EFFICIENCY OF Ocimum sanctum.L EXTRACTS FOR ANTIMICROBIAL
ACTIVITY AGAINST CERTAIN PATHOGENS
Melvinjoe, M1, Jayachitra. J1, Vijayapriya. M1 and Pandeeswari, N1
1. Department of Microbiology, Faculty of Agriculture, Annamalai University
ABSTRACT
The in vitro antimicrobial activity of Ocimum sanctum leaf, root and inflorescence
extracts were studied against selected pathogenic bacteria and fungi, following broth
dilution assay. Leaves, root and inflorescence extracts were prepared using absolute
alcohol, benzene, chloroform, methanol, petroleum ether, hot and cold water. Among
all the solvents used the leaf extracts prepared using ethanol was found to be more
effective against all the test microorganisms, which recorded minimum inhibitory
concentration (MIC) ranged between 1.0 to 4.0 mg/ml. Ethanol extracts showed MIC
of 1.0-4.0 mg/ml for bacteria and 2.0-4.0 mg/ml, for fungi. Extracts of benzene and
petroleum ether were ineffective in inhibiting the bacterial and fungal growth or
showed poor inhibition. The chloroform extract also showed a moderate antimicrobial
activity of 4.0-8.0 mg/ml against the bacterial pathogens and 4.0-6.0 mg/ml against
the fungal pathogens. The antimicrobial activity of Ocimum sanctum is discussed in
the present paper.
Keywords : Ocimum sanctum, minimum inhibitory concentration antimicrobial
activity, extracts and secondary metabolites.
INTRODUCTION
Ayurvedic medicinal plants are rich in secondary metabolites which are
potential source of drugs for approximately 25 per cent of prescribed medicines
(Farnsworth and Bunyapraphatsara, 1992). The plants known as medicinal plants are
rich in the secondary metabolites, which include alkaloids, glycosides, steroids and
relative active metabolites which are used as drugs in pharmaceutical industry (Sobli
and Pushphpangadan, 1982). The secondary metabolites are accumulated by plants in
their leaves, roots and other organs. Despite the fact their biosynthetic origin and role
in the plant are poorly understood, secondary metabolites are of considerable interest
because of their potential industrial, pharmacological and medicinal value (Farnsworth
and Bunyapraphatsara, 1992) Ocimum sanctum L. (Tulsi) or basil, queen of herbs has
been used in Indian sub continent for thousand of years as a house hold remedy.
The basil leaves are of high antimicrobial activity. The main components of
leaves are tannins (4.6 per cent) and essential oils (2 per cent) such as flavonoids and
phenolic acid. The amounts of the primary constituents of the essential oil vary
according to the geographical distribution and variety of the source plant material
such as Eugenol (upto 62 per cent), methyl eugenol upto 85 per cent) and and
 caryophyllene (upto 42 per cent) (Norr and Wagne 1992). The antimicrobial activity
of Ocimum sanctum of Laminaceae family was well documented in the literature.
The principle aim of the present work was to study antimicrobial activity of
leaf, root and inflorescence. Extracts were prepared in different solvents like absolute
alcohol, benzene, chloroform, methanol, petroleum ether, cold and hot water against
human pathogenic bacteria including Bacillus cereus, Escherichia coli, Klebsiella
pneumonia, Staphylococcus aureus, Vibrio parahaemolyticus and Salmonella typhi
and the pathogenic fungi including the yeast like Candida albicans, Blastomyces
dermatidis, Histoplasma capsulatum, Sporothrix schenckii, Cryptococcus neoformis.
MATERIALS AND METHODS
The first young plants of Ocimum sanctum were collected randomly during the
month of January from the outskirts of Kanyakumari district, Tamilnadu, India. The
plant species were identified by using the standard morphological characteristic
features (key) according to the flora of Madras Presidency.
Preparation of Extracts
Fresh plant material (i.e. leaf, root and inflorescence) were washed and surface
sterilized with 1 per cent ethanol and dried in hot air oven at about 35-40C and then
powdered using a mechanical grinder, sieved through a sieve and stored in individual
air tight bottles. 25 g of leaf, stem, root and inflorescence powder were extracted with
150 ml of solvent absolute alcohol, benzene, chloroform, methanol and petroleum
ether for 25 hrs by using Soxhlet apparatus. The extracts were dried in a flash
evaporator for 30 min and the left over powder was considered 100%. The hot and
cold water extracts were prepared by maceration using a mortar and pestle and then
filtering the extract by using Whatmann No.1 filter paper. Different concentrations
were prepared by redisolving the extract powder in the same solvent which was used
for the extraction.
Test microorganisms
Selected pathogenic bacteria Bacillus cereus MTCC 430, Klebsiella pneumoniae
MTCC 2405, Staphylococcus aureus MTCC 3160, Vibrio parahaemolyticus MTCC 451,
Salmonella typhi MTCC 734, Psuedomonas aerogenosa MTCC 4306 and fungi, the
yeast like Candida albicans MTCC 183, Blastomyces dermatidis ATCC 26199,
Histoplasma capsulatum ATCC 90723, Sporothrix schenckii ATCC 90723 and
Cryptococcus neoformis ATCC 2517 were obtained from Institute of Microbial
Technology India and American type cultural collection centre, U.S.A. respectively.
All test bacterial species were maintained on nutrient agar media. The 36 hrs
old bacterial culture were inoculated in to nutrient broth and incubated at 35  2C on
a rotary shaker (Labortechnik, Germany) at 100 rpm. After 36 hrs incubation the
bacterial suspension were centrifuged at 10000 xg for 15 min. The pellet was
resuspended in sterile distilled water and the concentration was adjusted to 1108
Cfu/ml by reading the OD to 0.4 at 610 nm using UV-Visible spectrophotometer
(Hitachi-U2000, Japan) and used for further studies. Fungal colonies were harvested
from 9-10 day old cultures which were maintained on sabourauds agar. The spores
were suspended in sterile distilled water and the spore suspensions were adjusted to
1108 spores/ml.
Antimicrobial assay
Antimicrobial assay were performed in 96 well sterile flat bottom microtiter
plates based on broth microdilution assay which is an automated colorimetric method,
uses the absorbance (optical density) of cultures. In a microtitre plate each well of
microtitre plates were filled with 200l of nutrient broth 1l of test organism and 15l
plant extract. The microtiter plates were incubated at 35  2C for 24hr. After the
incubation period the plates were read at 465 nm using ELISA reader (ELX 800 MS,
Biotek Instruments Inc. USA) MIC, determined as the lowest concentration of plant
extract inhibiting the growth of the organism were determined based on the readings.
RESULTS AND DISCUSSION
The result of the antimicrobial screening tests of leafs, roots and inflorescence
extracts of O. sanctum in different solvents were tested against human pathogenic
microorganisms using microdilution technique are depicted in Tables 1 and 2. The
extracts are found to be more effective against bacteria rather than fungi both benzene
and petroleum ether extracts were found to be ineffective or showed poor inhibition
on bacterial and fungal growth.
The microdilution assay gives MIC values ranging from 1.0-80.0 mg/ml for
different microorganisms (Tables 1 and 2) the lower MIC values were obtained for
leaf extracts obtained with ethanol which showed to 1.0 mg/ml for B. cereus,
S. aureus followed by chloroform extract which showed a least MIC of 1.0 against
S. aureus. The cold and hot a water extract also showed some, activity against some
of the pathogens. The results of the antimicrobial activity of the root extract showed a
moderate inhibitory effect against most of the pathogens which varied between
2.0 mg/ml to 80.0 mg/ml against the pathogens. The low MIC values were observed
in the chloroform extracts i.e. 2.0 for C. albicans and H. capsulatum the inflorescence
sample showed the least inhibitory effect against all the organisms tested which
showed a MIC ranging from 8.0 mg/ml to 80.0 mg/ml, in which the alcoholic extract
showed the least MIC of 6.0 for B. cereus and S. aureus.
The antimicrobial activity of O. sanctum extracts varied with plant parts and
the solvents used for extraction. The yields of the cold and hot water extracts were
relatively low. Maceration has generally reported to give low yield of plant extracts
as compared to Soxhlet extraction (Machado et al., 1999). The higher antimicrobial
property exhibited by the ethanol, benzene and chloroform extracts, over water
extracts show that higher proportion of plant components were water insoluble. It has
been found that most of the extracts contained the metabolites, though not in the same
proportions, and also the active principle were more soluble in analytical ethanol as
compared to the other solvents. The disparity in antimicrobial activity between the
cold and hot water extracts over other solvents is due to the fact that solvent extracts
have reported to contain higher amounts of plant constituents (Okete et al., 2001,
Okoli et al., 2002). But in some cases the cold water extract exhibited better antimicrobial
activity as compared to the hot water extract it may be due to the fact that heat inactivation
of some of the secondary metabolites in the hot water extract. The antibacterial activity of
the extracts against different pathogenic organisms is due to the presence of tannins,
essential oils, flavonoids, alkaloids and eugenol in varying proportions of O. sanctum
(Lakakso et al., 1990). The lower inhibitory effect of the root and inflorescence
samples may be attributed to the fact that the presence of low concentrations of
antimicrobial components as compared to the leaf extracts (Leven et al., 1999).
Phytochemicals exert their antimicrobial activity through different mechanisms,
tannins for example act by iron deprivation, hydrogen bounding or non-specific
interactions with vital proteins such as enzymes (Scalbert, 1991). The alkaloids act as
a DNA intercalator and an inhibitor of DNA synthesis through topoisomerase
inhibition (Passonen et al., 2002, Guittat et al., 2003). The lower inhibtiory effect of
the extracts against the fungal pathogens may be attributed to the fact that they are
resistant to the most commonly used antibiotics and also due to the fact that they have
rigid chitin in cell wall. It has been found that the gram positive bacteria were more
susceptible to the extracts as they have only on outer peplidoglycan layer, which is
not an effective barrier (Scherrer et al., 1971). The gram negative bacteria have an
outer phospholipidic membrane that makes the cell wall impermeable to lipophilic
solutes, to while the porins constitute a selective barrier to hydrophilic solutes with an
exclusion limit of about 600 Da (Nikaido and Varara, 1985). Many results confirmed
these observations, thus some plant extracts were found to be more active against
gram-positive bacteria than gram negative ones (Kelmanson et al., 2000, Mesika and
Atolsyane 2002). In conclusion ehtanolic and chloroform extracts of Ocimum sanctum
showed excellent antimicrobial activity against all the test microorganisms. Further
work is needed to isolate the active principle from the plant extracts to carry out
pharmaceutical studies.
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in
eastern
cape.
South
Afr.
Table 1. Minimum inhibitory concentration (MIC) of Ocimum sanctum for antibacterial activity
MIC (mg/ml)
Source
Solvents
Ocimum sanctum
Alcohol
Benzene
Chloroform
Leaf
Methane
Petroleum ether
Cool water
Hot water
Alcohol
Benzene
Chloroform
Root
Methane
Petroleum ether
Cool water
Hot water
Alcohol
Benzene
Chloroform
Inflorescence
Methane
Petroleum ether
Cool water
Hot water
Chromamphenical
B. cereus
K. pneumoniae
S. aureus
V.
parahaemolyticus
S. typhi
P. aeroginosa
1.0
1.0
*
30.0
4.0
6.0
80.0
6.0
8.0
*
4.0
2.0
6.0
*
*
*
8.0
10.0
20.0
20.0
6.0
1.0
1.0
1.0
4.0
6.0
*
*
6.0
6.0
2.0
2.0
8.0
*
10.0
20.0
8.0
30.0
4.0
2.0
6.0
1.0
10.0
30.0
4.0
30.0
80.0
4.0
6.0
4.0
8.0
20.0
80.0
80.0
*
4.0
Values are a mean 3 determinations
- = no value; * Shows poor inhibition of bacterial growth
Table 2. Minimum inhibitory concentration (MIC) of Ocimum sanctum for antifungal activity
MIC (mg/ml)
Source
Solvents
Ocimum sanctum
Alcohol
Benzene
Chloroform
Leaf
Methane
Petroleum ether
Cool water
Hot water
Alcohol
Benzene
Chloroform
Root
Methane
Petroleum ether
Cool water
Hot water
Alcohol
Benzene
Chloroform
Inflorescence Methane
Petroleum ether
Cool water
Hot water
Fuconazole
C. albicans
B. dermatidis
H. capsulatum
S. schenckii
C. neoformis
2.0
*
4.0
*
10.0
20.0
6.0
2.0
4.0
*
20.0
80.0
6.0
10.0
*
*
4.0
4.0
6.0
*
*
8.0
4.0
10.0
10.0
20.0
6.0
2.0
2.0
*
4.0
2.0
10.0
8.0
10.0
4.0
2.0
4.0
*
4.0
4.0
6.0
8.0
30.0
2.0
4.0
10.0
4.0
*
*
8.0
20.0
6.0
20.0
*
*
*
*
6.0
Values are a mean 3 determinations
- = no value; * Shows poor inhibition of bacterial