,31:194202.
TeessentialoilsandmethanolextractsofOcinunbasilicunL.,OcinunkilinandscharicunGuerke,Ocinungratis-
sinunL,OcinuncanunSims,andOcinuntenuiorunL.(greentype)wereexaminedfortheirpotentialantibacterial
activities.TechemicalcompositionofessentialoilsofOcinunspecieswasanalysedbyGC-MS.Teinhibitoryeects
ofessentialoilsandmethanolextractswerestudiedontwoGram-positive(Bacillussubtilis,Micrococcusluteus)andve
Gram-negative (Pseudononas aeruginosa, Shigella dysenteriae, Escherichia coli, Vibrio cholera, and Shigella exneri)
bacteria using disc-diusion method. Minimum inhibitory concentration (MIC) was assessed by micro broth dilution
method.TeantibacterialtestresultsshowedthattheessentialoilsofOcinunbasilicunL.,Ocinunkilinandscharicun
Guerke,andOcinungratissinunL.stronglyinhibitedthegrowthofallofthemicroorganismsstudied,especiallyofthe
Gram-negativestrains,whereasothertwoessentialoilsshowedmoderateactivities.Teresultmaysuggestthatthees-
sentialoilsofOcinunpossesscompoundswithantibacterialactivities,andthereforecouldbeusedasnaturalpreservative
ingredientsinfoodandiorpharmaceuticalindustries.
Keywords:antibacterialactivity,essentialoil,GC-MS,Ocinunspecies
Medicinalandaromaticplants(MAPs)havebeen
usedforcenturiesasremediesforhumandiseases
becausetheycontaincomponentsoftherapeutic
value.IthasbeenestimatedbyWHOthat80of
thepopulation,themajorityofthisinthedevelop-
ingcountries,stillrelyonplant-basedmedicine
forprimaryhealthcareneeds(WHO1993).For
thispurpose,variousstrategieshavebeendevel-
oped,e.g.biologicalscreening,isolation,aswell
asclinicaltrialsforavarietyofplants.
Followingtheadventofmodernmedicine,herbal
medicinesufferedasetback,butduringthelasttwo
orthreedecadestheadvancesinphytochemistry
andintheidentificationoftheplantcompounds,
providingeffectiveagainstcertainchronicdiseases
andemergenceofmultidrugresistantbacteria.
Thisawakeninghasledtoasuddendemandfor
herbalmedicine.Worldwideaswellasinthede-
velopingcountries,themosthumansdieddueto
infectiousbacterialdiseases(N:n2004).The
bacterialorganismsincludingbothGrampositive
andGramnegativeonesarethemaincauseofse-
vereinfectionsinhumans,becausetheyhavethe
abilitytosurviveinharshconditionsduetotheir
multipleenvironmentalhabitats(Anrvv:nuis
&Hovvvv2010).Nowadays,multipledrugresist-
anceisdevelopedduetotheindiscriminateuseof
commercialantimicrobialdrugscommonlyused
inthetreatmentofinfectiousdiseases(Guv:et
al.2008).Inadditiontothisproblem,antibiotics
are sometimes associated with adverse effects
onthehostincludinghypersensitivity,immune-
SupportbytheUniversityGrantCommission(UGC),NewDelhi,Govt.ofIndia,ProjectNo.F14-2(SC)i2007i(SA-III).
195
Czech J. Food Sci. Vol. 31, 2013, No. 2: 194202
suppression,andallergicreactions(Wvvvvetal.
1999).Ontheotherhand,environmentalsafetyis
a major concern in many countries, and the ap-
plicationofsyntheticagrochemicalsrunstherisk
ofcausingunacceptableenvironmentaldamage,
suchashealthhazardstohumans,toxicitytouseful
non-targetedanimalsandenvironmentalpollution.
Theuseofnaturalproductswouldbeahelpfulway
toreducethisrisk,andalsothesituationrequires
searchingfornewantimicrobialsubstances.The
screening of the active compounds from plants
hasleadtothediscoveryofnewmedicinaldrugs
which show efficient protection and treatment
roles against various diseases, including cancer
(Kurv et al. 2004, Snvvi & Ku::r 2007).
Therefore,thereisaneedtodevelopalternative
antimicrobialdrugsfrommedicinalplantsforthe
treatmentofinfectiousdiseases.
The genus Ocinun involves economically the
most important medicinal and aromatic herbs,
undershrubs or shrubs in the world. It belongs
tothefamilyLamiaceae,subfamilyOcimoideae,
andcomprisesmorethan30speciesdistributed
intropicalandsubtropicalregionsofAsia,Africa,
and Central and South America (P:o 1992).
Traditionally, the genus Ocinun is widely used
for the treatment of various ailments including
rheumatism,paralysis,epilepsy,highfever,diar-
rhea, sunstroke, influenza, gonorrhea, mental
illness,abdominalpains,colds,coughs,measles,
andhasalsoantipyretic,antihelmentic,stomatic,
anti-emetic,andantimalarialeffects(Ccvvvset
al.1990,Onvo-Orovietal.1998,Nvvoetal.
2002,Ezvvsiiietal.2004).Itisalsoasource
ofaromacompoundsandessentialoilscontain-
ing biologically active constituents that possess
insecticidal(Dvsnvovetal.1997),nematicidal
(Cn:vvivvetal.1982),andfungistaticproperties
(Rvuvvietal.1984).Theactivecompoundspre-
sentasvolatileoilfromtheleavesconsistmainlyof
eugenol,thymol,citrol,geraniol,camphor,linalool
l,andmethylcinnamate(Cnvivs&Siro1992,
Jivovv:z&Bucnnuvv2001,Mooviioetal.
2002,Vi&Muviiio2003,Poii&Vvv
2011,Sionetal.2011,Vvvretal.2011).The
seeds contain oil composed of fatty acids and
sitosterol.Therootscontainsitosterolandthree
triterpenes A, B, and C. Additionally, they also
containrosmarinicacid,thymol,methylchavicol
and,citraletc.(Dnvetal.1968),andvitamins
C, A, and minerals like calcium, zinc, and iron
(Anvsu&Viivisnri2007),aswellas
chlorophyllandmanyotherphytonutrients.Recent
interestinOcinunhasresultedfromitsinhibi-
toryactivityagainstHIV-Ireversetranscriptase
andplateletsaggregationinducedbycollagenand
ADP(adenosine5'-disphosphate)(Ozietal.
1998,Yrsietal.1998).However,theanti-
microbialactivityofOcinunessentialoilagainst
microorganisms has been investigated by some
researchers (Pvso et al. 1986, Nruv et
al.1999,Aovnoiu&Oioirvii2005,Aoiouzvi
et al. 2005, Moonooret al. 2011, Vvvr et
al.2011)usingdifferenttechniquesandtheirin-
vestigationsmostlycoveredoneindividualortwo
species.Unfortunately,thepublisheddataonthe
formersubjectaredifficulttocompare,because
thechemicalcompositionofessentialoilsisknown
tovarywiththelocalclimate,harvestperiod,and
environmentalconditions(Viviv&Siro2000),
andisalsodependentonthetypeofsolventusedin
theextractionprocedure(Cuetal.1989).Therefore,
thepresentstudyisaimedatevaluatingtheinvitro
antibacterialactivityofessentialoilsandmethanol
extractsoffivespeciesofOcinun,namelyOcinun
basilicunL.,OcinunkilinandscharicunGuerke,
Ocinun gratissinun L., Ocinun canun Sims.,
andOcinuntenuiflorunL.(greentype)against
theselectedmicroorganisms.
MATERIAL AND METHODS:
Plant material collection.FreshleavesofOci-
nun basilicun L., Ocinun kilinandscharicun
Guerke, Ocinun gratissinun L., Ocinun ca-
nun Sims., and Ocinun tenuif lorun L. (green
type)werecollectedfrommedicinalandaromatic
plant garden, Department of Botany, University
ofKalyani,Kalyani,WestBengal,India,inAugust
of2011(Table1),whichislocatedat2257'Nlati-
tude,8822'Elongitudewithanaveragealtitudeof
9.75ma.s.l.Thetaxonomicidentificationofthe
plantmaterialwasconfirmedbyDr.G.G.Maity,
TaxonomyandPlantsystematicUnit,Department
of Botany, University of Kalyani. The voucher
specimens (143, 148, 149, 150, and 163 KUH,
respectively) were deposited and preserved at
theDepartmentofBotany,UniversityofKalyani,
Kalyani,andWestBengal,India,forreference.
Preparation of methanolic plant extracts.Each
collectedplantmaterialwasdriedintheshadeand
groundinagrinderwitha2mmdiametermesh.
Thedriedandpowderedleaves(100g)weresucces-
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Vol. 31, 2013, No. 2: 194202 Czech J. Food Sci.
sivelyextractedwith500mlofmethanol(1:5wiv)
usingaSoxhletextractorfor72hatatemperature
notexceedingtheboilingpointofthesolvent(Li
etal.1999).TheextractswerefilteredusingWhat-
manfilterpaper(No.1)andthenconcentratedin
vacuo at 40C using a rotary evaporator (Buchi
RotavaporR-200,BuchiLabortechnikAG,Flawil,
Switzerland).Theextractswerethenlyophilised
andkeptinthedarkat+4Cuntiltested.
Extraction of the essential oils. Theair-dried
and ground aerial parts of the plants collected
weresubmittedtowaterdistillationfor2.5hus-
ingaClevenger-typeapparatus(Civvvovvetal.
1928)).Theessentialoilsobtainedweredriedover
anhydroussodiumsulphateand,afterfiltration,
storedat+4Cuntiltestedandanalysed.
GC-MS and GC-FID analysis.GC-MSanalyses
oftheessentialoilswereperformedonaHewlett-
Packardgaschromatograph,model6890,equipped
withaFID,usingHP-5MScapillarycolumn(30m
length x0.25mmi.d.x0.25mlmthickness)(Agi-
lentTechnologies,SantaClara,USA).Teinjector
wassetat250Candthedetectorat280C,respec-
tively.Teoventemperatureofthechromatogram
wasraisedfrom60Cto280C,respectively,atthe
heatingrateof10Cimin,andwasthenisothermally
heldfor5minutes.Teinjectorvolumewas1.0l.
Tesolventdelaywas2minanditwasinjectedina
splitratioof1:10.Heliumwasusedasthecarriergas
at15P.S.I.inletpressure.Teessentialoilconstitu-
entswereidentiedbythecomparisonoftheirGC
retentionindices(RI)andmassspectrawiththoseof
theauthenticstandardcompounds.Quantication
oftherelativeamountsoftheindividualcomponents
wasperformedaccordingtotheareapercentage
method(Tvicietal.2006)foreachplant.
Antibacterial activity
Test microorganism.Themethanolextractsand
essential oils were individually tested against a
panelofmicroorganismsincludingGram-positive
Bacillussubtilis(MTCC441),Micrococcusluteus
(MTCC2522),Gram-negativePseudononasaeru-
ginosa (MTCC741),Shigelladysenteriae(Clini-
calisolate),Escherichiacoli(MTCC443),Vibrio
cholera(MTCC3904),andShigellaflexneri(MTCC
1457). All the bacterial strains except Shigella
dysenteriae were obtained from the Institute of
Microbial Technology, Chandigarh, India. The
referencestrainsofbacteriaweremaintainedon
nutrientagar(HiMedia,Mumbai,India)slantsat
4Cwithasubcultureperiodof30days.
Preparation of McFarland standard. Teturbid-
itystandardwaspreparedbymixing0.5mlof1.75
(wiv)BaCl
2
2H
2
Owith99.5mlof1H
2
SO
4
BaSO
4
(viv).Testandardwasputintoscrewcaptesttube
tocomparetheturbidity.Tebacterialculturesof
theselectedstrainsweregrownovernightandwere
subsequentlymixedwithphysiologicalsaline.Tur-
biditywascorrectedbyaddingsterilesalineuntil
McFarland0.5BaSO
4
turbiditystandard10
8
Colony
FormingUnit(CFU)permlwasachieved.Tese
inoculawereusedforseedingofthenutrientagar.
Disc-diffusion assay.Theessentialoilsandmeth-
anolextractsweredissolvedinDMSO(dimethyl-
sulfoxide) to a final concentration of 30 mgiml
and sterilised through filtration using 0.45 m
membranefilters.Theantibacterialtestswerethen
carriedoutbydiscdiffusionmethod(Muvvvet
al.1995)using100lofthesuspensioncontaining
10
8
CFUimlofbacteriaonnutrientagar.Thediscs
(6mmindiameter)wereimpregnatedwith10l
ofessentialoilor30mgimlextracts(300gidisc)
placedontheinoculatedagar.Negativecontrols
were prepared using DMSO. Gentamicin (10 g
perdisc)wasusedaspositivereferencestandardto
determinethesensitivityofeachbacterialspecies
tested. The inoculated plates were incubated at
37Cfor24hours.Theantibacterialactivitywas
evaluated by measuring the zone of inhibition,
the diameters of these zones being measured in
millimetersagainstthetestorganisms.
Table1.Plantsandplantpartsused
No. Scienticname Commonname Family Partsused
1. OcinunbasilicunL. sweetbasil
Lamiaceae driedleaves
2. OcinunkilinandscharicunGuerke. camphorbasil
3. OcinungratissinunL. shrubbybasil
4. OcinuncanunSims. hoarybasil
5. OcinuntenuiorunL.(greentype) holybasil
197
Czech J. Food Sci. Vol. 31, 2013, No. 2: 194202
Determination of minimum inhibitory con-
centration. Theminimalinhibitoryconcentration
(MIC) values were followed with the bacteria
strains sensitive to the essential oils andior ex-
tracts in the disc diffusion assay. The inocula
ofthebacterialstrainswerepreparedfrom12h
brothculturesandthesuspensionswereadjusted
to0.5McFarlandstandardturbidity.Theessential
oilsandextractsofOcinunssp.dissolvedin10
DMSO,werefirstdilutedtothehighestconcen-
tration (500 giml) to be tested, and then serial
twofold dilutions were made in order to obtain
a concentration range from 7.8 to 500 giml in
10 ml sterile test tubes containing the nutrient
broth.MICvaluesoftheextractsagainstbacterial
strainsweredeterminedbasedonthemicrowell
dilutionmethodaspreviouslydescribed(Sorv
etal.2004).The96-wellplateswerepreparedby
dispensingintoeachwell95lofthenutrientbroth
and5loftheinoculum.Avolumeof100 lfrom
thestocksolutionsofOcinunspp.essentialoils
andextractsinitiallypreparedattheconcentration
of500gimlwasaddedintothefirstwells.Then,
100lfromtheirserialdilutionswastransferred
intosixconsecutivewells.Thelastwellcontaining
195lofthenutrientbrothwithoutthecompound
and5loftheinoculumoneachstripwasused
asnegativecontrol.Thefinalvolumeineachwell
was 200 l.Theplate wascovered with a sterile
platesealerandthenincubatedattheappropri-
atetemperature37Cfor24hours.Thebacterial
growthwasdeterminedbyabsorbancemeasured
at600nmusingtheELx800universalmicroplate
reader(BiotekInstrumentInc.,Winooski,USA)and
confirmedbyplating5lsamplesfromclearwells
onnutrientagarmedium.TheMICwasdefined
asthelowestconcentrationofthecompoundto
inhibitthegrowthofmicroorganisms.Eachtest
inthisstudywasrepeatedatleasttwice.
RESULTS AND DISCUSSION
Essential oil composition
Thepercentchemicalcompositionoftheessential
oilsoffivespeciesofOcinun,namelyO. basilicun,
O.kilinandscharicun,O.gratissinun.O. canun,
andO.tenuiflorun(greentype)isgiveninTable
2intheorderoftheretentiontimesofthecon-
stituents.Theyieldsoftheessentialoilsobtained
from dry leaves were 1.77, 1.92, 1.63, 1.81, and
1.73(viw),respectively.Themainconstituentsof
O. basilicunweregeraniol(34.89),citral(23.51),
linalool (2.21), and eugenol (1.33), O. kili-
nandscharicun camphor (21.65), eugenol
(9.65),cineole(2.07),andcitral(1.23),O. gratis-
sinun eugenol (47.45), citronellal (3.56),
cineole (1.97), geraniol (1.52), and vanillin
(1.52), O. canun camphor (3.47), cineole
(1.44),vanillin(1.03),andO.tenuiflorun(green
type) eugenol (8.81), citronellal (1.44), and
vanillin (1.16), respectively. The essential oil
compositionofO.basilicun,O.gratissinun,and
Table2. PercentagecompositionoftheessentialoilsofvespeciesofOcinuncultivatedinWestBengal
Compound
Retentionindices
a

(RI)(min)
Essentialoil()
b
O.basilicunL.
O.kilinandscha-
ricunGuerke
O.gratissinunL. O.canunSims.
O.tenuiorunL.
(greentype)
-Pinene 4.76 0.23 0.98 0.12 0.17
Camphor 4.87 0.64 21.65 0.15 3.47 0.05
Citral 5.26 23.51 1.23 0.52 0.33 0.22
Geraniol 5.76 34.89 0.49 1.52 0.14 0.83
Cineole 5.83 0.05 2.07 1.97 1.44
-Pinene 5.92 0.19 1.02 0.18 0.13 0.09
Citronellal 5.96 0.59 0.51 3.56 0.79 1.44
Eugenol 6.57 1.33 9.65 47.45 0.32 8.81
Vanillin 6.72 0.27 0.30 1.52 1.03 1.16
Linalool 7.41 2.21 0.23 0.12 0.02
a
identication of oil components was based on their relative retention indices (retention times) with those of authentic
standards,
b
quantitativeestimationwasdonebyanalysisofFIDareapercentdata
198
Vol. 31, 2013, No. 2: 194202 Czech J. Food Sci.
O.kilinandscharicunobtainedfromplantsgrown
innorthernIndia,wasfoundtoberichinO.ba-
silicunmethylchavicolandlinalool(21.9),O.
gratissinuneugenol(72.2),1.8-cineole(7.6),
germacrene D(2.7)and-caryophyllene(1.7),
and O. kilinandscharicun camphor (64. 9),
limonene (8.7), in accordance with previous
report(Poii&Vvvr2011).Similarresults
werefoundwithO.gratissinunandO.kilinand-
scharicun in sub tropical India (Vvvr et al.
2011). In the oils obtained from the aerial part
ofO.basilicungrowninColombiaandBulgaria,
linaloolandmethylcinnamatewerereportedas
the major components of volatile oils, respec-
tively ( Jivovv:z & Bucnnuvv 2001, Vi &
Muviiio 2003). It is interesting that the oils
extracted from Ocinun basilicun L. collected
from Bangladesh contain linalool and geraniol
as their main constituents (Mooviio et al.
2002), and the authors concluded that the oil
compositioncouldbedependentontheclimatic
conditions.Thechemicalcompositionofessential
oilsofOcinunspeciesshowsalargeinterspecies
variabilityand,withinthesamespecies,itseems
to depend on the genetic characteristics of the
plant and on the conditions under which it has
grown.Inthepresentstudy,ourfindingsonthe
majorcomponentsofO.kilinandscharicunand
Table3.AntibacterialactivityoftheessentialoilandmethanolextractofOcinunssp.againstthebacterialstrains
testedbasedondiscdiusionmethod
Microorganism
Inhibitionzoneindiameter(mm)aroundthediscsimpregnatedwith10lofessential
oilsandextracts(300gidisc)
essentialoil(10lidisc)
a
MeOHextracts
b
gentamicin
(10gidisc)
E1 E2 E3 E4 E5 M1 M2 M3 M4 M5
B.subtilis(MTCC441) 22 24 18 15 22 8 8 7
c
26
M.luteus(MTCC1541) 20 16 16 14 18 26
P.aeruginosa(MTCC741) 23 21 22 15 14 13
S.dysenteriae(clinicalisolate) 16 16 15 18 18
E.coli(MTCC443) 16 17 21 9 10 8 8 30
V.cholera(MTCC3904) 22 16 21 18 18 10 7 17
S.exneri(MTCC1457) 18 18 15 21 27
E1E5essentialoils:EIOcinunbasilicunL.,E2OcinunkilinandscharicunGuerke,E3OcinungratissinunL.,
E4OcinuncanunSims,E5OcinuntenuiorunL.(greentype),M1M5methanolextracts:M1Ocinunbasilicun
L.,M2OcinunkilinandscharicunGuerke,M3OcinungratissinunL.,M4OcinuncanunSims,M5Ocinun
tenuiorun L. (green type),

not active,
a
inhibition zone in diameter (mm) around the discs impregnated with 10 l of
essentialoil,
b
inhibitionzoneindiameter(mm)aroundthediscsimpregnatedwithextracts(300gidisc)
Table4. TeMICvaluesofessentialoilsofthevespeciesofOcinunagainstthebacterialstrainstestedinmicro-
dilutionassay(giml)
Microorganism
Essentialoil
O.basilicun
O.kilinand-
scharicun
O.gratissinun O.canun
O.tenuiorun
(greentype)
B.subtilis(MTCC441) 15.62 15.62 62.50 62.50 62.50
M.luteus(MTCC1541) 31.25 62.50 125 125 125
P.aeruginosa(MTCC741) 62.50 62.50 125 250 250
S.dysenteriae(clinicalisolate) 125 250 250 125 nt
E.coli(MTCC443) 15.62 31.50 31.50 nt 500
V.cholera(MTCC3904) 31.25 125 125 125 250
S.exneri(MTCC1457) 15.62 62.50 125 nt 62.50
ntnottested
199
Czech J. Food Sci. Vol. 31, 2013, No. 2: 194202
O.gratissinunoilswereinagreementwiththe
previousreport(Vvvretal.2011).According
totheliterature,themajorcompoundsconcerned
of O. basilicun, O. canun, and O. tenuif lorun
(green type) are different. The observed differ-
encesmaybeduetothedifferentenvironmental
andgeneticfactors,differentchemotypes,andthe
nutritional status of the plants as well as other
factorsthatcaninfluencetheoilcompositions.
Antibacterial activity (Disc diffusion test)
The in vitro antibacterial activities of the five
species of Ocinun essential oils and methanol
extracts against the microorganism employed
andtheirpotentialswerequalitativelyandquan-
titativelyassessedbythepresenceorabsenceof
inhibition zones and the zones diameters, MIC
values being showed in Tables 3 and 4. Accord-
ing to the result given in Table 3, the essential
oils of O. basilicun, O. kilinandscharicun, and
O. gratissinunhadagreatpotentialofantibacte-
rialactivities(inhibitiondiametersrangedfrom
1524 mm)againstall7 bacteriatested,whereas
thoseofO. canunandO.tenuiflorunhadsubstan-
tial activities (inhibition diameters ranged from
9 mmto22 mm)against5and6bacteriatested.
Ontheotherhand,thefractionsofthemethanol
extracts of O. basilicun, O. kilinandscharicun,
andO.gratissinunwerealsofoundtobeeffective
against B. subtilis, E. coli, and V. cholera out of
7 bacterialspeciesexamined(inhibitiondiameters
rangedfrom710 mm),respectively,probablydue
to the presence of similar compounds in these
methanol fractions, whereas those of Ocinun
canun Sims and Ocinun tenuiflorun L. plants
showednoantibacterialactivities(Table3).This
disagreement can be explained by that the bet-
terextractionofantimicrobialcompoundsfrom
various medicinal plants may require different
solvents.Whencomparedtothemethanolextracts,
theessentialoilsexhibitedastrongerandbroader
activityascomparedtothemethanolextracttested.
Based on these results of chemical composition
of the essential oils, it is possible to conclude
thattheantibacterialnatureoftheessentialoils
studiedisapparentlyrelatedtotheirhighphenolic
contents,particularlyoxygenatedterpenoidsand
phenolicterpenes,andthisfindingisinagreement
withpreviousreports(Buv:2004,Giiucciet
al.2009,Bssoiv&Juiii2012).Thisclaimis
furthersupportedbyourfindingsindicatinghigh
contents of terpenoids such as citral, geraniol,
eugenol, and camphor in the oils (Table 2). The
findings in this study support the observations
ofsomeotherresearchersaboutOcinunspecies
containing some substances with antibacterial
properties(Pvsoetal.1986,Nruvetal.
1999, Aovnoiu & Oioirvii 2005, Aoiouzvi
et al. 2005, Moonoor et al. 2011, Vvvr et
al.2011).However,itisdifficulttocomparethe
datawiththeliteraturebecauseseveralvariables
influencetheresults,suchastheenvironmental
andclimaticconditionsoftheplantandthechoice
oftheextractionmethodandantimicrobialtest.
Moreover,standardcriteriafortheevaluationof
the plant activity are missing and therefore the
results obtained by different authors are widely
different(Rvcioetal.1989,VovBuvonvet
al. 1991). This is the first study to provide data
about the extracts and essential oils of the five
species of Ocinun plants possessing potential
antibacterialactivitiesasevaluatedagainstseven
microorganisms.Theresultsindicatethatthees-
sential oils of Ocinun species can be used as a
naturalsourcethatmayleadtotheiruseassafe
alternatives to synthetic antimicrobial drugs. In
addition, the data in the present study support
theuseofOcinunspeciesasadditivesinfoods,
andastraditionalremediesforthetreatmentof
infectiousdiseases.
Minimum inhibitory concentration
All the essential oils tested were subjected to
MICstudiesagainstallthemicroorganisms.The
resultsinTable4interpretedasthelowestconcen-
trationsthatinhibitthevisiblemicrobialgrowth.
ThemaximalinhibitionzonesandMICvaluesfor
bacterialstrains,whichweresensitivetotheessen-
tialoilsofthefivespeciesofOcinun,wereinthe
rangeof924mmand15.62500giml(Table 4).
Basedontheseresults,itispossibletoconclude
thattheessentialoilshaveastrongerandbroader
spectrum of antimicrobial activity as compared
tothemethanolextractstested.Thisobservation
confirmedtheevidencegiveninapreviousstudy
reporting that the essential oils from medicinal
plantscontainmoreantimicrobialsubstancesthan
otherextractssuchaswater,methanol,ethanol,and
hexaneexstracts(Anroetal.1998,Eiorr1998).
Ourresultsalsoindicatedinthepresentstudythat
200
Vol. 31, 2013, No. 2: 194202 Czech J. Food Sci.
theO.basilicun,O.kilinandscharicun,andO.gra-
tissinunessentialoilsweremoreactiveagainst
allpathogenicbacteriaincludingGram-negatives
ones than the other two essential oils, probably
owingtothehighlevelsofphenoliccompounds
intheformerones(Table2).
According to a number of studies, the Gram-
positive bacteria are more sensitive to essen-
tialoilthanGram-negativebacteria.Apossible
explanation may reside in the possession of an
outer membrane, surrounding the cell wall of
Gram-negativebacteria,thusitislogicaltoexpect
thatthesebacteriawllbelesssusceptibletothe
antibacterialactivityofessentialoil.Thisouter
membrane may restrict the diffusion of hydro-
phobiccompoundsthroughitslipopolysaccharide
covering,presentingabarriertothepenetration
of numerous antibiotic molecule, and is also
associatedwiththeenzymesintheperiplasmic
space, which are capable of breaking down the
moleculesintroducedfromtheoutside(Niioo
1994,Goetal.1999).Gram-positivebacteriado
nothavesuchanoutermembraneandcellwall
structure.Theantibacterialsubstancescaneasily
destroytheirbacterialcellwallandcytoplasmic
membrane and cause leakage of the cytoplasm
anditscoagulation(Kivrn&Kuic2003).
However,thecurrentfindingsshowaremarkable
activityagainstallgram-negativebacteria.Thean-
tibacterialactivitiesofO.basilicun,O.kilinand-
scharicun, and O. gratissinun leaves extracts
essential oils may be due to high contents of
tannins and phenolic constituents. The most
active constituents (essential oils) rich in phe-
noliccompoundsarewidelyreportedtopossess
highlevelsofantimicrobialactivity(Pvsoet
al. 1986, Nruv et al. 1999, Dovr &
Dvs 2000), which has been confirmed and
extendedinthepresentstudy,althoughantimi-
crobial activities of phenolic compounds may
involve multiple modes of action. The mode of
actionofantimicrobialagentsalsodependsonthe
typeofmicroorganismsandismainlyrelatedto
theircellwallstructureandtheoutermembrane
arrangement.Medicinalplantscontaincomplex
phenolicsandthemechanismofactionofeach
phenolic compound against various bacteria is
also very complicated (Buv: 2004). Therefore,
itisnecessarytoinvestigatefurtherandunder-
standtherelationshipbetweentheantibacterial
activityandchemicalstructureofeachphenolic
compoundintheextractstested.
CONCLUSION
The growing tendency for replacing synthetic
additiveswithnaturaloneshasbroughtaboutgreat
interestintheevaluationofantimicrobialproper-
tiesoftheplantsproductsinbothacademicaland
industrial fields because of their relatively safe
status,wideacceptancebyconsumers,andtheir
exploitationforpotentialmultipurposefunctional
use.Tothebestofourknowledge,thisisthefirst
reportontheinvitrocomparativeevaluationof
theantibacterialactivitiesoftheessentialoilsand
methanolextractsoffivespeciesofOcinun.The
essentialoilsofOcinunspeciesprovedtopossess
interestingproperties,emergingfromboththeir
chemicalcompositionandfromtheevaluationof
theirinvitrobiologicalactivities.However,itis
verydifficulttoattributethebiologicaleffectofa
totalessentialoiltooneorafewactiveprinciples,
because in addition to the major compounds,
also minor compounds may make a significant
contributiontotheoilactivity.Fromtheresults
givenabove,wecouldinferthatOcinunessential
oils,indicatingstrongantibacterialactivities,are
very important botanical dietary supplements
that can be freely used in the food industry as
culinaryherbs.
Acknowledgement.TheauthorsaregratefultoDST-
FISTprogramme,Govt.ofIndia,DepartmentofBotany,
UniversityofKalyaniforInstrumentalfacilities.
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ReceivedforpublicationJune11,2012
AcceptedaftercorrectionsAugust22,2012
Correspondingauthor
Prof.Pv:novnGnosn,Ph.D.,UniversityofKalyani,CytogeneticsandPlantBiotechnologyResearchUnit,
DepartmentofBotany,741235,Nadia,WestBengal,India,E-mail:pdgbot@yahoo.co.in