Original Article

Antimicrobial Properties of Ocimum Species: An In Vitro Study

Malimone Chanthaboury1, Suwan Choonharuangdej2, Binit Shrestha1, Theerathavaj Srithavaj1

1
Maxillofacial Prosthetic Objective: This study aimed to determine the antimicrobial activity of ethanol-

Abstract
Unit, Department of extracts obtained from Ocimum gratissimum L. (clove or African basil,
Prosthodontics, Faculty Lamiaceae) and O. santum L. (holy basil) against some microorganisms present
of Dentistry, Mahidol
in oral cavity related to either medical or dental disease. Materials and Methods:
University, 2Department of
Oral Microbiology, Faculty Antimicrobial properties of both ethanol-extracts of Ocimum species against
of Dentistry, Mahidol Streptococcus mutans KPSK2, S. pyogenes ATCC 19615, Staphylococcus aureus
University, Bangkok, ATCC 16794, and Candida albicans ATCC 10231 were primarily determined
Thailand by agar disk diffusion method. The minimum inhibitory concentration (MIC)
and minimum bactericidal or fungicidal concentration (MBC or MFC) of these
herbal extracts were further determined by broth micro-dilution method. Results:
Ethanol-extracts of O. sanctum L. and O. gratissimum L. inhibited the growth
of all tested microorganisms in various degrees ranging from the strongest
antimicrobial activity of O. sanctum against S. pyogenes [MIC at 0.19% (w/v);
MBC at 0.78% (w/v)] to the least inhibitory activity of O. gratissimum against
C. albicans [MIC at 12.5% (w/v); undetectable MFC]. The ethanol-extract of
O. sanctum showed stronger antimicrobial property against the tested bacteria
and fungus than O. gratissimum. The ethanol-extracts of both Ocimum species
showed stronger antibacterial than antifungal activity. However, the ethanol-
extract of O. gratissimum even at a high concentration of 50% (w/v) was unable
to eliminate the tested fungus. Conclusion: Ethanol-extracts of Ocimum species
contain effective antibacterial and antifungal properties that may be beneficial
for further development of antimicrobial agents in medical and dental fields.
Received : 21-07-22
Revised : 04-11-22 Keywords: Antimicrobial activity, ethanol-extract, Ocimum gratissimum L.,
Accepted : 21-11-22
Published : 30-12-22
Ocimum sanctum L.

Introduction diseases. Among the commonly grown herbs in South

East Asia, Ocimum gratissimum L. (Lamiaceae,
A ll civilizations and religions from the ancient
period to the modern day have used various
medicinal plants and herbs in the health care of
clove or African basil) and O. sanctum L. (holy basil)
are known for their numerous pharmacological
properties including anti-oxidant, anti-inflammatory,
humans and animals. Moreover, these nature-derived
analgesic, antidiabetic, antihypertensive, antimicrobial,
products are generally well-accepted by the general
anticancer, hepatoprotective, and wound healing
population, who observed them as crucial aspects of
properties.[2] The family “Lamiaceae” grows all around
health care. These products also tend to have a high
the world, consisting of nearly 236 genera and 7200
success rate in treating diseases, as they are sources
of diverse molecules that show radical scavenger and
antimicrobial properties.[1] Many medicinal plants Address for correspondence: Dr. Theerathavaj Srithavaj,
and herbs have been extensively researched for their Maxillofacial Prosthetic Unit, Department of Prosthodontics,
Faculty of Dentistry, Mahidol University, Bangkok 10400, Thailand.
potential, mechanism, and effectiveness to cure several
E-mail: [email protected]
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How to cite this article: Chanthaboury M, Choonharuangdej S, Shrestha

DOI: 10.4103/jispcd.JISPCD_155_22
B, Srithavaj T. Antimicrobial properties of Ocimum species: An in vitro
study. J Int Soc Prevent Communit Dent 2022;12:596-602.

596 © 2022 Journal of International Society of Preventive and Community Dentistry | Published by Wolters Kluwer ‑ Medknow
 Chanthaboury, et al.: Antimicrob prop Ocimum spp

species. The genus Ocimum belongs to Lamiaceae (BD BBL) were purchased from Becton Dickinson and
family that comprises of more than 150 species. Company, France.
From the African to Asian continent, roots, leaves, barks, Herb collection and ethanol-extract preparation
seeds, and other parts of medicinal herbs have been Sun-dried leaves of O. gratissimum and O. sanctum
consumed either alone or mixed with drinks such as tea were minced and soaked in 95% ethanol (300 g minced
to cure fever, wounds, constipation, digestive problems, leaves/1 L ethanol) for 7 days at room temperature.[7]
hemorrhoid, and many others diseases.[2,3] Since the Spray drying technique was used to obtain a powder
emergence of antimicrobial resistance (AMR) strains form of ethanol-extracts of O. gratissimum and
of bacteria have made many infectious diseases difficult O. sanctum. The stock solutions of herbal extracts were
to manage[4] including opportunistic infections such as individually prepared by mixing 50 g of O. gratissimum
candidiasis that can affect the immunocompromised or O. sanctum powder in 100 mL of sterile distilled water
individuals. Many natural products with antimicrobial or 50% ethanol, respectively, to obtain a concentration
properties have been explored as alternative treatment.[5,6] of 50% (w/v).
Consecutively, several modern methodologies have also
Antimicrobial activity determination by agar disk diffusion
been developed to effectively extract and purify various
method
compounds from plants which can further enhance the
Streptococcus mutans KPSK2 and S. aureus ATCC
antimicrobial properties. However, it is also important
16794 were freshly subcultured on BHI agar, whereas
to determine whether the extraction process retains the
S. pyogenes ATCC 19615 was subcultured on SBA.
bioactive properties of the herbal compounds once used
They were then incubated at 37°C with 5% CO2 for
or consumed without any harmful effects. Therefore,
24–48 h. Candida albicans ATCC 10231 was subcultured
this study aimed to analyze the antimicrobial property
on SDA and incubated at 37°C for 24 h. A few colonies
of the ethanol-extracts obtained from the leaves of
of each microorganism were inoculated in BHI broth
O. sanctum and O. gratissimum [Figures 1A and B and
and SDB for bacteria and fungus, respectively, then
2A and B] against some pathogenic microorganisms of
incubated as described for 24 h. These bacterial and
oral and medical importance.
fungal suspensions were re-adjusted to obtain 1–5 x
Materials and Methods 108 colony-forming unit (CFU)/mL by measuring their
turbidity at λ=600 nm to obtain an optical density of
O. gratissimum and O. sanctum leaves were obtained 0.11 and 0.14, respectively, using cell densitometer. The
from an organic farm in Chonburi district, Thailand. inoculum was spread individually and evenly on MHA.
The tested microorganisms including Streptococcus
mutans KPSK2, S. pyogenes ATCC 19615, Each herbal extract solution [50% (w/v)] was
Staphylococcus aureus ATCC 16794, and Candida prepared in two-fold serial dilutions to obtain
albicans ATCC 10231 were provided by the Department various concentrations ranging from 12.5% to 50%
of Oral Microbiology, Faculty of Dentistry, Mahidol (w/v). Each herbal solution (20 μL) was applied on a
University. Brain heart infusion (BHI) agar and broth 6 mm-diameter paper disk and allowed to air dry prior
(BD BBL), Sabouraud dextrose agar (SDA) and broth to being placed on MHA. Sterile distilled water or 50%
(SDB) (Difco), trypticase soy agar with 5% sheep blood ethanol and 0.2% chlorhexidine were used as negative
(SBA) (BD BBL), and Mueller-Hinton agar (MHA) and positive controls, respectively. The disks containing

Figure 1: (A) Ocimum gratissimum L. plant. (B) Harvested sun-dried leaves of O. gratissimum

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 Chanthaboury, et al.: Antimicrob prop Ocimum spp

Figure 2: (A) Ocimum sanctum L. plant. (B) Harvested sun-dried leaves of O. sanctum

herbal extract were individually transferred and Inhibition zones of herbal extracts against the tested
placed on the MHA primarily inoculated with certain microorganisms were expressed in median of diameter
microbial suspension. The plates were then incubated (mm) with interquartile range. On the contrary, the
at appropriate environment for 24 h, after which the MIC values of the herbal extracts were depicted in
inhibition zones were observed and the diameters were mode values.
measured in millimeter.
Results
Minimum inhibitory concentration determination by broth
micro-dilution method Antimicrobial activity determination by agar disk diffusion
method
First, two-fold serial dilution of the herbal extracts was
prepared with sterile distilled water or 50% ethanol to The preliminary antimicrobial activity of ethanol
obtain various concentrations of the herbal extract extracts from O. sanctum and O. gratissimum leaves
ranging from 0.096% to 50% (w/v). Then, 100 μL were screened using agar disk diffusion method.
of herbal solution was added individually into each Four concentrations including 50%, 25%, 12.5%, and
well of 96-well plate that was previously filled with 6.25% (w/v) of each herbal extract were tested for
100 μL of BHI broth (2x). The final concentrations of their inhibitory activity against S. mutans KPSK2,
herbal solution ranged from 0.048% to 25%. Then, a S. pygogenes ATCC 19615, S. aureus ATCC 16794,
100 μL of bacterial or fungal suspension was added and C. albicans ATCC 10231. The ethanol extracts of
to each well (104 CFU/mL) and kept in an incubator both Ocimum species only inhibited the growth of the
at appropriate environment for 24 h. The lowest tested bacteria but did not have any inhibitory effect
concentration of herbal extract inhibiting the visible on the growth of fungus. The antimicrobial activity
growth of a microorganism was recorded as MIC. of O. sanctum and O. gratissimum extracts against the
A 0.2% chlorhexidine solution was used as the positive tested microorganisms was summarized in Tables 1
control whereas sterile distilled water or 50% ethanol and 2.
was used as the negative control. Minimum inhibitory concentration determination by broth
micro-dilution method
Minimum bactericidal or fungicidal concentration (MBC
or MFC) determination
Following the screening test, the ethanol-extracts of
A 20 μL of culture broth from the wells at MIC and O. sanctum and O. gratissimum leaves were further
examined for their MIC and MBC/MFC values by
higher concentrations were transferred, inoculated on
broth micro-dilution method. The ethanol-extract
appropriate culture agar plates, and then incubated
of O. sanctum inhibited the growth of the tested
at appropriate environment for 24 h. The lowest
bacteria and fungus with various degrees. O. sanctum
concentration of herbal extract required to kill the
extract strongly inhibited the growth of S. pyogenes
tested bacteria or fungus by 99.9% (noted as a complete
with MIC of 0.19% (w/v) but expressed relatively less
absence of bacterial or fungal colony) was recorded as
inhibitory activity against C. albicans with MIC of
the MBC or MFC.[8]
3.12% (w/v). Similar to O. sanctum, ethanol-extract of
Statistical analysis O. gratissimum inhibited the growth of both bacteria
Each experiment was conducted in triplicate and and fungus tested in this study. However, the inhibitory
repeated for five times at different periods of time. activity of O. gratissimum extract against the tested oral

598 Journal of International Society of Preventive and Community Dentistry ¦ Volume 12 ¦ Issue 6 ¦ November-December 2022
 Chanthaboury, et al.: Antimicrob prop Ocimum spp

Table 1: Antimicrobial activity of ethanol-extract of Ocimum sanctum leaves against the tested microorganisms
determined by agar disk diffusion method. Inhibition zone (diameter in mm) was expressed in median values and
interquartile range
Concentrations of herbal extracts (% [w/v]) Inhibition zone (median of diameter [mm] ± interquartile range)
Streptococcus Streptococcus Staphylococcus Candida albicans
mutans pyogenes aureus
50.0 7 (0) 9 (0) 7 (0) –
25.0 – 8 (0) – –
12.5 – 8 (1) – –
6.25 – 7 (8) – –
0.2% Chlorhexidine 15 (0) 13 (2) 14 (2) 14 (0)
50% Ethanol – – – –
– = absence of inhibition zone

Table 2: Antimicrobial activity of ethanol-extract of Ocimum gratissimum leaves against the tested microorganisms
determined by agar disk diffusion method. Inhibition zone (diameter in mm) was expressed in median values and
interquartile range
Concentrations of herbal extracts (% [w/v]) Inhibition zone (median of diameter [mm]± interquartile range)
Streptococcus Streptococcus Staphylococcus Candida albicans
mutans pyogenes aureus
50.0 8 (1.5) 0 (7.5) 7 (7) –
25.0 0 (6.5) – – –
12.5 – – – –
6.25 – – – –
0.2% Chlorhexidine 16 (1) 13 (1) 15 (1) 15 (0)
– = absence of inhibition zone

Table 3: Minimum inhibitory concentrate (MIC) and Table 4: Minimum inhibitory concentrate (MIC) and
minimum bactericidal concentrate (MBC)/minimum minimum bactericidal concentrate (MBC)/minimum
fungicidal concentrate (MFC) values of ethanol- fungicidal concentrate (MFC) values of ethanol-extract
extract from Ocimum sanctum leaves against the tested from Ocimum gratissimum leaves against the tested
microorganisms determined by broth micro-dilution microorganisms determined by broth micro-dilution
method. method
Microorganisms Ocimum sanctum 50% Ethanol Microorganisms O. gratissimum extract
extract dilution MIC [% (w/v)] MBC [% (w/v)]
MIC MBC MIC MBC Streptococcus mutans 1.56 12.5
[% [% [% [% Streptococcus pyogenes 1.56 6.25
(w/v)] (w/v)] (w/v)] (w/v)] Staphylococcus aureus 1.56 1.56
Streptococcus mutans 0.78 3.12 3.12 12.5 Candida albicans 12.5 >12.5
Streptococcus pyogenes 0.19 0.78 6.25 6.25 MIC and MBC/MFC values were expressed in mode values
Staphylococcus aureus 0.78 1.56 0.78 0.78
Candida albicans 3.12 12.5 6.25 12.5
of both herbs were tested for antimicrobial activity
MIC and MBC/MFC values were expressed in mode values.
(50% ethanol was used to dissolved O. sanctum extract powder)
against S. pyogenes ATCC 14615, S. aureus ATCC
16799, S. mutans KPSK2, and C. albicans ATCC
microorganisms was weaker than those of O. sanctum 10231. Ethanol-extract of O. sanctum leaves at 50%
extract. The MIC and MBC/MFC values of O. sanctum (w/v) concentration inhibited the growth of all three
and O. gratissimum extracts were summarized in bacteria with various degrees of effectiveness. By agar
Tables 3 and 4. disk diffusion method, S. pyogenes was sensitive most
to ethanol-extract of O. sanctum. This result was also in
Discussion agreement with the MIC value [0.19% (w/v)] determined
Ocimum sanctum (holy basil or ka-phrao in Thai) by broth micro-dilution method. In addition, the
and O. gratissimum (clove basil/African basil or yira/ growth of S. aureus and S. mutans were moderately
horapha chang in Thai), popular culinary herbs in Thai inhibited by this extract using agar disk diffusion and
cuisine, were selected for this study. Ethanol-extracts broth micro-dilution methods [MIC values of 0.78%

Journal of International Society of Preventive and Community Dentistry ¦ Volume 12 ¦ Issue 6 ¦ November-December 2022 599
 Chanthaboury, et al.: Antimicrob prop Ocimum spp

(w/v)]. The antimicrobial activity of ethanol-extract of (w/v)]. Furthermore, the antimicrobial property of
O. sanctum against the tested bacteria determined by O. gratissimum extract against S. aureus [MIC of
agar disk diffusion method was similar to a previous 1.56% (w/v)] found in this study was slightly stronger
study[9] but differed from several previous reports.[10-13] than the study by Amengialue et al.[18] [MIC of 3.0%
The ethanol-extract of O. sanctum at 50% (w/v) (w/v)]. However, the antimicrobial property against
concentration did not illustrate any inhibitory effect S. aureus of O. gratissimum ethanol-extract prepared
against C. albicans as primarily determined by agar by Soxhlet extractor[19] was much stronger [MIC of
disk diffusion method. The anti-Candida activity of 0.2 (w/v); MBC of 0.4% (w/v)] than the results of this
the ethanol-extract of O. sanctum prepared in this study. In addition, some reports have shown that herbal
study [MIC value of 3.12% (w/v)] was much weaker extracts with high quantity and quality can be obtained
than previously reported by Sivareddy et al.[14] [MIC through the ethanol-maceration with either microwave
value of 0.2% (w/v)]. This may be due to the amount of or ultrasonic method.[20,21]
raw material (dried leaves of herb) used for extraction The form of the extract is also an important factor
process. The amount of O. sanctum dried leaves used for affecting the biological properties of herbs dependent
extraction in the current investigation was four times on the diversity of active constituent released. Generally,
lower, compared with Sivareddy et al. (6 g in 20 mL of essential oils of O. sanctum and O. gratissimum have
solvent vs. 25 g in 20 mL of solvent, respectively). Other shown greater antimicrobial activities than other
notable differences were the extraction temperature forms including ethanol-, methanol- or n-hexane-
(room temperature vs. cold extraction) and storage extracts. Our previous study showed that the ethanol-
conditions (stored in auto-desiccator cabinet vs. at extracts of Ocimum species contain functional groups
-20°C). indicative of aliphatic primary amines, aromatic
Extraction method is a vital key factor involved in compounds, alkanes, and alkyl halides on Fourier
the release of active components from the herbs. The infra-red spectroscopy analysis.[22] Phenolic compounds
ethanol-extract of O. sanctum prepared with Soxhlet including phenolic acids, rosmarinic acid, caffeic acid,
extraction method showed the MIC and MBC against and anigenin commonly present in both essential oil and
S. mutans at 25 μg/mL [0.0025% (w/v)],[15] which was ethanol-extract of O. sanctum, are active constituents
much stronger than the values found in this study [MIC effective against several pathogenic bacteria.[23-25]
at 0.78% (w/v); MBC at 3.12% (w/v)]. Solvents used for Phenolic compounds can damage permeability of
extraction can also significantly affect the antimicrobial bacterial membrane through mechanisms such as
property of the extract. In 2016, Tantry et al.[16] found substrate complexing, membrane disruption, enzyme
that the methanolic extract of O. sanctum was effective inactivation, and metal chelation resulting in ion
against S. aureus at the MIC value of 18.68 ± 0.95 mg/ leakage that can cause loss of membrane proton motive
mL [1.868% (w/v)], which was slightly less effective force leading to bacterial death.[26,27] Furthermore,
than the ethanolic extract [MIC at 0.78% (w/v)] in this hydrophobic property allows the oil to incorporate into
study. This seems to show that the active constituents the membrane and mitochondria of microorganisms
of herb obtained may differ in quantity, quality, and increasing their permeability that leads to leakage of
categories dependent on the solvents used in the some cell contents and causes cell death.[26]
extraction process. In this study, the ethanol-extracts of the leaves of
The ethanol-extract of O. gratissimum leaves at the O. sanctum and O. gratissimum extract were prepared
concentration of 50% (w/v) expressed inhibitory effect and tested for antibacterial and antifungal effects
only on the tested bacteria as determined by agar disk against some important oral pathogens, and the
diffusion method. This result was further supported results showed the former to have relatively stronger
by the MIC values [1.56% (w/v)] of this herbal anti-bacterial effects. However, the study used powder
extract against all three tested bacteria. The ethanol- based herbal extract which requires a proper solvent
extract of O. gratissimum showed no inhibition zone to attain good dispersion and maximize its bioactive
against C. albicans by the agar disk diffusion method; properties. As with other studies of herbal extracts,
however, this herbal extract expressed the inhibitory there may be discrepancy in antimicrobial efficacy of
effect on the fungus with MIC value of 12.5% the extracts based on variations in plantation factors
(w/v). Moreover, both antibacterial and antifungal such as geographic location and climate,[28] duration
properties of O. gratissimum ethanol-extract in this of harvesting,[29] active ingredients,[22] extraction
study were similar to those reported by Biqiku et al.[17] processes,[23] strains of the tested microorganisms, and
with the MIC values at >1024 μg/mL [>0.1024% testing protocols. This study has shown promising

600 Journal of International Society of Preventive and Community Dentistry ¦ Volume 12 ¦ Issue 6 ¦ November-December 2022
 Chanthaboury, et al.: Antimicrob prop Ocimum spp

results for these herbal extracts but further investigations 4. Vikesland P, Garner E, Gupta S, Kang S, Maile-Moskowitz A,
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6. Sharaf MH, El-Sherbiny GM, Moghannem SA,
Conclusion Abdelmonem M, Elsehemy IA, Metwaly AM, et al. New
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effectively inhibited the growth of S. pyogenes, 7. Upadhyay A, Bhagwat D. In vitro antioxidant activity and
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University J of Nat Sci 2014;13:297-315.
more effective as antibacterial than antifungal agents.
8. Norris SJ, Edmondson DG. In vitro culture system to determine
In addition, O. sanctum ethanol-extract expressed MICs and MBCs of antimicrobial agents against Treponema
stronger antibacterial and antifungal properties than pallidum subsp. Pallidum (Nichols strain). Antimicrob Agents
those of O. gratissimum extract. Chemother 1988;32:68-74.
9. Xia KZ, Perveen N, Khan NH. Phytochemical analysis,
Acknowledgements antibacterial and antioxidant activity determination of Ocimum
The authors would like to thank Asst. Prof. Natchalee sanctum. Pharm Pharmacol Int J 2018;6:490-7.
Srimaneekarn for her assistance in statistical analysis. 10. Krishnan RJ, Nair SR. Preliminary study on the antibacterial
activity of six medicinal plants against two naso-pharyngeal
Financial support and sponsorship pathogens—Streptococccus pyogenes and Pseudomonas
Nil. aeruginosa. Am J Plant Sci 2016;7:907-15.
11. Kochikar Pai R, Bhat SS, Salman A, Chandra J. Use of
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The authors do not have any conflicts of interest to anticariogenic agent: An in vitro study. Int J Clin Pediatr Dent
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12. Seleman NS, Amri E. Antibacterial activity of aqueous,
Authors’ contribution ethanol and acetone extracts of Ocimum sanctum Linn. AJBIO
SC and TS were involved in study conception, MC was 2015;3:256-61.
13. Subramanian G, Tewari BB, Gomathinayagam R. Studies
responsible for data collection and data acquisition, of antimicrobial properties of different leaf extract of tulsi
SC and TS were involved in data analysis and data (Ocimum tenuiflorum) against human pathogens. Am Int J
interpretation, MC, BS, and SC were involved Contemp Res 2014;4:149-57.
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read and approved the final version of the manuscript Subrahamanyam G. Antifungal activity of solvent extracts of
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