Isolation of Antibacterial Compound from Ocimum sanctum (Tulsi) against Enteropathogenic Bacteria

Abstract

Hexane, petroleum ether, chloroform, ethyl acetate, acetone and methanol extracts of Tulsi (Ocimum sanctum) were analyzed for determination of antibacterial activity against enteric-pathogenic bacteria Escherichia coli, Shizella dysenteriae, Salmonella typhi, Pseudomonas aeruginosa and Vibrio cholerae using agar well diffusion method. Only acetone extract of all of them exhibited an inhibiting effect on the development of the tested strains. By using the disc diffusion method, the chosen pathogenic bacteria were examined for their pattern of antibiotic susceptibility. A comparison of antibiotics and acetone extract was performed. It was discovered that acetone extract found to be remarkable sensitivity against the test pathogens, total 37 fractions were obtained from the fractionization of acetone extract through column chromatography and found 4 major and 13minor compounds were obtained through Thin Layer Chromatography method. Antibacterial activity of different fractions was determined by spot assay technique. The fractions 1, 7, 13, and 21 demonstrated the greatest antibacterial activity when tested against the test pathogens out of all the fractions collected. Octadecanoic acid and methyl ester were found in the fraction number 13 according to the results of the Gas Chromatography Mass Specrometry. Stearic acid methyl ester obtained from the acetone extract of Ocimum sanctum has been recommended for human trials against various bacterial infections.

Full text

_____________________________________________________________________________________________________
++ Research Scholar;
*Corresponding author: E-mail: sarveshmicro@gmail.com;
Curr. J. Appl. Sci. Technol., vol. 42, no. 14, pp. 1-11, 2023
Current Journal of Applied Science and Technology
Volume 42, Issue 14, Page 1-11, 2023; Article no.CJAST.99622
ISSN: 2457-1024
(Past name: British Journal of Applied Science & Technology, Past ISSN: 2231-0843,
NLM ID: 101664541)
Isolation of Antibacterial Compound
from Ocimum sanctum (Tulsi) against
Enteropathogenic Bacteria
Sarvesh Kumar Mishra a++*, Krishna Ash a++
and Shailendra Kumar Srivastava a++
a Department of Microbiology and Microbial Technology, AAIDU, Allahabad, Uttar Pradesh, India.
Authors’ contributions
This work was carried out in collaboration among all authors. All authors read and approved the final
manuscript.
Article Information
DOI: 10.9734/CJAST/2023/v42i144116
Open Peer Review History:
This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers,
peer review comments, different versions of the manuscript, comments of the editors, etc are available here:
https://www.sdiarticle5.com/review-history/99622
Received: 23/03/2023
Accepted: 25/05/2023
Published: 03/06/2023
ABSTRACT
Hexane, petroleum ether, chloroform, ethyl acetate, acetone and methanol extracts of Tulsi
(Ocimum sanctum) were analyzed for determination of antibacterial activity against enteric-
pathogenic bacteria Escherichia coli, Shizella dysenteriae, Salmonella typhi, Pseudomonas
aeruginosa and Vibrio cholerae using agar well diffusion method. Only acetone extract of all of them
exhibited an inhibiting effect on the development of the tested strains. By using the disc diffusion
method, the chosen pathogenic bacteria were examined for their pattern of antibiotic susceptibility.
A comparison of antibiotics and acetone extract was performed. It was discovered that acetone
extract found to be remarkable sensitivity against the test pathogens, total 37 fractions were
obtained from the fractionization of acetone extract through column chromatography and found 4
major and 13minor compounds were obtained through Thin Layer Chromatography method.
Antibacterial activity of different fractions was determined by spot assay technique. The fractions 1,
7, 13, and 21 demonstrated the greatest antibacterial activity when tested against the test
Original Research Article

Mishra et al.; Curr. J. Appl. Sci. Technol., vol. 42, no. 14, pp. 1-11, 2023; Article no.CJAST.99622
2
pathogens out of all the fractions collected. Octadecanoic acid and methyl ester were found in the
fraction number 13 according to the results of the Gas Chromatography Mass Specrometry. Stearic
acid methyl ester obtained from the acetone extract of Ocimum sanctum has been recommended for
human trials against various bacterial infections.
Keywords: Compound isolation; Ocimum sanctum; E. coli; S. dysenteriae; S. typhi; P. aeruginosa;
V. cholera.
1. INTRODUCTION
“Tulsi is the medicinal plant and three types of
Tulsi are found in India; the green leafed (Sri or
Rama Tulsi) is the most common; the second
type (Krishna Tulsi) bears dark to purple leaves
and the third type is a forest variety (Vana Tulsi)
that often grows in wild. India is the richest
source of medicinal plants, which constitutes an
effective source of both traditional and modern
medicine. Our rural population has great faith in
herbal medicine; about 80% of the population
depends on it as their primary health care” [1].
Bradu [2] found that “among the enormous
number of medicinal plants, members of Genus
Ocimum sanctum belonging to the Family
Lamiaceae is a group of aromatic shrub
distributed mainly in tropical and warm temperate
regions of the world”. “Among the 176 species of
Ocimum identified, only six species have been
reported from India, O. gratissimum L., O.
basilicum L., O. canum L. and O. adscendens L.
(Only four out of six have been listed). Of these
only O. gratissimum L., O. basilicum L. and O.
sanctum have been reported to have medicinal
properties” [3].
Tulsi (Holy basil or sweet basil) in Sanskrit
means “one that is incomparable” one that does
not tolerate or permit similarity. The extract of
tulsi is commonly known for the treatment of
cold, headache, stomach disorders etc [4]. “Tulsi
has been recognized for thousands of years to
be one of India’s greatest healing herbs that
nourishes a person’s growth to a perfect health
and nurtur for long-life. The goal of Ayurveda is
to develop good health and protect the health of
the healthy and alleviate disorders in the
diseased person. Herbal products are produced
to maintain good health and to treat disease” [5].
“Tulsi has a long history of medicinal use; it is
mentioned in the oldest ancient Ayurvedic
Charak Samhita and also mentioned in Puranas
and Rig Veda. For its religious sanctity, Tulsi has
been grown in Indian sub-continent” [6]. The
diverse medicinal properties of tulsi such as
hypoglycemic, anti-hyperlipedimic, hypotensive,
anti-inflammatory and antimicrobial are
mentioned in an Indian traditional medicine
scripture “Charak Samhita”. “The various
compounds like beta-caryophyllene, derivatives
of eugenol, rosmarinic acid, vanillin, gallic acid,
ursolic acid and vanillic acid are present in tulsi
leaves” [6]. “The Tulsi has ties with the Hindu
God Vishnu and his worship. Tulsi is grown in
every house of Hindus in India and the leaves of
Tulsi are used in the routine worship. Similarly,
the rosary beads made from the stem of Tulsi
plant is used for prayers and meditation” [1].
“About 85% population of the whole world
partially or wholly is dependent on herbal
medicines for the treatment of primary health
related issues. According to traditional medical
system, herbal medicines are the major
remedies” [7]. “Ocimum is used in medicine to
treat different diseases of respiratory tract
infection, diarrhea, headache, ophthalmic, skin
diseases, and pneumonia, also as a treatment
for cough, fever and conjunctivitis. Methanol
extract an aqueous suspension of O. sanctum
leaves have been shown to exhibit anti-
inflammatory activity against carrageenan
induced paw edema in rats and analgesic
activity. Antihistaminic, antispasmodic and
resistant stress induced cholesterol lowering
activities of Ocimum sanctum are available” [8].
“In addition to therapeutic significance of O.
sanctum reports on their antimicrobial and
antibacterial varieties of Ocimum species against
different pathogenic bacteria i.e. E. coli, Shigella
sp. and Salmonella typhi etc. were reported by”
[9]. Tulsi contains vitamins A, C and also four
marker compounds, viz., eugenol, luteolin,
ursolic acid and oleanolic acid in the leaf of green
and black varieties of O. sanctum. One important
component of Tulsi, eugenol was shown to lower
blood levels of stress hormone in rats.
The aim of the present study was to compare the
activity of extract obtained from fresh and dried
leaves of Ocimum sanctum against clinical
isolates of enteric bacteria, identify the
antibacterial compound and compare the
sensitivity of enteric pathogenic bacteria against
antibiotics and extract.

Mishra et al.; Curr. J. Appl. Sci. Technol., vol. 42, no. 14, pp. 1-11, 2023; Article no.CJAST.99622
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2. MATERIALS AND METHODS
Test organism: The test organisms in the study
were obtained from the culture collection bank of
the Department of Microbiology and Microbial
Technology, SHUATS, Prayagraj, India. The
enteric pathogens are given (Table 1).
Preparation of extract: Fresh leaves of Ocimum
sanctum were collected and wash with distilled
water and air dried. Twenty gram of powder was
transferred in to the clean conical flask, 100 ml
hexane added to it, left for 24 h and the extract
was filtered, collected and dried at room
temperature. The residue of the Ocimum
sanctum powder dried and dispended in the
petroleum ether. The extraction process was
followed with petroleum ether, chloroform, ethyl
acetate, acetone and methanol in a similar
manner.
Antibacterial activity of Ocimum sanctum
Extract: Antibacterial activity of Ocimum
sanctum extracts was tested using Agar well
diffusion technique as described by Agarry et al.
[10]. For this melted and cooled sterile Nutrient
Agar media was poured into sterilized Petri
dishes and was allowed to solidify. Using
sterilized cotton swabs, overnight broth culture of
the test organisms was swabbed on the Nutrient
Agar plates uniformly. Wells (5mm) were cut on
the inoculated Nutrient Agar plates with the aid of
a sterile stainless steel cork borer. About 0.1 ml
of the Ocimum sanctum extracts were filled into
each of the wells. These plates were then
incubated at 37°C. The zone of inhibition was
recorded as the presence of antibacterial action.
Each extract of Ocimum sanctum was tested
against each organism in triplicates.
Isolation of antibacterial agents: The screening
of major and minor compounds present in the
extract was done by Thin Layer Chromatography
(TLC) by using Silica gel G. Further the selection
of solvent combination for mobile phase required
to isolate the antibacterial agent through Column
Chromatography by TLC.
Thin Layer Chromatography: TLC plates were
prepared by pouring the slurry of silica gel G
prepared with distilled water on the sterilized
glass plates and then drying at 60-80OC. The
plates have prepared to spot the sample with
capillary. These plates were then kept in different
concentrations of different solvent combinations
(on the basis of polarity) in a vertical position to
run the compound, the plates were dried and
sprayed with H2SO4/ Methanol Solution (1:9v/v)
and kept in oven at 60-80OC for 5min to develop
the TLC plates. The separated compounds on the
TLC plates detected as clear spots
(chromatogram). The distance of these
compounds from the origin and distance of
solvent front were calculated as retention factor
(Rf) value [11].
Rf value = Distance travelled by the solute (cm) /
Distance traveled by the solvent (cm)
Thus, the total number of compounds in the
particular extract as well as the extract solvent
combination to isolate each compound was
identified.
Column Chromatography: Column was packed
with silica gel (60-120 mesh) slurry prepared in
higher polar solvent and on the column the
extract was loaded along with the slurry. Different
concentrations of the solvent combinations were
run through the column one after another till
entire fraction of compound of particular mobile
phase is accomplished. A different fraction
obtained from column chromatography was used
for analysis of their antibacterial properties [11].
Evaluation of antibacterial activity of different
compounds: The different fractions obtained from
the column chromatography were allowed to
stand at room temperature until complete
evaporation of solvent and then the dried fraction
was dissolved in di-methyl sulfoxide (DMSO) that
were used for screening antibacterial activity
against the selected pathogenic bacteria by
using spot assay technique as described by Jack
[12].
Table 1. Microbial culture from culture collection bank
S. No.
Test Organism
Culture Number
1
Escherichia coli
MCCB0017
2
Salmonella typhi
MCCB0038
3
Shigella dysenteriae
MCCB0042
4
Pseudomonas aeruginosa
MCCB0034
5
Vibrio cholerae
MCCB0049

Mishra et al.; Curr. J. Appl. Sci. Technol., vol. 42, no. 14, pp. 1-11, 2023; Article no.CJAST.99622
4
Table 2. List of antibiotics with concentration
S. No.
Antibiotics
Concentrations
1
Ciprofloxacin
5 µg
2
Chloramphenicol
30µg
3
Gentamycin
10µg
4
Kanamycin
30µg
5
Norfloxacin
10µg
6
Oflaxacin
5 µg
7
Piperacillin
10µg
8
Streptomycin
10µg
9
Tobramycin
10µg
10
Trimethaoprim
5 µg
Spot Assay Technique: The sterilized Nutrient
Agar media was poured into the sterilized Petri
dishes and was allowed to solidify and then the
overnight broth culture of the selected pathogenic
bacteria was swabbed on the surface of the
media uniformly, with the sterile cotton swabs.
The isolated dried fractions dissolved in DMSO
and then spotted with the capillary. These plates
were incubated at 37+1 OC for 24h. The
presence of zones of inhibition was noted after
24h of incubation.
Identification of antibacterial compound: The
maximum antibacterial activity against the test
organisms was further identified with the help of
Gas Chromatography Mass Spectroscopy
(GCMS) and Proton Nuclear Magnetic Resonance
(1HNMR) for which the sample was sent to
Central Drug Research Institute (CDRI),
Lucknow. Identification of respective compound
was done with the help of peak values and mass
spectrum data generated during analysis [13,14].
Antibiotic susceptibility test: For conducting
the Antibiotics susceptibility test the following
antibiotic disc were used (Table 2).
Antibiotic susceptibility test was done using disc
diffusion method [15].
Disc diffusion method: In this method the
microorganisms were swabbed on the agar plate
under sterile condition and antibiotic discs were
placed on the surface of the inoculated agar
plate. The plates were incubated at 37°C for 24 h
after completing incubation the zone of inhibition
were measured and recorded in mm then the
values was compared with national committee for
clinical laboratory standards [16] to determine the
sensitivity resistance pattern of the test
microorganism.
Statistical analysis: The data obtained was
subjected to analysis of varience (two way
classification) at 5% level of significance and
interpreted accordingly.
3. RESULTS AND DISCUSSION
Antibacterial activity of Ocimum sanctum
extract against selected enteric pathogenic
bacteria: The Ocimum sanctum extracts
obtained from different solvents viz. hexane,
petroleum ether, chloroform, ethyl acetate,
acetone and methanol showed a varying degree
of activity against selected gram negative
pathogens (Table 3).
While categorizing each extract against selected
pathogenic bacteria, it was found that acetone
showed highest antibacterial activity (15.33mm)
against S.dysenteriae and lowest (9.66mm)
against V. cholera. Ethyl acetate extract showed
maximum antibacterial activity (13.66mm)
against S. dysenteriae, S. typhi and minimum
(8.33mm) against V. cholerae while no
antibacterial activity against P. aeruginosa
(p<0.05). Methanol extract showed maximum
antibacterial activity (9.33 mm) against S.
dysenteriae and minimum activity (8.33mm)
against V. cholerae. Using hexane, petroleum
ether and chloroform extract zero zone of
inhibition was found. With respect to individual
pathogen, it was found that acetone showed
significantly highest antibacterial activity than
ethyl acetate, methanol, hexane, petroleum ether
and chloroform extract. However, significantly
lower antibacterial activity were observed with
ethyl acetate and methanol extract respectively.
In a similar study of Perez et al. [17] as adopted
earlier Ahmad and Beg [18] was used “0.1ml of
diluted inoculums (105CFU/ml) of test organism
was spread on agar plates. Well of 8mm
diameter were punched into the agar medium
and filled with 100µl (150mg/ml) of plant extract,
solvent blanks and antibiotic (Chloramphenicol,

Mishra et al.; Curr. J. Appl. Sci. Technol., vol. 42, no. 14, pp. 1-11, 2023; Article no.CJAST.99622
5
100µg/ml conc.) to which the test bacteria were
sensitive”. “It was also indicated by results that
the best solvent for extracting antibacterial
substances from the leaves of tulsi plant is
methanol” [19].
Antibiotic susceptibility test of the test
organisms: The selected pathogenic bacteria
were tested for antibiotic susceptibility pattern by
disc diffusion method. All the organisms viz.
Escherichia coli, Shigella dysenteriae,
Salmonella typhi, Vibrio cholerae and
Pseudomonas aeruginosa were found to be
resistant towards Kanamycin and Piperacillin
(Table 2). It was observed that Escherichia coli
showed intermediate activity towards
Chloramphenicol, Ciprofloxacin and Ofloxacin
while it was found to be resistant towards other
antibiotics i.e. Gentamicin, Kanamycin,
Norfloxacin, Piperacillin, Streptomycin,
Tobramycin and Trimethoprim. Shigella
dysenteriae showed intermediate activity only
towards Chloramphenicol and was found to be
resistant to all other antibiotics viz. Ciprofloxacin,
Gentamicin, Kanamycin Norfloxacin, Ofloaxacin,
Piperacillin, Streptomycin, Tobramycin and
Trimethoprim. Similarly Salmonella typhi was
found to be sensitive against Ciprofloxacin,
Norfloxacin and Trimethoprim while it showed
intermediate activity towards Chloramphenicol,
Gentamicin, Ofloxacin, Streptomycin and
Tobramycin and showed resistance towards
Kanamycin and Piperacillin. Vibrio cholera was
the only microorganism which showed resistan
ceto all the antibiotics used. Pseudomonas
aeruginosa was sensitive to Tobramycin showed
intermediate activity towards Ciprofloxacin,
Gentamicin, Norfloxacin and Streptomycin
while it showed resistance to all other
antibiotics.
Effect of Acetone extract of Ocimum sanctum
in comparison with antibiotics against
selected pathogenic bacteria: A comparative
study was done between the antibiotics and
acetone extract and it was observed that both
showed an equal pattern of sensitivity against the
test pathogens (Table 5).
Table 3. Antibacterial activity of Ocimum sanctum extract against selected enteric pathogenic
bacteria
Test
Extracts
Results
Organisms
Zone of inhibition(mm)*
Hexane
Petroleum
Chloroform
Ethyl
Acetone
Methanol
ether
acetate
E.coli
0.00
0.00
0.00
11.33
13.00
0.00
S,p<005
S.dysenteriae
0.00
0.00
0.00
13.66
15.33
9.33
S,p<0.05
S.typhi
0.00
0.00
0.00
13.66
12.33
0.00
S,p<0.05
V.cholerae
0.00
0.00
0.00
8.33
9.66
8.33
S,p<0.05
P.aeruginosa
0.00
0.00
0.00
0.00
0.00
0.00
Results
S,
S,
S,
p<0.05
p<0.05
p<0.05
*includes well size of 5mm diameter, Due to organisms: Fcal=12.72>Ftab(5%)=2.4683; CD=1.577 Due to
solvents: Fcal=47.81>Ftab(5%)=2.1482; CD=1.866 *S means significant
Table 4. Antibiotic susceptibility of selected pathogenic bacteria
Organisms
Antibiotics
C
Cf
G
K
Nx
Of
Pc
S
Tb
Tr
E.coli
++
++
–
–
–
++
–
–
–
–
S.dysenteriae
++
–
–
–
–
–
–
–
–
–
S.typhi
++
+++
++
-
+++
++
-
++
++
+++
V.cholerae
–
–
–
–
–
–
–
–
–
–
P.aeruginosa
–
++
++
–
++
–
–
++
+++
–
(-):resistant, (++):intermediate, (+++):sensitive,
C-Chloramphenicol, Cf-Ciprofloxacin, G-Gentamicin, K-
Kanamycin, Nx-Norfloxacin, Of-Oflaxacin, Pc-Piperacillin, S-Streptomycin, Tb-Tobramycin,Tr-Trimethoprim

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6
Table 5. Effect of Acetone extract of Ocimum sanctum in comparison with antibiotics against
selected pathogenic bacteria
Organisms
Antibiotics
Extract
C
Cf
G
K
Nx
Of
Pc
S
Tb
Tr
Acetone
E.coli
++
++
–
–
–
++
–
–
–
–
+++
S.dysenteriae
++
–
–
–
–
–
–
–
–
–
+++
S.typhi
++
+++
++
-
+++
++
-
++
++
+++
+++
V.cholerae
–
–
–
–
–
–
–
–
–
–
+++
P.aeruginosa
–
++
++
–
++
–
–
++
+++
–
–
(+++): Sensitivity,
C-Chloramphenicol,
(++
Cf-Ciprofloxacin,
):intermediate,
G-Gentamicin,
K-Kanamycin,
(-)-
resistant
Nx-Norfloxacin,
Of-Oflaxacin,
Pc-Piperacillin,
S-Streptomycin,
Tb-Tobramycin,
Tr-Trimethoprim
Table 6. Major and minor compounds detected in the Acetone extract of Ocimum sanctum
Extract
Major
compound
Mobilephase
R
-
value
Minor
compound
Mobilephase
R
f
value
Acetone 1
EthylAcetate:
Petroleum (7:3)
0.91
1
Methanol: Ethylacetate
(9:1)
0.73
2
Ethyl Acetate:
(9:1)
0.90
2
Methanol:
Acetone(8:2)
0.72
3
Acetone:
Methanol (5:5)
0.73
3
Methanol:
Acetone(9:1)
0.70
4
Acetone:
Methanol (3:7)
0.72
4
Acetone: Ethylactate
(1:9)
0.67
5
Methanol: Ethylactate
(7:3)
0.66
6
Methanol: Acetone
(6:4)
0.66
7
Methanol: Acetone
4:6)
0.63
8
Chloroform:
Ethylacetate(2:8)
0.61
9
Petroleumether:
Acetone(4:6)
0.42
10
Hexane: Ethylacetate
(2:8)
0.42
11
Petroleumether:
Acetone 2:8)
0.38
12
Acetone: ethylacetate
(6:4)
0.33
13
Hexane:Acetone (9:1)
0.15
Identification of the major and minor
compounds in the acetone extract of Ocimum
sanctum: For the identification of major and
minor compounds in the acetone extract of
Ocimum sanctum various solvent combinations
hexane, petroleum ether, chloroform,
ethylacetate, methanol and acetone were used.
On the basis of spots and retention factor (Rf)
value four major and thirteen minor compounds
were identified at different combinations of
acetone: hexane, acetone: petroleum ether,
acetone: chloroform, acetone: ethylacetate and
acetone: methanol in the acetone extract
(Table 6).
Isolation of different compounds in the
acetone extract of Ocimum sanctum: Isolation
of various compounds in the acetone extract of
Ocimum sanctum was determined by eluting in
the step wise gradient solvent system by TLC
technique with 10-90% Acetone with hexane
(v/v), 80-20% acetone with petroleum ether (v/v),
20-80% hexane with ethyl acetate (v/v), 60-40%
acetone with ethyl acetate (v/v)and 40-60%

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petroleum ether with acetone (v/v). Increasing in
polarity in acetone extract, a total of 37 fractions
were obtained. The fractions with similar Rf
values were pooled together and labeled
accordingly (Table 7).
Antibacterial activity of different fractions
obtained from column chromatography: A
varying degree of antibacterial activity of different
extracts of Ocimum sanctum was done in the
present study against enteric pathogenic
bacteria. A high degree of antibacterial activity
for the fraction number 1, 7, 13, 21 was obtained
from the acetone extract of Ocimum sanctum
against the test pathogens.
Moderate antibacterial activity of one (fraction no.
12) was observed in the acetone extracts where
no antibacterial activity was observed from there
twenty three fractions (Table 8). Further, the
fraction number13 with maximum antibacterial
activity showed same zone of inhibition against
Escherichia coli, Shigella dysenteriae,
Salmonella typhi and Vibrio cholera but showed
no zone of inhibition against Pseudomonas
aeruginosa.
Identification of compound in the acetone
extract of Ocimum sanctum: For the
identification of the fraction number 1, 7, 13, 21
showing maximum antibacterial activity, the
fraction was subjected to Gas
Chromatography Mass Spectrometry (GC-MS)
by diluting them in Acetone having a
dilution factor of 10-6. The range of spectrogram
were observed to be from 40-299,
hence the peaks having m/z ratio less than 40
were not visible. Moreover smaller molecules like
-CH3, -C2H5 and -C3H7 are so facile that the
molecular ion peak was observed in the
spectrum.
Table 7. Rf values of acetone extract of Ocimum sanctum observed using different mobile
phase system
S. N.
Solvent system(Mobilephase)
Rfvalue
Fractionno.
1.
Hexane:Acetone(9:1)
0.15
1,7
2.
Petroleumether:Acetone (2:8)
0.38
11,12,13
3.
HexaneEthylacetate (2:8)
0.42
9,18
4.
Acetone:Ethylacetate(6:4)
0.33
20,21
5.
Petroleumether:Acetone (4:6)
0.42
23
6.
Chloroform:ethylacetate(2:8)
0.61
25
7.
Methanol:acetone(4:6)
0.63
28
8.
Methanol:Ethylacetate(7:3)
0.66
16,29,
9.
Methanol:Ethylacetate(9:1)
0.73
34
10.
Chloroform:Ethylacetate (1:9)
0.90
35
11.
Petroleumether:Ethylacetate(3:7)
0.91
36
Table 8. Antibacterial activity of different fractions obtained from acetone extract of
Ocimum sanctum
Extract
Fraction No.
Antibacterial activity
Acetone
1,7,13,21
+++
12
++
2,3,4,5,6,8,9,10,11,14,15,16,17,18,19,20,22, 23,24,25,
26,27,28,29,30,31,32,33,34,35,36,37
–
*+++ = maximum antibacterial activity, ++ = moderate activity, – = no activity
Table 9. Antibacterial compounds isolated from the acetone extract of Ocimum sanctum.
Test Organisms
Antibacterialcompounds Acetoneextract
Escherichiacoli
7,13,21
Salmonellatyphi
3,11,12,13,21
Shigelladysenteriae
1,13,12,13,21
Vibriocholera
7,13,11,21
Pseudomonasaeruginosa
–

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GCMS of fraction number 13: The molecular
ion identified by the final peak in the mass
spectrogram at m/z =298 undergoes
fragmentation with the removal of -CH3 molecule
(mol. mass=15) giving rise to the formation of a
C18H35O2 molecule (mol. mass=283) due to
which a peak at m/z=283 was observed (Fig. 3).
In the second step C18H35O2 again gets
fragmented into C16H30O2 molecule
(mol.mass=254) and a -C2H5 molecule (mol.
mass= 29) giving rise to the large peak at
m/z=254 in mass spectrogram. Further the
cleavage of the C16H30O2 molecule takes place
giving rise to the removal of an -OCH3 molecule
(mol. mass = 31) and in the formation of a
C15H27O molecule (mol. mass = 223) showing
the peak at m/z=223. In the fourth step
C15H27O molecule gets cleaved into C12H20O
molecule (mol. mass=180) and a C3H7 molecule
(mol. mass=42) giving rise to the peak at
m/z=180 in mass spectrogram. In the next step
C12H20O again gets fragmented into C10H18
molecule (mol. mass=138) and a CH2=C=O
molecule (mol. mass=42) giving rise to the small
peak at m/z=138 in mass spectrogram. Finally
the C10H18 molecule gets cleaved into the C6H9
molecule (mol. mass =81) due to the removal of
a C4H9 molecule (mol. mass =57). Therefore a
peak at m/z = 81 was observed in the mass
spectral data (Fig. 1). The comparison of the
fragmentation pattern of the fraction number 13
with that of Octadecanoic acid having almost the
same mol. wt. (mol. mass=298) (Fig. 2) the
compound was identified as eugenol as also
reported previously [20,21]. The compound
eugenol has been demonstrated to have both
antibacterial (Naamura et al. 1999) and
antifungal [21-24] activities.
Fig. 1. Schematic representation of the fragments obtained in the mass Spectrum of fraction
number13

Mishra et al.; Curr. J. Appl. Sci. Technol., vol. 42, no. 14, pp. 1-11, 2023; Article no.CJAST.99622
9
Fig. 2. Mass Spectrum of Stearic acid, Methylester or Octadecanoic acid, Methylester
Fig. 3. Structure of Stearic acid, Methyl ester or Octadecanoic acid, Methylester
However, in similar study the eugenol
compounds have antifungal activity was isolated
and identified. The antifungal activity of eugenol
was evaluated against aspecies of Alternari from
tomato and Pennicilium chrysogenum. The
unmatched data of fraction could be due to the
effects of the solvent used during the analysis of
the sample. Further, slight variations in the peaks
are observed and are within the acceptable
limits. The higher range of mismatch in the
sample due to various handling and processing
techniques.
The leaves of Ocimum sanctum were dried and
powdered. The powder was thus subjected to
solvent extraction viz. hexane, petroleum ether,
chloroform, ethyl acetate, acetone and methanol.
Further, the antibacterial activities of these
extracts were performed by agar well diffusion
method against the five selected enteric
pathogen. Moreover, the antibacterial
susceptibility pattern was determined by disc
diffusion method against the selected test
organisms, thereafter a comparative study was
done between the extract and the selected
antibiotics. The extract showing maximum activity
by agar well diffusion method was further
subjected to thin layer and column
chromatography and the fractions thus obtained
were evaluated by spot assay technique to
determine the antibacterial activity. Fraction
number 13 was identified as Octadecanoic acid,
Methyl ester or Stearic acid, Methyl ester (Figs. 2
and 3).
4. CONCLUSION
Many microorganisms, which cause damage to
human health, exhibit drug resistance due to
inadequate use of antibiotics. Thus there is need
for the discovery of new substances from natural
sources including plants a high proportion of
health problem in developing countries like India
has become a global concern. Resistance, too
many antibiotics has created immense clinical
problem in the treatment of many major
diseases. Thus this situation has compelled for
the search of new antimicrobial compounds that
could be used as an alternative remedy for the
treatment of infectious diseases. Hence, as per
the result observed in the present study Ocimum
sanctum has shown promising efficacy. Since the
compound extracted is reported to have a
number of advantages on human health, it can
be concluded that this particular medicinal plant
could be recommended for treatment over
various human diseases and can also be used in
the medicinal purposes.

Mishra et al.; Curr. J. Appl. Sci. Technol., vol. 42, no. 14, pp. 1-11, 2023; Article no.CJAST.99622
10
ACKNOWLEDGEMENT
The authors are thankful to the management,
director of institution and laboratory staff for
providing me the infrastructure facilities for the
study.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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