Original Article

Assessment of potential antimicrobial

activity of Ocimum basilicum extract
Department of Public
and chlorhexidine against Socransky’s
Downloaded from http://journals.lww.com/jisp by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW

Health Dentistry, KLE

Vishwanath Katti Institute
of Dental Sciences,
KLE Academy of Higher
complex pathogens of oral cavity: An
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 01/22/2024

Education and Research,

1
Dr. Prabhakar Kore in vitro study
Basic Science Research
Centre {BSRC}, Atrey J. Pai Khot, Anil V. Ankola, Suneel Dodamani,1 Roopali M. Sankeshwari,
Nehrunagar, Belagavi, Ram Surath Kumar, Varkey Nadakkavukaran Santhosh
India

The work belongs to Abstract:

the Department of
Background and Objective: Periodontitis is a multifactorial disease initiated by periodontal pathogens and
Public Health Dentistry, progresses further in destruction of periodontium. Hence, the objective of this study was to test the efficacy of
KLE Vishwanath Katti Ocimum basilicum seeds extract on periodontal pathogens. Materials and Methods: O. basilicum seeds were
Institute of Dental authenticated from a recognized taxonomist. They were coarsely powdered; ethanol‑based extract preparation
Sciences, KLE Academy was done by the Soxhlet method and aqueous‑based extract by hot infusion procedure. Extracts so obtained
of Higher Education were assessed for minimum inhibitory concentration, minimum bactericidal concentration, zone of inhibition,
and Research, and time‑kill assay of O. basilicum seeds extract on periodontal pathogens, and comparatively evaluated the
Belagavi - 590 010, effectiveness against 0.12% chlorhexidine (CHX) gluconate in triplicates. Kruskal-Wallis Test was employed
wherein the statistical significance was set at P ≤ 0.05. Results: The concentration of O. basilicum ethanolic
India
extract against periodontal pathogens was determined to be 10 mg/ml, whereas 4.7 mg/ml of aqueous extract
was proven effective against periodontal pathogens. Similarly, aqueous extract of O. basilicum developed a wider
zone against periodontal pathogens compared to ethanol‑based O. basilicum extract. Statistically significant
Access this article online difference found in the effectiveness between both extract and CHX. Conclusion: The antibacterial activity was
evident in both the extracts of O. basilicum against anaerobic periodontal pathogens. However, it was more
Website:
https://journals.lww.com/jisp pronounced in aqueous extract, but lower compared to CHX.
Key words:
DOI:
10.4103/jisp.jisp_406_22 Antimicrobial activity, basil seeds, herbal extract, Ocimum basilicum, periodontal pathogens
Quick Response Code:

INTRODUCTION actinomycetemcomitans), Porphyromonas gingivalis (P.

gingivalis), and Tannerella forsythia (T. forsythia) are

P eriodontal disease is one of the most crucial

Address for
correspondence:
Dr. Ram Surath Kumar,
Department of Public
Health Dentistry, KLE
Vishwanath Katti Institute
of Dental Sciences,
KLE Academy of Higher
Education and Research,
Belagavi - 590010, India.
E‑mail: ramsurathkumar
1996@gmail.com
Submitted: 09‑Sep‑2022
Revised: 07‑Feb‑2023
Accepted: 19‑Feb‑2023
Published: 01-Sep-2023
© 2023 Indian Society of Periodontology | Published by Wolters Kluwer - Medknow
issues for dentists and patients. It is identified
as a major public health problem throughout the
world and is the common reason of tooth loss in
adults. Periodontitis is characterized by microbially
associated, host‑mediated inflammation that
results in loss of periodontal attachment.[1] The
periodontium is a connective tissue organ, protected
by epithelium, that attaches the teeth to the bone
of the jaws and provides a continually adapting
apparatus for their support during function.[2] A
variety of triggering factors like bacterial species,
dyscrasias, avitaminosis, etc., cause inflamed
gums leading to gingivitis.[3] Salivary tartar has
an additive effect to these factors in causing
gingivitis.[4] Aggressive periodontitis, chronic
periodontitis, and those resulting from conditions
such as acquired immunodeficiency syndrome,
diabetes, malnutrition, and immunosuppression
are the other types of periodontitis.[5] Researchers
found that Aggregatibacter actinomycetemcomitans (A.
likely to cause aggressive periodontitis.[6] Deep
pockets are caused by bacteria such as P. gingivalis
and A. actinomycetemcomitans, as well as other
species, and are linked with resistance to traditional
therapies.[7]
This is an open access journal, and articles are
distributed under the terms of the Creative Commons
Attribution‑NonCommercial‑ShareAlike 4.0 License, which
allows others to remix, tweak, and build upon the work
non‑commercially, as long as appropriate credit is given and
the new creations are licensed under the identical terms.
For reprints contact: WKHLRPMedknow_reprints@
wolterskluwer.com
How to cite this article: Pai Khot AJ,
Ankola AV, Dodamani S, Sankeshwari RM,
Kumar RS, Santhosh VN. Assessment of potential
antimicrobial activity of Ocimum basilicum extract
and chlorhexidine against Socransky’s complex
pathogens of oral cavity: An in vitro study. J Indian
Soc Periodontol 2023;27:479-86.
479
 Pai Khot, et al.: Antimicrobial activity of the Ocimum basilicum extract against selective periodontal microorganisms

Despite major breakthroughs in disease biology and treatment
approaches during the last century, the periodontal disease is
still unmanageable over the world. Chlorhexidine (CHX) is a
commonly prescribed medication for the treatment of dental
disease.[8] The proof of its efficacy in plaque control is beyond
dispute. It has been used by dental practitioners for nearly three
decades. However, the use of CHX for dental disease prevention
has been controversial owing to its various side effects such as
Downloaded from http://journals.lww.com/jisp by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW
and The study was approved by the Institutional Research
and Ethics Committee of KAHER’s KLE Vishwanath Katti
Institute of Dental Sciences (reference number: 1499 and
date of approval: 25th October 2021). O. basilicum seeds were
authenticated from a recognized taxonomist. The coarse
powder of O. basilicum seeds was procured from Ayurveda
pharmacy of a recognized institute (AYUSH Approved Central

Research Facility and Drug testing laboratory for Ayurveda,

dry mouth, irritation, teeth staining, and taste disturbances.[9]
The use of herbal products offers a valuable alternative to CHX
owing to their medicinal values and no side effects.[10,11]
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 01/22/2024
Siddha, and Unani Drugs).
The seeds were crushed with a pestle in a mortar to much
finer particles, of which 100 g was soaked in 1 L of boiled hot

The practice of medicine has evolved over many centuries water for 48 h, and the mixture obtained was filtered with
to reach its current state. India, since time immemorial, Whatman number 1 filter paper (Sigma Aldrich® Chemicals
is a land where natural herbs and derived products have Pvt. Ltd., Bangalore, India) which was adapted according to
been used to cure various diseases. Herbal and natural hot infusion method on a thermostatically water bath (Labline®
folk medicine products have been utilized for generations Stock Center, Mumbai, India). The filtrate was concentrated at
for betterment of humankind. The most common herbal 40°C in a rotary evaporator (IKA™), at 190–220 rpm for 48 h and
ingredients to be incorporated into oral care products stored in the refrigerator at 4°C in air‑tight sterile container till
(e.g., toothpaste and mouth rinse) are sanguinarine, propolis, further use [Figure 1].
Azadirachta indica (neem), charcoal clove, and miswak.[12]
Basil, popularly known as the “King of Herbs,” is high in The Soxhlet method was followed to prepare crude extract
phytochemicals that have substantial nutritional, antioxidant, of the O. basilicum seeds, where in 99.9% ethanol (Changshu
and health advantages. Basil seeds are authenticated as Hongsheng Fine Chemicals Co. Ltd, China) was used as a
“Ocimum basilicum” belonging to the family “Lamiaceae,” which solvent. The seed powder (100 g) was placed in a muslin
is an annual plant.[13,14] Sweet basil seeds contain polyphenolic cloth bag which was kept in the body of Soxhlet extractor,
flavonoids, particularly orientin and vicenin; essential oils such and to this, ethanol (800 ml) was added. The entire
as eugenol, citronellol, linalool, limonene, citral, and terpineol; procedure ran for 6 h at a boiling temperature of ethanol
high levels of beta carotene, lutein, zeaxanthin, Vitamin A,
Vitamin C, and Vitamin K; and minerals such as potassium,
manganese, copper, calcium, magnesium, and folates.[14]
Furthermore, different research has shown that sweet basil
seeds provide health benefits such as weight loss, healthy
skin, cooling effect, acidity prevention, anti‑inflammatory,
and anticancer properties.[15,16] Active compounds found in
O. basilicum seeds are planteose, mucilage, and polysaccharides.
Moreover, secondary metabolites of seeds, including phenolic
and flavonoid, have been shown to exhibit pharmacological
properties such as antioxidant, antibacterial, antiviral,
antidiabetic, anti‑inflammatory, antiallergic, anticancer,
neurodegenerative, and vasodilatory effects.[17]
a b
A thorough literature search found limited studies reporting
the antibacterial activities of diverse species of Ocimum
against cariogenic pathogens. Thus, there is an impending
need for assessing the antimicrobial properties of a novel
O. basilicum extract against selective periodontal pathogens.
However, periodontal disease control is urgent, and there
are considerable gaps in the study field. Hypothesis states
that there is a difference in the effectiveness of O. basilicum
seeds extract and 0.12% CHX gluconate against P. gingivalis,
A. actinomycetemcomitans, and T. forsythia. Hence, the aim and
objective of this study was to assess the potential antimicrobial
efficacy of ethanolic and aqueous solvent‑based O. basilicum
seed extract against periodontal pathogens in comparison with
CHX gluconate, so that its formulation can be effectively used
to reduce the pathogenic microorganisms in the oral cavity.
c d
MATERIALS AND METHODS Figure 1: Ocimum basilicum extract preparation. (a) Hot infusion method for
Ocimum basilicum AE preparation; (b) Filtration of the supernatant O. basilicum
The present study was an in vitro study; it was conducted AE; (c) Soxhlet method Ocimum basilicum ethanolic extract preparation; (d) Rotary
according to the guidelines of good laboratory practice[18] evaporator (IKA™) to concentrate the filtrate of extract. AE – Aqueous Extract

480 Journal of Indian Society of Periodontology - Volume 27, Issue 5, September-October 2023
 Pai Khot, et al.: Antimicrobial activity of the Ocimum basilicum extract against selective periodontal microorganisms
78° C (173° F) completing 24 cycles. Once the process was
finished, the ethanol was entirely evaporated using rotary
evaporator (IKA™), at 40°C, leaving a yield of extracted plant
material (about 20 mg). The yield was resuspended in dimethyl
sulfoxide (DMSO) (Qualigens, Thermo Fisher Scientific Pvt.
Ltd, Mumbai, India) in 20 mg/ml ratio to obtain a stock
solution. The extracts obtained from both the techniques
were subjected to preliminary phytochemical screening for
Downloaded from http://journals.lww.com/jisp by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW
qualitative detection of phytoconstituents using standard
procedures as described by Trease and Evans.
The standard fresh strains of P. gingivalis – ATCC
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 01/22/2024
33277, A. actinomycetemcomitans – ATCC 29523, and T.
forsythia – ATCC 43037 were procured from LGC Promochem
India Pvt. Ltd., Bangalore, India. The strains were cultivated
anaerobically in 5 ml sterile brain heart infusion (BHI)
broth (Sigma Aldrich® Chemicals Pvt. Ltd., Bangalore, India)
supplemented with 1% horse serum, 0.5 mg/ml of hemin,
and 5 mg/ml of Vitamin K, and inoculated in anaerobic
chamber (N2 80%, H2 10%, and CO2 10%) at 37°C for 48 h until
it achieved the turbidity of 0.5 McFarland standards (1 × 108
colony‑forming unit (CFU)/ml).
Twelve mgs of CHX salt was mixed and diluted in
10 ml of distilled water (1.2 mg/ml) in a centrifuge tube
(Tarsons Products Ltd., Kolkata, India). The mixture was
vortexed vigorously using a MixMate ® Vortex agitator
(Eppendorf, Sydney, Australia) for 30 s at 1000 rpm (maximum
setting) to obtain a final concentration of 10 ml of 0.12% of
CHX solution. Following which 96‑well microtiter plate (NEST
Biotechnology, Jiangsu, China) were taken, and 10 wells were
allotted for each extract in triplicates. A volume of 100 µl broth
was added in all the wells, and 100 µl of each extract was added
in the first well; serial doubling dilutions were implied for the
extract to requisite concentrations up to the 10th well. Therefore,
the concentration in subsequent wells reduced by 50%. A
volume of 10 μl of inoculum was added to all 10 wells and
kept for incubation at 37°C in McIntosh and Fildes’ anaerobic
jar for 2 days. After 48 h, 10 µl resazurin dye (5 mg/10 ml
distilled water) (Hi‑Cert™ HiMedia® Laboratories, Pvt. Ltd,
Mumbai, India) was added and observed for color change
from blue/violet to slight pink/magenta except positive
control by incubating in anaerobic condition at 37°C for 4 h.
The concentration at which resazurin reduces to resafurin
compound by color change from blue to pink color was taken
as a minimum inhibitory concentration (MIC) value.[19] This
procedure was repeated for three periodontal pathogens and
two extracts separately. The results recorded in triplicate
outcomes and mean value were taken.
The spread plate method was used to determine minimum
bactericidal concentration (MBC). The bacterial suspension
from alternate wells with dilutions of O. basilicum ethanolic
extract (20, 5, 1.25, 0.312, and 0.078 mg/ml), O. basilicum aqueous
extract (10, 2.5, 0.625, 0.156, and 0.039 mg/ml), and CHX
(0.12, 0.03, 0.0075, 0.0018, and 0.00046 mg/ml) concentrations
higher than the MIC value was inoculated on BHI agar plates
and incubated for 48 h at 37°C. The concentration at which
no bacterial growth was seen, considered bactericidal values.
The MBC is defined as the minimum concentration of drug
which kills 99.9% of the test microorganisms in the original
inoculum.[20]
Journal of Indian Society of Periodontology - Volume 27, Issue 5, September-October 2023
Agar well diffusion (punch well diffusion) was used to test
antimicrobial susceptibility. Lawn culture of test organism
was made on respective media. The whole surface of the
agar plate was swabbed three times with the cotton swab.
Following which, individual 4 mm depth and 6 mm diameter
wells were cut in agar plates with sterile borer. These wells
were filled with 100 μl of stock solution (20 mg/ml) for
each extract and control to determine the antimicrobial
activity against the pathogens. CHX gluconate (0.12%)
(ICPA Health Products Ltd, Mumbai, India) was used
as a positive control and saline (0.90% w/v of NaCl, 308
mOsm/L) (Amanta Healthcare Ltd., Gujarat, India) as a
negative control. After 48 h of incubation period, the zone
of inhibitions against the extracts was measured using a
Scienceware® Vernier Caliper. The diameters of the clear
inhibition zones were measured to the nearest millimeter.
A time‑kill assay was performed by the broth microdilution
method, according to the Clinical and Laboratory Standards
Institute guidelines. MBC values obtained were used for
time‑kill assay; 48 h grown inoculum was diluted in fresh BHI;
and optical density (OD) was adjusted at 540 nm. The increase
of value by <0.05 indicates no change in OD. Extract deficient
was considered a negative control. Further, these tubes were
incubated at 37°C, and aliquot of the cultures was streaked on
agar plates at a time interval of 0, 3, 6, 12, and 24 h. After 48 h
of incubation, colony formation was observed. The killing time
was recorded when the cell density decreased by ≥3 log10 in
CFU/ml compared to cell density at 0 h.[21]
The 3‑[4,5‑dimethylthiazol‑2‑yl]‑2,5‑diphenyl tetrazolium
bromide (MTT) (HiMedia ® Laboratories, Pvt. Ltd,
Mumbai, India) reagent was prepared using 5 mg in
1 ml of phosphate buffered saline – pH 7.4 (HiMedia ®
Laboratories, Pvt. Ltd, Mumbai, India), and a cytotoxicity
assay was performed on gingival fibroblast.[22,23] In vitro
growth inhibition effect of O. basilicum extract was assessed
byenzyme-linked immunosorbent assay (ELISA) reader
(Epoch, BioTek® Instruments, Inc., USA) by the determination
of conversion of MTT into “formazan blue” by living cells. Fifty
microliter of 4000 cells/ml cell suspension was seeded into
each well in a 96‑well microtiter plate, and the final volume
was made up to 150 µl by adding Dulbecco’s Modified Eagle
Medium (DMEM) (Gibco™ Life Technologies, Bangalore,
India). One hundred microliter of the O. basilicum extract was
added to the wells and incubated for 24 h, in the presence of
5% CO2, at 37°C in a CO2 incubator (New Brunswick™ Galaxy®
170 R, Eppendorf, Germany). After 24 h, 20 µl of 5 mg/ml MTT
reagent was added to the wells. The supernatant was carefully
removed without disturbing the precipitated formazan
crystals, and 100 µl of DMSO was added to dissolve the crystals
formed. The OD was measured at a wavelength of 570 nm.
The experiment was repeated in triplicates for both ethanolic
extract and aqueous extract of O. basilicum to ascertain the
antimicrobial activity. The collected data were entered in MS
Excel and analyzed using IBM‑SPSS® Statistics‑Version 21
(IBM Corp. Released 2012. IBM SPSS Statistics for Windows,
Version 32.0, Armonk, NY, USA: IBM Corp.). The Kruskal–Wallis
test was applied to test the significance among both extracts and
CHX for three different organisms. The statistical significance
was set at P ≤ 0.05 for the test.
481
 Pai Khot, et al.: Antimicrobial activity of the Ocimum basilicum extract against selective periodontal microorganisms

RESULTS of exposure, the O. basilicum ethanolic extract also exhibited a

MIC of ethanolic extract and aqueous extract of O. basilicum was
determined by resazurin broth dilution method. P. gingivalis
was the most sensitive organism to aqueous extract of
O. basilicum with the lowest MIC value of 2.5 mg/ml, followed
by A. actinomycetemcomitans and T. forsythia with MIC value
Downloaded from http://journals.lww.com/jisp by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW
similar bactericidal activity. At 24 h, no growth was observed
with CHX, while the growth declined up to ~1 log10 with the
O. basilicum extracts [Figure 4].
Statistical analysis was performed on the results collected.
The antibacterial activity of O. basilicum against A.

of 4 mg/ml and 10 mg/ml, respectively. While ethanolic
extract showed a significant MIC value of 10 mg/ml for all
three periodontal pathogens. A. actinomycetemcomitans and
T. forsythia were the most sensitive organisms to CHX gluconate
with MIC value of 0.0093 mg/ml, followed by P. gingivalis
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 01/22/2024
actinomycetemcomitans, P. gingivalis, and T. forsythia was
determined using the Kruskal–Wallis test. The P value
difference between both extracts and CHX against three
periodontal pathogens was found to be statistically

significant [Table 3].

having MIC value of 0.075 mg/ml [Figure 2].

It was found that concentration of 5 mg/ml, aqueous extract

O. basilicum restricted A. actinomycetemcomitans growth on
the plate, while MBC value of aqueous extract of O. basilicum
against P. gingivalis and T. forsythia was 2.5 mg/ml and
10 mg/ml, respectively. However, ethanolic extract of
O. basilicum showed antibacterial property at 12.5 mg/ml
against A. actinomycetemcomitans, while the average MBC
value for O. basilicum ethanolic extract against P. gingivalis and
T. forsythia was 10 mg/ml. MIC and MBC of test sample against
periodontal pathogens are depicted in Table 1.

The zone of inhibition for A. actinomycetemcomitans against

O. basilicum ethanolic extract was 12 mm which was lower
than aqueous extract at 16 mm and CHX at 18 mm. For
P. gingivalis, both the extracts had a smaller zone of inhibition
compared to CHX. The zone of inhibition was produced by
aqueous extract at 13 mm, ethanolic extract at 11 mm, and
CHX at 16 mm against P. gingivalis. The zone of inhibition
for T. forsythia after 48 h of incubation was broader for CHX
with zone of inhibition of 18 mm, followed by aqueous
extract 17 mm and ethanolic extract with zone of inhibition
of 13 mm, as shown in Table 2. The zone of inhibition of
ethanolic, aqueous‑based extract of O. basilicum and 0.12%
CHX against periodontal pathogens by well diffusion method
is represented in Figure 3.

An almost similar killing profile was observed with O. basilicum

aqueous extract and CHX. Both the cases showed a rapid and
considerable decrease in the bacterial density after 4 h. At 7 h

Table 1: Minimum inhibitory concentration and minimum

bactericidal concentration of ethanolic, aqueous‑based
extract of Ocimum basilicum and 0.12% chlorhexidine
against periodontal pathogens
Samples MIC (mg/mL) MBC (mg/mL)
PG AA TF PG AA TF
Ethanolic extract of 10 10 10 10 12.5 10
O. basilicum 10 10 10 10 12.5 10
10 10 10 10 12.5 10
Average 10 10 10 10 12.5 10
Aqueous extract of 2.5 5 5 2.5 5 10
O. basilicum 2.5 2.5 5 2.5 5 10
2.5 5 10 2.5 5 10
Average 2.5 4 6.7 2.5 5 10
Chlorhexidine 0.075 0.0093 0.0093 0.075 0.0093 0.0093
MIC – Minimum inhibitory concentration; MBC – Minimum bacterial
concentration; PG – Porphyromonas gingivalis; AA – Aggregatibacter Figure 2: MIC for antimicrobial activity using resazurin method. EE = Ethanolic
actinomycetemcomitans; TF – Tannerella forsythia; O. basilicum – Ocimum Extract of Ocimum Basilicum, AE – Aqueous Extract of Ocimum basilicum,
basilicum CHX – Chlorhexidine, MIC – Minimum inhibitory concentration

482 Journal of Indian Society of Periodontology - Volume 27, Issue 5, September-October 2023
 Pai Khot, et al.: Antimicrobial activity of the Ocimum basilicum extract against selective periodontal microorganisms
Downloaded from http://journals.lww.com/jisp by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW

a b
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 01/22/2024

c
Figure 3: The zone of inhibition of ethanolic, aqueous‑based extract of
Ocimum basilicum and 0.12% CHX against periodontal pathogens by
well diffusion method; (a) Porphyromonas gingivalis; (b) Aggregatibacter
actinomycetemcomitans; (c) Tannerella forsythia. CHX – Chlorhexidine

Table 2: Zone of inhibition of ethanolic, aqueous‑based

extract of Ocimum basilicum and 0.12% chlorhexidine
against periodontal pathogens by well diffusion method
Samples Zone of inhibition (mm)
PG AA TF
Ethanolic extract of O. basilicum 11 12 13
Aqueous extract of O. basilicum 13 16 17
Chlorhexidine 16 18 18
PG – Porphyromonas gingivalis; AA – Aggregatibacter
actinomycetemcomitans; TF – Tannerella forsythia; O. basilicum – Ocimum
basilicum

The O. basilicum extract was nontoxic to the human gingival

fibroblast, against which the cytotoxic activity has been
tested [Table 4].

DISCUSSION

India is a land of beautiful natural flora, with an abundance

of medicinal plants and seeds which are available
throughout the country. A few extracts are known to
possess antimicrobial activity, especially against bacterial
pathogens. O. basilicum (sabja seeds) is one such seed which
is predominantly present in the Indian subcontinent. The
present study was carried out to test the antibacterial
activity of O. basilicum extract against common oral
anaerobic organisms, especially periodontal pathogens.
Ethanol and water were used as a solvent for extract
preparation. Although both the extracts showed promising
results, against the various bacterial strains, the effect of
aqueous extract was much more pronounced than ethanolic
extract. In contrast, Kalra et at.[10] found that O. basilicum
oil in aqueous base had less efficacy against periodontal
pathogens compared to ethanol‑based extract. The active
constituent present in Ocimum species is flavonoids and
perhaps it is absorbed more in aqueous extract, primarily
Figure 4: Time‑kill assay of ethanolic, aqueous‑based extract and CHX on three
different periodontal pathogens at various time intervals. X‑axis = Time duration,
Y‑axis = Concentration of substrate in mg/ml. CHX – Chlorhexidine
responsible for the therapeutic potentials of Ocimum
species.[17,24]
A few studies done in India have already reported the
antibacterial activity of O. basilicum, but there is a marked
void in exploring the properties of O. basilicum. The current
study thoroughly evaluated the antibacterial properties against
periodontal pathogens; similarly, sweet basil essential oils
show excellent in vitro anticariogenic bacteria and anti‑plaque
activities, according to Wiwattanarattanabut et al.,[11] and may
be offered as alternative and effective supplements to maintain
oral health status.

Journal of Indian Society of Periodontology - Volume 27, Issue 5, September-October 2023 483
 Pai Khot, et al.: Antimicrobial activity of the Ocimum basilicum extract against selective periodontal microorganisms

Table 3: Comparison of minimum inhibitory concentration, minimum bactericidal concentration values of ethanolic,
aqueous‑based extract and chlorhexidine on three different periodontal pathogens
Procedure Samples PG AA TF
Mean P Mean P Mean P
rank rank rank
MIC Ethanolic extract of O. basilicum 8.00 0.018* 8.00 0.020* 7.50 0.029*
Aqueous extract of O. basilicum 5.00 5.00 5.50
Downloaded from http://journals.lww.com/jisp by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW

Chlorhexidine 2.00 2.00 2.00

MBC Ethanolic extract of O. basilicum 8.00 0.018* 8.00 0.018* 6.50 0.018*
Aqueous extract of O. basilicum 5.00 5.00 6.50
Chlorhexidine 2.00 2.00 2.00
*P≤0.05 – *P-value ≤ 0.05 is considered statistically significant. Test applied – Kruskal–Wallis test; level of significance. O. basilicum – Ocimum
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 01/22/2024

basilicum; MIC – Minimum inhibitory concentration; MBC – Minimum bactericidal concentration; PG – Porphyromonas gingivalis; AA – Aggregatibacter
actinomycetemcomitans; TF – Tannerella forsythia

Table 4: Mean of optical densities of surviving cells of two study groups, namely, chlorhexidine gluconate and
Ocimum basilicum extract at a wavelength of 570 nm for different concentrations
Concentration OD Mean Percentage viability(%) Results as observed
NC 0.164 0.287 100 No lysis
0.439
0.259
OBE 0.269 0.274 95.59 No lysis
0.245
0.31
CHX 0.154 0.217 75.52 No lysis
0.264
0.233
NC – Negative control; O. basilicum – Ocimum basilicum; OBE – O. basilicum extract; CHX – Chlorhexidine; OD – Optical density; nm – Nanometer

Analyzing the efficacy of O. basilicum extract against oral
periodontal microorganisms, the maximum activity was
shown for P. gingivalis, which is supported by Ahmed et al.,[25]
concluding that the inhibition zones were always varied and
had significantly risen with the concentration of O. basilicum
extract, and the growth was entirely prevented at the greatest
concentration. On some bacteria, O. basilicum shows an
inhibitory action comparable to antibiotics. It would be possible
to produce new effective antibiotics using the effective material
to be refined from this plant.
As demonstrated in the current investigation, O. basilicum
extract had proven to be an efficient antimicrobial against
periodontal infections. Another study by Hanif et al.[26] found
that Omani basil essential oil exhibited a strong antibacterial
activity against all the bacteria tested, except Pseudomonas
putida and Pseudomonas aeruginosa. Linalool (69.9%) was
identified as the major component present in Omani basil oil
which was found highly active against human pathogens.
The current evidence obtained from this study shows a
significant inhibitory effect of O. basilicum extract on anaerobic
microorganisms. The effect produced was equivalent or less
than that produced by CHX in the case of anaerobic bacteria.
When compared to CHX, O. basilicum is a natural substance
with no associated negative effects. It has a better chance of
being accepted by the people because it is native to the country
and has a religious significance. These findings indicate the
possibility of using O. basilicum in oral health care products
for reducing microbial load in the oral cavity.
In summary, periodontitis is a multifactorial disease, but
periodontal pathogens are primarily believed in initiation
and progression of the disease. For a long time, the use of
chemical therapeutic agents with the mechanical debridement
is considered a treatment of choice with improved results.[27,28]
Commonly used therapeutic agents like CHX is chemically
derived and is considered the gold‑standard therapeutic agent
used as an adjunct to mechanical debridement for plaque
control. However, certain adverse effects have been reported
with the long‑term use of CHX. These factors triggered the need
for the development of an alternative agent which is equally
effective and help in minimizing all the adverse effects seen
with the existing agents.[29,30] Herbal medicine is an upcoming
alternative and is considered the most acceptable form of
therapy by many researchers due to its diverse medicinal
properties with a fewer side effects.[31,32] Plants with medicinal
properties and therapeutic uses are gaining popularity among
the researchers to explore the different uses of plants and their
compounds responsible for specific properties.[33]
This study being an in vitro provides just preliminary evidence
of antibacterial efficacy of O. basilicum extract. Periodontal
disease is a complex illness whose etiology has been linked to a
various pathogenic bacteria, but the use of only three organisms
is one of the study’s drawbacks. However, predominant
periodontal microorganisms were selected in the present study.
Future clinical trials will be carried out to demonstrate its effect
on the various microorganisms and to ascertain cytotoxicity
against the periodontal tissues.
CONCLUSION
Recognizing plants that have medicinal properties becomes
important and needs to be developed because it is believed
that drugs derived from natural materials are relatively
safe and inexpensive. The antibacterial activity was evident
in both the extracts of O. basilicum against anaerobic

484 Journal of Indian Society of Periodontology - Volume 27, Issue 5, September-October 2023
 Pai Khot, et al.: Antimicrobial activity of the Ocimum basilicum extract against selective periodontal microorganisms
periodontal pathogens. However, it was more pronounced in
aqueous‑based extract, but its efficacy was lesser compared
to CHX. Thus, it can be formulated in the mouthwash and
effectively used as an alternative to standard considering its
long‑term use and lesser side effects. This herbal mouthwash
would bring significant changes when used as an integral part
in oral health in a community.
Downloaded from http://journals.lww.com/jisp by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW
Acknowledgment
The authors would like to thank Vinuta Hampiholi, Ritiha
Uppin, Shivani Tendulkar, Thamil Selvan Muthuraj, Vijay
Kumbar, and Preetam Mehetri for providing subject insights
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 01/22/2024
during the course of the study. The authors also thank
KAHER’s Dr. Prabhakar Kore Basic Science Research Center
for providing resources for the study.
Financial support and sponsorship
The sponsorship was obtained from ICPA Health Products
Ltd, Mumbai, India, in the form of materialistic support
(Chlorhexidine gluconate salt - Batch No: 20BPLS/CHH001).
Conflicts of interest
There are no conflicts of interest.
REFERENCES
1. Tonetti MS, Greenwell H, Kornman KS. Staging and grading of
periodontitis: Framework and proposal of a new classification
and case definition. J Periodontol 2018;89 Suppl 1:S159‑72.
2. BowenWH, MelcherAH, editors. Biology of the Periodontium.
London, Academic Press; 1969.
3. Kala BS, Gunjan C, Disha N, Shobha P. Treatment of periodontal
disease‑a herbal approach. Int J Pharm Sci Rev Res 2015;33:126‑36.
4. Reddy PD, Satyanarayana T, Latha S, Purushothaman M. Local
drug delivery of herbs for treatment of periodontitis. J Innov
Trends Pharm Sci 2010;1:245‑51.
5. Kornman KS. Controlled‑release local delivery antimicrobials
in periodontics: Prospects for the future. J Periodontol
1993;64:782‑91.
6. Oettinger‑Barak O, Sela MN, Sprecher H, Machtei EE. Clinical
and microbiological characterization of localized aggressive
periodontitis: A cohort study. Aust Dent J 2014;59:165‑71.
7. Chenni M, El Abed D, Rakotomanomana N, Fernandez X,
Chemat F. Comparative study of essential oils extracted
from Egyptian basil leaves (Ocimum basilicum L.) using
hydro‑distillation and solvent‑free microwave extraction.
Molecules 2016;21:E113.
8. de Oliveira RR, Schwartz‑Filho HO, Novaes AB Jr., Taba M Jr.
Antimicrobial photodynamic therapy in the non‑surgical treatment
of aggressive periodontitis: A preliminary randomized controlled
clinical study. J Periodontol 2007;78:965‑73.
9. Jones CG. Chlorhexidine: Is it still the gold standard? Periodontol
2000 1997;15:55‑62.
10. Kalra K, Vasthare R, Shenoy PA, Vishwanath S, Singhal DK.
Antibacterial efficacy of essential oil of two different varieties
of Ocimum (Tulsi) on oral microbiota‑an in vitro study. Indian J
Public Health Res Dev 2019;10:188‑93.
11. Wiwattanarattanabut K, Choonharuangdej S, Srithavaj T. In vitro
anti‑cariogenic plaque effects of essential oils extracted from
culinary herbs. J Clin Diagn Res 2017;11:C30‑5.
12. Janakiram C, Venkitachalam R, Fontelo P, Iafolla TJ, Dye BA.
Effectiveness of herbal oral care products in reducing dental
plaque and gingivitis – A systematic review and meta‑analysis.
BMC Complement Med Ther 2020;20:43.
Journal of Indian Society of Periodontology - Volume 27, Issue 5, September-October 2023
13. Rezzoug M, Bakchiche B, Gherib A, Roberta A, FlaminiGuido,
Kilinçarslan Ö, et al. Chemical composition and bioactivity of
essential oils and ethanolic extracts of Ocimum basilicum L. and
Thymus algeriensis boiss. and Reut. From the Algerian Saharan
Atlas. BMC Complement Altern Med 2019;19:146.
14. Hussain AI, Anwar F, Hussain Sherazi ST, Przybylski R.
Chemical composition, antioxidant and antimicrobial activities
of Basil (Ocimum basilicum) essential oils depends on seasonal
variations. Food Chem 2008;108:986‑95.
15. Shafique M, Khan SJ, Khan NH. Study of antioxidant and
antimicrobial activity of Sweet Basil (Ocimum basilicum) essential
oil. Pharmacologyonline 2011;1:105‑11.
16. Marwat SK, Khan MS, Ghulam S, Anwar N, Mustafa G, Usman K.
Phytochemical constituents and pharmacological activities
of Sweet Basil‑Ocimum basilicum L. (Lamiaceae). Asian J Chem
2011;23:3773.
17. Bucktowar K, Bucktowar M, Bholoa LD. A review on Sweet Basil
seeds: Ocimum basilicum. World J Pharm Pharm Sci 2016;5:554‑67.
18. Kendall G, Bai R, Błazewicz J, De Causmaecker P, Gendreau M,
John R, et al. Good laboratory practice for optimization research.
J Oper Res Soc 2016;67:676‑89.
19. Sarker SD, Nahar L, Kumarasamy Y. Microtitre plate‑based
antibacterial assay incorporating resazurin as an indicator of cell
growth, and its application in the in vitro antibacterial screening
of phytochemicals. Methods 2007;42:321‑4.
20. MacFarlane WT, Samaranayake LP. Clinical Oral Microbiology.
Ch. 13. London, England: Butterworth‑Heinemann; 1989.
p. 187‑203.
21. Vysakh A, Midhun SJ, Jisha N, Jayesh K, Vijeesh V, Jyothis M,
et al. Rotula aquatica lour. Inhibits growth and biofilm formation
of clinically isolated uropathogenic Escherichia coli. Asian Pac J
Trop Biomed 2020;10:547.
22. Coelho AS, Laranjo M, Gonçalves AC, Paula A, Paulo S,
Abrantes AM, et al. Cytotoxic effects of a chlorhexidine
mouthwash and of an enzymatic mouthwash on human gingival
fibroblasts. Odontology 2020;108:260‑70.
23. Amirinia F, Salehi Rad H, Pourhajibagher M. In vitro antimicrobial
and cytotoxicity activities of some medicinal plant extracts
against oral microbial pathogens. Folia Med (Plovdiv) 2021;63:932‑40.
24. Gajendiran A, Thangaraman S, Thangamani V, Ravi D, Abraham J.
Antimicrobial, antioxidant and anticancer screening of Ocimum
Basilicum seeds. Bull Pharm Res 2016;6:114‑9.
25. Ahmed RI, Mohammed A, Hatem ZA. The effects of ethanolic
extract of seed Sweet Basil (Ocimum Basilicum) against different
gram negative and positive Bacteria and Fungi. Asian Acad Res J
Multidiscip 2016;3:201‑6.
26. Hanif MA, Al‑Maskari MY, Al‑Maskari A, Al‑Shukaili A,
Al‑Maskari AY, Al‑Sabahi JN. Essential oil composition,
antimicrobial and antioxidant activities of unexplored Omani
Basil. J Med Plant Res 2011;5:751‑7.
27. Brennan‑Krohn T, Kirby JE. Antimicrobial synergy testing by
the inkjet printer‑assisted automated checkerboard array and the
manual time‑kill method. J Vis Exp 2019;18:58636.
28. Rubab S, Bahadur S, Hanif U, Durrani AI, Sadiqa A, Shafique S,
et al. Phytochemical and antimicrobial investigation of methanolic
extract/fraction of Ocimum basilicum L. Biocatal Agric Biotechnol
2021;31:101894.
29. Joshi RK. Chemical composition and antimicrobial activity
of the essential oil of Ocimum basilicum L. (Sweet Basil) from
Western Ghats of North West Karnataka, India. Anc Sci Life
2014;33:151‑6.
30. Kaya I, Yigit N, Benli M. Antimicrobial activity of various extracts
of Ocimum basilicum L. And observation of the inhibition effect
on bacterial cells by use of scanning electron microscopy. Afr J
Tradit Complement Altern Med 2008;5:363‑9.
31. Bilal A, Jahan N, Ahmed A, Bilal SN, Habib S, Hajra S.
Phytochemical and pharmacological studies on Ocimum basilicum
485
 Pai Khot, et al.: Antimicrobial activity of the Ocimum basilicum extract against selective periodontal microorganisms

Linn‑A review. Int J Curr Res Rev 2012;4:73‑83. doi: 10.4103/jomfp.jomfp_174_23.

32.
Downloaded from http://journals.lww.com/jisp by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AW
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 01/22/2024
Pai Khot AJ, Ankola AV, Naik VV, Sankeshwari RM, Kumar RS, 33. Palombo EA. Traditional medicinal plant extracts and natural
Shah MA. Remineralising potential of Ocimum basilicum varnish products with activity against oral Bacteria: Potential application
and fluoride varnish on initial enamel caries: An in vitro in the prevention and treatment of oral diseases. Evid Based
microscopic study. J Oral Maxillofac Pathol 2023. [Ahead of print] Complement Alternat Med 2011;2011:680354.

486 Journal of Indian Society of Periodontology - Volume 27, Issue 5, September-October 2023