JPAM Vol 15 Issue1 P 285-299

Sowmya & Raveesha | J Pure Appl Microbiol | 15(1):285-299 | March 2021
Article 6618 | https://doi.org/10.22207/JPAM.15.1.22

Print ISSN: 0973-7510; E-ISSN: 2581-690X
Research Article OPEN ACCESS
Antibacterial Activity and Time-kill Assay of

Terminalia catappa L. and Nigella sativa L. against
Selected Human Pathogenic Bacteria
Sowmya1 and Koteshwar Anandrao Raveesha 1,2*

1
Center for Innovative Studies in Herbal Drug Technology, Department of Studies in Botany, University of
Mysore, Mysuru - 570 006, Karnataka, India.
2
Department of Water and Health, Faculty of Life Sciences, JSS Academy of Higher Education and Research
Sri Shivaratreeshwara Nagara, Mysuru - 570 015, Karnataka, India.
Abstract
The current investigation aims to test the susceptibility of human pathogenic clinical isolates and MTCC
strains to leaf and seed extracts of Terminalia catappa and Nigella sativa. Disc diffusion assay, micro
dilution assay and minimum Bactericidal Concentration investigated the susceptibility of bacteria
to the test extracts. The active extract was subjected to phytochemical screening, separation of the
phytochemicals by Thin Layer Chromatography, bioactivity guided assay and Time- kill assay. Acetone
and methanol extracts of T.catappa revealed, significant inhibition of clinical origin Staphylococcus
aureus followed by Proteus vulgaris and the MTCC strains Staphylococcus aureus, Salmonella typhi,
Pseudomonas aeroginosa and Bacillus subtilis. Nigella sativa inhibited the growth of clinical origin
Staph.aureus and MTCC strain of Staph.aureus, Salmonella typhi and B.subtilis. Minimum inhibitory
concentration for all the test bacteria was reported in the range of 5000μg/ml to 9 μg/ml in T. catappa
extract. Most sensitive being the clinical isolate Staph. aureus and Proteus vulgaris. The bactericidal
concentration for the test bacteria was found to be between 5000μg/ml and 625μg/ml. Phyto-chemical
analysis of leaf extracts of T. catappa found to have dominated by polyphenols (Terpenoids, steroids,
flavonoids, flavones, saponins and tannins) and N.sativa extracts recorded the presence of alkaloids,
proteins and oils and fats. TLC profiling of the acetone extract revealed many antibacterial active bands.
Bands having Retention factor 0.47 and 0.52 were active against the test bacteria. Time kill assay of the
acetone extract of T. catappa were carried out for the first time. The extract exhibited dose dependent
bactericidal and bacteriostatic activity against the clinical isolates.
Keywords: Combretaceae, Terpenoids, Polyphenols, Thymoquinone, Bioautography, Time- kill assay
*Correspondence: [email protected]
(Received: August 31, 2020; accepted: January 26, 2021)
Citation: Sowmya, Raveesha KA. Antibacterial Activitiy and Time-kill Assay of Terminalia catappa L. and Nigella sativa L. against
Selected Human Pathogenic Bacteria. J Pure Appl Microbiol. 2021;15(1):285-299. doi: 10.22207/JPAM.15.1.22
© The Author(s) 2021. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which
permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons license, and indicate if changes were made.
Journal of Pure and Applied Microbiology 285 www.microbiologyjournal.org

Sowmya & Raveesha | J Pure Appl Microbiol | 15(1):285-299 | March 2021 | https://doi.org/10.22207/JPAM.15.1.22
Introduction growth of infectious microbes, many plants are

Recently there has been increase in the still being used for their antimicrobial properties
spread of untreatable microbial infections and and many other biological activities. Inspite of
bacteria cause 90% of the infections1. World Health existence of vigorous antimicrobial drugs, the
Organization reports that infectious diseases cause emergence of resistance microbes has created
50% deaths. The non-selective use of synthetically an immense interest in the exploration and the
originated antibiotics, microorganisms have led to outcome of efficacious drugs13.
the development of multidrug resistance, which Terminalia catappa is a large tropical tree
poses a serious health concern2. The resistance of the family Combretaceae1. Different parts of the
to drug may be caused mostly by the unregulated plant are used in folklore medicine and studies have
use of antibiotics and poor hygienic conditions revealed various medicinal properties15. Various
and affects severely in every aspect of life 3. The research disclose the medicinal uses like microbial
escalating resistance in microorganisms is due to inhibition of leaf aqueous and methanolic extract
the phenomenon of genetic mutations or acquired of the plant16. Bacterial inhibitory activity of leaf
antibiotic resistant genes influenced by ill-suited aqueous extract17, anti-inflammatory property of
use of antibiotics1. Additionally antibiotics are leaf ethanolic extract18, wound healing activity of
associated with unfavorable consequences on the bark, antioxidant and radical scavenging activity
host including hypersensitivity, allergic reactions of leaf aqueous extract, anticancer and anti-
and immune- suppression4. The multiple drug aging activity of ethanol and aqueous extract of
resistance has enforced researchers in search leaves have also been reported19. Anti-methicillin
of new drugs with antimicrobial property from potential of phytochemicals of T.catappa have also
different sources like medicinal plants, which been evident20. Insilico studies of compounds from
serve as acceptable source of novel antimicrobial T. catappa are responsible for hepatoprotective
agents 5. Rational localization of biologically and hepatotoxic properties 21. Extracts of the
active components from folk medicines and leaves of the plant also possess anticancer, anti-
systemic evaluation will result in finding of novel HIV reverse transcriptase, hepatoprotective,
efficacious drugs, which are potentially active Anti-inflamatory, Anti-hepatitis and Aphrodisiac
against pathogens 6. Antimicrobial drugs with effects 22 . Phytochemicals include tannins
efficacious mode of action should be developed (Punicalagin, Punicalin, Chebulagic acid, geranin,
in order to surpass the downside of current granitin B), flavonoids (Vitexin, Rutin, Isovitexin),
antimicrobial drugs7. A large number of medicinal and Terpenoids (Ursolic acid, Asiatic acid) which
plants are reported to exhibit antimicrobial are responsible for therapeutic activity23 .
activity2. Reports of WHO states that medicinal Nigella sativa, belongs to the family
plants would be the best root to acquire diverse Rananculaceae24. It is native to Mediterranian
of drugs1. In this context, plants promise a source regions such as South west Asia, Southern Europe
of natural antimicrobial agents8. Antimicrobial and North Africa25. Seeds contain yellowish volatile
activities of plants are ascribed to the presence oil, fixed oil, alkaloids, saponins, minerals and
of phytochemicals like tannins, phenols, alkaloids, vitamins26. The reported studies related to Nigella
terpenoids and flavonoids 9. The application sativa have illustrated wide range of biological
of plant based drugs in the treatment against activities such as antioxidant27, anti-inflamatory28,
pathogens is obtaining great acceptance because antidiabetic 29, anti prostate cancer effects 30,
of scientific interest and non toxicity properties 10. antibacterial31, immunomodulatory effect32, and
Approximately 119 drugs isolated from hepatoprotective33.
plants are used in treating infectious diseases Secondary metabolite analysis found that
worldwide11. Not less than 50% of the drugs seeds contain two types of alkaloids, isoquinone
which are used in treating clinically infectious alkaloids (Nigellimine – N oxide) and pyrazole
diseases accounts to have originated from alkaloids (Nigellicine and Nigellidine)34. Essential
natural and natural product derivatives12. Since phytoconstituents also include thymoquinone,
phytochemicals have the ability to inhibit the

P-cymene, 4- Terpineol, carvacrol, t- anethol, Microbroth dilution method for determining

longifoline, thymol, thymohydroquinone and Minimum inhibitory concentration (MIC) and
dithymoquinone .addtionaly they have a water Minimum Bactericidal Concentration (MBC)
soluble pentacyclic triterpene, alpha- hedarin and MIC was established by employing
saponins as anticancer agents 35. 96 well microtiter plate according to the CLSI
M07-A9 document37 . The test plant extract, which
Materials and methods showed the inhibitory activity, were selected
Preparation of the plant extract for determining MIC and MBC. The test plant
Mature leaf material was collected from extract were serially diluted two fold to obtain
fields in and around Mysuru, Karnataka. The leaves concentration of 5000-9μg/ml. The experimental
were shade dried and powdered using laboratory set up included reference drug controls and solvent
blender. N. sativa seeds were collected from the as positive and negative controls respectively.
local market, powdered and used for successive Aliquot of standardized inoculum (1.5 X 108 colony
solvent extraction by soxhlet extractor using forming units/ml) was added to all the wells. The
polar and non-polar solvents. After extraction micro dilution plate was covered to avoid drying
the solvents were evaporated to dryness under followed by incubation. Inhibition of bacterial
reduced pressure and preserved the extracts at growth was confirmed by addition of 20μl of
4°C for future analysis. aqueous solution of 2, 3,5-Triphenyl tertrazolium
Microorganisms and growth conditions: chloride (TTC) and re-incubated for 4-5h. The
Human pathogenic bacteria were obtained colorless well was designated as the MIC, which
from MTCC Chandigarh, Viz, B.subtilis (MTCC inhibited the growth of bacteria. Change of colour
121), B.cereus (MTCC 1272), Pseudo.aeruginosa, from colourless to pink indicated the presence of
(MTCC424) Salm. typhi (MTCC 733), Escherichia viable cells. MBC was ascertained by sub culturing
coli (MTCC 7410) and Staph.aureus (MTCC 7443). 10μl of test dilution from the lowest concentration
Human pathogenic clinical isolates were provided well by streaking on the previously sterilized and
by Microbiology department, Government Medical solidified MH agar with overnight incubation
College, Mysuru, Viz, Escherichia coli, Proteus at optimum temperature. The well, which gave
vulgaris, Klebsiella pneumoniae, Staphylococcus no bacterial colony on the agar medium, was
aureus and Salmonella typhi. The pathogens were designated as MBC.
maintained on Muller Hinton agar in glass tubes. Phytochemical composition of the active test
Bacteria were resuscitated by sub culturing from extracts
stock onto Muller Hinton agar plates followed by The test extracts were subjected to
incubation overnight for their optimum growth. qualitative phytochemical analysis according to the
Antibacterial activity of the plant extracts established procedures to determine the presence
Antibacterial susceptibility test by Disc diffusion of different class of secondary phytochemicals38,39.
assay Thin layer chromatography (TLC) of the active
Antibacterial potency of the test plant extracts
extracts were done by disc diffusion method The active extracts were subjected for
employing the CLSI M02-A document36. Inoculums separation of phytochemicals. Based on the
were prepared and adjusted to McFarland review of literature and slight modification, EMW
standard with cell density of 1to 1-2 X108 CFU/ (Ethyl acetate: methanol: water) with 40:5.4:4
ml. Standardized inoculum (0.1ml) was swabbed ratio showed better separation of the compounds
to the previously solidified MH agar plate. Sterile and it was best-suited eluent system for the
discs were loaded with test plant extract (100mg/ separation of phytocompounds from members
ml) and impregnated onto the plates followed by of Combretaceae. The retention factor (Rf) of the
incubation for 18-24 hours at 35 ±2°C. Standard separated phytochemicals was calculated40.
antibiotics and solvents were employed as positive TLC bioautography
and negative control. All experiments were carried Agar overlay technique41 was adopted
out in triplicates. Zone of inhibition (ZOI) around with minor modifications to locate the antibacterial
the disc was measured in mm. bands. The TLC plates (silica gel G f254, Merk) were
spotted with test plant extract(acetone extract) One milliliter of standardized test inoculum was
and developed in the pre saturated developing mixed with 10ml molten Muller Hinton agar. TLC
chamber using EMW as eluent system. After chromatograms were placed in the sterilized plates
the solvent front reached the optimum distance and thin layer of media inoculated with the test
the chromatogram was removed and visualized bacteria was flooded on the chromatogram. Plates
under long and short UV to locate the separated were incubated overnight at 37°C. ZOI around the
compounds. The chromatogram was dried separated phytochemicals can be seen as a clear
overnight and subjected to overlay bioautography. area without the growth of the test bacteria. As
Fig. 1 & 2. Antibacterial activity of Acetone extract (Fig:1) and Methanol extract (Fig :2) of T.catappa on human
pathogenic clinical isolates. Data are represented in mean ±SEM. Different letters in the bars depict the significant
difference at P<0.05. Error bars signify standard error. Inhibition zones are presented in millimeter (mm).
Fig. 3 & 4. Antibacterial activity of Methanol extract (Fig:3) and Acetone extracts (Fig :4) of T.catappa on human
pathogenic bacteria (MTCC). Data are represented in mean ±SEM. Different letters in the bars indicate statistical
difference (P<0.05). Error bars signify standard error. Inhibition zones are presented in millimeter (mm).
a confirmatory test the chromatograms were Satatitical analysis

flooded with microbiological agar containing TTC Values are presented as Mean ±SEM.
and further incubates for 30 minutes for optimum ANOVA was employed to determine the difference
colour development. The ZOI is seen as white zone in specific means followed by tukey’s post hoc
against the pink background. test at P<0.05. Graphpad prism (version 8) and
Time kill assay Microsoft excel 2007 was used in generating
Protocol of kill time assay was adopted42 graphs and analyzing the data.
with slight modifications. The active extract of T.
catappa was set at a concentration of 4MIC and Results
8MIC. A control tube was maintained without Disc diffusion assay
the test extracts. Each tube was then inoculated The test organisms exhibited different
with standardized suspension of clinical isolates sensitivity for the extracts tested which is shown
of S.aureus and P.vulgaris At prefixed time points in Fig. 1 to 4. All the test pathogens exhibited
aliquots were withdrawn from each concentration significant sensitivity towards acetone and
diluted serially (10-1 to 10- 4) and aliquots were methanol extracts of T.catappa. Acetone extract
plated on agar plates. Colony counts were had good inhibitory activity against the test
performed after overnight incubation at 37°C in organisms in contrast with the methanol extract.
ambient air. Colony counts were averaged and Significant inhibition was observed against the
expressed as log10 cfu/ml. clinical isolate Staph.aureus (22.5mm) followed
Fig. 5. MIC of active extracts of T. catappa on human pathogenic bacteria.
Fig. 6. MIC of active extracts of T. catappa on human pathogenic clinical isolates.

by P.vulgaris (20.5mm), Salm.typhi and Kleb. Staph.aureus (MTCC 7443) and Salm.typhi (MTCC
pneumoniae with inhibition zone 16.4mm and 733) with zone inhibition 11.25mm and 15.9mm
12.4mm respectively. The MTCC strains also respectively.
exhibited sensitivity towards the acetone and Micro dilution assay
methanol extracts with zone of inhibition ranging Considering the results obtained from
between 11.7mm and 23.3mm. Hexane extract disc diffusion assay, deducing the lowest inhibitory
of N.sativa inhibited the growth of clinical isolate concentration (MIC) of active extracts was
of Staph.aureus with inhibition zone 19.08mm, considered necessary and the results are shown
in Fig. 5 and 6.
Of all the organisms tested, the clinical
isolate Staph.aureus and P. vulgaris was the
Fig. 7. Chromatogram of acetone extract of T.catappa

leaf eluted wih Ethyl acetate, Methanol and Water
solvent system (40:5.4:4) showing clear seperation of
phytocompounds.
Table 1. Minimum Inhibitory and Minimum Fig. 8. Agar overlay bioautography of acetone extract of
Bactericidal Concentration and of acetone T.catappa leaf showing inhibition of P. vulgaris(Clinical
isolate) at Rf values 0.71. 0.59 and 0.47.
and methanol extract of T.catappa on human
pathogenic bacteria (MTCC)
Table 2. Minimum Inhibitory and Minimum Bactericidal
Concentration of acetone and methanol extract of
MTCC Acetone Methanol T.catappa on human pathogenic clinical isolates
strains extract extract
MIC MBC MIC MBC Clinical Acetone Methanol
isolates extract extract
S.aureus 9 * 78 * MIC MBC MIC MBC
S.typhi 9 625 78 5000
B.cereus 1250 2500 2500 5000 S.aureus 39 1250 78 5000
P.vulgaris 312 5000 312 5000
B.subtilis 1250 * 1250 *
K.pneumoniae 1250 5000 1250 5000
P.aeruginosa 625 5000 625 5000 S.typhi 625 5000 1250 5000
FN: Values presented in μg/ml; *: Indicates no MBC FN: Values presented in μg/ml
established
most sensitive to the acetone extract with

lowest concentration of 39μg/ml and 312μg/ml
respectively, Kleb.pneumoniae and Salm.typhi
were less susceptible with concentration 1250μg/
ml and 625μg/ml respectively. The acetone
extract also exhibited inhibitory effect at the
lowest concentration against all the MTCC strains
tested except E.coli with concentrations between
1250μg/ml and 9μg/ml.
Methanol extract showed inhibitory
activity towards the test pathogens but slightly
less effective compared to the acetone extract
having concentration between 5000μg/ml and
625μg/ml for clinical isolated bacteria. The
minimum concentration of methanol extracts
required against B.subtilis (MTCC 121) Salm.typhi
(MTCC 733), Staph.aureus (MTCC 7443), Pseudo.
aeruginosa (MTCC 424), and B. cereus (MTCC
1272) was in the range between 1250μg/ml and
78μg/ml.
Minimum Bactericidal Concentration
Acetone extract reported bactericidal Fig. 9. Agar overlay bioautography of acetone extract
effect at 1250μg/ml for clinical isolate Staph. of T.catappa leaf showing inhibition of S.aureus (MTCC
aureus where as the bactericidal effect for Kleb. 7443) at Rf values 0.47, 0.2 and 0.07.
Fig. 11. Agar overlay bioautography of acetone

Fig. 10. Agar overlay bioautography of acetone extract
of T.catappa leaf showing inhibition of S.typhi (MTCC
extract of T.catappa leaf showing inhibition of
733) at Rf values 0.92, 0.79, 0.59, 0.52, 0.47, 0.39, 0.2 S.aureus (clinical isolate) at Rf values 0.47, 0.39,
and 0.07. 0.2 and 0.07.
pneumoniae, Salm.typhi and P. vulgaris was 0.92, 0.79, 0.59, 0.52, 0.47, 0.39, 0.2 and 0.07.
5000μg/ml. MBC against Salm.typhi (MTCC 733), Bands with Rf value of 0.47, 0.2 and 0.07 strongly
B.cereus (MTCC 1272) and Pseudo.aeroginosa inhibited the Staph.aureus (MTCC 7443) (Fig. 9).
(MTCC 424) was at the concentration of 625 μg/ Clear zone of inhibition around the separated
ml, 2500 μg/ml and 5000 μg/ml respectively. bands was indicative of antibacterial property of
On the contrast bactericidal action of methanol the compounds present.
extract was 5000μg/ml for all the bacterial clinical Colorless areas indicate inhibition of the
isolates and the MTCC strains except Staph.aureus test organisms by the separated phytocompounds.
(MTCC 7443) and B.subtilis (MTCC 121), which did Kill time assay
not show any bactericidal activity at the tested The time kill profile for clinical isolates
concentration. Staph. aureus and P.vulgaris are presented in
Results of phytochemical analysis Table 4 showing the reduction in viable cell counts
presented in Table 3. Broad group of phytochemicals at particular time intervals. For both bacterial
were present in the acetone and methanol extract strains a separate time kill profile was produced
like terpenoids, flavonoids, saponins, cardiac over a period of 24 hours of incubation following
glycosides, phenols and tannins. Hexane extract inoculation. Difference in the viable counts for
of Nigella sativa was dominated by alkaloids, oils both bacterial strains were more after 4h post
and fats. inoculation. At concentration corresponding to
TLC profiling and bioautography 8MIC bactericidal activity as observed at time
Thin layer chromatography of acetone point of 2-4h for Staph.aureus (Fig. 13) after
extract using EMW solvent system was capable of which regrowth of the bacteria was observed
eluting bands with Rf values 0.07, 0.20, 0.39, 0.47, indicating concentration dependent killing of the
0.52, 0.59, 0.71, 0.79, 0.88 and 0.92. test bacteria. However, 4MIC concentration did
Agar overlay bioautography of acetone not kill the bacteria but slowed down the growth
extract of T. catappa showed clear zone of compared to initial control. For P. vulgaris (Fig. 12)
inhibition at Rf values 0.47, 0.2 and 0.07 against maximum reduction of viable cell counts was seen
clinical isolate S. aureus (Fig. 11) and Proteus after 24h of incubation at 8 MIC concentration
vulgaris (Fig. 8) was inhibited by the bands with Rf with near bactericidal effect of 3.37 log10 Cfu/
values 0.71, 0.59 and 0.47. S.typhi (MTCC 733) (Fig. ml and 4MIC concentration reduced the viable
10) was also inhibited by the bands with Rf value cell counts after 8h of incubation with 3.9log10
Table 3. Qualitative phytochemical analysis of the test plants
Secondary Tests T.catappa N.sativa

metabolites
Acetone Methanol N-hexane
Terpenoids Salkowski test + + -

Liebermann test + + -
Alkaloids Mayers test - - +
Wagners test - - +
Flavonoids Lead acetate test + + -
Shinoda test + + -
Alkaline test + + -
Saponins Froth test + + -
Tannins Gelatin test + + -
Oils and fats Oils and fats - - +
Phenols Fecl3 test + + -
Cardiac glycosides Killer-kellani test + + -
FN: +: Present; --: Absent

Table 4. Time kill profile of acetone extract of Terminalia catappa showing reduction of viable cell counts at
different time intervals.
Time intervals 0hr 2hr 4hr 6hr 8hr 24hr 0hr 2hr 4hr 6hr 8hr 24hr
Test bacteria S.aureus (Log10cfu/ml) P.vulgaris (Log10cfu/ml)
Concentration
Control 2.89 3.19 3.32 4.85 5.87 6.19 3.5 4.38 4.91 5.78 5.8 6.07
4MIC 2.82 3.20 2.1 3.38 3.74 6.24 3.21 4.2 4.02 4.07 3.9 5.76
8MIC 2.38 3.0 3.0 3.25 3.27 6.11 3.40 3.4 3.74 3.68 3.84 3.37
Fig. 12. Time kill curve of Acetone extract on P. vulgaris (Clinical isolate).
Fig. 13. Time kill curve of Acetone extract on Staph.aureus (Clinical isolate).
Cfu/ml exhibiting the bacteriostatic action of dose and time dependent killing effect irrespective
the extract. For both the bacteria tested the of the bacteria.
viable colonies were reduced post inoculation
after which the colonies regrew until the end Discussion
of time course. However, there was delay in the The present investigation reports the
exponential growing phases of the test bacteria. acetone and methanol extract of T.catappa leaf
8MIC concentration was more effective than 4MIC and hexane extract of N.sativa seeds demonstrated
for both the test bacteria indicating the extract is the antibacterial activity against the human

pathogenic bacteria and clinical isolates. The bacteria. We report strong antibacterial inhibition
preliminary method for evaluating the sensitivity by polar solvent extracts (acetone and methanol)
of the test bacteria towards the plant extract is of T.catappa against clinical isolate Staph.aureus
inhibition zone testing by disc and well diffusion followed by Salm.typhi (MTCC 733) and clinical
assay. However, to test the efficacy of the extracts, isolate P.vulgaris.
broth dilution assays are also used preferably The hexane extract of N.sativa exhibited
microbroth dilution assay41. inhibitory activity against clinical isolate Staph.
Acetone and methanol extract of aureus, Salm. typhi (MTCC 733), Staph. aureus
T.catappa demonstrated varying level of activity (MTCC 7443) and B. subtilis (MTCC 121). The
with inhibition zones ranging between 11.66 and hexane extract of N.sativa showed susceptibility
23.33 mm for all the test bacteria. against gram positive and negative bacteria
The results are in accordance with the indicating wide range inhibitory activity. The
previous reports where in polar solvent (methanol results are in agreement with other workers
and aqueous) extracts of T. catappa leaves in where methanol and N-hexane extract of N.
addition to twenty other plants were evaluated sativa showed considerable good activity against
against bacteria (Gram positive and gram negative) the test pathogens showing varying zone of
and found that majority of the test strains were inhibition with varying dilutions. Gram-positive
susceptible to methanol extract having inhibition S.epidermidis was most sensitive to the extracts52.
zone 5-18mm16. The differences in the antibacterial Aqueous and alcoholic extract of seeds of N.sativa
activity can be due to the different chemical against bacteria and fungi was reported and all the
composition and the distinct mechanism of action extracts posed different efficacy against the test
of their bioactive constituents43. The results can organisms53.
also be attributed with previous reports, which Evaluation of essential oil for antibacterial
recorded the aqueous extracts of T.catappa activity, which showed the suppression of test
exhibited inhibitory activity against Staph. pathogens exhibiting varying zone of inhibition54.
aureus, Kleb.pneumoniae, E.coli and Candida The results can be related to other reports where
albicans 44. Several other reports have revealed the essential oil and oleoresins exhibited the
the antimicrobial activity of leaves and fruits of significant inhibition of test organisms55. N-hexane
T.catappa on different gram positive and negative extract and seed oil of N.sativa on selected human
bacteria45. The susceptibility results obtained by pathogenic bacteria was evaluated and found that
disc diffusion assay of leaf methanol extract of T. the test microbes were inhibited by both extract
catappa against Staph.aureus, pseudo. aeroginosa, and oil56. The results obtained in the study are
B.subtilis and a clinical isolate Staph.aureus in concordance with the previous studies. The
demonstrated that gram-positive organisms were methanol extracts obtained from the seeds were
more susceptible46. Polyphenols are reported to evaluated for antibacterial activity by and found
have antibacterial activity47. Various polyphenols that Staph. aureus and Pseudo.aeroginosa were
isolated from T.catappa like catappanin, geranin, sensitive to the extracts57. N-hexane, methanol and
gallantonic, leutolin, apigenin, orientin, isovitexin, aqueous seed extract of N.sativa was evaluated for
catachien, kampferol, genistein, querccetin, ellagic their invitro susceptibility activity where methanol
acid, chlorogenic acid, ferulic acid , arjunetin,, extract showed considerable inhibition of Staph.
ursolic acid, gallic acid, arjunoilc acid, betulinic aureus, Escherichia coli and Salm. enterica58. The
acid and several compounds from T.catappa have efficacy of N.sativa oil is attributed to its quinone
been reported. The above isolated compounds constitutes in the fixed and essential oil, which
from the plant could be acreditted to the invitro is, endowed with thymoquinone a significant
susceptibility of bacteria found in another study48. bioactive constituent making up 30-48% of total
Several other workers49-51 have also reported the constituents. Other functional constituents include
antibacterial properties of various other parts of p-cymeme, carvacrol, thymohydroquinone,
T. catappa against human pathogenic bacteria. dihydrothymoquinone, thymol, α-thujene,t-
Our results exhibit a wide spectrum inhibitory anthole, β-pinene, α pinene and γ-terpinene59.
activity towards both gram-positive and negative
The MIC values of T.catappa showed, that
acetone extract was more potent in inhibiting the several continents for treating numerous diseases
test bacteria and methanol extract was slightly less including cardiovascular effects, wound healing,
potent than the acetone extract. However, hexane abdominal disorders, conjunctivitis, hypertension,
and chloroform extract had no activity against the pneumonia, gastric ulcers, jaundice, leprosy,
test strains owing to the fact that polar component edema and skin diseases64. Various species of
of the extract is posing the antibacterial action. Terminalia have been utilized in treating infections
The acetone extract showed strong inhibition caused microbes and many current researchers
of clinical isolate Staph. aureus (MIC; 39μg/mL) have documented their antibacterial potential.
and P. vulgaris (MIC; 625μg/mL). MIC values of Indian and southern Asian Terminalia species
acetone extract for MTCC bacteria ranged between have been particularly studied well. Antibacterial
9μg/ml and 1250μg/ml. Methanol extract posed susceptibility of wide panel of organisms has been
the lowest inhibitory concentration (MIC) from specifically documented in Terminalia arjuna,
78μg/ml to 1250μg/ml for both clinical isolates Terminalia belllirica Terminalia catappa and
and MTCC strains. However, the methanol extract Terminalia chebula65.
exhibited moderate inhibitory activity compared to
Qualitative secondary metabolite
acetone extract. MBC values were more than the screening of active extracts (acetone and
MIC. When higher concentration used there might methanol) exhibited the dominance of terpenoids,
result in bactericidal effect. Some researchers phytosterols, flavonoids, flavones and saponins.
have reported the antimicrobial agents having These compounds have been previously reported
MIC values between 1.60mg/ml to 8mg/ml as frail by earlier workers66-69. The leaves of Terminalia
microbial inhibitors60. None of the extracts showed catappa contain chebulagic acid corilagin,
MIC values greater than 8mg/ml. In agreement gentisic acid, geranin, granatin B, kaempferol,
with our study the previous reports where the punicalagin, punicalin, quercetin, terfavin A and
crude extracts of leaves of T. catappa and eight terflavin B 70. The study is in agreement with
other plants were evaluated for MIC against human the 71 wherein quercetin was identified in the
pathogenic bacteria and reported that pathogens leaves of Terminalia catappa along with other
were inhibited at the concentration from 16mg/ml components like flavonoids, carotenoids and other
to 0.015mg/ml61. The MIC of leaves of T.catappa phenolic compounds, which is responsible for the
was evaluated at concentration ranging from traditional use of the plant.
100mg/ml to 350mg/ml against a panel of Hexane extract of N. sativa revealed the
bacteria. Bacteria included Staph. aureus, Bacillus presence of alkaloid, which is the major component
subtilis, Bacillus cereus, Pseudomonas aeroginosa, of the seeds. The results are in accordance with
Proteus mirabilis, Salmonella typhi and Shigella the earlier work72, which reported the occurrence
dysentriae 62. Comparative evaluation of leaf of tannins, alkaloids, flavonoids and sterols.
aqueous and methanol extracts of Combretum and TLC bioautography helps in locating the
Terminalia spp. of southern Africa were evaluated, antibacterial active spots on the chromatogram.
which reported that both the species had broad EMW solvent system was capable in eluting
spectrum antimicrobial activity inhibiting both the antibacterial compound in acetone extract
bacteria and fungi. The MIC values inhibiting the of with Rf value of 0.59, 0.52, 0.47 and 0.39.
test strains were less than 1000 μg/ml where in Bioautography studies revealed that most of the
Bacillus subtilis had least MIC ranging from 124- separated compounds had antibacterial potential,
578μg/ml Staphylococcus aureus (395-770 μg/ which can be due to the occurrence of polyphenols
ml), K.pneumoniae (318-531μg/ml) P.aeroginosa in the test plant extract, which is also confirmed
(36-512μg/ml)63. Our work reports that the test from the qualitative phytochemical analysis. Time
strains were inhibited at the lowest concentration kill studies are important because they provide
with special reference to clinical isolate of information about pharmacodynamics of the
Staph. aureus and Proteus vulgaris. Members antibacterial agent73. Extracts showed variable
of the Terminalia genus have a long history in kinetics against test bacteria. Time kill findings
traditional system of medicines and are used in displayed different levels of time dependent

and concentration dependent inhibition of the providing the facilities and to the Department
organisms. Bacteriostatic and bactericidal activity of Microbiology, Mysore Medical College and
was displayed by the extract mainly against Research Center for providing the clinical bacterial
clinical isolates but regrowth of the test bacteria isolates.
occurred after specific time interval which could be
attributed to the use of lesser concentration of the CONFLICT OF INTEREST
extracts and also the gram positive and negative The authors declare that there is no
bacteria differ in cell wall and the membrane conflict of interest.
composition which regulate their susceptibilities
to plant metabolites. At higher concentration AUTHORS’ CONTRIBUTION
bactericidal activity may be achieved invitro. Time All authors have made substantial, direct
kill kinetics of T.catappa against selected human and intellectual contribution to the work and
pathogenic bacteria was carried out for the first approved it for publication.
time, however there are fewer reports related to
time kill kinetics of fungi74 but, reports on bacteria FUNDING
are scarce to the best of our knowledge. Authors are thankful to ICMR-New
Delhi and VGST-CISEE, Government of Karnataka
Conclusion for financial assistance. funding grant number
The results achieved in this study indicate No.45/19/2018/TM/BMS
that T.catappa and N.sativa are potential sources
of antibacterial agents against the bacterial DATA AVAILABILITY
clinical isolates in particular. The acetone extract All datasets analyzed during this study
of T.catappa exhibited wide spectrum activity are included in the manuscript.
against (gram-positive and gram-negative)
MTCC and clinical isolates. The extracts also ETHICS STATEMENT
inhibited the test bacteria at a very minimal Not applicable.
concentration. Bioautography profile of acetone
extract resulted in potent antibacterial bands, References
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Sowmya & Raveesha | J Pure Appl Microbiol | 15(1):285-299 | March 2021

Article 6618 | https://doi.org/10.22207/JPAM.15.1.22

Research Article OPEN ACCESS

Antibacterial Activity and Time-kill Assay of

Sowmya1 and Koteshwar Anandrao Raveesha 1,2*

(Received: August 31, 2020; accepted: January 26, 2021)

Journal of Pure and Applied Microbiology 285 www.microbiologyjournal.org

Introduction growth of infectious microbes, many plants are

Journal of Pure and Applied Microbiology 286 www.microbiologyjournal.org

P-cymene, 4- Terpineol, carvacrol, t- anethol, Microbroth dilution method for determining

a confirmatory test the chromatograms were Satatitical analysis

Fig. 5. MIC of active extracts of T. catappa on human pathogenic bacteria.

Fig. 6. MIC of active extracts of T. catappa on human pathogenic clinical isolates.

Fig. 7. Chromatogram of acetone extract of T.catappa

most sensitive to the acetone extract with

Fig. 11. Agar overlay bioautography of acetone

Table 3. Qualitative phytochemical analysis of the test plants

Secondary Tests T.catappa N.sativa

Terpenoids Salkowski test + + -

FN: +: Present; --: Absent

Journal of Pure and Applied Microbiology 292 www.microbiologyjournal.org

Journal of Pure and Applied Microbiology 293 www.microbiologyjournal.org

Journal of Pure and Applied Microbiology 295 www.microbiologyjournal.org

Journal of Pure and Applied Microbiology 296 www.microbiologyjournal.org

J Nat Med. 2008;62(2):259-262. doi: 10.1007/s11418- 7847.162103

Journal of Pure and Applied Microbiology 297 www.microbiologyjournal.org

Journal of Pure and Applied Microbiology 298 www.microbiologyjournal.org

Journal of Pure and Applied Microbiology 299 www.microbiologyjournal.org

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