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Jurnal Kedokteran Brawijaya Vol. 33, No. 1, February 2024, pp. 1-5
Article History: Received 10 April 2023, Accepted 29 February 2024

Research Article

Antibacterial and Antifungal Properties of Citronella oil Against Streptococcus mutans and
Candida albicans by In Vitro Study

Sifat Antibakteri dan Anti-fungal Minyak Sereh terhadap Streptococcus mutans dan Candida
albicans Secara In Vitro

Vinna Kurniawati Sugiaman1, Wahyu Widowati2, Hanna Sari Widya Kusuma3, Nindia Salsabila4, Rizal Rizal5
1
Faculty of Densitry Universitas Kristen Maranatha Bandung
2
Faculty of Medicine Universitas Kristen Maranatha Bandung
3,4
Aretha Medika Utama Biomolecular and Biomedical Research Center Bandung
5
Biomedical Engineering Department of Electrical Engineering Faculty of Engineering Universitas Indonesia Jakarta

ABSTRACT
Streptococcus mutans and Candida albicans are the main microorganisms that cause dental cavities. It can cause infection,
damaged tissue around teeth, abscesses, and focal infection to other organs in the body. Natural products are currently
widely used as products or as additives in the prevention of dental caries which have more anti-bacterial and anti-fungal
activities than antibiotics that can cause resistance. Citronella (Cymbopogon citratus) is abundant and easy to grow. This
study was conducted to determine the effectiveness of citronella (Cymbopogon citratus) oil on the growth of S. mutans and
C. albicans microorganisms by Minimum Inhibitory Content (MIC) and Minimum Bactericidal Content (MBC). The method
of MIC is broth microdilution by making chlorhexidine concentration levels of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and
0.2% with 4 replications each. The MIC value was determined based on absorbance spectrophotometry and the MBC value
was determined from the agar plate using the spread method. Biofilm eradication test was conducted by crystal-violet
staining and measuring the absorbance. The results of MIC and MBC were obtained on S. mutans, namely at
concentrations of 25% and 100%, respectively. Meanwhile, the results of MIC and MBC on C. albicans were obtained at
concentrations of 50% and 100%, respectively. Furthermore, the citronella oil has antibacterial and antifungal activities.
Keywords: Antimicrobial, Candida albicans, Minimum Bactericidal Content, Minimum Inhibitory Concentration,
Streptococcus mutans

ABSTRAK
Streptococcus mutans dan Candida albicans merupakan mikroorganisme utama penyebab gigi berlubang. Hal ini dapat
menyebabkan infeksi, kerusakan jaringan di sekitar gigi, abses dan infeksi fokal ke organ tubuh lainnya. Produk alami saat
ini banyak digunakan sebagai produk atau sebagai bahan tambahan dalam pencegahan karies gigi yang memiliki aktivitas
anti bakteri dan anti jamur dibandingkan antibiotik yang dapat menyebabkan resistensi. Citronella (Cymbopogon citratus)
melimpah dan mudah tumbuh. Penelitian ini bertujuan untuk mengetahui efektivitas minyak serai wangi (Cymbopogon
citratus) terhadap pertumbuhan mikroorganisme S. mutans dan C. albicans berdasarkan Minimum Inhibitory Content
(MIC) dan Minimum Bactericidal Content (MBC). Metode MIC adalah mikrodilusi kaldu, dengan membuat kadar
konsentrasi klorheksidin 100%, 50%, 25%, 12,5%, 6,25%, 3,125%, dan 0,2% dengan masing-masing 4 ulangan. Nilai MIC
ditentukan berdasarkan spektrofotometri absorbansi dan nilai MBC ditentukan dari lempeng agar menggunakan metode
sebar. Uji eradikasi biofilm dilakukan dengan pewarnaan kristal-violet dan diukur absorbansinya. Hasil MIC dan MBC
diperoleh pada S. mutans yaitu pada konsentrasi masing-masing 25% dan 100%, serta hasil MIC dan MBC pada C. albicans
diperoleh pada masing-masing konsentrasi 50% dan 100%. Selain itu, minyak serai memiliki aktivitas antibakteri dan
antijamur.
Kata Kunci: Antimikroba, Candida albicans, Kandungan Bakterisida Minimum, Konsentrasi Hambat Minimum,
Streptococcus mutans

Correspondence: Wahyu Widowati. Faculty of Medicine Universitas Kristen Maranatha Bandung, Jl. Prof. Drg. Surya Sumantri No. 65,
Bandung, West Java, Indonesia Tel. +62022-2102186/+6222-70753665 Email: wahyu_w60@yahoo.com

DOI: http://dx.doi.org/10.21776/ub.jkb.2024.033.01.1

1
 Antibacterial and Antifungal Properties of... 2

INTRODUCTION METHOD
Dental cavities in people have been linked to Preparation of Citronella Oil
Streptococcus mutans as the main causative agent. The The extraction was carried out at the Pharmaceutical
bacterium's capacity to create the biofilm known as dental Company PT Indesso Aroma, Baturaden, Purwokerto,
plaque on teeth surfaces is a crucial aspect of its Central Java with CoA No Batch. 895850017. The
pathogenicity (1). This organism also has surface proteins extraction was carried out for the plan to develop
that work together to form tooth plaque and cause dental standardized herbal medicines from natural ingredients
cavities, including glucosyltransferases, numerous glucan- consisting of Citronella extract as an anti-bacterial and
binding proteins, protein antigen C, and collagen-binding anti-fungal. The extraction method started by washing the
protein (2). Candida albicans colonization caused lemongrass and cutting them to ¼cm x ¼cm x ¼cm.
significant microbial dysbiosis characterized with distinct Methanol was used as maceration solvent with ratio of
microbial composition and structure as compared to the 1:10 between solute and solvent. Maceration was carried
biofilm in the absence of C. albicans. These findings is in out for 10 hours (13). The resulting extract is in the form of
agreement with previous studies showing that C. albicans oil.
influenced the microbial composition of saliva biofilms
Research on the MIC and MBC tests of citronella oil on the
(3). The primary bacterial pathogen in tooth caries, growth of S. mutans and C. albicans was carried out at the
especially in early-childhood caries (ECC), is frequently Aretha Medika Utama Bandung Biomolecular and
identified as S. mutans. Candida albicans cells are Biomedical Laboratory, with work procedures using the
commonly found in plaque biofilms from ECC-affected 2012 CLSI (Clinical Laboratory Standard Institute) method
infants, along with heavy infection by S. mutans, with modifications. This research is pure experimental
suggesting that S. mutans may not act alone. laboratory in vitro by comparing the sample groups
Exopolysaccharide (EPS) synthesis is increased by the containing citronella oil with concentrations 100%, 50%;
presence of C. albicans, causing cospecies biofilms to 25%; 12.5%; 6,25%, and 3.125% and positive control in the
accumulate more biomass and contain more live S. form of 0.2% chlorhexidine (Minosep) with 4 replication.
mutans cells than single-species biofilms (4). Citronella oil dilution was carried out by adding n-hexane.
Nowadays, the demand for natural preservatives is solely Chlorhexidine was acted as the positive control because it
related to the increasing interest in healthy lives and was reported that Chlorhexidine was an antiseptic that
health issues. Additionally, the use of antibiotics leads to could go averse to both types of bacteria, viruses, and
the development of multidrug resistant microorganisms, facultative aerobes and anaerobes (14). The growth
so research into these natural components has gained control was used as the negative control.
more focus in the scholarly community (5). Numerous Antibacterial and Antifungal Assay
useful natural compounds derived from various plant The isolates of S. mutans and C. albicans were obtained by
species have been found to possess antimicrobial qualities sub culture in MHA media at 37°C for 24 hours. Some of the
(6). Among them, some essential oils (EOs) have the colonies were inoculated to 10ml MHB. McFarland 0.5
potential to be useful food stabilizers, making them standard was used to adjust the turbidity. Inoculum was
appealing substitutes for manufactured compounds (7). obtained by conducting dilution to the solution with
Additionally, EOs are not too difficult to obtain, physiological solution. Media MHB with ratio of 1:20 was
ecologically favorable (they degrade rapidly in soil and then used to conduct dilution. The inoculum was then
water), and not too harmful to people (8). obtained in the range of 1-5 x 105 CFU/ml. The MIC of
Tropical and subtropical areas of the globe are home to Citronella oil against S. mutans and C. albicans was
Cymbopogon citratus, more commonly known as "Lemon determined by the broth microdilution method as
Grass" (9). C. citratus is native to Asia (Indochina, recommended by the Clinical and Laboratory Standards
Indonesia, and Malaysia), Africa, and the Americas, but is Institute (CLSI) M07-A9.
widely cultivated in temperate and tropical regions of the At the final concentration, 100μL of Citronella oil was
world (10). Alpha citral (geranial), beta citral (neral), and added into 96-well microplate. For the positive control we
myrcene are the three primary constituents of this C. added 100µl of 0.2% chlorhexidine (Minosep) to the well.
citratus essential oil. While studies have proven the The sample of each bacterial and fungal suspension (24h
antibacterial activity of alpha and beta citral against gram- incubated S. mutans and C. albicans) respectively with a
positive and gram-negative bacteria as well as fungistatic concentration of 1.5 × 106CFU/mL with Mueller Hinton
activity against Candida species, myrcene has not Broth (Himedia, M403-500G) were also added to the wells.
exhibited any antimicrobial activity (11). The therapeutic The microplate was incubated for 24 hours at 37°C in
actions of C. citratus include anti-amoebic, antibacterial, anaerobic condition then measured the absorbance with
antidiarrheal, antifilarial, antifungal, and anti- Spectrophotometry (Multiskan GO Thermo Scientific
inflammatory effects. Antimalarial, antimutagenic, anti- 51119300) at 415nm for S. mutans and 510nm for C.
mycobacterial, antioxidant, hypoglycemia, and albicans. The MIC was determined as the lowest
neurobehavioral benefits, among others, have also been concentration of Citronella oil in which visible bacterial
researched (12). With all of the benefits of C. citratus and fungi growth was inhibited. Also, the quantitative
stated above, this study were to determine the evaluation of microbial growth was determined based on
effectiveness of citronella (C. citratus) oil on the growth of the previous study (15).
S. mutans and C. albicans microorganisms as antibacterial The MBC was considered to be the lowest concentration of
and antifungal activity. Citronella oil which prevented the growth and reduced the

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 Antibacterial and Antifungal Properties of... 3

inoculum by >99.9% within 24 hours, irrespective of Table 1. Table of optical density of S. mutans and C. albicans
counts of survivors at higher sample concentrations. For
this purpose, 20μL of contents of the well determined as Optical Density (OD)
Samples
the MIC and also the wells with a concentration of 2 S. mutans C. albicans
×103CFU/ml were cultured in spread inoculation in the Chlorhexidine 0.2% 0.0012 ± 0.0001 0.0012 ± 0.0014
Mueller Hinton Agar by pour plate method (Himedia, Citronella oil 100% 0.0023 ± 0.0002 0.0012 ± 0.0004
M096-500G) and incubated for 24 hours in anaerobic Citronella oil 50% 0.0415 ± 0.0002 0.0337 ± 0.0004
Citronella oil 25% 0.0939 ± 0.0002 0.0567 ± 0.0010
condition with the temperature of 37°C and 5% CO2 (15).
Citronella oil 12.5% 0.1917 ± 0.0003 0.0775 ± 0.0002
Colony counter was used to count the total colony. The Citronella oil 6.25% 0.1961 ± 0.0002 0.1057 ± 0.0007
optical density (OD) measurement utilized microplate Citronella oil 3.125% 0.2613 ± 0.0004 0.1192 ± 0.0002
reader at wavelength of 625 nm. Treatments' OD were Note: *Data provided as mean ± STD
compared to Blank's OD.

RESULT
Table 2. Table of total colony count of S.Mutans and C.
The measurement of optical density of Citronella oil albicans
against S. mutans results are presented in the table I, that
showed Citronella oil 3.125% has the highest average Total Colony (CFU/ml)
Samples
value of absorbance with 0.2613 and lowest in the S. mutans C. albicans
chlorhexidine control with mean values absorbance Chlorhexidine 0.2% 0±0 0±0
0.0011. On the results of various measurements extract Citronella oil 100% 0±0 0±0
treatment, showed that the average value the highest Citronella oil 50% 20250 ± 957 4500 ± 1290
corrected absorbance is found in the concentration of Citronella oil 25% 103750 ± 2500 28750 ± 2217
Citronella oil 12.5% 139500 ± 2380 43500 ± 4434
Citronella oil 100% with the absorbance value is 0.0023,
Citronella oil 6.25% 191500 ± 5447 53500 ± 5322
This shows that S. mutans can be inhibited by Citronella oil.
Citronella oil 3.125% 253250 ± 2217 61750 ± 4645
The measurement of optical density of Citronella oil Note: *Data provided as mean ± STD
against C.albicans results are presented in the Table 1. The
data showed that Citronella oil 3.125% has the highest
average value of absorbance with 0.1192 and lowest in the Concentration of an antibacterial agent in the lowest value
chlorhexidine control with mean values absorbance that could inhibit an organism development visually is
0.0012. Based on the measurement result of various called MIC (16). In this research, MIC value of Citronella oil
extract treatments, Citronella oil 100% exhibited the against S. mutans and C. albicans was taken by the
highest absorbance value, which is 0.0022. It means that concentration of Citronella oil 25% with 75.08% and
C. albicans could be inhibited by Citronella oil. The result 53.66% repectively. MBC value was determined by
of MBC S.mutans and C. albicans were shown in Table II. It concentration that could inhibit the growth of 99.9%
was showed that both Chlorhexidine 0.2% and Citronella organism (17). In this research, the MBC value of Citronella
oil 100% resulted in zero total colony (0 CFU/ml). It means oil against S. mutans and C. albicans was taken by the
that Citronella oil 100% exhibited the same result as the concentration of Citronella oil 100% with 99.38% and
positive control. Meanwhile, the highest total colony 99.04%, respectively (Figure 1).
count came from Citronella oil 3.125% with average
253250 CFU/ml. In C. albicans, it is shown that the lowest
DISCUSSION
total colony count at Citronella oil 100% with 0 CFU/ml and
the highest at Citronella oil 3.125% with 61750 CFU/ml. Terpenes, alcohols, ketones, aldehydes, and esters make

120.00 g g g g
100.00 f
e f
Inhibition (%)

80.00
c d e
60.00
b d
40.00
c
20.00 b a a
0.00
-20.00

TREATMENTS
C. albicans S.mutans

Figure 1. Citronella oil (CO) inhibition against S. mutans and C. albicans

Note: *Data was presented in mean ± STD. Difference letters (a,b,c,d,e,f,g) were shown significantly difference among treatments in each sample
(p<0.05) based on Tukey Post Hoc Test.

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 Antibacterial and Antifungal Properties of... 4

up the majority of the substances found in Cymbopogon microorganisms (21). Kaempferol targets the stability of
citratus. Essential oils that contain Citral, Nerol Geraniol, the bacterial membrane to produce its antibacterial
Citronellal, Terpinolene, Geranyl acetate, Myrcene, and properties (22).
Terpinol Methylheptenone are a few of the documented Secondary compounds of plants with antifungal action
phytoconstituents. The plant is also said to contain
include flavonoids by denaturing the extracellular proteins
phenolic substances like luteolin, isoorientin 2'-O-
of the fungus cell walls, flavonoids cause harm to the fungal
rhamnoside, quercetin, kaempferol, and apigenin as well
cell walls and block the activity of enzymes. Denatured
as flavonoids. Numerous pharmaceutical actions,
including anti-amoebic, antibacterial, antidiarrheal, proteins disturb the process of how cells are made,
antifilarial, antifungal, and anti-inflammatory resulting in modifications to the protein structure and an
characteristics, have been linked to Cymbopogon citratus, increase in cell membrane porosity. This rise results in
according to studies. Antimalarial, antimutagenic, anti- intracellular material leaks, cellular ATP shortage,
mycobacterial, antioxidant, hypoglycemia, and metabolism disturbance, growth suppression, and cell lysis
neurobehavioral benefits, among others, have also been (23). Although the exact method by which terpenoid
researched (12). compounds prevent the growth of fungus is still unknown,
the existence of lipophilic or hydrophobic characteristics in
Citronellal compounds, citronellol, geraniol, geranyl terpenoid compounds causes cell coagulation and
acetate, and citronellyl acetate were terpenoids. In order cytoplasmic membrane damage in fungal cells (24).
to reduce the permeability of the bacterial cell wall and
cause the cells to lack nutrients, inhibit bacterial growth, Based on the antibacterial and antifungal assay, Citronella
or die, terpenoid compounds damage the membranes of oil 100% showed the best inhibition than any other
lipophilic compounds by forming strong polymer bonds concentrations. In addition, Citronella oil showed better
and harming porins (18). Monoterpene substances work inhibition to S. mutans in overall concentrations. However,
as an antibiotic by having a toxic impact on the the MIC obtained at concentration of 25% for both S.
composition and operation of bacterial cell membranes. mutans and C. albicans. Previous study showed that C.
Since monoterpenes are lipophilic, they transition from nardus essential oil exhibited S. mutans activity at 25%
the polar phase to the non-polar bacterial cell membrane (25). Meanwhile, inhibition of C. albicans was showed at
structure (19). concentration of 6.25%. This means that the result of this
Luteolin's ability to block DNA topoisomerase I and II study is comparable to prior study. This study was hindered
activity, which led to a reduction in the production of by some limitations, one of which is that the
nucleic acids and proteins, is what gives it its antibacterial characterization of citronella oil has not been able to be
properties (20). An important flavonoid compound, called conducted, thus restraining the ability to know the exact
quercetin has a wide range of medicinal effects. compound that is responsible for these bioactivity.
Numerous studies examine its antibacterial effects and Citronella oil has the potency for antibacterial and
potential mechanisms of action. It has been antifungal treatment, through inhibit S. mutans and C.
demonstrated that quercetin prevents the development albicans growth.
of various Gram-positive and Gram-negative bacteria,
fungus, and viruses. The method of its antimicrobial ACKNOWLEDGEMENT
action involves mitochondrial failure, cell membrane
injury, altered membrane permeability, suppression of We are gratefully acknowledging Aretha Medika Utama,
protein and nucleic acid synthesis, downregulation of Biomolecular and Biomedical Research Center, Bandung,
virulence factor expression, and prevention of biofilm West Java, Indonesia, that had sponsored, facilitated, and
formation. A powerful antimicrobial agent against drug- supported this research. We also appreciate PT Indesso
resistant strains, quercetin, has also been shown to Aroma, Baturaden, Purwokerto, Central Java, for preparing
suppress the development of a number of drug-resistant the Citronella extract.

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