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International Journal of Secondary Metabolite
2022, Vol. 9, No. 3, 258–267
https://doi.org/10.21448/ijsm.1059886
Published at https://dergipark.org.tr/en/pub/ijsm Research Article
258
Anti-quorum sensing and cytotoxic activity of elemi essential oil
Ahu Soyocak 1,
*
, Ayse Ak 2, Ebru Onem 3
1Istanbul Aydın University, Faculty of Medicine, Department of Medical Biology, İstanbul, Türkiye
2Kocaeli University, Vocational School of Health Services, Department of Medical Imaging Techniques, Kocaeli,
Türkiye
3Suleyman Demirel University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Isparta,
Türkiye
Abstract: Essential oils have several biological activities such as
antimicrobial, antioxidant, proliferative, and anti-inflammatory. This study
aimed identification of bioactive compounds found in Elemi essential oil (EO)
and to determine the anti-quorum sensing and cytotoxic activities of EO. In this
study, bioactive compounds of EO were analyzed using GC-MS, and the
antibacterial activity of elemi was screened against Staphylococcus aureus
ATCC 25923, Methicillin-Resistant Staphylococcus aureus ATCC 43300,
Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853,
Pseudomonas aeruginosa PAO1. Anti-biofilm activity and pyocyanin
production on P. aeruginosa PAO1 were also investigated. The effect of EO
on cell viability was also analyzed by thiazolyl blue tetrazolium bromide
(MTT) and neutral red uptake (NR) assay in fibroblast cells. According to GC
results, the major component of EO was determined as limonene (55%). A
sub-MIC of elemi essential oil inhibited biofilm formation and pyocyanin
production by 43% and 56%, respectively. On the other hand, EO also had an
acute effect on the mitochondrial and lysosomal activities of fibroblast cell
lines. Mitochondrial and lysosomal activities were significantly decreased
when EO concentrations were applied for 24 and 48 hours (p<0.05). In
conclusion, EO has inhibitory activity on biofilm formation and pyocyanin
production, and also the lower doses of oil have no toxic effects on fibroblast
cells. However, higher doses of EO have more cytotoxic effects on
mitochondrial activity rather than the lysosomal activity of fibroblast cell lines.
It is thought that EO exhibits these activities due to the amount of limonene in
its content.
ARTICLE HISTORY
Received: Jan. 19, 2022
Revised: May 24, 2022
Accepted: July 12, 2022
KEYWORDS
Essential oil,
Elemi oil,
Anti-bacterial,
Anti-quorum,
Cellular activity
1. INTRODUCTION
The history of therapeutics usage of plants dates back to ancient times and still maintains its
popularity today. Essential oils are obtained from different parts of the plant such as resin, bark,
flower, leaf, seed, root, and woody parts, and have a wide range of uses for many years,
especially in cosmetics, medicine, food industry, aromatherapy and phytotherapy (Bhuiyan et
al., 2020; Hyldgaard et al., 2012). The main components of essential oils are mono and
*
CONTACT: Ebru Önem ebruonem@sdu.edu.tr Süleyman Demirel University, Faculty of
Pharmacy, Department of Pharmaceutical Microbiology, Isparta, Türkiye
ISSN-e: 2148-6905 / © IJSM 2022
Soyocak, Ak & Onem
259
sesquiterpenes and these components have some biological activities such as antimicrobial,
antioxidant, proliferative, and anti-inflammatory (Bilenler & Gökbulut, 2013; Prabuseenivasan
et al., 2006; Tahir et al., 2020). Numerous studies have been conducted on the antimicrobial
effects of essential oil components. In addition to the antibacterial activity of the essential oil,
anti-quorum sensing activity has also been reported in many studies (Onem et al., 2021;
Sobrinho et al., 2020). In recent years, anti-quorum sensing activity is an approach that is
thought to be effective in combating infectious diseases due to antibiotic resistance (Algburi et
al., 2017; Alva et al., 2019; Millezi et al., 2016; Roy et al., 2018). This system provides
communication between bacteria through signal molecules called autoinducers and enables
many behaviors to be exhibited (Gürağaç et al., 2022).
P. aeruginosa is an opportunistic pathogen that causes chronic infections, especially in
severe hospital infections. These diseases are caused by virulence factors whose release occurs
under the control of the quorum sensing (QS) system (Abisado et al., 2018). Inhibition of this
system is seen as a promising new approach to combating bacteria. Synthetic and natural
molecules are being researched for this purpose (John et al., 2017; Langeveld et al., 2014;
Morohoshi et al., 2007). Elemi oil (EO) which has been used in the form of an ointment as a
stomach stimulant and as an expectorant (Mogana & Wiart, 2011), is a collective term used for
oleoresin obtained from the bark of the Canarium luzonicum (Blume) A Gray (Nikolic et al.,
2016). C. luzonicum is commonly known in the Philippines as 'pisa' and 'basiad' and is used for
its oleoresin (known locally as 'sahing') that flows from the stem. When processed, it is called
'brea blanca' (white pitch) and exported as Manila elemi. Anti-bacterial, anti-fungal, and
hepatoprotective activities of Canarium L. species are generally known and it is demonstrated
that Canarium patentinervium Miq. extracts had antitumor activity in cancer cells lines
(Mogana & Wiart, 2011). In a study conducted with Canarium album Raeusch, it was found
that scopoletin and isocorilagin, which is one of its phytochemical components, showed strong
inhibition on influenza A and their IC50 values were 22.9 ± 3.7 and 5.42 ± 0.97 μg/ml,
respectively (Yang et al., 2018).
In this study, activity of EO obtained from C. luzonicum was examined on Gram-positive
and Gram-negative bacteria. Biofilm inhibitory effect and pyocyanin production of P.
aeruginosa PAO1 were also investigated. On the other hand, its effect on the cellular activity
of normal fibroblast cells was examined with thiazolyl blue tetrazolium bromide (MTT) and
neutral red uptake (NR). MTT and NR assays are the most commonly used for the detection of
cytotoxicity or cell viability following exposure to toxic substances (Fotakis & Timbrell, 2006).
The reduction of tetrazolium salts as MTT enables them to transform into a structure called
formazan and brings about a color change (Tokur & Aksoy, 2017). The tetrazolium ring can
only be broken by active mitochondria so that only living cells can produce the purple color
(Mosmann, 1983; Perez et al., 2017). On the other hand, dead cells lose their ability to reduce
tetrazolium compounds and do not cause any color change (Riss & Moravec, 2006). NR is a
compound that interacts with the lysosomes of living and uninjured cells. Both assays are based
on different physiological endpoints (Borenfreund et al., 1988). In this study, the effect of EO
on L929 cell viability was evaluated in a controlled manner through these assays that measure
cytotoxicity by two different pathways. In the literature review, no similar studies were found
on the anti-quorum sensing activity in PAO1 and cytotoxicity of elemi oil.
2. MATERIAL and METHODS
2.1. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
Compounds of EO (commercially available) were analyzed by Shimadzu GC-MS QP 5050
(Kyoto, Japan) gas chromatograph-mass spectrometer system and the results of the analysis are
shown in Table 1.
Int. J. Sec. Metabolite, Vol. 9, No. 3, (2022) pp. 258-267
260
Table 1. GC/MS analysis conditions.
Column
Cp WAX 52 CB capillary column
(50 m x 0.32 mm ID, df:1.2 µm)
Carrier gas
Helium (99.999%)
Flow rate
10 p.s.i.
Injection volume
1 µL
Oven temperature
60°C
raised 220°C’at 2°C/min -220ºC 20 min
Injection block
240ºC-250ºC
2.2. Anti-bacterial activity
The anti-bacterial effect of EO on Staphylococcus aureus ATCC 25923, Methicillin-Resistant
Staphylococcus aureus (MRSA) ATCC 43300, Escherichia coli ATCC 25922, P. aeruginosa
ATCC 27853 was evaluated and P. aeruginosa PAO1 strains were tested using the agar well
method (Holder & Boyce, 1994) and microdilution method was used to determine the minimum
inhibitory concentration (MIC) values. According to the method, 100 μL of EO was added to
96-well microplates containing 100 μL of medium and serial dilutions were made in two folds,
respectively. The microplates were incubated overnight at 37 °C by adding the bacteria
suspension prepared according to 0.5 McFarland (108/mL). Following the incubation,
microplates were evaluated and the lowest concentration without growth was determined as the
minimum inhibitory concentration (MIC).
2.3. Pyocyanin Assay
Pyocyanin assay was carried out as described before (Essar et al., 1990). PAO1 culture was
incubated overnight in Luria Bertani Broth. Pyocyanin was extracted from the culture with 3
mL chloroform and separated organic phase fixed with 1mL of 0.2 N HCl. The absorbance of
the solution was measured at 520 nm.
2.4. Biofilm Formation Assay
Biofilm formation was conducted by crystal violet (CV) assay (O’Toole, 2011). A volume of
20 µL of EO, 180 µL of medium, and 10 µL of PAO1 culture were added to each well of 96-
well microplate. After 48 hours of incubation, the plate was poured and washed 3-5 times with
distilled water. Then the crystal violet at 0.1% concentration is added to the wells for 30
minutes. The plate was again washed 3-5 times with distilled water. After the washing process
200 µL of 95% ethanol was added to each well and after 15 minutes resolving CV read at 570
nm (Biotek-Epoch 2-Microplate Spectrophotometer).
2.5. Cell Culture and Cellular Activity
Mouse fibroblast cells (L929) were maintained in DMEM medium with 10% Fetal Bovine
Serum. Cells were trypsinized with 0.05% Trypsin/EDTA solution and cells (2x104) were
seeded in a 96-well plate and cultured for 24 hours. After 24 hours of incubation, EO
concentrations were applied to the each well. EO was dissolved in dimethylsulphoxide (DMSO)
and diluted in complete DMEM to 630, 420, 210, and 105 μg/mL concentrations. The final
DMSO concentration was below 1%. Concentrations of EO were removed after 24 and 48 h,
and 100 µL of MTT in 5 mg/mL was added to the wells. The formazan crystals after 3 hours of
incubation were dissolved with 100 µL DMSO and absorbances at 570 nm were measured by
a microtiter plate reader spectrophotometer (Multiskan GO-Thermo).
After EO application for 24 and 48 h, 100 μL neutral red medium was added, and the plate
was washed after 3 h incubation at 37 °C. The dye was extracted with 100 μL acidified ethanol
solution (Repetto et al., 2008). The optical density (OD) of neutral red extract at 540 nm was
measured in a microtiter plate reader spectrophotometer (Multiskan GO-Thermo).
Soyocak, Ak & Onem
261
2.6. Statistical Analysis
The data obtained from the study were evaluated with IBM SPSS 21 package program. The
compliance of the data to normal distribution was determined by the Shapiro-Wilk test. Group
comparisons were made using the independent sample tests one-way ANOVA. Results are
given as mean ± standard deviation. The statistical significance level was accepted as p <0.05.
3. RESULTS
3.1 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The compounds of EO determined by GC-FID and GC/MS techniques are shown in Table 2,
and 38 compounds were determined in different percentages. It was seen that the highest value
belongs to limonene with 55.88%; followed by elemol with 17.54%, phellandren alfa with
10.96%, and beta phellandrene with 4.51% rate.
Table 2. GC–MS analysis of extracts % major components and their retention times.
No
Name of compound
rt (min)
%
1
alpha pinene
6.689
0.55
2
beta phellandrene
8.112
4.51
3
pinene beta
8.314
0.20
4
beta myrcene
8.758
0.90
5
phellandrene alpha
9.544
10.96
6
delta 3-carene
9.636
0.05
7
alpha terpinene
10.002
0.25
8
p-cymene
10.386
2.84
9
limonene
10.781
55.88
10
cis-ocimene
10.929
0.20
11
beta ocimene y
11.432
0.20
12
gamma terpinene
12.023
0.19
13
trans sabinene hydrate
12.669
0.05
14
alpha terpinolene
13.481
1.38
15
1 -methyl-4-isopropenylbenzene
13.697
0.10
16
linalool
14.316
0.04
17
*
15.697
0.04
18
cis p-mentha 2,8-dien 1 ol
16.424
0.04
19
*
16.788
0.04
20
4-terpineol
19.098
0.48
21
cis p-mentha-1 8-dien-2-ol
19.335
0.01
22
dmbca
19.513
0.26
23
beta fenchyl alcohol
20.055
1.72
24
a-phellandrene epoxide
20.536
0.26
25
trans-carveol
21.594
0.04
26
cis-sabinol
22.930
0.05
27
d-carvone
23.126
0.06
28
piperitone
23.783
0.08
29
alpha cubebene
29.910
0.03
30
copaene alpha
31.700
0.15
31
beta elemene
32.655
0.11
32
methyleugenol
33.451
0.37
33
caryophyllene
34.432
0.19
34
alpha humulene
36.675
0.10
35
spathulenol
37.663
0.03
36
germacrene-d
38.310
0.08
37
alpha muurolene
39.535
0.02
38
elemol
42.726
17.54
Total
100
*Unknown, rt: retention time
Int. J. Sec. Metabolite, Vol. 9, No. 3, (2022) pp. 258-267
262
3.2. Antibacterial Activity and Minimum Inhibitory Concentration
According to agar well diffusion results, different inhibition zone has detected at the test
concentration, which was statistically significant compared to a positive control (gentamicin 40
μg/disc) (p<0.001) (see Table 3). The lowest MIC value was determined against P. aeruginosa
(MIC = 2.7 μg/mL) and MIC values of elemi were in the range of 2.7 μg/mL to 21.85 μg/mL
(MRSA 5.4 μg/mL; S. aureus 21.85 μg/mL; E. coli 5.4 μg/mL).
Table 3. Zone diameter of Elemi on strains.
S. aureus
ATCC 25923
MRSA
ATCC 43300
E. coli
ATCC 25922
P. aeruginosa
ATCC 27853
P. aeruginosa
PAO1
Control
(Gentamicin)
15.00±0.00 ns
15.00±0.00 ns
14.00±0.00 a*
16.00±0.00 a*
16.00±0.00 a**
Elemi
14.67±1.53
18.00±2.00
11.00±1.00 b
10.33±1.15 b
9.33±0.58 b
*The differences within columns signed with different letters are significant (p<0.05); **The differences within
columns signed with different letters are significant (p<0.01); SD ± mean
3.3. Pyocyanin and Biofilm Inhibition
The discovery of bacterial communication and especially the knowledge that some
microorganisms cause diseases by using this QS system has led to an increase in studies on the
inhibition of the system (Banu & Mary, 2016).
EO was tested for the inhibition effect of pyocyanin production and biofilm formation which
were QS-related virulence factors in P. aeruginosa PAO1. The sub-MIC concentration of EO
inhibited pyocyanin production by 56%. (p<0.01) (Figure 1).
Figure 1. Inhibition effect of EO on pyocyanin production and biofilm formation in PAO1.
**The differences signed different letters are significant (p<0.01); SD ± mean
3.4. Effects of Elemi oil on Cellular Activity of Fibroblast Cells
As seen in Figure 2a, the mitochondrial activities of the cells decreased significantly compared
to the control at the end of 24 hours at all the concentrations applied (p<0.05). The concentration
leading the greatest reduction was 630 μg/mL. Concentrations of 210 and 105 μg/mL caused
inhibition of mitochondrial activity, similarly. When the effects of the concentrations applied
at the end of 24 and 48 hours on the lysosomal activities of the cells were examined, it was
determined that the 630 and 420 μg/mL had similar effects, and also the 210 and 105 μg/mL
doses had similar effects. All concentrations suppressed lysosomal activity relative to control
(p<0.05) (Figures 2b and 2d). In Figure 2c, all concentrations caused suppression of
mitochondrial activities of cells compared to control (p<0.05), while doses of 420 and 210
μg/mL had similar effects in manner time-dependent. With the longer exposure to
concentrations, all of them exhibited more inhibitory activity.
Soyocak, Ak & Onem
263
Figure 2. Effects of EO concentrations on mitochondrial (a and c) and lysosomal activity (b and d) of
L929 cell lines for 24 and 48 hours. Different letters show statistically significant differences between
groups.
4. DISCUSSION and CONCLUSION
The use of plants in health has a history of many years. They are used in different forms such
as extraction, boiling, and obtaining essential oil. Essential oils are obtained from different parts
of plants and are known to have therapeutic effects. In this study, we evaluated the antibacterial,
antibiofilm, and cytotoxic activities of EO. In addition to these, the phytochemicals have tried
to be determined. The results showed that the major component of the Elemi is limonene
(55.88%) and the similar studies with elemi, limonene is found major component but it was
observed at 36.40 % and 36.38 % rates (Galovičová et al., 2020; Kačániová et al., 2020). The
composition of essential oils may differ depending on the region where they are grown, the time
of harvest, and the procedure used to extract the oils (Nannapaneni et al., 2009; Paibon et al.,
2011).
Major components of essential oils are in the group of hydrocarbons and refer to the terpenes
consisting only of carbon and hydrogen. Limonene is also one of the monoterpenes included in
hydrocarbons. In a study conducted with limonene, α-pinene, β-pinene, p-cymene, it was
observed that the antibacterial effects of these monoterpenes were at different rates
(Koutsoudaki et al., 2005). This can be explained by the fact that the contents of essential oils
have a synergistic effect and thus show more antibacterial properties (Hyldgaard et al., 2012).
The antibacterial activity of essential oils has been linked to their hydrophobicity. This feature
permits the EO to enter the bacterial cell membrane, damaging it and making it more permeable
(Dănilă et al., 2018). In a similar study, the essential oil extracted from the resin of Canarium
strictum Roxb. showed antimicrobial activity against S. aureus, P. aeruginosa, E. coli,
Klebsiella pneumonia. The most effective results were seen on MRSA and MDR E. coli strains
with >0.66 mg/mL MIC value (Tahir et al., 2020).
Pyocyanin is one of the important virulence factors produced by Pseudomonas for the
control of the QS system. Another factor that plays an important role in virulence, biofilm, is
the 3-dimensional structure that bacteria build by exhibiting coordinated behavior (Millezi et
al., 2016). The biofilm formation is important for the treatment of infectious diseases because
more than 60% of persistent and chronic infections are known to involve biofilm and it is more
Int. J. Sec. Metabolite, Vol. 9, No. 3, (2022) pp. 258-267
264
difficult to treat with antibiotics than planktonic form (Ceylan et al., 2014; Lewis, 2001). EO
reduced biofilm formation 43% rate (p<0.01) (Figure 1) and no similar research on
Pseudomonas was found in the literature review.
In this study, it was determined that cytotoxic activities of EO on L929 cells. Similar activity
was observed in HT-29 epithelial cells exposed to increasing concentrations of essential oils
which include EO (25–200 g/mL) and it was determined that EO exhibited strong cytotoxicity
on the mitochondrial activity of cells (Senthil Kumar et al., 2020). According to GC-MS
analysis, it was determined that limonene was the most abundant compound (%55.88) in EO.
Since limonene is the major constituent of EO, limonene inevitably contributes to EO’s
cytotoxicity. In human neuroblastoma cells, limonene caused cytotoxicity and mitochondrial
damage in a previous study (Russo et al., 2013). Limonene also reduced cell viability and
triggered mitochondrial-dependent apoptosis in human colon cancer cells LS174T in a dose-
dependent manner (ranging from 0.4 to 3.2 mol/L) (Mukhtar et al., 2018; Vieira et al., 2018).
Limonene extracted from Citrus sinensis also decreased cell viabilities of human
adenocarcinoma (SW480 and HT-29) cells (Murthy et al., 2012). Limonene also exhibited
cytotoxic activity by inducing the autophagy-lysosomal pathway (Russo et al., 2014; Yu et al.,
2018). In this study, it was seen that EO had a more suppressive effect on mitochondrial activity
than lysosomal activity in fibroblast cells.
In conclusion, EO has inhibitory activity on biofilm formation and pyocyanin production,
and also lower doses of oil have no toxic effects on fibroblast cells. However, higher doses of
EO have more cytotoxic effects on mitochondrial activity rather than lysosomal activity of
fibroblast cell lines. It is thought that EO exhibits these activities due to the excessive amount
of limonene in its content. More research is needed to determine the efficacy of EO in vivo for
dermatological applications.
Declaration of Conflicting Interests and Ethics
The authors declare no conflict of interest. This research study complies with research and
publishing ethics. The scientific and legal responsibility for manuscripts published in IJSM
belongs to the authors.
Authorship contribution statement
Ahu Soyocak, Ebru Onem: Investigation, Resources, Visualization, Software, Formal
Analysis, and Writing -original draft. Ayse Ak: Investigation, Writing, Methodology,
Supervision and Validation.
Orcid
Ahu Soyocak https://orcid.org/0000-0003-0999-2774
Ayse Ak https://orcid.org/0000-0002-1385-5847
Ebru Onem https://orcid.org/0000-0002-7770-7958
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