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ISSN - 0974-2441
ResearchArticle
LEAFEXTRACTOFCENTRATHERUMPUNCTATUMEXHIBITSANTIMICROBIAL,ANTIOXIDANT
ANDANTIPROLIFERATIVEPROPERTIES
NAVEENKUMARPAWARANDNEELAKANTANARUMUGAM*1
DepartmentofBiotechnology,SchoolofLifeSciences,PondicherryUniversity,RVenkataramaNagar,Kalapet,Puducherry,India
Email:n_arumugam@hotmail.com
ABSTRACT
Centratherum punctatum, the Brazilian button flower, is very closely related to its counterpart C. anthelmethicum a plant known for its high
medicinal value. To validate if C. punctatum would also be of any such value, the leaf extract of the plant was evaluated for antimicrobial,
antioxidant,humancelltoxicitypropertiesandanalyzedforthepresenceofphytochemicalconstituents.Powderedleafofthe plantwasextracted
withdifferentorganicsolventsandtestedforantimicrobialactivitybytheagarwelldiffusionmethod.Theantioxidantactivitywasanalyzed by
Ascorbicacidmethod.ThetoxicityoftheextractwastestedbytheMTTassayusinghumanperipheralbloodmononuclearcells(PBMCs).Extracts
werethensubjectedtobioautographyandthephytochemicalconstituentsisolatedandtestedforantimicrobialactivity.TLCfractionsthattested
positiveforantimicrobialactivitywerepartiallycharacterizedforfunctionalgroupidentificationbyKBrmethodusingFourierTransformInfrared
Spectroscopy.Acetone,methanolandethylacetateextractsofleafshowedinhibitoryactivityagainstfouroutoffivepathogenicbacteriaincluding
themultidrugresistant(MDR)AcinetobacterbaumaniiandStaphylococcusaureustested.Antifungalactivitywasexhibitedbyacetoneandethyl
acetate extracts. Phytochemical analyses revealed the presence of flavonoids, tannins and cardiac glycosides, of which flavonoids showed anti
bacterialactivity.TheIC50valuefortheacetoneextractwasfoundtobe10.63g/ml.FTIRanalysisrevealedthepresenceofalkene,alkane,aliphatic
amine and aromatic functional groups among others. We conclude that the present study adds credence to the ethnomedicinal properties of C.
punctatum.Furthercharacterizationofphytochemicalcompoundsfromthisprolificherbmayyieldpotentialantimicrobialagents.
Keywords:Centratherumpunctatum,Acinetobacterbaumanii,Staphylococcusaureus,multidrugresistance,antioxidant,flavanoids,tannins,cardiac
glycosides.
INTRODUCTION
Nature has been a valuable source of medicine and has helped
human in the maintenance of his health since time immemorial1.
AccordingtotheWorldHealthOrganization(WHO),almost80%of
the worlds population relies on traditional medicines for their
health needs due to better cultural acceptibility, fewer side effects
andbettercompatibilitywiththehumanbody2,3.
Indiscriminateuseofantibioticsappearstopromotedevelopmentof
pathogens showing resistance to multiple drugs and this scenario
continues to become grave day by day. Exhibition of multidrug
resistance by pathogenic bacteria conveys a great challenge ahead
for the society thereby causing a renewal of interest in ethno
botanicmedicines4,5.Oflatetherehavebeenseveralattemptsmade
fordiscoveryofnewantimicrobialsubstancesfromnaturalsources
68.
Centratherum punctutam Cass. (Asteraceae), the Brazilian bachelor
button,isoneamong33speciesofthetypegenusCentratherumand
isaperennialbushyplantof4560cmheight(Fig.1).Ithasawell
branched stem with refreshing scented foliage and purple flower
heads. Recently an essential oil containing nearly 59 different
compoundshasbeenisolatedfromleavesofthisplant 9.
Centratherin, a sesquiterpene lactone, has been isolated from C.
punctatum but its medicinal properties have not yet been
established conclusively10. A related species C. anthelminticum is
known for antifilarial11 and antihyperglycemic properties12.
Thereforeinthisstudyweattemptedtoseekascientificjustification
fortheuseofC.punctatumasasourceofherbalmedicinebytesting
theextractonsomeclinicallyimportanthumanandplantpathogens
forantimicrobialactivity.Phytochemicalanalysiswasperformedto
detect the presence of bioactive constituents and an attempt was
alsomadetocharacterizethebioactiveTLCfractionsbyFTIR.
MATERIALSANDMETHODS
Plantmaterials
Centratherum punctutam Cass. (Asteraceae) (Fig.1) was obtained
from herbal collections of Indian Council of Agricultural Rearch
Krishi Vigyan Kendra, Puducherry, and maintained in the
experimental garden of the Department of Biotechnology,
PondicherryUniversity.Theidentityoftheplantwasauthenticated
by Prof. N. Parthasarathy, a taxonomist of our University, and a
voucher specimen of the plant bearing the number DBTAZ002 has
been deposited in the Herbaria maintained by him in the
Department of Ecology and Environmental Sciences of our
University.
Chemicals
Solvent and other chemicals used for this study were of AR grade
quality and were procured from Merck (India) and HiMedia,
Mumbai. Precoated silica gel 60 F254 plates used for thin layer
chromatography(TLC)wereobtainedfromMerck,Germany.
Bacterialcultures
The test organisms (Pseudomonas aeruginosa (ATCC 2706),
Acinetobacter baumanii (ATCC 19606), Staphylococcus aureus,
Escherichia coli, Bacillus subtilis and Providencia rettgeri) were
obtained from the culture collections of our Department. Fungal
plant pathogens, Fusarium spp. and Curvularia spp., were obtained
fromIMTECH, Chandigarh, India.Except for B. subtilis the bacterial
culturesweregrownandmaintainedonMuellerHintonagarslants.
B. subtilis was maintained as a spore suspension in sterile distilled
water. Fungal cultures were maintained on potato dextrose agar
(PDA)slants.
Culturemedia
The culture media used for testing antimicrobial activity were
MuellerHintonagar,DaviesSyntheticMinimalmedium(onlyfor B.
subtilis) and Potato dextrose agar for fungal strains. The culture
media were prepared and sterilized following the manufacturers
(Himedia,India)instructions.
Preparationofplantextracts
Ten grams of airdried leaf samples were powdered and soaked in
100ml of hexane or ethyl acetate or acetone or methanol and
incubated at room temperature for 48 hours with intermittent
shaking. The solventleaf mixtures were then centrifuged at
6,000rpm at 4C for 10 mins to obtain the supernatant and the
supernatant was concentrated using a Rotavapour (BuchiR
Switzerland)at50C.Theresidueobtainedafterdryingwasweighed
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and reconstituted into suspension by dissolving in the respective
solventswithwhichtheywereextracted.
Testforantimicrobialactivitybyagarwelldiffusionmethod
The efficacy of the plant extracts for antimicrobial activity was
evaluated in terms of zone of inhibition by the agar welldiffusion
method3. The target bacteria (except B.subtilis) were cultured
separatelybyinoculationonMuellerHintonagar.ForB.subtilis,the
spore suspension was mixed with Davies Synthetic Minimal Agar
medium prior to plating for solidification and the plates were
subjectedtoagarwelldiffusionassay.
Antifungal activity was determined by culturing the spores of test
fungi on PDA plates. Wells with 6mm diameter and 5mm depth
were cut out from the agar plates using a sterile cork borer. 30l
plantextractsfromeachofthesolventextractsadjustedto1,5,10,
15 and 20mg/ml was injected into the wells. Pure solvents served
asnegativecontrols.Thecultureplateswereincubatedat37Cand
after 48 hours the diameter of the inhibition zone was measured
usingmillimeterscale.Thetestswerecarriedoutatleasttwice.
Phytochemicalanalysis
Analysisforthepresenceofalkaloids,tannins,saponins,flavonoids
and cardiac glycosides in the leaf extract was done following the
methodsdescribedinHarborne13.
Bioautographyofphytochemicalcompounds
Themetabolitestestedpositiveforthephytochemicalanalysiswere
isolated and tested for antibacterial activity by the agar well
diffusion method. Flavonoids were isolated by heating 0.5g of leaf
samplewith5mlmethanolonwaterbathat40Cfor10min.
Thefiltratewasconcentratedbyevaporationto1/4 thofitsoriginal
volume. Tannins were isolated by heating 0.5g of the sample with
10ml 2M HCl in a boiling water bath for 30min. The filtrate was
mixed thoroughly with 1ml ethyl acetate, and ethyl acetate layer
was then discarded. Five drops of amyl alcohol were added to
aqueousphaseandshakenthoroughly.
The alcoholic layer was retained for antibacterial assay. Cardiac
glycosideswereisolatedbyheating0.5gofthesamplewith5mlof
50% (v/v) methanol and 10ml of 10% (w/v) lead (II) acetate
solutioninawaterbathat40Cfor10min.Thefiltratewascooled
andextractedtwicewith10mldichloromethane/isopropanol(3:2).
Thelowerorganic or aqueous phaseswerecombined, filtered over
anhydroussodiumsulphateandevaporatedtodryness.Theresidue
was dissolved in 1ml dichloromethane/isopropanol (3:2) and this
solutionwasusedforantibacterialassays.Thetestorganismsused
for testing antibacterial activity were A.baumanii, S.aureus and
B.subtilis.
Antioxidantassay
AntioxidantassaywasperformedfollowingtheprotocolofPrietoet
al 14.Ascorbicacidintherangeof10100gwasusedasstandard
to make a standard curve for antioxidant activity. 300l of the
standard and test samples (leaf extracts) were mixed with 3ml of
thereagentsolution(0.6MH2SO4,28mMSodiumPhosphate,4mMof
Ammonium molybdate) taken in a test tube. The test tubes were
covered with aluminium foil and incubated in a thermal block at
95Cfor90min.
Antiproliferativeassay(MTTassay)
MTT assay was performed following Alley et al15, by plating
phytohemagglutinin (PHA; 1g/ml) induced human peripheral
bloodmononuclearcells(PBMCs)in96wellplatescontaining200l
ofRPMI1640mediumperwellalongwithconcentrationsofeachof
the crude leaf extracts adjusted to 0.1, 1,10,25, 50 and 100g/ml
andincubatedat37Cfor24hours.
Followingthecompletionofincubation,10lofMTTfromthestock
solution(5mg/mlMTTdyeinPBS)wasaddedtoeachwell,andthe
plate was incubated at 37C in a 5% CO2 atmosphere for 4 hours.
After4hours,thesupernatantwasremovedwithoutdisturbingthe
cell pellet, 50l DMSO was added to each well and after thorough
mixing(todissolvethedyecrystals),theabsorbancewasmeasured
using an ELISA plate reader set at 570nm. The controls for this
assay consisted of untreated cells (positive control), mitogen
induced cells, mitogeninduced cells treated with DMSO and
mitogeninducedcellstreatedwithTritonX100(negativecontrol).
ThinLayerChromatographyandBioautography
The crude leaf extracts obtained using different solvents were
subjected to thinlayer chromatography (TLC) by loading on
precoatedTLCsilicagel60F254plates(8cmx6cm;Merck).Ethyl
acetatehexane(1:9v/v)mixturewasusedasthemobilephase.TLC
chromatograms were scanned under UV light at 254nm and the
fluorescentbandsweremarked.ThebandsfromTLCperformedon
acetone extract were scraped separately, dissolved in 1ml of
acetone, vortexed, and centrifuged at 6,500xg for 15 min. The
supernatants were collected and used to evaluate antibacterial
activitybyagarwelldiffusionassay.
FTIRanalysisofbioactiveTLCfractions
TLC fractions that showed antibacterial activity were further
subjected to spectroscopic analysis for identification of the
functionalgroupsinthebioactivecompounds.Samplewasprepared
asdescribedinNaumannetal16.
AknownweightofTLCpurifiedfractionsoftheacetoneleafextract
(1mg)wastakeninamortarandpestleandgroundwith2.5mgof
drypotassiumbromide(KBr).Thepowdersoobtainedwasfilledin
a 2mm internal diameter microcup and loaded onto FTIR set at
26C1C.Thesampleswerescannedusinginfraredintherangeof
4000400cm1 using Fourier Transform Infrared Spectrometer
(Thermo Nicolet Model6700). The spectral data obtained were
comparedwiththereferencecharttoidentifythefunctionalgroups
presentinthesample.
Statisticalanalysis
The data obtained were analyzed statistically and the results are
presentedasMeanStandarddeviation.
RESULTS
Antimicrobialspectrumofleafextracts
The organic solvent extract of leaf when used at a minimal
concentrationof1mg/mlinhibitedfouroutoffivebacterialstrains
tested (Table 1). The hexane extract was poor in exhibiting anti
microbial activity which may be attributed to polar nature of the
antibacterialsubstances.Bacillussubtilisinparticularwasinhibited
byallfourextracts.
Thereactionmixwasallowedtocooltoroomtemperatureandthe
absorbance of the solution was measured at 695nm against a
blank containing 3ml of reagent solution and the appropriate
volume of the corresponding solvent and incubated under the
same conditions as the test samples. Butylated hydroxytoluene
(BHT),awellknownantioxidantusedasfoodadditive,wasuseda
positive control (dilutions prepared from 1mg/ml BHT stock
solutioninethanol).
Phytochemicalanalysisandbioautography
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AsianJPharmClinRes,Vol4,Issue3,2011,7176
pathogenicbacteria.Itissignificanttonotethattheflavonoidfrom
C. punctatum was able to inhibit even the multidrug resistant
(MDR)AcinetobacterbaumaniiandStaphylococcusaureus(Table3).
Theactivityofflavonoidsispossbilyduetotheirabilityto complex
withextracellularandsolubleproteins,oftenleadingtoinactivation
of the protein and loss of function. The probable targets for
flavonoids are the proteins present in the bacterial cell wall with
some of the lipophilic flavonoids disrupting even the bacterial
membranes16. The other two phytochemicals, namely, tannins and
cardiacglycosides,inhibitedonlyBacillussubtilis(Table3).
AntioxidantAssay
The residues of leaves obtained by extraction procedure described
in this paper exhibited antioxidant activities that ranged from
210g/ml to 353g/ml (Table 4). Among the four solvents the
acetoneextractshowedthemaximumactivityof353.36g/ml.The
antioxidant activity was however found to be lower than the
strandardantioxidantbutylatedhydroxyltoluene(BHT).
AntiproliferativeAssay
Antiproliferative effect of the leaf extracts at different
concentrations (0.1, 1, 10, 25, 50 and 100g/ml) was tested using
PBMCs.After24hoursexposure,thePBMCsweresubjectedtoMTT
assayandtheresultsarepresentedinFigure3.Untreatedcellsand
cells treated with DMSO served as positive controls, while cells
treated with TritonX100 served as negative control. A
concentrationdependent increase in inhibition of cell proliferation
wasobservedforallthefourextracts.IC50valuewaslowestforthe
acetone extract at 10.63g/ml, while the hexane extract showed a
higherIC50valueof95.54g/ml(Fig.3).
CONCLUSION
TLCandbioautography
ACKNOWLEDGMENTS
Inordertoresolvedifferentcomponentsfromthecrudeextractand
test their efficacy for bioactivity, TLC of the leaf extract was
performed. Depending on the solvent used for extraction, the
residues resolved into 7, 8, 10 and 13 different bands for ethyl
acetate, methanol, hexane and acetone respectively. Since acetone
extractoftheleafgavethemaximumnumberofbandsitwastaken
for further investigation. The bands of acetone extract resolved on
TLCwereelutedindividuallyandtestedforantibacterialactivity.Of
the 13 TLC bands the fractions eluted from bands 1, 2, 3, 5 and 6
were observed to show antibacterial activity against the three
pathogenicbacteriaB.subtilis,A.baumaniiandS.aureusandthedata
ispresentedinTable5.
Of the four different leaf extracts tested, the acetone extract was
found to exhibit the highest antimicrobial, antioxidant and anti
proliferative capabilities. Presence of flavonoids might be
responsiblefortheantimicrobialpropertiesofC.punctatum.These
compoundsneedfurtherevaluationbeforethey canbe declared as
potential medicinal agents. We are now attempting to purify the
antimicrobial compounds identified in large quantities for further
characterizationandtesting.
FourierTransformedInfraredSpectroscopy(FTIR)
SpectroscopicanalysiswasperformedforthefiveTLCfractionsthat
showed antibacterial activity. A comparison of the FTIR spectrum
obtained with that of the reference chart revealed the presence of
functional groups such as alkanes, amides, amines aromatics
aliphaticamines,alkylhalidesetcintheleafextract(Fig.4;Table6).
DISCUSSION
Oneofthecommonexercisedonetolabelaplantasanewsourceof
drugistotestanextractoftheplantforantimicrobialactivity.The
antimicrobial extract is then subjected to biophysical and
biochemicalanalysistoidentifytheactiveprinciplethatmighthave
beenresponsibleforsuchbiologicalactivity.Thoughafewcytotoxic
compounds have been isolated and identified from Centratherum
punctatum,ithasnotbeenproventobeamedicinalplantsofar10,17.
Fig.1:AplantofCentratherumpunctatumatfloweringstagein
theexperimentalgardenoftheDepartmentofBiotechnlogy,
PondicheryUniversity
Table2:Phytochemicalconstituentspresentintheleafextracts
ofc.Punctatum
S.No.
1.
Phytochemical
Alkaloids
Occurrence
2.
Flavonoids
3.
Saponins
4.
Tannins
5.
CardiacGlycosides
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Table1:Dataonzoneofinhibitionbyextractsofleafofcentratherumpunctatumobtainedusingdifferentorganicsolvents.
Organism
ZoneofInhibition(mm)
Name
Characteristic
Hexane
Acetone
EthylAcetate
Methanol
Pseudomonasaeruginosa
Gramnegative
Acinetobacterbaumanii
Gramnegative
22
Escherichiacoli
Gramnegative
4.67 0.58
3.33 0.58
1.671.53
2.67 0.58
Providenciarettgeri
Gramnegative
3.340.58
Staphylococcusaureus
Grampositive
3.6670.58
51
Bacillussubtilis
Grampositive
7.334 0.58
71
4.67 1.53
Fusariumspp.
Plantpathogen
21
Curvularia
Plantpathogen
Theleafextractswereusedat1mg/mlbydryweight.
Table3:Antibacterialactivityofphytochemicalsisolatedfromc.Punctatum
ZoneofInhibition(mm)
Flavanoids
21
9
10
Organism
Bacillussubtilis
Acinetobaterbaumanii
Staphylococcusaureus
Tannins
8
CardiacGlycosides
7
Table4:AntiOxidantActivityofLeafExtractsOfC.Punctatum
S.no.
1.
2.
3.
4.
5.
Sample
Acetoneextract
Methanolextract
EthylAcetateextract
Hexaneextract
ButylatedHydroxytoluene(BHT)
Concentrationofantioxidant(g/ml)
353.36
287.46
258.50
210.69
889.91
Table5:Dataonzoneofbacterialgrowthinhibitionbyfractions(15)elutedfromTLCofleafextractofc.Pucntatum
Organism
ZoneofInhibition(mm)
B.subtilis
A.baumanii
S.aureus
1
7
3
2
5
4
3
4
4
4
4
5
5
5
5
Table6:FunctionalgroupsidentifiedbyFTIRspectroscopyinthefractionfiveofthetlcofleafextractofc.Pucntatum
Wavenumber
2927.8
1639.4
1564.3
1426.2
1098.6
798.8
466.3
Bond Functionalgroup(cm1)
CHstretch
Alkanes
NHoutofplane
Amides
Amines
NH2inplanebend
CCstretch(inring)
Aromatics
CNstretch
Aliphaticamines
CHoutofplanebending
1,2,3,4tetrasubstitutedbenzene
CIstretch
AlkylHalide
85
80
75
70
65
% Inhibition
60
55
50
45
40
35
30
25
20
15
0
20
40
60
80
100
C oncentration (ug/m l)
Fig.3:Antiproliferativeactivityofhexane(black),methanol(blue),ethylacetate(red)andacetone(green)extractsofCentratherum
punctatumleafextractsonhumanPBMCs
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Fig.2:ZoneofInhibitioncausedbyacetone(A,B),methanol(C)andhexane(D)extractsofCentratherumpunctatumonthepathogenic
microbesAcinetobacterbaumanii(A),Providenciarettgeri(B),Staphylococcusaureus(C)andBacillussubtilis(D)
Fig.4:FTIR(FourierTransformedInfrared)SpectrumofTLCFraction5ofleafextractofCentratherumpunctatum
(fordetailspleaserefertoTable6)
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