Biyoloji Bilimleri Araştırma Dergisi 2 (2): 97-101, 2009
ISSN:1308-3961, www.nobel.gen.tr 97
In vitro evaluation of antimicrobial activity of crude extracts of medicinal plants against multi
drug resistant pathogens
Nanda Kumar RAJENDRAN¹, Jayapradha RAMAKRISHNAN²
¹School of Chemical and Biotechnology, SASTRA University, Thanjavur, India
Corresponding author Geliş Tarihi : 10 Ekim 2008
e-mail: firstname.lastname@example.org Kabul Tarihi : 15 Kasım 2008
In vitro antibacterial activity of aqueous and methanol extracts of some medicinal plants were screened against multi-drug resistant bacteria including
Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Streptococcus pyogenes isolated from
clinical specimen. The selection of medicinal plants was based on the fact that most of these plants were not previously screened against multi-drug resistant
pathogenic organisms. Methanol extracts of W.somnifera showed higher antibacterial activity compared to other plant extracts tested. The minimum inhibitory
concentration (MIC) of methanol extracts of W.somnifera was in the range of 50 to 100 mg/ml. The antibacterial activity of the extracts was not signiﬁcantly
affected by heating at 100oC for 1 h but the activity was lost at pH 8. Phytochemical studies on the leaf extracts of medicinal plants revealed the presence of
alkaloids, saponins, Cardiac glycosides, steroids, tannins and flavonoids.
Keywords: Antimicrobial activity, W.somnifera, multidrug resistant pathogens, MIC, Phytochemical studies
INTRODUCTION MATERIALS AND METHODS
There is a continuous and urgent need to discover new antimicrobi- Plant materials
al compounds with diverse chemical structures and novel mechanisms The plant leaves were collected locally and were further
of action because there has been an alarming increase in the incidence identiﬁed and the respective voucher specimens have been de-
of new and re-emerging infectious diseases. Another big concern is the posited in the Department of Environment and herbal science,
development of resistance to the antibiotics in current clinical use. In re- Tamil University, Thanjavur, India.
cent years, drug resistance to human pathogenic bacteria has been com-
Extraction of Plant Materials and their extractive value
monly reported from all over the world. The drug-resistant bacteria and
fungal pathogens have further complicated the treatment of infectious Aqueous Extraction
diseases in AIDS and cancer patients [3, 14]. In the present scenario For aqueous extraction, 10 g of air dried powder was placed
of emergence of multiple drug resistance to human pathogenic organ- in 100 ml of distilled water (1:10 ratio) and boiled for 6 hours and
isms, this has necessitated a search for new antimicrobial substances then ﬁltered. The ﬁltrate was condensed in boiling water bath and
from other sources including plants. Higher plants produce hundreds used for further analysis .
to thousands of diverse chemical compounds with different biological Methanol extraction
activities . The antimicrobial compounds produced by plants are 10 g of air dried powder was placed in 100 ml of methanol
active against plant and human pathogenic microorganisms . The in a conical flask and kept in rotary shaker at 150 rpm for 24 h.
substances that can either inhibit the growth of pathogens or kill them After 24 h, it was ﬁltered and the solvent was evaporated to make
and have no or least toxicity to host cells are considered candidates for the ﬁnal volume one-fourth of the original volume. It was stored
developing new antimicrobial drugs. It is expected that plant extracts at 4oC for further studies. The extractive values of aqueous and
showing target sites other than those used by antibiotics will be active methanolic plant extracts were analysed .
against drug-resistant microbial pathogens. However, very little infor-
Preliminary Phytochemical Analysis
mation is available on such activity of medicinal plants [6, 8].
Qualitative phytochemical analysis of the of the plants col-
In the present study, we have selected some medicinal plants to be
lected was determined as follows: Phytochemical analysis for
screened against multi-drug resistant bacteria including Staphylococ-
major phyto constituents of the plant extracts was undertaken us-
cus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escher-
ing standard qualitative methods as described by various authors
ichia coli, Proteus mirabilis and Streptococcus pyogenes. The selection
[4, 7, 12, 15, 17]. The plant extracts were screened for the pres-
of medicinal plants is based on the fact that most of these plants were
ence of biologically active compounds like glycosides, alkaloids,
not previously screened against multi-drug resistant pathogenic organ-
tannins, flavonoids, saponins and steroids.
isms. Phytochemical analysis of active plant extracts for their major
group of phytoconstituents and the active group of certain extracts is Microrganisms used
also reported here. Clinical isolates of Escherichia coli, Klebsiella Pneumoniae,
98 N. K. Rajendran ve J. Ramakrishnan / Bibad, 2 (2): 97-101, 2009
Proteus mirabilis, Pseudomonas aeruginosa, Staphy- ously adjusted to a concentration of 105 cells/ml was then
lococcus aureus, and Streptococcus pyogenes obtained introduced. A set of test tubes containing broth alone were
from the Department of Microbiology, Thanjavur Gov- used as control. All the test tubes and control were then
ernment Medical College were used in this study. All incubated at 37oC for 18 h. After the period of incuba-
cultures were bio chemically tested for purity. tion, the tube containing the least concentration of extract
Culture media and inoculum preparation showing no visible sign of growth was considered as the
minimum inhibitory concentration 
Clinical isolates were frequently sub cultured and
maintained in Nutrient Agar plates. For Antimicrobial as- Stability tests
say, microbial cultures freshly grown at 37°C were appro- pH and temperature stability of the extracts were de-
priately diluted in sterile normal saline solution to obtain termined using the method of Sammuelson . 50 mg/
the cell suspension at 105CFU/ml. ml concentration of the constituted extracts was treated
Antibiotic resistance of test strains at 60oC and 100oC separately in a water bath for 60
min. The antimicrobial activity of each of the treated
Antibiotic sensitivity of test strains was determined by
and non-treated samples was determined as described
the standard Disc diffusion against a number of antibiotics
earlier. For determination of effect of pH, 50 mg/ml dilu-
. The potency of antibiotics per disc was as follows,
tions of the samples were adjusted to pH ranges between
Amoxyclav (Ac), Clindamycin (Cd) (10 µg/ disc each);
6.0 and 8.0 using 0.1 N HCl and 0.1N NaOH. The antimi-
Clarithromycin (Cw) (15 µg/ disc each); Cefaclor (Cj),
crobial activity of each of treated and non-treated extracts
Cloxacillin (cx) (30 µg/ disc). All antibiotic discs were
purchased from the Hi-Media Pvt.Ltd. (Bombay, India).
Antimicrobial susceptibility testing
The agar well diffusion method  as adopted ear- RESULTS AND DISCUSSION
lier  was used; 0.1 ml of diluted inoculum (105CFU/
ml) of test organism was spread on MHA plates. Wells of Phytochemical Analysis
8 mm diameter were punched into the agar medium and The Phytochemical screening of the plant extracts re-
ﬁlled with 100 µl (150 mg/ml) of plant extract and solvent vealed the presence of tannins, alkaloids and flavanoids
blanks. The plates were incubated for 18 h at 37°C. Anti- in all the plant extracts studied as shown in Table 1. The
microbial activity was evaluated by measuring the zone of presence of saponins, tannins, alkaloids, flavanoids, ste-
inhibition against the test organism. roids and cardiac glycosides provoked for an in-depth
Determination of the minimum inhibitory concen- study on the plant. The metabolites are of various phar-
tration (MIC) macological importance. Many triterpene saponins and
The methanol extract of W.somnifera which showed their aglycones have been reported to have varied uses as
signiﬁcant antimicrobial activity were selected for deter- anti-ulcerogenic, anti-inflamatory, ﬁbrinolytic, antipyret-
mination of MIC. A stock solution of 100 mg/ml was pre- ic, analgesic and anti-edematous in action. The presence
pared. This was serially diluted to obtain various ranges of tannin in most of plant extract could be responsible for
of concentrations between 5mg/ml to 100mg/ml. 0.5 ml possible antitumor and anti oxidant activities [7, 11].
of each of the dilutions of different concentrations was Antibiotic resistance of test strains
transferred into sterile test tube containing 2.0 ml of nutri- Results in Table 2 showed that test organisms exhib-
ent broth. To the test tubes, 0.5 ml of test organism previ- ited varying degrees of multi drug resistance to standard
Table 1. Phytochemical screening of the plant extracts
+ indicates Presence and - indicates Absence
N. K. Rajendran ve J. Ramakrishnan / Bibad, 2 (2): 97-101, 2009 99
antibiotics disc used in this study. The test organisms pneumoniae, Proteus mirabilis, P. aeruginosa, S. aureus
used in this study were resistant to Clindamycin (Cd) and Streptococcus pyogenes) were studied. The results
, Cloxacillin (cx) and Clarithromycin (Cw) at 10 µg/disc, presented in the table 3, showed that MeOH extract in-
30 µg/disc and 15 µg/disc respectively ,but susceptible to hibited the growth of most of the organisms used. But
Amoxyclav (Ac) at 10 µg/disc and Cefaclor (Cj) 30 µg/ aqueous extract showed least activity against the micro
disc . When compared to the antibacterial activity of organisms.
the extracts it was observed that the inhibitory activity Minimum inhibitory concentration of the active ex-
of the methanol extracts of W.somnifera at 50 mg/ml tract against the test organisms
was greater than those of the standard antibiotics. Most
The MIC results are presented in Table 4. The crude
interestingly these extracts exerted inhibitory properties
extract of the Withania somnifera inhibited and fully
on β-lactamase positive isolates.
prevented the growths of Proteus mirabilis at a concen-
Antimicrobial susceptibility testing tration of 50 mg/ml, E.coli and P.aeruginosa at a concen-
In the present investigation crude extracts were de- tration of 60 mg/ml, K.pnemoniae at a concentration of 80
rived from the plant leaves and their efﬁcacy to inhibit mg/ml, S.aureus and Streptococcus pyogenes at a concen-
the growth of Multi drug resistant organisms (E. coli, K.
Table 2. Antibiogram of clinical isolates against various standard drugs
Conc. = Concentration
Table 3. Effect of aqueous and methanolic extract on microbicidal activity
-- indicates no zone of inhibition
Aq = aqueous extracts
MeOH = Methanolic extracts
100 N. K. Rajendran ve J. Ramakrishnan / Bibad, 2 (2): 97-101, 2009
tration of 100 mg/ml as recorded in Table. Hence the crude ing as reported that many natural antimicrobials including
leaf extract contains maximum active phyto compounds. honey are active against animal pathogens at acidic pH .
The stability of the antimicrobial activity of the extracts
of Withania somnifera was analysed by heating at 60oC
and 100oC for one hour and the results from the table 5 and
 Ahmad I, Mehmood Z, Mohammad F. 1998. Screening
6 depicted that the stability of crude extract showed slight de-
of some Indian medicinal plants for their antimicrobial
crease with increased temperature treatment. Also, the stabil- properties. Journal of Ethnopharmacology. 62: 183–193.
ity of the antimicrobial property at acidic pH is not surpris-
Table 4. MIC of methanolic extract of W. somnifera against human drug resistant pathogens
*= MIC concentration, --= No growth, += cloudy solution (slight growth), ++= Turbid
solution (strong growth), +++= Highly turbid solution (dense growth)
Table 5. Effect of temperature on stability of the antimicrobial activity of the extracts of Withania somnifera
+++= Highly stable, ++= Moderately stable, += Slightly stable and --= Not stable
Table 6. Effect of pH on stability of the antimicrobial activity of the extracts of Withania somnifera
++= Stable and --= not Stable
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