IOSR Journal of Dental and Medical Sciences (IOSR-JDMS)

e-ISSN: 2279-0853, p-ISSN: 2279-0861.Volume 22, Issue 4 Ser.5 (April. 2023), PP 28-33
www.iosrjournals.org

Antimicrobial efficacy of essential oils of clove, neem and

cinnamon on Streptococcus mutants and lactobacillus
acidophilus in comparison with 0.2% chlorhexidine – An
in vitro study
Bharath Shivakumar1, Sunitha S1, Chandrashekar BR1, Mirunalini
Sundaravadivelu1
1
(Department of Public Health Dentistry, JSS Dental College and Hospital, JSSAHER, India)

Abstract:
Background:Dental caries is the most common, chronic infectious disease impacting around 3.5 billion
people around the world according to Global Burden of diseases (2019). In-vitro studies of essential oils of
neem, clove and cinnamon and their antimicrobial efficacy against S.mutans and L.acidophilus at different
concentrations will help develop Phytochemical drugs with better synergistic effect as an adjunct to
maintaining good oral hygiene.
Materials and Methods: Authenticated commercially available pure clove oil, neem oil and cinnamon oil were
purchased and diluted to obtain 1%, 5%, and 10% concentrations. The ATCC (American Type Culture
Collection) strains of S.mutans (25175), and L. acidophilus (4356) was procured, the bacterial cultures were
maintained on BHI (Brain Heart Infusion) agar and sheep blood agar slants respectively, with periodic
subculturing and stored at 4° C. Antimicrobial efficacy was assessed using the agar well diffusion method. 0.2%
chlorhexidine was used as the positive control and DMSO (Dimethyl sulfoxide) as a negative control.
Results: The essential oils of clove, neem and cinnamon showed antimicrobial efficacy against S.mutans and L.
acidophilus at different concentrations. The cinnamon oil at 10% concentration showed a 22.54±0.69
(Mean±SD) zone of inhibition againstL. acidophilus similar to 0.2% Chlorhexidine. The antimicrobial efficacy
of all three essential oils increased as there was an increase in concentration.
Conclusion:The cinnamon essential oil had overall superior antimicrobial activity against both bacteria as
compared to the other two essential oils, clove and neem. Cinnamon oil showed a mean zone of inhibition
similar to 0.2% chlorhexidine against L. acidophilus at 10% concentration.
Key Word: Clove oil, Neem oil, Cinnamon oil, Streptococcus mutans, Lactobacillus acidophilus.
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Date of Submission: 03-04-2023 Date of Acceptance: 14-04-2023
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I. Introduction
Oral health is very important for general health since it affects a person’s ability to consume food,
communicate, and live life to the fullest. Oral health has its implications on systemic conditions like the
cardiovascular system, diabetes-related gum disease, and respiratory illnesses [1]. The prevalence of oral
disorders among chronic diseases is high and expensive treatment costs make them significant social and
economic issues. The human oral cavity contains more than 700 different bacterial species [2]. The oral
microbiota of different people, however, may differ significantly. Sometimes, the defence mechanism against
the invasion of foreign germs is assisted by microbial interactions within the oral cavity. The oral health of an
individual may suffer negatively due to these invading microbes. Oral illnesses such as dental caries, periodontal
disease, and mucosal and systemic diseases are influenced by unbalanced microbial flora [3]. Over the course of
several decades, dental caries has spread over the globe to become one of the most widespread chronic
infectious oral diseases. The oral microbiota and dental plaque both are thought to be primarily composed of
Streptococcus mutans a gram-positive coccus, facultative anaerobe and Lactobacillus acidophilus is a rod-
shaped, anaerobic gram-positive microorganism [3,4].Caries and periodontal diseases can be prevented by
brushing teeth often and implementing a variety of other oral hygiene practices, even though the creation of
biofilm in the oral environment is a normal and continuous process. Due to the inadequacy of mechanical means
of plaque removal, patients and the general population may seek additional benefits from chemotherapeutic
antiplaque medications [5].
Since the dawn of human history, essential oils made from plants and herbs have been utilized
medicinally [9]. There are several biological characteristics of essential oils, including larvicidal activity,
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 Antimicrobial efficacy of neem, clove and cinnamon oil against S mutants and L. acidophilus.

analgesic, anti-inflammatory, antifungal, and antioxidant capabilities[10,11]. Utilizing different essential oils
that include a wide range of phytochemicals might inhibit or limit bacterial development. The antibacterial,
antibiotic, and antiviral activities of neem (Azadirachta Indica)are well-known in the medicinal plant world.
The main components of neem oil in terms of phytochemistry are oleic acid, linoleic acid, hexadecanoic acid
and alkaloids like nimbin, tannins and nimbidin [12]. As spices, clove (Syzygium aromaticum) and cinnamon oil
(Cinnamomum verum), are used to improve the flavour of food. Their fragrant, carminative, antibacterial and
antioxidant qualities are well established [13]. Due to their widespread availability, low cost, biodegradability,
and safety, essential oils are widely used [14,16].
A thorough analysis of these essential oils provides the opportunity to examine their potential for use as
antibacterial agents against the most common microorganisms responsible for tooth decay and gum disease. In
light of this, the current study aims to compare the antibacterial effects of essential oils of neem, clove, and
cinnamon at different concentrations with those of 0.2% chlorhexidine against Streptococcus mutans and
Lactobacillus acidophilus.

II. Material And Methods

The in-vitro study was carried out to test the antibacterial activity of clove oil, neem oil and cinnamon
oil against streptococcus mutans and lactobacillus acidophilus. The protocol of the study was approved by the
institutional ethical committee (RP No: 46/2020). The study was carried out in an integrated collaborative
biopharmaceutical laboratory which is recognized by the department of scientific and industrial research (DSIR)
Government of India.

Bacterial Strain and Inoculum

We obtained strains of Streptococcus mutans (25175) and Lactobacillus acidophilus (4356), both of
which are available from the American Type Culture Collection (ATCC). The bacterial cultures were
maintained on Brain heart infusion agar and on sheep blood agar culture slants, respectively, by performing
periodic subculturing while being kept at a temperature of 4 degrees Celsius. The cell suspensions of the
cultures were adjusted to the concentration of 1-2 x 106 cells per millilitre. The bacterial strains were then plated
on Soyabean casein-digested agar.

Chemicals and reagents

Commercially available pure authenticated clove oil, neem oil and cinnamon oil were purchased. The
essential oils purchased were examined for their purity and then dilution was carried out. The essential oils were
diluted using 5% tween 20 solution. The concentrations prepared were 1%, 5% and 10% for all the three
essential oils. All other chemicals used were of the highest purity available from commercial sources.

Dilution of the essential oils

The essential oils were diluted using 5% tween 20 solution. 990µl of tween 20 was diluted with 10µl of
each essential oil separately to obtain 1% concentration. 950µl of tween 20 was diluted with 50µl of each
essential oil separately to obtain 5% concentration. 900µl of tween 20 was diluted with 100µl of each essential
oil separately to obtain 10% concentration.

Soyabean casein digested agar plates preparation

The Soyabean casein digested agar was prepared using the manufacturer’s instruction (HIMEDIA
Soyabean Casein Digest Medium – M1838-500G; LOT: 0000413018; EXP: 2014-23). 35.7 grams of powder
was added to 1000 ml distilled water after which it was boiled to ensure complete dissolution of the medium.
The media was autoclaved at 15lbs pressure (118- 121°C respectively) for 15 minutes.

Assessment of mean zone of inhibition for clove oil, neem oil and cinnamon oil making use of agar well
diffusion method
The antimicrobial characteristics of clove, neem and cinnamon oil were evaluated by making use of the
agar well-diffusion method. For this, the prepared Soyabean casein digested agar was poured into Petri plates
and allowed to cool in a laminar airflow unit under aseptic conditions.
10µl of the standardized bacterial suspension of S.mutans and L.acidophilus was dispensed and spread
over the agar Petri plates. 5mm wells were then dug out from each Petri plate of the set agar. The wells were
filled with Samples (clove, neem and cinnamon of 1%, 5%, 10% each) (25µl) and Positive control 0.2%
Chlorhexidine (25µl), Negative control Dimethyl sulphoxide (DMSO) (25µl). The treated plates with S.mutans
and L.acidophilus were incubated at 37°C for 24hrs.
The zone of inhibition (ZOI) surrounding the samples were measured by taking diameters from 3
different points after 24 hours. All the experiments were done in triplicates sets (Figure 12-17). The zone of

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 Antimicrobial efficacy of neem, clove and cinnamon oil against S mutants and L. acidophilus.

inhibition was computed by subtracting 5mm as the diameter of the testing sample from the average values
obtained.

Statistical analysis
The statistical analysis was done using SPSS software version 24 (IBM, Chicago, USA). Quantitative data is
presented as mean and standard deviation.
 The comparison of mean zone of inhibition of different group of agents was done using one-way
ANOVA with Turkey’s Post-hoc Test.
 The statistical significance was fixed at 0.05.

III. Result
The antimicrobial efficacy of all the samples and their concentrations was determined by the well diffusion
method. The level of antimicrobial activity is determined by the size of the inhibition zone shown by the
essential oil.

Mean inhibition zone for S.mutans

The mean zone of inhibition for S.mutans against all the agents is given in Table 1. The mean inhibition zone
increased as the concentrations of the essential oils increased. Cinnamon oil at 10% concentration showed
13.50±3.10 (mean±SD) which was the highest inhibition zone among three essential oils.

Table no 1: The Mean zone of inhibition for Streptococcus mutans against Clove, neem and cinnamon oils
at 1%, 5% and 10% concentrations in comparison with Chlorhexidine 0.2%.

Extract / Standard Group 1% concentration 5 % concentration 10 % concentration

Clove oil G1 0.23±0.10 5.09±0.65 8.90±0.56

Neem oil G2 0.60±0.00 6.91±1.24 10.87±0.60

Cinnamon oil G3 0.88±0.14 7.80±0.54 13.50±3.10

0.2% Chlorhexidine G4 23.40±1.16 23.03±0.51 23.42±0.38

* Statistical inference F value: 3462.10 F value: 777.10 F value: 115.23

df : 3 df : 3 df : 3
p value: 0.001 p value: 0.001 p value: 0.001

Post hoc analysis G1 vs G2 – 0.55 G1 vs G2 – 0.96 G1 vs G2 – 0.23

G1 vs G3 – 0.62 G1 vs G3 – 0.25 G1 vs G3 – 0.15
G1 vs G4 – 0.00 G1 vs G4 – 0.00 G1 vs G4 – 0.00
G2 vs G3 – 0.99 G2 vs G3 – 0.10 G2 vs G3 – 0.26
G2 vs G4 – 0.00 G2 vs G4 – 0.00 G2 vs G4 – 0.00
G3 vs G4 – 0.00 G3 vs G4 – 0.00 G3 vs G4 – 0.00

* one way ANOVA with Post hoc Statistical analysis

Table 1: Mean inhibition zone for S.mutans against clove, neem and cinnamon oils at different
concentrations

16
14
12
Axis Title

10
8
6
4
2
0
1% concentration 5% concentration 10% concentration
Clove oil 0.23 5.09 8.9
Neem oil 0.6 6.91 10.87
Cinnamon oil 0.88 7.8 13.5

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 Antimicrobial efficacy of neem, clove and cinnamon oil against S mutants and L. acidophilus.

Mean inhibition zone for L.acidophilus

The mean zone of inhibition for L.acidophilus against clove, neem, cinnamon oil and 0.2%
chlorhexidine is given in Table 2. The mean inhibition zone increased as the concentrations of the essential oils
increased. The 0.2% chlorhexidine showed the highest mean zone of inhibition. Cinnamon oil at 10%
concentration showed mean zone of inhibition (22.54±0.69) similar to 0.2% chlorhexidine (23.78±0.64) the
Tukey’s post hoc test revealed that there is no statistically significant difference between both the groups.

Table 2: Mean inhibition zone for L.acidophilus against clove, neem and cinnamon oils at different
concentrations
Extract / Standard 1% concentration 5 % concentration 10 % concentration

Clove oil 8.56±0.83 14.56±1.06 15.73±1.28

Neem oil 3.24±0.41 8.23±0.54 10.58±0.92

Cinnamon oil 15.78±1.01 18.87±1.04 22.54±0.69

0.2% Chlorhexidine 24.33±0.75 24.73±0.59 23.78±0.64

* Statistical inference F value: 542.05 F value: 413.40 F value: 65.21

df : 3 df : 3 df : 3
p value: 0.00 p value: 0.00 p value: 0.00

Post hoc analysis G1 vs G2 – 0.01 G1 vs G2 – 0.00 G1 vs G2 – 0.00

G1 vs G3 – 0.01 G1 vs G3 – 0.00 G1 vs G3 – 0.00
G1 vs G4 – 0.00 G1 vs G4 – 0.00 G1 vs G4 – 0.00
G2 vs G3 – 0.03 G2 vs G3 – 0.00 G2 vs G3 – 0.01
G2 vs G4 – 0.00 G2 vs G4 – 0.00 G2 vs G4 – 0.00
G3 vs G4 – 0.00 G3 vs G4 – 0.00 G3 vs G4 – 0.40

* one way ANOVA with Post hoc Statistical analysis

20

15
Axis Title

10

0
1% concentration 5% concentration 10% concentration
Clove oil 8.56 14.56 15.73
Neem oil 3.24 8.23 10.58
Cinnamon oil 15.78 18.87 13.5

IV. Discussion
Oral health in its entirety is best achieved with regular hygiene practices and making use of different
oral hygiene aids. Over the period of time, there are different oral hygiene aids available in the market. There
are a variety of toothbrushes available, some of which use motorized technology. Additionally, there are new-
age mouthwashes available that combine different genre chemicals and bioactive agents to work effectively and
address different problems.
The incorporation of organic compounds in conventional medical and dental treatments has been
ascribed the name "phytotherapeutics" or "ethnopharmacology," and it is well-known that these treatments have
a number of advantages over traditional pharmaceuticals 14. The versatility of medicinal plants' health-
promoting functionalities, cost-effectiveness, availability, and minimal adverse effects related to the drugs
derived from these plants continue to pique the interest of researchers. This interest has been sustained by the
fact that medicinal plants are readily available. Essential oils are well-known for the antibacterial, antioxidant,
antifungal, and antiviral qualities that they possess. Essential oils are basically procured from natural
resources and contain a wide variety of chemical compounds that are known for their therapeutic potential.
Cinnamon oil showed antimicrobial efficacy against Streptococcus mutans and Lactobacillus
acidophilus. The antimicrobial properties of cinnamon oil is well established and proved in the study conducted
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 Antimicrobial efficacy of neem, clove and cinnamon oil against S mutants and L. acidophilus.

by Prabuseenivasan S et al (15), where seven essential oils were selected to determine the minimum inhibitory
concentration against six bacterial species and cinnamon oil showed promising antimicrobial activity even at a
lower concentration. Which was in accordance to this study.
In contrast to this investigation, the research carried out by Rajini Kanth et al(16)found that cinnamon
essential oil had a smaller mean zone of inhibition in comparison to clove oil. This may be due to the difference
in the methodology of the study, the bacterial culture was obtained by decayed tooth and the culture of the
bacteria did not have differentiation among different species.
The study conducted by M. Fadli et al (17) evaluated the antimicrobial activity of the combination of
essential oil and traditional antibiotics. The synergetic effects of the combination of essential oils and antibiotics
were promising increasing the antimicrobial activity of the drug and reducing the minimum effective dose. This
can contribute towards lowering the toxic side effects and treatment costs. The evaluation of the synergistic
effects must be done both in vitro and in vivo since they can vary depending on the active ingredients in the
essential oils, their composition, and the length of exposure. Studies on the mechanisms of action of essential
oils have not yet been extensively established.

V. Conclusion
The cinnamon essential oil had overall superior antimicrobial activity against both bacteria as compared to the
other two essential oils, clove and neem. Cinnamon oil showed a mean zone of inhibition similar to 0.2%
chlorhexidine against L.acidophilus at 10% concentration.

STRENGTHS AND LIMITATIONS

The highlights of the present study are
 The invitro observations were made in well-monitored strict lab conditions.
 This was the first of its kind study, where three different essential oils were evaluated for antimicrobial
activity at three different concentrations (1%, 5%, 10%) in comparison with 0.2% chlorhexidine against
Streptococcus mutans and Lactobacillus acidophilus.
Limitations
 Because it is an in-vitro study, it is not possible to reproduce biological variables seen in the human
body.
 The diversity in drug distribution to infection sites within the body may vary in comparison to in-vitro
conditions.
Recommendation
 Many other essential oils can be tested for antimicrobial efficiency and can be compared with the three
mentioned in this study
 Antimicrobial efficacy can be assessed further in in-vivo conditions for the formulation of drugs
The combination effects and synergistic effects of different essential oils can be studied further.

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Bharath Shivakumar, et. al. “Antimicrobial efficacy of essential oils of clove, neem and cinnamon
on Streptococcus mutants and lactobacillus acidophilus in comparison with 0.2% chlorhexidine –
An in vitro study.” IOSR Journal of Dental and Medical Sciences (IOSR-JDMS), 22(4), 2023, pp.
28-33.

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