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Semillas de Occimum Basilicum Como Antibacteriano Oral
Semillas de Occimum Basilicum Como Antibacteriano Oral
Despite major breakthroughs in disease biology and treatment and The study was approved by the Institutional Research
approaches during the last century, the periodontal disease is and Ethics Committee of KAHER’s KLE Vishwanath Katti
still unmanageable over the world. Chlorhexidine (CHX) is a Institute of Dental Sciences (reference number: 1499 and
commonly prescribed medication for the treatment of dental date of approval: 25th October 2021). O. basilicum seeds were
disease.[8] The proof of its efficacy in plaque control is beyond authenticated from a recognized taxonomist. The coarse
dispute. It has been used by dental practitioners for nearly three powder of O. basilicum seeds was procured from Ayurveda
decades. However, the use of CHX for dental disease prevention pharmacy of a recognized institute (AYUSH Approved Central
has been controversial owing to its various side effects such as
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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 Agar well diffusion (punch well diffusion) was used to test
finished, the ethanol was entirely evaporated using rotary antimicrobial susceptibility. Lawn culture of test organism
evaporator (IKA™), at 40°C, leaving a yield of extracted plant was made on respective media. The whole surface of the
material (about 20 mg). The yield was resuspended in dimethyl agar plate was swabbed three times with the cotton swab.
sulfoxide (DMSO) (Qualigens, Thermo Fisher Scientific Pvt. Following which, individual 4 mm depth and 6 mm diameter
Ltd, Mumbai, India) in 20 mg/ml ratio to obtain a stock wells were cut in agar plates with sterile borer. These wells
solution. The extracts obtained from both the techniques were filled with 100 μl of stock solution (20 mg/ml) for
were subjected to preliminary phytochemical screening for each extract and control to determine the antimicrobial
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qualitative detection of phytoconstituents using standard activity against the pathogens. CHX gluconate (0.12%)
procedures as described by Trease and Evans. (ICPA Health Products Ltd, Mumbai, India) was used
as a positive control and saline (0.90% w/v of NaCl, 308
The standard fresh strains of P. gingivalis – ATCC mOsm/L) (Amanta Healthcare Ltd., Gujarat, India) as a
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 01/22/2024
33277, A. actinomycetemcomitans – ATCC 29523, and T. negative control. After 48 h of incubation period, the zone
forsythia – ATCC 43037 were procured from LGC Promochem of inhibitions against the extracts was measured using a
India Pvt. Ltd., Bangalore, India. The strains were cultivated Scienceware® Vernier Caliper. The diameters of the clear
anaerobically in 5 ml sterile brain heart infusion (BHI) inhibition zones were measured to the nearest millimeter.
broth (Sigma Aldrich® Chemicals Pvt. Ltd., Bangalore, India)
supplemented with 1% horse serum, 0.5 mg/ml of hemin, A time‑kill assay was performed by the broth microdilution
and 5 mg/ml of Vitamin K, and inoculated in anaerobic method, according to the Clinical and Laboratory Standards
chamber (N2 80%, H2 10%, and CO2 10%) at 37°C for 48 h until Institute guidelines. MBC values obtained were used for
it achieved the turbidity of 0.5 McFarland standards (1 × 108 time‑kill assay; 48 h grown inoculum was diluted in fresh BHI;
colony‑forming unit (CFU)/ml). and optical density (OD) was adjusted at 540 nm. The increase
of value by <0.05 indicates no change in OD. Extract deficient
Twelve mgs of CHX salt was mixed and diluted in was considered a negative control. Further, these tubes were
10 ml of distilled water (1.2 mg/ml) in a centrifuge tube incubated at 37°C, and aliquot of the cultures was streaked on
(Tarsons Products Ltd., Kolkata, India). The mixture was agar plates at a time interval of 0, 3, 6, 12, and 24 h. After 48 h
vortexed vigorously using a MixMate ® Vortex agitator of incubation, colony formation was observed. The killing time
(Eppendorf, Sydney, Australia) for 30 s at 1000 rpm (maximum was recorded when the cell density decreased by ≥3 log10 in
setting) to obtain a final concentration of 10 ml of 0.12% of CFU/ml compared to cell density at 0 h.[21]
CHX solution. Following which 96‑well microtiter plate (NEST
Biotechnology, Jiangsu, China) were taken, and 10 wells were The 3‑[4,5‑dimethylthiazol‑2‑yl]‑2,5‑diphenyl tetrazolium
allotted for each extract in triplicates. A volume of 100 µl broth bromide (MTT) (HiMedia ® Laboratories, Pvt. Ltd,
was added in all the wells, and 100 µl of each extract was added Mumbai, India) reagent was prepared using 5 mg in
in the first well; serial doubling dilutions were implied for the 1 ml of phosphate buffered saline – pH 7.4 (HiMedia ®
extract to requisite concentrations up to the 10th well. Therefore, Laboratories, Pvt. Ltd, Mumbai, India), and a cytotoxicity
the concentration in subsequent wells reduced by 50%. A assay was performed on gingival fibroblast.[22,23] In vitro
volume of 10 μl of inoculum was added to all 10 wells and growth inhibition effect of O. basilicum extract was assessed
kept for incubation at 37°C in McIntosh and Fildes’ anaerobic byenzyme-linked immunosorbent assay (ELISA) reader
jar for 2 days. After 48 h, 10 µl resazurin dye (5 mg/10 ml (Epoch, BioTek® Instruments, Inc., USA) by the determination
distilled water) (Hi‑Cert™ HiMedia® Laboratories, Pvt. Ltd, of conversion of MTT into “formazan blue” by living cells. Fifty
Mumbai, India) was added and observed for color change microliter of 4000 cells/ml cell suspension was seeded into
from blue/violet to slight pink/magenta except positive each well in a 96‑well microtiter plate, and the final volume
control by incubating in anaerobic condition at 37°C for 4 h. was made up to 150 µl by adding Dulbecco’s Modified Eagle
The concentration at which resazurin reduces to resafurin Medium (DMEM) (Gibco™ Life Technologies, Bangalore,
compound by color change from blue to pink color was taken India). One hundred microliter of the O. basilicum extract was
as a minimum inhibitory concentration (MIC) value.[19] This added to the wells and incubated for 24 h, in the presence of
procedure was repeated for three periodontal pathogens and 5% CO2, at 37°C in a CO2 incubator (New Brunswick™ Galaxy®
two extracts separately. The results recorded in triplicate 170 R, Eppendorf, Germany). After 24 h, 20 µl of 5 mg/ml MTT
outcomes and mean value were taken. reagent was added to the wells. The supernatant was carefully
removed without disturbing the precipitated formazan
The spread plate method was used to determine minimum crystals, and 100 µl of DMSO was added to dissolve the crystals
bactericidal concentration (MBC). The bacterial suspension formed. The OD was measured at a wavelength of 570 nm.
from alternate wells with dilutions of O. basilicum ethanolic
extract (20, 5, 1.25, 0.312, and 0.078 mg/ml), O. basilicum aqueous The experiment was repeated in triplicates for both ethanolic
extract (10, 2.5, 0.625, 0.156, and 0.039 mg/ml), and CHX extract and aqueous extract of O. basilicum to ascertain the
(0.12, 0.03, 0.0075, 0.0018, and 0.00046 mg/ml) concentrations antimicrobial activity. The collected data were entered in MS
higher than the MIC value was inoculated on BHI agar plates Excel and analyzed using IBM‑SPSS® Statistics‑Version 21
and incubated for 48 h at 37°C. The concentration at which (IBM Corp. Released 2012. IBM SPSS Statistics for Windows,
no bacterial growth was seen, considered bactericidal values. Version 32.0, Armonk, NY, USA: IBM Corp.). The Kruskal–Wallis
The MBC is defined as the minimum concentration of drug test was applied to test the significance among both extracts and
which kills 99.9% of the test microorganisms in the original CHX for three different organisms. The statistical significance
inoculum.[20] was set at P ≤ 0.05 for the test.
Journal of Indian Society of Periodontology - Volume 27, Issue 5, September-October 2023 481
Pai Khot, et al.: Antimicrobial activity of the Ocimum basilicum extract against selective periodontal microorganisms
of 4 mg/ml and 10 mg/ml, respectively. While ethanolic actinomycetemcomitans, P. gingivalis, and T. forsythia was
extract showed a significant MIC value of 10 mg/ml for all determined using the Kruskal–Wallis test. The P value
three periodontal pathogens. A. actinomycetemcomitans and difference between both extracts and CHX against three
T. forsythia were the most sensitive organisms to CHX gluconate periodontal pathogens was found to be statistically
with MIC value of 0.0093 mg/ml, followed by P. gingivalis
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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
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a b
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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
DISCUSSION
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
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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 is considered a treatment of choice with improved results.[27,28]
periodontal microorganisms, the maximum activity was Commonly used therapeutic agents like CHX is chemically
shown for P. gingivalis, which is supported by Ahmed et al.,[25] derived and is considered the gold‑standard therapeutic agent
concluding that the inhibition zones were always varied and used as an adjunct to mechanical debridement for plaque
had significantly risen with the concentration of O. basilicum control. However, certain adverse effects have been reported
extract, and the growth was entirely prevented at the greatest with the long‑term use of CHX. These factors triggered the need
concentration. On some bacteria, O. basilicum shows an for the development of an alternative agent which is equally
inhibitory action comparable to antibiotics. It would be possible effective and help in minimizing all the adverse effects seen
to produce new effective antibiotics using the effective material with the existing agents.[29,30] Herbal medicine is an upcoming
to be refined from this plant. alternative and is considered the most acceptable form of
therapy by many researchers due to its diverse medicinal
As demonstrated in the current investigation, O. basilicum properties with a fewer side effects.[31,32] Plants with medicinal
extract had proven to be an efficient antimicrobial against properties and therapeutic uses are gaining popularity among
periodontal infections. Another study by Hanif et al.[26] found the researchers to explore the different uses of plants and their
that Omani basil essential oil exhibited a strong antibacterial
compounds responsible for specific properties.[33]
activity against all the bacteria tested, except Pseudomonas
putida and Pseudomonas aeruginosa. Linalool (69.9%) was
This study being an in vitro provides just preliminary evidence
identified as the major component present in Omani basil oil
of antibacterial efficacy of O. basilicum extract. Periodontal
which was found highly active against human pathogens.
disease is a complex illness whose etiology has been linked to a
The current evidence obtained from this study shows a
various pathogenic bacteria, but the use of only three organisms
significant inhibitory effect of O. basilicum extract on anaerobic
is one of the study’s drawbacks. However, predominant
microorganisms. The effect produced was equivalent or less
than that produced by CHX in the case of anaerobic bacteria. periodontal microorganisms were selected in the present study.
When compared to CHX, O. basilicum is a natural substance Future clinical trials will be carried out to demonstrate its effect
with no associated negative effects. It has a better chance of on the various microorganisms and to ascertain cytotoxicity
being accepted by the people because it is native to the country against the periodontal tissues.
and has a religious significance. These findings indicate the
possibility of using O. basilicum in oral health care products CONCLUSION
for reducing microbial load in the oral cavity.
Recognizing plants that have medicinal properties becomes
In summary, periodontitis is a multifactorial disease, but important and needs to be developed because it is believed
periodontal pathogens are primarily believed in initiation that drugs derived from natural materials are relatively
and progression of the disease. For a long time, the use of safe and inexpensive. The antibacterial activity was evident
chemical therapeutic agents with the mechanical debridement 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 13. Rezzoug M, Bakchiche B, Gherib A, Roberta A, FlaminiGuido,
aqueous‑based extract, but its efficacy was lesser compared Kilinçarslan Ö, et al. Chemical composition and bioactivity of
to CHX. Thus, it can be formulated in the mouthwash and essential oils and ethanolic extracts of Ocimum basilicum L. and
Thymus algeriensis boiss. and Reut. From the Algerian Saharan
effectively used as an alternative to standard considering its
Atlas. BMC Complement Altern Med 2019;19:146.
long‑term use and lesser side effects. This herbal mouthwash
14. Hussain AI, Anwar F, Hussain Sherazi ST, Przybylski R.
would bring significant changes when used as an integral part Chemical composition, antioxidant and antimicrobial activities
in oral health in a community. of Basil (Ocimum basilicum) essential oils depends on seasonal
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during the course of the study. The authors also thank Phytochemical constituents and pharmacological activities
KAHER’s Dr. Prabhakar Kore Basic Science Research Center of Sweet Basil‑Ocimum basilicum L. (Lamiaceae). Asian J Chem
2011;23:3773.
for providing resources for the study.
17. Bucktowar K, Bucktowar M, Bholoa LD. A review on Sweet Basil
seeds: Ocimum basilicum. World J Pharm Pharm Sci 2016;5:554‑67.
Financial support and sponsorship
18. Kendall G, Bai R, Błazewicz J, De Causmaecker P, Gendreau M,
The sponsorship was obtained from ICPA Health Products John R, et al. Good laboratory practice for optimization research.
Ltd, Mumbai, India, in the form of materialistic support J Oper Res Soc 2016;67:676‑89.
(Chlorhexidine gluconate salt - Batch No: 20BPLS/CHH001). 19. Sarker SD, Nahar L, Kumarasamy Y. Microtitre plate‑based
antibacterial assay incorporating resazurin as an indicator of cell
Conflicts of interest growth, and its application in the in vitro antibacterial screening
There are no conflicts of interest. of phytochemicals. Methods 2007;42:321‑4.
20. MacFarlane WT, Samaranayake LP. Clinical Oral Microbiology.
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486 Journal of Indian Society of Periodontology - Volume 27, Issue 5, September-October 2023