Int. J. Pharm. Sci. Rev. Res., 53(2), November - December 2018; Article No.

05, Pages: 24-28 ISSN 0976 – 044X

Research Article

Phytochemical Analysis and Antimicrobial Activity of Ocimum tenuiflorum (Tulsi), A Known

Indian Folk Medicinal Plant
P. Saravanakumar, S. Thangapandiyan*, R. Dharanipriya, S. Gowri Shankar
PG & Research Department of Zoology, PSG College of Arts & Science, Coimbatore, Tamil Nadu, India.
*Corresponding author’s E-mail: stp.nano@gmail.com

Received: 20-08-2018; Revised: 15-10-2018; Accepted: 18-11-2018.

ABSTRACT
In recent years researchers have been paid attention to find out new alternative sources of antimicrobial agents especially
from plant sources. Ocimum tenuiflorum (tulsi) has served human kind as sources of medicinal agents since its earliest
beginnings. Phytoconstituents present in leaves, barks& flower spikes and to characterize the compounds responsible for
microbial activity. Phytochemical screening of the plant leaves reveals that the presence of saponins, alkaloids, flavonoids,
steroids, phenols, tannins and glycosides. Different leaf extracts of O. tenuiflorum L were prepared and its antimicrobial
activity were evaluated by agar well diffusion method against both gram positive &gram negative bacterial strains as well as
three fungal pathogens. Ethanol extract had maximum zone of inhibition (18±1.25) against salmonella gallinarum, whereas
ethanol& methanol extracts were exhibited minimum zone of inhibition (7±0.47) against staphylococcus aureus. Acetone
extract had maximum zone (17±0.81) of antifungal activity against Aspergillus Niger. Antimicrobial activity of various extract
of leaves of O. tenuiflorum was carried in attempt to develop a new pharmaceutical drug from natural origin for the
treatment of pathogenic microbes.
Keywords: O. tenuiflorum leaves, phytoconstituents, antimicrobial activity.

INTRODUCTION common sugars, amino acids, proteins and chlorophyll

while secondary constituents consist of alkaloids,

S ynthetic drugs are not only expensive and

inadequate for the treatment of diseases but also
often associated with adulterations and side effects.
Therefore, there is need to search new infection fighting
terpenoids, steroids and flavonoids.5
O. tenuiflorum or Ocimum sanctum is the most sacred
herb and it is otherwise called Krishna tulsi.6 Tulsi is
strategies to control microbial infections. However, the known as queen of medicinal plants. It is known to be an
past record of rapid, widespread emergence of resistance important medicinal plant from ancient period in India.
to newly introduced antimicrobial agents indicates that Studies show that many Ocimum species are useful for
even new families of antimicrobial agents will have a the treatment of disorders in central nervous system
short life expectancy while there are some advantages of (CNS) and also as antidepressant.7 In traditional medicine,
using medicinal plants, such as often fewer side effects, aqueous extract of Ocimum teneiflorum leaves is used for
better patient tolerance, relatively less expensive, common cold and fever. Whole plant is also used for
8
acceptance due to long history of use and being treating jaundice and for alleviating blood pressure.
renewable in nature. For these reasons, researchers are
The novel medicinal plant Ocimum sanctum has been
increasingly turning their attention to herbal products,
reported for its antimicrobial activity and it exhibited
looking for new leads to develop better drugs against
profound activity against various pathogenic
multiple drug resistant microbial strains. 1,2 The primary
microorganisms. The antimicrobial activities of the plant
benefits of using plants derived medicines are that they
extract can be attributed to the presence of secondary
are relatively safer than synthetic alternatives offering
metabolites including glycosides, alkaloids, steroids,
profound therapeutic benefits and more affordable
3 terpenoids, flavonoids and saponins. These active
treatment. Phytoconstitutens are the natural bioactive
components usually interfere with the growth or
compounds found in plants. Phytochemicals have
metabolism of microorganisms in a negative manner
antioxidant or hormone-like effect which helps in fighting
resulting in cell death.9
against many diseases including cancer, heart disease,
diabetes, high blood pressure and preventing the Antimicrobial drug resistance is a global problem today as
formation of carcinogens on their target tissues. 4 These the resistant microorganisms have emerged and spread
phytoconstituents work with nutrients and fibers to form throughout the world because of their genetic plasticity.
an integrated part of defense system against various Natural products of plants have been considered as the
diseases and stress conditions. Phytochemicals are active ingredients of most of the modern medicines.
basically divided into two groups, i.e. primary and Therefore, the aim of the current study has been focussed
secondary constituents according to their functions in to screen the effect of O. tenuiflorum on diversified
plant metabolism. Primary constituents comprise pathogenic microorganisms such as Staphylococcus

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Int. J. Pharm. Sci. Rev. Res., 53(2), November - December 2018; Article No. 05, Pages: 24-28 ISSN 0976 – 044X

aureus, Bacillus subtilis, Pseudomonas flurescense, Assay to evaluate antibacterial activity of plant extracts
Salmonella gallinarum and fungal pathogens Candida
The antibacterial activity of plant extract was evaluated
albicans, Aspergillus niger, Aspergillus flavus, and
using agar well diffusion method.11 Pure cultures of each
Trichodserma viride. The microorganisms were chosen in
bacterial strain were sub cultured in nutrient broth on a
this study are ubiquitous in our environment and are the
rotary shaker at 200 rpm for 24 hours at 37oC. For
primary causative agents of most common infections.
preparing Mueller Hinton agar (MHA) plates, the MHA
MATERIALS AND METHODS medium was boiled to dissolve completely and sterilized
o
by autoclaving at 15 lbs pressure (121 C) for 30 minutes.
Collection and identification of plant
After sterilization, 20 ml of MHA media was poured into
Healthy, disease free, mature leaves of O. tenuiflorum the sterile petri plates and kept at room temperature for
were collected from the region of Annur, Coimbatore solidification. Then, each strain was swabbed uniformly
District, and Tamil Nadu. The collected plant material was onto the individual Mueller Hinton agar plates using
identified and authenticated by Botanical Survey of India, sterile cotton swabs. Wells of 6 mm diameter were made
Tamil Nadu Agricultural University, Tamil Nadu, where the on Mueller Hinton agar plates using sterile cork borer and
voucher sample was preserved. 50 µl of plant extracts were poured into each well on all
o
plates. The plates were incubated overnight at 37 C and
Preparation of leaf material
the results were observed by the presence of bacterial
Leaves of selected plant were plucked and washed growth inhibition zone around the sample loaded well
thoroughly with running tap water. It was again washed and their diameters (mm) were measured using
with sterile distilled water to remove dirt prior to drying measuring scale. The assay was performed in triplicates.
process. The leaves were dried in shade at room
RESULTS AND DISCUSSION
temperature for a week to remove the moisture content
and powdered using mixer grinder. Finally, powdered Collection and identification of plant
sample was stored at room temperature for further
Healthy, disease free, mature leaves of O. tenuiflorum
studies.
were collected from the region of Annur, Coimbatore
Preparation of plant extract District, and Tamil Nadu. Fig.1 shows the O. tenuiflorum
plant with fresh leaves. O. tenuiflorum is commonly
2.5 g of powdered sample was taken in air tight bottles.
known as Tulsi is cultivated for religious and medicinal
To this, 50 ml of different solvents such as ethanol,
purposes. It is widely known across the Indian
methanol, acetone and distilled water was added. After 2
Subcontinent as a medicinal plant and an herbal tea.
days, the contents were stirred well thoroughly and
filtered using Whatmann No.1 filter paper. The filtrate
was collected and stored in sterile bottle at 4°C until
further use. For antibacterial studies, each extract was
prepared by dissolving 250 mg in 5 ml of 10% (v/v)
aqueous dimethyl sulphoxide (DMSO).
Phytochemical analysis
Freshly prepared leaf extracts were subjected to standard
phytochemical analyses using standard procedure.10 In
order to find out the presence of various
phytoconstituents such as alkaloids, terpenoids,
flavonoids, tannins, steroids, anthroquinones, saponins, Figure 1: Morphology of O. tenuiflorum
resins, glycosides and phenols.
Preparation of leaf material
Antimicrobial Studies of Plant Extracts
Fresh Leaves of O. tenuiflorum were collected and
Bacterial culture washed thoroughly with running tap water. It was again
The following bacterial pathogens namely Bacillus subtilis, washed with sterile distilled water to remove dirt prior to
Stphylococcus aureus, Pseudomonas flurescens, drying process. Afterwards the leaves were dried in shade
Salmonella gallinarum, and Proteus valgaris, were at room temperature for a week to remove the moisture
procured from the Microbial Type Culture Collection content and powdered using mixer grinder. Finally,
(MTCC), Chandigarh, India. All the cultures were grown on powdered sample was stored at room temperature for
nutrient agar plates and maintained in the nutrient agar further studies. Fresh drying powdered from O.
slants at 4°C. Overnight culture in the nutrient broth was tenuiflorum leaves were presented in Figs 2a, 2b
used for the present experimental study.

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Int. J. Pharm. Sci. Rev. Res., 53(2), November - December 2018; Article No. 05, Pages: 24-28 ISSN 0976 – 044X

extracts (ethanol, methanol, acetone and aqueous)

contains the presence of alkaloids, terpenoids, flavonoids,
tannins, steroids, anthroquinones, saponins, resins,
glycosides and phenols. Phytochemical analysis revealed
that O. tenuiflorum contained rich source of bioactive
compounds such as alkaloids, terpenoids, flavonoids,
tannins, steroids, anthroquinones, saponins, resins,
glycosides and phenols. Phytochemicals are divided into
two groups, which are primary and secondary
Figure 2: Fresh leaves (a) Powder form of O. tenuiflorum
(b) constituents; according to their functions in plant
metabolism. Primary constituents comprise common
Preparation of plant extracts sugars, amino acids, proteins and chlorophyll while
The leaf extracts of Ocimum tenuiflorum was prepared secondary constituents consist of alkaloids, terpenoids
with four different solvents such as ethanol, methanol, and phenolic compounds.12 O. tenuiflorum leaf extracts
acetone and distilled water. 2.5g of leaf powder was contained various phytochemical compounds such as
dissolved in 50ml of each solvent. These four leaves saponins, alkaloids, flavonoids, cardiac glycosides,
extract with different solvents. steroids, phenols and tannins.13 The presence of tannins
suggests the ability of this plant to play a major role as
Phytochemical analysis antidiarrhoec and antihaemorrhagic agent. 14 The
The results of the phytochemical composition of the presence of glycosides has been used for over two
15
extracts of O. tenuiflorum is given in Table 1. The results centuries as stimulants in cases of cardiac failure.
of the phytochemical studies showed that all tested
Table 1: Qualitative phytochemical analysis of O. tenuiflorum leaf extracts
Ethanol Methanol Acetone Aqueous
Compounds Test Adopted
Extract Extract Extract Extract
Alkaloids Mayer’s Test + + + +
Terpenoids Salkowski Test + + + +
Flavonoids Sodium hydroxide Test + + - -
Tannins Lead Acetate Test + + _ +
Steroids Chloroform Test + + _ +
Anthraquinones Free anthraquinones Test _ _ _ _
Saponins Foam Test + + _ +
Resins Sodium hydroxide Test + + + +
Glycosides Keller Killiani’s Test + + + _
Phenols Ferric chloride Test + + + +

+ = Present; - = Absent

Antimicrobial study Of O. tenuiflorum Leaf Extracts Stphylococcus aureus. Regarding antifungal effect of
ethanolic extract of O. tenuiflorum, maximum inhibition
The antimicrobial effect various leaf extracts of (ethanol,
zone (14 mm) and minimum inhibitory zone (8 mm) was
methanol, acetone and aqueous extracts) O. tenuiflorum
observed against Trichoderma viridae and Aspergillus
were studied against selected bacterial and fungal
flavus respectively.
pathogenic strains such as Escherichia coli, Proteus
vulgaris, Pseudomonas fluorescens, Salmonella Methanolic extract of O. tenuiflorum against selected
gallinarum, Staphylococcus aureus, Aspergillus flavus, bacterial pathogens exhibited maximum inhibition zone
Aspergillus niger and Trichoderma viridea by agar well (16 mm) against Proteus vulgaris followed by inhibitory
diffusion method and the results are presented in Figs zone of about 15 mm against Pseudomonas fluorescens,
3&4. Organic extracts of Ocimum sanctum L. showed Salmonella gallinarum. Least inhibitory zone of 7 mm was
wide zones of inhibition against Escherichia coli, observed against Staphylococcus aureus. Meanwhile, the
Staphylococci sp., Shigella sp., Staphylococcus aureus and antifungal effect of O. tenuiflorum methanolic leaf
Enterobacteria sp.16 The antibacterial effect of ethanolic extracts exhibited maximum activity against Aspergillus
extract of O. tenuiflorum exhibited maximum inhibition niger (14 mm), Aspergillus flavus (12 mm) and
zone (18 mm) against Salmonella gallinarum. Whereas Trichoderma viride (9 mm) respectively. Alcoholic extract
the minimum inhibitory zone (7 mm) was noticed against showed wider zone for Vibrio cholera.17

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Int. J. Pharm. Sci. Rev. Res., 53(2), November - December 2018; Article No. 05, Pages: 24-28 ISSN 0976 – 044X

The antimicrobrial effect of acetone extracts of O. CONCLUSION

tenuiflorum demonstrated greatest susceptibility towards
The frequency of life threatening infections caused by
bacterial pathogens (17 mm for Escherichia coli, 16 mm
pathogenic microorganisms has increased worldwide and
for Salmonella gallinarum, 13 mm for Pseudomonas
is becoming an important cause of morbidity and
fluorescens, 9 mm for Staphylococcus aureus and 7 mm
mortality in immune compromised patients in developing
for Proteus vulgaris) whereas the least susceptibility was
countries like our India. Results on bactericidal effect
noticed on fungal pathogens (17 mm for Aspergillus niger,
revealed that leaf extracts of O. tenuiflorum showed
12 mm for Aspergillus flavus and 11 mm for Trichoderma
varying degrees of antibacterial property against the
viridae). The aqueous extract of O. tenuiflorum showed
selected pathogens used. In conclusion, the present
greatest inhibitory zone against Pseudomonas fluorescens
findings clearly indicated that O. tenuiflorum leaves
(17 mm) followed by Escherichia coli (16 mm),
possess the capabilities of being a good candidate in the
Trichoderma viridae (14 mm), Aspergillus niger (13 mm),
search for a natural antibacterial agent against diseases
Salmonella gallinarum and Staphylococcus aureus (12
caused by both gram-positive and gram-negative
mm), Aspergillus flavus (10 mm) and Proteus vulgaris (8
pathogenic strains.
mm). Aqueous and acetone extracts of Ocimum sanctum
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Source of Support: Nil, Conflict of Interest: None.

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