British Microbiology Research Journal
1(3): 70-78, 2011
SCIENCEDOMAIN international
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In vitro Evaluation of Antibacterial Activity of
Various Crude Leaf Extracts of Indian Sacred
Plant, Ocimum sanctum L.
Pankaj Goyal*1 and Purshotam Kaushik1
1
Department of Botany and Microbiology, Gurukul Kangri University, Hardwar,
Uttarakhand, India.
Received 13th May 2011
Research Article Accepted 28th June 2011
Online Ready 16th July 2011
ABSTRACT
The medical world is on an immense requirement to discover novel antibiotics due to
widespread emergence of resistance among microbial pathogens against currently available
antibiotics. Traditional plants have been proved to be better source in the search for novel
antimicrobial compounds. In such effort, we accessed the susceptibilities of some clinically
significant bacterial species against various extracts made up from leaves of Ocimum
sanctum L. (family: Lamiaceae). Antibacterial activity of crude extracts was found to be
reliant on the nature of extract and the bacterial strains evaluated. Methanol extract was
found to have comparatively higher activity than other organic and aqueous extracts. Gram-
positive bacteria showed variable susceptibilities while Gram-negative Salmonella typhi has
shown to be completely resistance to all the tested extracts. Minimum inhibitory
concentration data showed hopeful results as some of the extracts exhibited significant
inhibitions of bacteria even at low concentrations. This study indicated that leaves of
Ocimum sanctum L. have significant antibacterial activity and it could be very useful in the
discovery of novel antibacterial/antimicrobial agents.
Keywords: Ocimum sanctum; Indian sacred plant; resistance; antibiotics; natural products;
antibacterial potential; susceptibility assay; minimum inhibitory concentration;
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British Microbiology Research Journal, 1(3): 70-78, 2011
ABBREVIATIONS
CFU: Colony Forming Unit; GC-MS: Gas Chromatography & Mass Spectrometry; IR: Infra-
Red Spectroscopy; LC-MS: Liquid Chromatography & Mass Spectrometry; MDRTB:
Multidrug-Resistant Mycobacterium tuberculosis; MHA: Mueller Hinton Agar; MIC: Minimum
Inhibitory Concentration; mm: Millimeter; ml: Milliliter; MRSA: Methicillin-resistant
Staphylococcus aureus; MTCC: Microbial Type Culture Collection; NCCLS: National
Committee for Clinical Laboratory Standards; NCR: National Capital Region of India; NMR:
Nuclear Magnetic Resonance; µg: Microgram; µl: Microliter; VRE: Vancomycin-resistant
Enterococcus faecalis.
1. INTRODUCTION
In recent years, there has been a relentless increase in the occurrence of antibiotic
resistance to many common bacterial pathogens such as Staphylococcus aureus,
Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Escherichia
coli, Klebsiella pneumonia, Pseudomonas aeruginosa etc. Furthermore, methicillin-resistant
Staphylococcus aureus (MRSA), penicillin-resistant Pneumococcus, vancomycin-resistant
Enterococcus faecalis (VRE), and multidrug-resistant Mycobacterium tuberculosis (MDRTB)
are now commonplace pathogens that are proving difficult to treat effectively (Russell, 2002).
Antibiotic resistance among bacterial pathogens is also increased in the hospital
environment which is being considered in the context of its source, effects on individual
patients and on hospital practice (Cohen, 1992; Gold and Moellering, 1996; Russell, 2002).
This phenomenon of resistance among diverse bacterial pathogens against currently
available antibiotics leads researchers to discover novel sources of antimicrobial drugs; thus,
there has been a renewed interest in natural products from plants. These natural products
would be able to provide a unique element of molecular diversity and biological functionality,
which is indispensable for drug discovery (Perez et al., 1990; Nisbet and Moore, 1997).
Historically, plants have been placed at top among the sources of novel drugs with
antimicrobial activity, as traditional medicines based on plants and plant extracts have made
considerable contributions to human health and well-being. Plants provide a natural blueprint
for the development of new drugs (Cragg et al., 1997; Iwu et al., 1999). It is estimated that
plant materials have provided the models for more than 50% Western drugs today (Robbers
et al., 1996). Plant based antimicrobials represent a vast untapped source for medicines and
they provide enormous therapeutic potential. They are effective in the treatment of infectious
diseases while simultaneously mitigating many of the side effects that are often associated
with synthetic antimicrobials, and they offer more affordable treatment (Murray, 1995; Iwu et
al., 1999).
India is a land of biodiversity in terms of plant species. Various plants have been mentioned
in Ayurveda, an ancient Indian Sanskrit literature, for their therapeutic advantages (Kaushik,
1988). Many herbs used by Ayurvedic practitioners show promising results in the treatment
of various ailments and these herbs could be appropriate for large randomized trials. Many
potent drugs have been purified from medicinal plants having anti-rheumatic, anti-
thrombotic, antimalarial, anticancer, antidiabetic and antimicrobial properties (Kaushik and
Dhiman, 2000).
Tulasi (Holy Basil) is a traditional plant considered sacred by the Hindus. This religion links
the plant with the Goddess figure as described in the Puranas. Hindus regard it as an earthly
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manifestation of goddess Vrindavani, who is dear to Lord Vishnu. The name “Tulasi” in
Sanskrit means “the incomparable one.” The Shyama Tulasi or Krishna Tulsi (Ocimum
sanctum L. syn. Ocimum tenuiflorum) possesses great medicinal value as mentioned in
Charak Samhita, an ancient Indian literature. It is a most common household plant in India
and grows wild in tropics. Native to India, it is a short lived perennial herb or small shrub of
Mint family Labiatae (Lamiaceae). It has small leaves with a strong smell and purple flowers.
The foliage is green or purple, strongly scented. Oil extracted from leaves of this plant
possesses significant insecticidal properties (Nanasombat and Lohasupthawee, 2005).
Ocimum sanctum has been extensively studied for therapeutic potentials in various areas
like immuno-stimulation, anticancer antioxidant, as adjuvant to radiotherapy, antiulcer,
analgesic and antidiabetic (Hammer et al., 1999).
The present study aimed to investigate the susceptibility of several clinically significant
bacterial strains against various crude extracts prepared from the leaves of Ocimum
sanctum (Tulsi). The minimum inhibitory concentrations (MICs) were also determined for the
crude extracts showing significant activity against the bacterial strains selected for the
susceptibility assay.
2. MATERIAL AND METHODS
2.1 Collection of Suitable Plant Material
Ocimum sanctum (Figure 1) was collected in the month of May & June of 2007 from semi-
arid, unshaded land of Ghaziabad (NCR), India. The plant was taken to the laboratory and
was authenticated by Prof. P. Kaushik, Gurukul Kangri University. Leaves suitable for
extraction were plucked and were washed under running tap water followed by sterilized
distilled water. Leaves were air-dried, powdered and were subjected to the following
extraction protocols.
2.2 Aqueous Extraction
Air-dried powder of Tulsi leaves (10 g)
was boiled in 400 ml distilled water till one
fourth of the extract, initially taken, was
left behind after evaporation. The solution
was then filtered using muslin cloth.
Filtrate was centrifuged at 5000 rpm for
15 minutes. The supernatant was again
filtered using Whatman filter no. 1 under
strict aseptic conditions and the filtrate
was collected in fresh sterilized bottles
and stored at 4°C until further use.
Fig. 1. Ocimum sanctum plant
2.3 Organic Solvent Extraction
Air-dried powder (10 g) was thoroughly mixed with 100 ml organic solvent viz., ethanol,
methanol, hexane or ethyl acetate. The mixture was placed at room temperature for 24 h on
shaker with 150 rpm. Solution was then filtered through muslin cloth and then re-filtered
through Whatman filter no. 1. The filtrate thus obtained was concentrated by complete
evaporation of solvent at room temperature to yield the pure extract. Stock solutions of
various organic crude extracts were prepared by mixing well the appropriate amounts of
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dried extracts and suitable solvent to give rise a final concentration of 100 mg/ml. Each
solution was stored at 4°C after collecting in sterilized bottles until further use.
2.4 Test Bacteria for Susceptibility Assay
Tulsi extracts were evaluated for their antibacterial potential against both Gram-positive and
Gram-negative bacteria. The bacterial strains were collected from Microbial Type Culture
Collection (MTCC), Institute of Microbial Technology (IMTech), Chandigarh, India. Bacterial
species examined were Escherichia coli MTCC 739, Salmonella typhi MTCC 531, Bacillus
cereus MTCC 430, Bacillus subtilis MTCC 736, Streptococcus pyogenes MTCC 442 and
Staphylococcus aureus MTCC 740. These bacterial cultures were maintained in nutrient
agar slants at 37°C. Each of the bacteria was reactivated prior to susceptibility testing by
transferring them into a separate test tube containing nutrient broth and incubated overnight
at 37°C.
2.5 Bacterial Susceptibility Assay
In vitro antibacterial activities of all aqueous and organic extracts from dried leaves of Tulasi
plant were determined by standard agar well diffusion assay (Perez et al., 1990). Petri
dishes (size 100 mm diameter) containing 18 ml of cool and molten Mueller Hinton Agar
(MHA) (at 40°C) were seeded with 100 µl inoculum of bacterial strain (inoculum size was
adjusted so as to deliver a final inoculum of approximately 1.0 x 108 CFU/ml). Media was
allowed to solidify and then individual Petri dishes were marked for the bacteria inoculated.
Wells of 6 mm diameter were cut into solidified agar media with the help of sterilized core-
borer. Aliquot 100 µl of each extract was poured in the respective well and the plates were
incubated at 37°C overnight. Organic solvents, in which extracts were prepared, were used
as negative control while tetracycline antibiotic of one unit strength was used as positive
control. The experiment was performed in triplicate under strict aseptic conditions. The
antibacterial activity for each of the extract evaluated was expressed in terms of the average
of the diameter of zone of inhibition (in mm) produced by the respective extract at the end of
incubation period. Standard deviations were also calculated and represented in the
respective table against each extract.
2.6 Determination of Minimum Inhibitory Concentration
Extracts producing an inhibition zone ≥15 mm in diameter were screened to determine
minimum inhibitory concentrations (MICs) by standard two-fold microbroth dilution
methodology given by NCCLS (1997). A stock solution of each active extract was serially
diluted in 96-wells microtiter plate with Mueller Hinton broth to obtain a concentration ranging
from 8 µg/ml to 4096 µg/ml. A standardized inoculum for each bacterial strain was prepared
so as to give inoculum size of approximately 5 x 105 CFU/ml in each well. Microtiter plates
were then kept at 37°C for an overnight incubation. Following incubation, the MIC was
calculated as the lowest concentration of the extract inhibiting the visible growth of bacterial
strain.
All the chemical ingredients used in present study were of analytical grade, and were
purchased from Hi Media, India.
3. RESULTS AND DISCUSSION
Data of antibacterial activity of various crude extracts prepared from dry leave of Ocimum
sanctum (Tulsi) are demonstrated in Table 1. Data indicated that the most active extract was
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found to be of methanol which inhibited a total of four bacteria studied in the range of 11.86
mm to 18.50 mm size of inhibition zone. Ethanol, ethyl acetate, and aqueous extracts were
found to be effective only against Staphylococcus aureus and Escherichia coli but, with
comparatively lower activity than that of methanol extract. Hexane extract was found
completely inactive against all the organisms tested.
Staphylococcus aureus (a Gram-positive bacterium) was observed as most susceptible
bacterium as it was inhibited by almost all the extracts except hexane extract. Streptococcus
pyogenes and Salmonella typhi was found to be resistant to all the extracts tested.
MICs of different active extracts from leave of tulsi had been demonstrated in Table 2.
Staphylococcus aureus was inhibited at 1024 µg ml-1 by methanol extract, while this
bacterium was inhibited at 2048 µg ml-1 concentration of ethanol and aqueous extracts.
Inhibitions of Staphylococcus aureus, Escherichia coli, and Bacillus subtilis were not seen at
even the maximum concentrations of ethyl acetate, ethanol and methanol extracts,
respectively (i.e. MIC >4096 µg ml-1).
Some earlier studies have found the inhibitory effect of methanol extracts of leaves of
Ocimum basilicum in the range of 10 mg ml-1 against Staphylococcus aureus and 40 mg ml-1
against Escherichia coli. Our findings are in agreement with the results of these studies done
with other species of Ocimum (Grosvenor et al., 1995; Navarro et al., 1996). In another
study, ethanol extracts of Ocimum sanctum leaves have been found completely inactive
against studied microorganisms (Nanasombat and Lohasupthawee, 2005).
It is in agreement with our investigation, since ethanol extract was found to have very weak
activity against Staphylococcus aureus and Escherichia coli while other bacteria used in the
current study were found to show complete resistance in this extract. The leaf extracts of
Ocimum gratissimum prepared by using cold water, hot water, and steam distillation
methods, were evaluated against Salmonella typhi, Salmonella typhimurium,
Staphylococcus aureus and Escherichia coli. Only the steam distillation extract inhibited the
growth of the organisms with zone size ranging from 30 mm to 39 mm for different bacteria
and MIC was evaluated just 0.1% for Staphylococcus aureus (Adebolu and Oladimeji, 2005).
The resistance of the organisms to the other extracts could be due to the evaporation of the
oil during the boiling or due to insufficient release of the oil during cold extraction. The
antimicrobial property present in the oil was probably due to the eugenol. Essential oil of
Ocimum gratissimum was found significant inhibitory, while the oil from Ocimum basilicum
and Zingiber officinale was found to have less activity (Nguefack et al., 2004).
Variations in the activity level might be attributed to the low affinity between less polar
components of the plant extract and the polar substrate (agar). Opalchenova & Obreshkova
(2003) observed the antibacterial effect of Ocimum basilicum (basil) essential oil against
multidrug resistant clinical isolates of Staphylococcus, Enterococcus and Pseudomonas with
MIC reported between 0.0030% and 0.0007% (v/v).
Basil oil has limited solubility in aqueous media, thus showing very limited activity in aqueous
extract. Variable antibacterial activity in different extracts of Ocimum species against
different bacterial strains were evaluated (Nakamura et al., 1999; Adiguzel, 2005; Mbata and
Saikia, 2005).
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Table 1. In vitro antibacterial activity of aqueous and organic extracts of Ocimum sanctum leaves
Zone of Inhibition* ( in mm diameter)
Gram-Negative Bacteria Gram-Positive Bacteria
Type of Extract
Escherichia Salmonella Streptococcus Staphylococcus Bacillus Bacillus
coli typhi pyogenes aureus subtilis cereus
Ethanol 12.50±0.50 NI NI 13.83±0.58 NI NI
Organic Methanol 14.67±0.58 NI NI 18.50±0.87 12.70±0.75 11.86±0.81
Extract Ethyl Acetate 10.27±1.42 NI NI 13.20±0.72 NI NI
Hexane NI NI NI NI NI NI
Aqueous Extract 11.93±1.01 NI NI 15.23±0.40 NI NI
Tetracycline 29.50±0.50 25.83±1.61 29.83±1.89 32.50±1.50 34.17±1.76 32.16±1.04
Ethanol NI NI NI NI NI NI
Controls
Methanol NI NI NI NI NI NI
Ethyl Acetate NI NI NI NI NI NI
Hexane NI NI NI NI NI NI
*
Mean of three values ± Standard Deviation. ‘NI’-No Inhibition was observed.
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Table 2. Minimum inhibitory concentration of active crude extracts of Ocimum sanctum leaves
Type of Concentration of Extracts*
(in µg ml-1) MIC
Active Crude Test Microorganisms
(in µg ml-1)
Extracts
4096 2048 1024 512 256 128 64 32 16 8
Ethanol Staphylococcus aureus - - + + + + + + + + 2048
Ethanol Escherichia coli + + + + + + + + + + >4096
Methanol Bacillus subtilis + + + + + + + + + + >4096
Methanol Staphylococcus aureus - - - + + + + + + + 1024
Methanol Escherichia coli - + + + + + + + + + 4096
Ethyl Acetate Staphylococcus aureus + + + + + + + + + + >4096
Aqueous Staphylococcus aureus - - + + + + + + + + 2048
Tetracycline Staphylococcus aureus - - - - - - - - - - <8
* (-) represents ‘No Growth Observed’; (+) represents ‘Growth Observed
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4. CONCLUSION
The current scenario of antibiotics is very threatening with significant emergence of
resistance among bacterial pathogens against available antibiotics. The present
investigation reveals that Ocimum sanctum and other plants would be the major source in
finding metabolites with greater efficacy against resistant bacteria pathogens. The present
study focused to the discovery of novel antibacterial/antimicrobials that can lead to the
development of pharmaceuticals of plant origin. Since, the present study was focused only to
examine the antibacterial potential of crude extracts; therefore further study can be made to
isolate the pure compounds responsible for the activity from the extracts with the help of
numerous advanced technologies such as GC-MS, LC-MS, IR and NMR.
ACKNOWLEDGMENTS
Authors are highly thankful to Dr. Abhishek Chauhan (Gurukul Kangri University, Hardwar,
Uttarakhand, India) for his assistance during the research work and manuscript preparation.
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© 2011 Goyal & Kaushik; This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted
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