Int.J.Pharm.Sci.Rev.Res.

,14(2),2012;n03,2529ISSN0976044X

ResearchArticle

SCREENINGOFANTIBACTERIALACTIVITYOFMEDICINALPLANTS

Nasrullah1,Suliman1,KhaistaRahman1,MuhammadIkram1,MohammadNisar1&2andImranKhan2
DepartmentofBotany1/Biotechnology2,UniversityofMalakand,Chakdara,Dir(L),KhyberPakhtunkhwa,Pakistan

Acceptedon:06052012;Finalizedon:25052012.
ABSTRACT
Antibacterialagentsaretheremarkablesourceagainstbacterialinfectionbutcontinuousincreasesinantibioticsresistantbacteria
threaten human health, especially for immunosupressed patients since last three decades. The selected plants widely used in
folklore remedies, showed broad spectrum activity. Methanol and nhexane extracts of fruits of Solanum nigrum L., leaves of
DodonaeaviscosaJacqandCannabissativaL.,werescreenedforantibacterialactivities,againstdifferentGrampositiveandGram
negativebacterialstrains.TheresultsshowedthatnhexaneextractsofplantswereinactiveagainstPseudomonasaeruginosawhile
methanolicextractsofallplantsexceptSolanumnigrumL.wereactiveagainstallthetestedorganisms.Theresultsindicatedthatall
medicinalplantshaveeffectiveantibacterialactivitiesandshouldbeanewsourceofantibiotics.Furtherworkisneededtoisolate
theactiveantimicrobialagents.
Keywords:Solanumnigrum,Dodonaeaviscosa,Cannabissativa,Grampositive/Gramnegativebacteria.

INTRODUCTION
The rich flora of Pakistan includes medicinal plants that
are being used for therapeutic purposes. Developing
countries like Pakistan depend on plant resources for
food, shelter, fodder, agriculture and herbal medicines.
Out of about 258,650 species of higher plants reported
from the world; more than 10% are used to cure ailing
communities.MajorityofthepeopleinPakistanrelieson
medicinalplantstofindtreatmentfortheirminor,evenin
somecasesmajordiseases.1,2
Allovertheworld,hundredsofplantsarerichsourcesof
medicine3, which are use as potent agents for powerful
drug discovery in different countries4. The therapeutic
value of plants is due to the presence of some
chemical substances within the plant tissues which
produceadefinitephysiologicalactiononthehuman
body; include alkaloids, flavonoids, glucosides, tannins,
gums, resins, essential oils, fatty oils, carbon
compounds,hydrogen,oxygen,nitrogensaltsofsome
chemicalsetc. 5.Awiderangeofmedicinalplantpartsis
used for extract as raw drugs6. This awareness is
tremendously increased finding of new antimicrobial
bioactive7,8.
The control of bacterial infection has been remarkably
impressive since the discovery of antibacterial drugs.
However, some of the pathogens rapidly become
resistant to many of the first discovered effective drugs
(WHO, 2002)9, hence, actions must be taken to reduce
thisproblemsuchascontrollingthemisuseofantibiotics
and continuing investigations aimed at the development
ofdrugsfromnaturalsources10.Effortsareontopromote
noveltechnologiesandtointensifyresearchincombating
drug resistant microbes7, 8. Therefore, researchers are
increasingly turning their attention to ethnomedicine,

looking for new leads to develop more effective drugs

againstmicrobialinfections11.
Solanumnigrum(L.,FamilySolanaceae)iscommonlyused
in the traditional medicine as a remedy for treating
variousdiseases.Theberriespossessmedicinalproperties
such as sedative, diaphoretic, diuretic, hydragogue,
expectorant and are useful in the disease of liver, heart
and eyes and are also effective at piles, fever and
dysentery12. The leaves are used to heal open wounds
andareknowntopossesshypotensiveeffect13.
Dodonaeaviscosa(J.)belongstofamily,Sapindaceaeand
similarly, Dodonaea viscosa (J.) is a popular medicinal
plant. Its leaves D. viscose is used as antiinflammatory,
antiulcer, antibacterial and antifungal agents and in the
treatment of fractures14. Cannabis sativa L. belongs to
Cannabaceae family and used inflammation, nausea,
headache,hematochesia,diarrhea,andalopecia15,16.
The present study aims to evaluate the antibacterial
activityofmethanolandnhexaneextractsofthefruitof
Solanum nigrum L, leaves of Dodonaea viscosa Jacq and
CannabissativaL.Theplantswereselectedonthebases
oftheirimportanceintraditionalmedicine,sothisstudy
was carried out to check the antimicrobial activities of
theseimportantplantsgrowinginPakistan.
MATERIALSANDMETHODS
Plantmaterials
Solanumnigrum(L.),Dodonaeaviscosa(J.)andCannabis
sativa(L.)werecollectedfromdifferentareasofBatkhela,
MalakandAgency,Pakistan.Theplantswereidentifiedby
Madam Zakia Ahmad, Lecturer in the Department of
Botany, University of Malakand, Chakdara Dir (L), KPK,
Pakistan.

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Int.J.Pharm.Sci.Rev.Res.,14(2),2012;n03,2529ISSN0976044X
Extractionofplantmaterials
Prior extraction, plant parts materials were cleaned 23
timeswithrunningwaterandoncewithsterilizeddistilled
water then surface sterilized with 1%mercuric chloride.
The materials were dried under shade at room
temperature(2535C).Theairdriedplantmaterialswere
grounded into coarse powder form through electric
blender.
Three hundred grams of each airdried plant material
were soaked in 70% methanol and 90% nhexane
respectively. They were regularly shaken for maximum
extraction at 80rpm for seven days. After 7 days, the
extract was filtered usingWhattman filterpaper(No. 1).
Theextractssolutionswereevaporatedtodrynessunder
reducedpressureattemperatureof45Cusingavacuum
pump with the rotary evaporator. The paste obtained
after rotary evaporation contained some water content
which was further dried in water bath at 60C for one
hour. The thick pastes obtained are known as the crude
extract. The extracts were kept in sterile bottles at 5C
untiluse.
Testmicroorganismsandmicrobialculture
Five bacterial strains in which two were Gram positive:
Bacillus cereus and Bacillus subtilis and three Gram
negative: E. coli, Pseudomonas aeruginosa and
Salmonellatyphi,wereusedinthisstudy.Allthebacterial
strains were clinical isolates obtained from the
Department of Pharmacy, University of Malakand,
Chakdara,KhyberPukhtunkhwa,Pakistan.
Growthmedia
Nutrient agar media is best growth media for bacteria.
TheMediawascomposedofBeefextract3.0g,Agar15.0g
and Peptone 5.0g. One liter media was prepared by
dissolving40gofnutrientagarin700mlofdistilledwater.
Aftercompletedissolution,thefinalvolumeofthemedia
wasraisedto1000mlbyaddingmoredistilledwater.The
mediawasboiledusingahotplate.ThePHwasadjusted
to 7.0 at 25 C, using 0.1M NaOH and 0.1M HCl. The
needed media and all glassware were sterilized through
autoclavingat15psiat121for20minutes.

culture media using sterile cork borers to make three to

five uniform wells in each Petri dish. A drop of molten
nutrientagarwasusedtosealthebaseofeachwell.The
wellswerefilledwith50oftheextractconcentrationof
25mg/ml, 50mg/ml and 100mg/ml. In the central well
50 of 90% nhexane and 70% methanol was use as
negativecontrolforrespectiveextracts.Theantibacterial
activities were determined after 24 hours at 37C
incubation in the incubator. The diameter of zone of
inhibition produced by the extract were measured and
compared with standard. Each sample was used in
triplicate for the determination of antibacterial activity.
Theworkwascarriedoutinlaminarflow.
RESULTSANDDISCUSSION
ThemethanolandnhexaneextractsofS.nigrumL.fruits,
leaves of D. viscosa Jacq and C. sativa L. were screened
for antibacterial activity against two Grampositive
bacteria; B. subtilis, B. cereus and three Gramnegative
bacteria;E.coli,P.aeruginosaandS.typhi.Themethanol
crudeextractsshowedgoodantibacterialactivitythanthe
nhexanecrudeextracts.Thenhexaneextractsofallthe
tested plants were inactive against P. aeruginosa. The
methanol extracts of the plants showed good
antibacterialactivities.
The methanol fruit extract of S. nigrum L. showed
significant antibacterial activity against all the tested
strainsexceptB.subtilis.Thelowestactivitywasrecorded
against B. cereus while moderate activity with 16.6 mm
ZOIwasrecordedagainstS.typhiat100mg/mlwhichisin
consensus with Rani and Khullar, (2004)20that S. nigrum
L. seeds possess active constituents against S. typhi. Our
results showed that the extract has active constituents
against other bacterial strains like E. coli and P.
aeruginosa with highest ZOI (20mm) at 100 mg/ml. It is
shownintable1andFig.1.

Antibacterialactivity
Antimicrobial was checked by antibacterial susceptibility
test(AST).ASTstandardtestscanbeconvenientlydivided
into diffusion and dilution methods. Common diffusion
tests include agar well diffusion, agar disk diffusion and
bioautography, while dilution methods include agar
dilution and broth micro/macrodilution. The broth and
agar based methods are the conventional reference
methods for AST17. Agar well diffusion assay is used to
check crude extract of different plant parts for
antibacterialactivities18,19.
The20mlofnutrientagarwasplatedinPetridishesand
allowed to solidify for 30 minutes. Wells of 6mm in
diameter and about 2 cm apart were punctured in the

The nhexane extract of S. nigrum L. fruit showed good

activityagainstE.coli(14mm)butdidnotinhibitgrowthof
otherstrains.Thisshowsthatnhexanehavenoabilityto
extract the active constituent from S. nigrum L. fruit to
inhibit B. subtilis, B. cereus, P. aeruginosa and S. typhi.
Kaushik et al., (2009)21 report is in divergence with our
resultswhocheckedethanolextractoffruitofS.nigrum
against S. aureus and B. subtilis, and inhibition was
recordedatallthetestedconcentrations(100,75,50and
25mg/ml).Itmaybeduetoethanoluseforextractionby
Kaushiketal.,(2009)21.DataisshowninFig.2.

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Int.J.Pharm.Sci.Rev.Res.,14(2),2012;n03,2529ISSN0976044X
Table1:AntibacterialActivityofSelectedPlants
Plants

Solvents
Methanol

SolanumnigrumLinn.
nhexane

Methanol
CannabissativaL.
nhexane

Methanol
DodonaeaviscosaJacq.
nhexane

Concentrations(mg/ml)
25
50
100
25
50
100
25
50
100
25
50
100
25
50
100
25
50
100

Ciprofloxacin(100g/ml)

Diameterofzoneofinhibition(mm)
B.s
B.c
E.c
P.a
S.t

130.1 12.60.2 16.10.1 100.3

14.70.1 140.4 17.20.4 15.20.3

16.30.4 200.2
200.2 16.60.2

100.1

110.3

14.60.5

13.60.1 13.10.3 11.30.2

13.50.1
190.5 15.20.2 120.1 11.40.2 140.2
19.30.1 160.2 23.30.2 14.20.2 240.4
12.50.1

13.70.2

150.5
110.2
100.3

12.20.5

100.2 11.50.1

11.80.1

10.50.4 13.60.1 14.70.5 14.30.1

100.4 15.40.2 18.20.2 17.30.1 14.60.1

11.10.1 120.3

13.10.3

13.20.1 180.1

150.2
31.10.2 26.90.2 30.20.1 25.10.3 28.40.1

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Int.J.Pharm.Sci.Rev.Res.,14(2),2012;n03,2529ISSN0976044X
The methanol extract of C. sativa L. leaves was active
against all the bacterial strains except P. aeruginosa at
25mg/ml concentration. The extract showed highest
activityagainstallstrainswhichisshowninFig.3.
The nhexane extracts of C. sativa L leaves were active
against B. subtilis at all concentrations and showed
activityagainstB.cereus,E.coliandS.typhiat100mg/ml.
P. aeruginosa showed resistance at all concentrations of
nhexane extract. Wasim et al., (1995)22, published that
ethanolandpetroleumetherextractsoftheleavesofC.
sativaL.exhibitedactivitybothagainstGrampositiveand
Gramnegativebacteria.
B. subtilis showed highest resistance to methanol leaves
extract of D. viscosa Jacq. and highest lethality was
recordedforE.coliandP.aeruginosa.
The nhexane extracts (Fig. 4) of D. viscosa Jacq. leaves
completelyfailedagainstB.subtilisandP.aeruginosabut
active against B. cereus, E.coli and S. typhi at 50mg/ml
and100mg/mlconcentrations.Previously,Khurrametal.,
(2009)23 reported that the nhexane shoot extracts of D.
viscosa Jacq. showed inhibition against E. coli and P.
aeruginosa, B. subtilis and S. typhi. Getie et al., (2003)24
also reported that the crude extracts of the leaves of
D.viscosa Jacq possess antibacterial activity against
Streptococcus pyogenes and staphylococcus aureus. Our
results correlated with Thring et al., (2007)25 previously
reported that the aqueous, methanol, ethanol and ethyl
acetate leaf extracts of D. viscosa Jacq showed activity
againstP.aeruginosa.ThedataisshowedinFig.5and6.
CONCLUSION
The fruit of S. nigrum L, leaves of D. viscosa J. and C.
sativa L. showed antibacterial activity. These plants
containedactiveantibacterialconstituents.Furtherwork
isneededtoisolatetheseactiveagentswhichmaybeuse
asagoodsourceforantibiotics.
Acknowledgment:Theauthorsoweadepthofgratitude
totheDepartmentofPharmacy,UniversityofMalakand,
Chakdara, KPK, Pakistan for facilitating the research and
also thankful to Madam Zakia Ahmad, Lecturer in the
DepartmentofBotany,UniversityofMalakand,Chakdara,
KPK,Pakistanforherassistanceinidentifyingplants.
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