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CHEMICAL ENGINEERING TRANSACTIONS
VOL. 56, 2017
A publication of
The Italian Association
of Chemical Engineering
Online at www.aidic.it/cet
Guest Editors: Jiří Jaromír Klemeš, Peng Yen Liew, Wai Shin Ho, Jeng Shiun Lim
Copyright © 2017, AIDIC Servizi S.r.l.,
ISBN 978-88-95608-47-1; ISSN 2283-9216
Synergistic Benefit of Eugenia Caryophyllata L. and
Cinnamomum Zeylanicum Blume Essential Oils against Oral
Pathogenic Bacteria
Siti Nurazwa Zainola, Shahida Mohd Saidb, Zamirah Zainal Abidinb, Nuramirah
Azizand, Fadzilah Adibah Abdul Majid*,c, Ibrahim Jantand
aDepartment of Bioprocess Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310
Johor Bahru, Malaysia
bFaculty of Dentistry, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia
cInstitute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia
dFaculty of Pharmacy, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia
f.adibah@umt.edu.my
Eugenia caryophyllata L. and Cinnamomum zeylanicum Blume essential oils offer great potential against the
pathogens of oral cavity. Both of these essential oils have recently being used as antibiotics and antiviral agents.
This study investigates the synergistic effect of E. caryophyllata and C. zeylanicum oils against known resistant
bacteria in oral infection namely Enterococcus faecalis, Aggregatibacter actinomycetemcomitans,
Streptococcus mutan and Streptococcus salivarius. The combination effect E. caryophyllata and C. zeylanicum
were evaluated using checkerboard assays. Amoxicillin at concentration 0.1 mg/mL was used as positive control
while each bacteria suspension (105 CFU/mL) was used as negative control. The minimum inhibitory
concentration (MIC) and fractional inhibitory concentration index (FIC) were calculated to characterised
interaction between the combinations. Both E. caryophyllata and C. zeylanicum oils possess antimicrobial
activity against all the four bacteria when used on its own or in combination. In combination, the MIC values
were reduced for all bacteria compared when in single form. The synergistic antibacterial effect was significant
toward E. faecalis, A. actinomycetemcomitans and S. mutans with FIC index is 0.5 and below. Partial synergy
was produced in S.salivarus. The acceptable range for synergistic effect is between 0.16 mg/mL and 0.31
mg/mL. Above from 0.63 mg/mL are considered as additive and antagonistic effect. The combinations of these
essential oils gave a stronger antibacterial activity and directly can replace the common use of antibiotics like
Amoxicillin. This finding suggests a potential therapeutic benefit using the combination of E. caryophyllata and
C. zeylanicum oils in the future management of oral infection.
1. Introduction
Oral disease including tooth decay and periodontal disease is a major health problem especially in the
developing countries and about 60 - 90 % of children and adults are affected (Petersen, 2004). Although there
are some successful conventional clinical approaches, herbal medicine is also popularly used as preventive
therapy. Essential oils has been used especially as mouth rinse either traditionally or clinically to treat oral
diseases including periodontal disease (Moran, 2008). Natural antimicrobial agents has been used in recent
years as a strategies to control the resistant food-borne bacteria and other pathogenic microorganism (Yap et
al., 2014). Interaction between essential oils may lead to synergistic, additive, indifferent (non-interaction) or
antagonistic effect. Synergistic effect is observed when the effect of combined substances is greater than the
sum of the individual effects. An additive is when the combined effect is equal to the sum of the individual effects
while the absence of interaction is defined as indifference. Antagonistic is defined when the effect of one or both
substances is less when they applied together than when individually applied (Bassolé and Juliani, 2012).
Clove (Eugenia caryophllata L.) oil has a pleasant aroma and is useful as an essential food flavour,
pharmaceutical and dentistry. It has been used for a long time by dentists as its main active ingredient, eugenol,
DOI: 10.3303/CET1756239
Please cite this article as: Zainol S.N., Said S.M., Abidin Z.Z., Azizan N., Majid F.A.A., Jantan I., 2017, Synergistic benefit of eugenia
caryophyllata l. and cinnamomum zeylanicum blume essential oils against oral pathogenic bacteria, Chemical Engineering Transactions, 56,
1429-1434 DOI:10.3303/CET1756239
1429
is primarily responsible either as a bacteriocidal or bacteriostatic agent to fight dental infection on teeth (Bankar
et al., 2012). It is also used traditionally in many cultures for relieving toothache, sore gums and oral ulcers
beside as a dressing in dentistry for minor wounds (Aneja and Joshi, 2010).
Cinnamon (Cinnamomum zeylanicum Blume) oil on the other hand, has been reported to have high antioxidant
activity and antimicrobial properties (Li et al., 2013;). It is also been used in toothpastes, mouthwash or chewing
gum for dental caries prevention (Pandita et al., 2014).
While the antimicrobial of E. caryophyllata and C. zeylanicum oils has been intensively studied only a few studies
have been reported on antimicrobial effect of combined application of E. caryophyllata and C. zeylanicum oils.
However, there are only few studies demonstrated the activity of combined E. caryophyllata or C. zeylanicum
oils with other essential oils. For example, the combination of E. caryophyllata with rosemary oils (Fu et al.,
2007), C. zeylanicum with Thyme or clove oil (Lu et al., 2011) and C. zeylanicum with Lavandula angustifolis
oils (de Rapper et al., 2013). All of these studies shown antimicrobial effect on Gram-positive and Gram-negative
strains. Moreover, they also limited information on the scientific data of essential oils including Eugenia
caryphyllata L. and Cinnamomum zeylanicum Blume oils on oral pathogens. Thus, synergistic effect of Eugenia
caryphyllata L. and Cinnamomum zeylanicum Blume oils is carried out as an alternative treatment for potential
treatment of oral infections.
2. Methodology
2.1 Essential oils and chemicals
Eugenia Caryophyllata L. and Cinnamomum Zeylanicum Blume essential oils were provided by Department of
Periodontology, Faculty of Dentistry, Universiti Kebangsaan Malaysia (UKM). Dimenthysulfoxide (DMSO,
100 %) (Sigma), ethanol (75 %) (Merck, Germany)
2.2 Bacteria cell and culture media
All bacteria strains used were provided by Department of Periodontology, Faculty of Dentistry, Universiti
Kebangsaan Malaysia (UKM) namely Enterococcus faecalis (ATCC® 29212TM), Streptococcus mutans (ATCC®
25175TM), Streptococcus salivarius (ATCC® 13419TM) and Aggregatibacter actinomycetemcomitans (ATCC®
29522TM). These are anaerobic and facultative anaerobic bacterial commonly found in periodontal tissue and
associate with periodontal disease. Brain Heart Infusion (BHI) agar and broth was used as a media culture. Mitis
salivarius (MS) agar was used to maintain the growth of streptococci spp. and tryptic soy-serum-bacitracin-
vancomycin (TSBV) agar was prepared for A. actinomycetemcomitans culture.
2.3 Antibacterial assay
2.3.1 Minimal inhibitory concentration (MIC)
The microdilution minimum inhibitory concentration assay was carried out as described by de Rapper et al.
(2013) with slight modifications. This method was used to determine the lowest concentration that inhibit the
bacteria and used as references to calculate fractional inhibition concentration (FIC) index. The E. caryophyllata
and C. zeylanicum oils were diluted to a concentration 250 mg/mL using dimethylsulfoxide (DMSO) as a diluent.
Both essential oils were serially diluted to concentrations 1.25 mg/mL, 0.62 mg/mL,
0.31 mg/mL, 0.15 mg/mL, 0.078 mg/mL, 0.039 mg/mL, 0.019 mg/mL and 0.00 mg/mL. The volume of essential
oils was added at a same volume 50 : 50 µL for combination test and dispended in 96-microtiter plate. The
selected bacteria suspension was prepared by diluting it in broth media culture to get an approximate
concentration of 1 × 105 colony forming units (CFU)/mL. Bacteria suspensions were added to all well of the
microtiter plates at a volume of 100 µL. Then, the microtiter plates were sealed with parafilm, to prevent any
loss of essential oils due to their inherent volatility. The plates were incubated at 37 °C in anaerobic environment
for 24 h. The reading of absorbance was taken using a plate reader at wavelength 590 nm. The negative control
used was bacteria suspension in broth only while the positive control was Amoxicillin (0.1 mg/mL). All assays
were done in triplicates in three independent experiment.
The percentage of bacterial inhibition was calculated using Eq(1):
% inhibition of concentration
= [(Mean OD590 control well – Mean OD590 sample well)/ Mean OD590 control well] × 100 %
(1)
where Control well = bacteria suspension in broth medium only , and Sample well = concentration of essential
oils combination in broth medium
1430
2.4 Fractional inhibition concentration (FIC)
The Fractional inhibition concentration was used to determine the interaction between the oils by calculating the
fractional inhibitory concentration index (∑FIC) (Sadiki et al., 2014) using the following formula:
(2)
(3)
(4)
∑FICI values interpreted as follows:
≤ 0.5 = synergistic;
0.5 - 0.75 = partial synergy;
0.76 - 1.0 = additive;
> 1.0 - 4.0 = non-interactive;
> 4.0 = antagonistic
2.5 Gas chromatography test
The sample preparation was done by diluted 250 µL of each essential oils in 250 µL ethyl acetate and 1.0 µL
was injected into the column. Shimadzu GC2000 gas chromatography equipped with column DB-5 (1 µm
thickness, 30.0 m length, 0.25 mm diameter), was used for the analysis of oils. Injector and flame ionisation
detector (FID) temperature were maintained at 250 °C. Nitrogen was the carrier gas used with a flow rate of 1.0
mL/min. Initial temperature of the oven was programmed at 75 °C for the initial 10 min, then 3 °C /min to 230 °C
/min for 5 min. Total program time was 73.33 min (Ahmad et al., 2010).
3. Result and discussion
The synergistic effect of E. caryophyllata and C. zeylanicum oils was shown in Table 1. In combination, the MIC
values were reduced for all bacteria and showed synergistic effect, defined FICI as ≤0.5. This was shown when
the combination oils interacted with E. faecalis, A. actinomycetemcomitans and S. mutans. However, only partial
synergy effect was shown for S. salivarius, E. faecalis and S. mutans with the FICI values 0.38 and 0.31. The
finding from our study was similar to other reported studies where E. caryophyllata and C. zeylanicum oils was
found gave a greater inhibition of some Gram positive and Gram negative bacteria when tested alone (Hussein
et al., 2014). We also found similar finding which showed E. caryophyllata having lesser antibacterial activity on
S. mutans than other species (Cai and Wu, 1996). Meanwhile, a different finding was seen with C. zeylanicum,
where S. mutans were observed to be the most sensitive at a lower MIC value of 12.8 µg/mL (Kohanteb and
Fani, 2011). When in combinations, the synergy effects were achieved against S. mutans in equal volume ratios
even though a predominant non-interactive effect was noted. Here, S. salivarius antibacterial activity was less
sensitive towards combination of E. caryophyllata and C.zeylanicum oils.
Table 1: Synergistic effect of E. caryophyllata and C. zeylanicum oils against 4 selected bacteria
Strains
Essential Oils
MIC (mg/mL)
FIC
FICI
Outcome
Alone
Combination
E. faecalis
E. caryophyllata
1.25
0.16
0.13
0.38
Synergistic
C. zeylanicum
1.25
0.31
0.25
A. actinomycetemcomitans
E. caryophyllata
1.25
0.31
0.25
0.5
Synergistic
C. zeylanicum
0.63
0.16
0.25
S. mutans
E. caryophyllata
1.25
0.08
0.06
0.31
Synergistic
C. zeylanicum
0.63
0.16
0.25
S.salivarius
E. caryophyllata
1.25
0.63
0.5
0.75
Partial
synergy
C. zeylanicum
1.25
0.31
0.25
Figure 1 showed a summary of synergistic study for E. caryophyllata and C. zeylanicum oils, the acceptable
range concentration when to do a further study in term of product development. The synergistic range was
between 0.16 mg/mL and 0.31 mg/mL. The partial synergy was produced when the concentration of the oils in
between 0.31 mg/mL and 0.63 mg/mL. Beyond this outcome, results were recorded as additive or antagonistic
effects. The antibacterial activity of this combination was good as antibiotics, like Amoxicillin. The antibacterial
activity of the Amoxicillin was reported when the concentration at 0.1 mg/mL.
1431
Essential oils have been used for many years to treat human health from infectious diseases and against
notorious pathogens. More recently, there are increasing evidence indicating that essential oils and plant
extracts are useful for specific medical treatment. The antibacterial activity of E. caryophyllata and C. zeylanicum
have been reported, but in the present study tested on combination of these two essential oils have not reported
yet especially on oral health perspective. Previous study reported that E. caryophllata and C. zeylanicum
exhibited strong antibacterial activity against for both gram-positive and gram-negative bacteria (Hussein et al.,
2014). The investigation on the essential oils is necessary to improve the quality of healthcare because there
are potentially useful source for antimicrobial compounds, especially against bacterial pathogens
(Prabuseenivasan et al., 2006).
Our finding has the potential to assist researchers and clinicians to explore further the use of herbal oils as
alternative to the commonly used antibiotics. Antibiotics are commonly prescribed in dental practice for oral
infections and the total percentage of global antibiotic costs was 75 % (Roda et al., 2007). It has been reported
that there are about 5 % of hypersensitivity and diarrhoea occurred when patients were prescribed with
amoxicillin (Jorger and Miriam, 2002). The combinations of these essential oils gave stronger antibacterial effect
and may be beneficial to replace common use of antibiotics like Amoxicillin in the clinic.
Figure 1: Summary for synergistic study for E. caryophyllata (Ec) and C. zeylanicum (Cz) oils
It is believed the chemical composition of the E. caryophyllata L. and C. zeylanicum Blume oils contributed to
the anti-microbial properties and been evaluated. About 42 and 24 compounds of the oils were identified with
the aid of gas chromatography (GC) and literature comparison. In this study, (Z)- Methyl cinnamate was detected
as major compound in C. zeylanicum Blume oils with amount 38.57 % followed by (E)-cinnamaldehyde (16.71
%). The other constituents are bornyl acetate (6.33 %), methyl nonanate (1.25 %), benzaldehyde (1.29 %),
benzene acetaldehyde (1.48 %), borneol (0.95 %), 1, 8-cineole (0.19 %),
(E)-isoeugenol (0.41 %) and (Z)-cinnamyl acetate (0.059 %). As reported from previous study,
(E)-cinnamaldehyde was found as major compounds with the percentage amount 68.95 % (Unlu et al., 2010)
and 97.7 % (Singh et al., 2007). However, in this study the percentage amount (E)-cinnamaldehyde detected is
low. This is because cinnamaldehyde was easily oxidised and has high volatility properties (Gunkel et al., 2011).
The composition in C. zeylanicum oil shows the noticeable amount of aromatic components.
The major components of E. caryophyllata L. oil was isobornyl propanoate (53.15 %), (E)-isoeugenol
(11.39 %), γ-muurolene (1.26 %), (E )-γ bisbolene (1.35 %), cis-beta-elemenone (2.99 %), dihydro-eugenol
acetate (0.60 %), (Z)-isoeugenol (0.25 %), (E )-methyl isoeugenol (0.19 %), (E )-caryophyllene (0.09 %) and
eugenol (0.07 %). In contrast, Nassar et al. (2007) identified eugenol (71.56 %) and eugenol (8.99 %) acetate
as a major volatile constituents in E. caryophyllata oil with the same approaches. Based on literature, eugenol
was claimed as a major compound in E. caryophyllata oil but in this study, eugenol is a minor compound. Since
E. caryophyllata oil is volatile oil, this could be a one of the reason the decline of eugenol compounds during
analysis. The characteristic of volatile oils is easily to oxidise when the oils exposed to light and air. The oils
also will evaporate when subjected to heat.
4. Conclusions
E. carypohyllata L. and C. zeylanicum Blume oils when used in combination showed a potential benefit in
inhibiting oral infection involving E. faecalis, A.actinmyocetemcomitans, S.mutans and S.salivarius from the oral
cavity. These essential oils combination at suitable concentration could be an alternative to use of synthetic
antimicrobial agents and may lead a new research on natural products in future management of oral infection.
Cz 0.16 mg/mL
Ec 0.08 mg/mL
Cz 0.31 mg/mL
Ec 0.63 mg/mL
Synergistic range
Partial synergy
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Acknowledgements
This study was supported by Young Investigator Initiative Grant (GGPM-2014-020) from UKM, UTM-Research
University Grant (RUG) (03H95) and Ministry of Agriculture Grant (MOA-NRGS) (4H016).
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