Journal of Science and Arts Year 19, No. 3(48), pp. 715-722, 2019 [611271]

Journal of Science and Arts Year 19, No. 3(48), pp. 715-722, 2019
ISSN: 1844 – 9581 Chemistry Section ORIGINAL PAPER
ANTIOXID ANT AND ANTIBACTERIAL ACTIVITY OF
CYMBOPOGON CITRATUS AND SYZYGIUM AROMATICUM
ESSENTIAL OILS ALONE AND IN COMBINATION
MARICICA STOICA1, CRISTIAN DIMA2*, GIGI COMAN2, PETRU ALEXE2,
ANDRA NEAGOIE2
_________________________________________________
Manuscript received: 19.07.2019; Accepted paper: 06.08.2019;
Published online: 30.09.2019.

Abstract. Essential oils are now getting more importance in food industry , being used
to prevent food spoilage and to promote food preservation. The present study was carried out
so as to evaluate the antioxidant and antimicrobial effect of the essential oils of two aromatic
plants , namely Cymbopogon citratus (Lemongrass) and Syzygium aromaticum (Clov e), and to
investigate a possible synergistic effect of the combination of these oils. The evaluation was
performed for individual oils and the combination of the two oils in 1:1, 2:1 and 1:2 ratios,
against two bacterial pathogens , namely Escherichia coli and Staphylococcus aureus. The
study showed promising results for the use of Cymbopogon citratus and Syzygium
aromaticum oils, as antioxidant (individual ly and in combination) and antimicrobial
(individual) agents. Cymbopogon citra tus and Syzygium aromaticum oils are good candidat: [anonimizat]: Cymbopogon citratus essential oil, Syzygium aromaticum essential oil,
antioxidant activity, antibacterial activit y

1. INTRODUCTION

The International Organization for Standardization (ISO) defines essential oil as a
product obtained from a natural raw material of plant origin, obtained by steam distillation, by
mechanical processes from the epicarp of citrus fruits, or by dry distillat ion, after the
separation of the aqueous phase [ 1]. Essential oils (EOs) consist of lipophilic and highly
volatile secondary plant metabolites, reaching a mass below a molecular weight of 300, which
can be physically separated from other plant components o r membranous tissues [2]. EOs are
considered a green alternative in the nutritional, pharmaceutical, and agricultural fields due to
their antimicrobial (antibacterial, antifungal, and antiviral), insecticidal, and antioxidant
properties [2, 3 ]. Nowadays, EOs have acquired great popularity over the years as consumers
have developed a growing awareness toward the use of natural ingredients, especially in foods
and cosmetics [3]. EOs are natural mixtures consist ing of about 20 -60 components at
extremely different concentrations, some components ( e.g. terpenes, terpenoids) being present
at fairly high concentrations (20 -70), while other components are present in trace amounts [4].
The compon ents at high concentrations like terpenes and terpenoids possess a major role in

1 Dunarea de Jos University of Galati, Cross -Border Faculty, 800008 Galati, Romania .
E-mail: [anonimizat] .
2 Dunarea de Jos University of Galati , Faculty of Food Science and Engineering , 800001 Galati, Romania.
E-mail: [anonimizat] ; [anonimizat] ; [anonimizat] ;
*Corresponding author: cristian.dima@ugal.ro .

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the antimicrobial and biological effect of EOs, promoting food preservation, and alternatives
to treat ing infectious diseases [4]. Cymbopogon citratus (lemongrass) is a perennial plant that
grows spontaneously around the world, mainly in the tropical and savannah regions.
Cymbopogon citratus essential oil is used in the food industry, fragrances, cosmetics and
pharmaceuticals , and has a strong antibacterial activity against numerous bacteria such as
Acinetobacter baumanii, Aeromonas veronii, Enterococcus faecalis, Escherichia coli,
Klebsiella pneumonia, Salmonella enterica serotip typhimurium, Serratia marcesens, Proteus
vulgaris, Enterobacter aerogenes, Corynebacteri um equii, Staphylococcus aureus, and
Candida albicans [5-7]. Syzygium aromaticum (clove) is one of the most valuable spices that
has been used for centuries as food preservative and for many medicinal purposes. This plant
is one of the richest source s of phenolic compounds like eugenol, eugenol acetate and gallic
acid, and possesses a great potential for pharmaceutical, cosmetic, food and agricultural
applications [8]. Syzygium aromaticum essential oil inhibits many microbes including
Lactobacillus sp., Bacillus thermoacidurans , Salmonella sp., Corynebacterium michiganense ,
Pseudomonas striafaciens , Clostridium botulinum , Alternaria sp., Aspergillus sp.,
Canninghamella sp., Fusarium sp., Mucor sp., and Penicillium sp. [9-11]. Most of the
antimicrobial act ivity of EOs is found in the oxygenated compounds ( e.g. alcohols, phenolic
terpenes), while some hydrocarbons also have an timicrobial effects [12 -21]. The interactions
between these components may lead to antago nistic or synergistic effects [12 -17]. As a r ule,
combinations, either single EO s or combinations of purified primary components, influence
many biochemical processes in the bacteria, producing a plethora of interactive antibacterial
effects [12]. In recent years, there has been an increased interest in the use of natural
antimicrobials from EOs, and the use of EOs combinations is an important strategy in
control ling food-borne bacteria and other pathogenic microorganisms [12]. In EOs
combination s, the interaction between antimicrobials can result in three different outcomes
i.e., synergistic ( the antibacterial activity greater than the sum of the antibacterial activity of
individual oil), additive ( the antibacterial activity is equal to the sum of the individual
compounds of individual oil), or antagonistic (a decreased antimicrobial activity of the
combination as compared to their individual antimicrobial activity). The aim of this study is
to evaluate the antioxidant and antimicrobial activities of Cymbopogon citratus and Syzygium
aromaticum EOs, and to investigate a possible synergistic effect of the mixture s of these oils.

2. MATERIALS AND METHOD S

2.1. MATERIALS

EOs. The EOs used in this s tudy were commercial samples of Cymbopogon citratus
and Syzygium aromaticum . The Cymbopogon citratus EO was obtained from Herbavit
(Romania) , while the Syzygium aromaticum EO was obtained from Fares (Romania). Bot h
EOs were manufactured by hydro distillation.
Reagents. 2.2 diphenyl -2-picrylhydrazyl (DPPH), Folin -Ciocalteu, gallic acid, sodium
nitrite, and sodium molybdate were supplied by Sigma -Aldrich (Germany). The aqueous
solutions were prepared with ultra -pure water. All reagents were of analytical grade and
highest purity available.
Bacteria and culture media. The EOs and their combination were tested on the
Escherichia coli and Staphylococcus aureus. Escherichia coli and Staphylococcus aureus
were cultured on Muller Hinton Agar and Chapman medium , respectively .

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2.2. METHODS

2.2.1. Determination of total phenolic compounds (TPC)

The content of TPC in Cymbopogon citratus and Syzygium aromaticum EOs and their
combinations were determined using a modified El-Maati’s method [ 22]. Briefly, an amount
of 400 μL of every dilu ted essential oil (10 mg in 10 mL solvent), 2.5 mL of Folin -Ciocalteu
reagent (diluted 10 times with distilled water) and 2 mL of Na 2CO 3 were added to a tube and
then homogenized. The reaction was kept for 30 minutes in the dark, after which the
absorbance was read at 760 nm by means of a Camspec spectrophotometer. For the control
sample, 400 μL of distilled water was used. The TPC content expressed as the gallic acid
equivalent (GAE) was calculated based on the calibration curve u sing the linear equation (1).

(1)

where y is the absorbance ; x is the concentration (mg GAE ·g−1 extract) ; R2 – correlation
coefficient. The concentration of phenolic compounds was measured five times. Th e results
were reported as mean standard deviation and expressed as mg of gallic acid equivalents
(GAEs) per 100 g sample (essential oils alone or in combination).

2.2.2. Determination of phenolic acids

The c ontent of phenolic acids was measured by a spectrophotometric method with
Arnov's reagent (10.0 g sodium molybdate, 10.0 g sodium nitrite in 100 .0 mL water). The
sample (1.0 mL ) was pipetted into 10.0 mL volumetric flask containing 5.0 mL water; next,
1.0 ml HCl (18 g/L ), 1.0 mL Arnov's reage nt and 1.0 mL NaOH (40g/L ) were added. The
volume was brought to 10.0 mL with distilled water. The total phenolic acid content was
calculated acco rding to the e quation (2 ).

(2)

where A is the abs orbance of the mixture analysed at 490 nm , and m is the mass of the
mixture , in grams. The results are averages of five measurements, expressed as the caffeic
acid equivalent (mg CAE / 100g ) [23].

2.2.3. Determination of antioxidant activity ( DPPH• radical scavenging activity)

The antioxidant properties of Cymbopogon citratus and Syzygium aromaticum EOs
and their combinations were also investigated by determining the free radical -scavenging
activity of the DPPH• radical based on the method proposed by El-Maati et  al. [ 13]. Briefly,
100 µL of each oil (10 mg extract/10 mL solvent) was added to 3 mL of 0.1 mM DPPH•
dissolved in ethanol according to the solvent used for extraction. After the incubation time o f
30, 60 and 120 minutes at room temperature, the absorbance was measured against the control
at 517 nm. The p ercentage of the antioxidant potential of DPPH• radicals was calculated as
follow s, according to equation (3 ).

(3)

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where A control is the absorbance of the control, and A sample is the absorbance in the presence of
the sample . BHT (butylated hydroxytoluene) was used as a reference substance .

2.2.4. Disk diffusion assay

The antibacterial activity of Cymbopogon citratus and Syzygium aromaticum EOs and
their combinations was evaluated as in the traditional antibiotic susceptibility testing using the
disc diffusion method . An amount of 20 mL Mueller -Hinton Agar Medium was transferred to
a Petri dish. After cooling and solidification, inoculation was performed by spreading
Drigolski dis c of 1 μL of cell suspension in 0.9% NaCl. Then the sterile paper discs (6 mm in
diameter ) were impregnated with 5 µL essentia l oil and placed on the medium surface .
Erythromycin and Norfloxacin were used as controls. Plates were then incubated at 37 °C for
24 h using a Stericell thermostat (Germany ). Following incubation, the growth inhibition
zones were observed. Each experiment was done in triplicate.

2.2.5. Statistical analysis

All assays were carried out five times and results were reported as mean ± standard
error. The statistical significance between the phenolic content, antioxidant activity and
antibacterial values of the essential oils and their mixtures was evaluated with ANOVA.
Values of p lower than 0.05 were considered to be statistically significant.

3. RESULTS AND DISCUSSI ON

3.1. RESULTS

To evaluate the antioxidant and antimicrobial activities of Cymbopogon citratus and
Syzygium aromaticum EOs , and to investigate a possible synergistic effect of the mixtures
consist ing of these oils, the following samples were used in the experiment (Table 1).

Table 1. The samples used in experiment.
Sample Cymbopogon
citratus
EO Syzygium
aromaticum EO Combinations of Cymbopogon
citratus and Syzygium
aromaticum EOs
P1 X – –
P2 – X –
P3 – – 1:1
P4 – – 2:1
P5 – – 1:2

Table 2 shows the total phenolic content (TPC) and phenolic acids amount of the
analysed EOs and their combinations .

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Table 2. Total phenolic content (TPC) and phenolic acids of EOs and their mixtures.
Sample TPC
[mg GAEa / 100 g ] Phenolic acids
[mg CAEb / 100 g ]
P1 146.27 ±0.51 2.84±0.74
P2 138.71±0.93 2.59±0.98
P3 143.93 ±1.05 3.44±1.42
P4 133.34 ±0,84 3.20±1.11
P5 132.44 ±0.56 3.05±0.65
aGallic acid equivalent ; bCaffeic acid equivalent

Figs. 1 and 2 show the DPPH• radical scavenging activity of Cymbopogon citratus and
Syzygium aromaticum EOs alone and in combination.

Figure 1. DPPH• radical scavenging activity of Cymbopogon citratus and Syzygium aromaticum EOs.

Figure 2 . DPPH• radical scavenging activity of mixtures consisting of Cymbopogon citratus and Syzygium
aromaticum EOs.

0 20 40 60 80 100
0 30 60 120 A A (%)
Time (minutes)
BHA P1 P2
0 20 40 60 80 100
0 30 60 120 A A (%)

Time (minutes)
BHA P3 P4 P5

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Table 3 shows the different inhibition zone diameters of Cymbopogon citratus and
Syzygium aromaticum EOs and their combinations.

Table 3 . Antibacterial activities of Cymbopogon citratus and Syzygium aromaticum EOs and their
mixtures against Escherichia coli and Staphylococcus aureus. Values are the means of five measurements.

Sample Inhibition zone diameters (mm)
Escherichia
coli Staphylococcus aureus
P1 29.0 12.0
P2 12.0 16.5
P3 16.5 12.0
P4 16.0 7.5
P5 15.0 9.0
Control 31.0 26.5

3.2. DISCUSSION

The total phenol content is a very important indicator in the ch aracterization of EOs
because most phenolic compounds have both antioxidant and antibacterial properties. The
total phenol content was estimated by the Folin -Ciocalteu colorimetric method in compa rison
with standard gallic acid.
The essential oils studied and their combinatio ns had an important load of phenols
(Table 2). Among these, the TPC ranged from 138.71±0.93 (Syzygium aromaticum oil) to
146.27 ±0.51 (Cymbopogon citratus oil) gallic acid equivalents (GAE mg / 100 g). Both
essential oil s under study should be considered as a very good source of phenolic compounds.
According to Khadri et al. [5], who reported a total phenolic content of 129 mg GAE / 100 g
in the Cymbopogon citratus oil, an amount of 146.27 ±0.51 mg GAE / 100 g could be
considered as high.
In the case of Syzygium aromaticum oil, El -Maati et al. have reported a total phenolic
content ranging from 58.5 to 293 mg GAE / 100g [13]. Many researchers who have been
concerned with the antioxidant activity of essential oils have highlighted the correlation
between t he nature of compo unds and their ability to inhibit the oxidation process [14]. For
the essential oil of Cymbopogon citratus it has been established that the main components
responsible for the antioxidant action are : E- Citral/ Geraniol (37.7%), Z – Citral / Neral
(21.2%), Selina – 6- en-4-ol (8.9%), Myrcene (2.5%), and Sesquiterpene (1.7%) [7].
Eugenol (the active substance) makes up 90 -95% of the Syzygium aromaticum oil.
EOs of Cymbopogon citratus and Syzygium aromaticum and their combinations have a
relatively high content of phenolic acids (Table 2). Phenolic compounds are secondary
metabolites that can act as antibacterial agents. The antibacterial activity could be attributed to
the hydrophobic character of phenolic content .
Assays based on the use of DPPH• radicals are among the most popular
spectrophotometric methods for determination of the antioxidant capacity of essential oils.
The DPPH• scavenging methods have been used to evaluate the antioxidant activity of
compounds due to their simple, rapid, sensitive, and reproducible procedure . The DPPH•

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radical scavenging activity of Cymbopogon citratus and Syzygium aromaticum EOs and their
mixtures was shown in Fig s. 1 and 2 , respectively.
Figs. 1 and 2 it show that the samples containing a large amount of phenols manifested
a high antioxidant activity similar to that of BHA . For the P1 sample (essential oil of
Cymbopogon citratus ) the antiradical activity was 90.3% after 12 0 minutes , and for the
sample P3 the antiradical activity was 91.85% after 120 min . A high antioxidant activity is
influenced by the high content of phenolic acids [24].
The a ntimicrobial activity was determined by using the agar well diffusion assay. In
the present study , the Cymbopogon citratus and Syzygium aromaticum EOs and their mixtures
were tested against two resistant bacteria , i.e. Escherichia coli and Staphylococcus aureus.
The antibacterial activity of Cymbopogon citratus and Syzygium aromaticum EOs and their
combinations and their potency was quantitatively assessed by the presence or absence of the
inhibition zone and its diameter .
The antibacterial activity of Cymbopogon citratus and Syzygium aromaticum EOs and
their combinations can be classified into three levels such as: weak activity (inhibition zone ≤
12 mm), moderate activity ( inhibition zone 12 mm – 20 mm), and strong act ivity (inhibition
zone ≥ 20 mm) [ 25].
The results indicate the different inhibition zone diameters of Cymbopogon citratus
and Syzygium aromaticum EOs and their mixtures (Table 3 ). Significant difference in the
activities of the investigated samples against the tested bacteria was observed (p<0.05). The
Cymbopogon citratus EO showed a strong act ivity against Escherichia coli (the highest
diameter inhibition – 29 mm), and a weak activity against Staphylococcus aureus (low zone of
inhibition – 12 mm) (Table 3 ). At the same time , the Syzygium aromaticum EO showed a
weak activity against Escherichia coli (low zone of inhibition – 12 mm), and a moderate
activity against Staphylococcus aureus (the diameter of inhibition – 16.5 mm) (Table 3 ).
The Syzygium aromaticum EO showed a higher inhibitory activity against
Staphylococcus aureus than Cymbopogon citratus EO. The results are consistent with those
presented by Mohd et al., 2010 and Zulfa et al., 2016 [7, 26]. All combinations have no
synergistic effect (the antibacterial activity is lower than the sum of the antibacterial activity
of the oils taken individually ).

4. CONCLUSIONS

The results clearly demonstrate a good antioxidant activity of the Cymbopogon
citratus and Syzygium aromaticum essential oils, similar to that exhibited by the synthetic
antioxidant BHT. The results illustrate the effectiveness of Cymbopogon citratus and
Syzygium aromaticum oils alone and in combination as potential natural antioxidants in
replacing synthetic anti oxidants.
The Cymbopogon citratus oil alone exhibited a strong antimicrobial activity against
Escherichia coli, while Syzygium aromaticum oil showed a higher inhibitory activity against
Staphylococcus aureus than Cymbopogon citratus oil. No combinations of these oils have any
synergistic effect. The a ntibacterial activity of the combinations consisting of Cymbopogon
citratus and Syzygium aromaticum oils is lower than the sum of the antibacte rial activity of
the oils considered individually .
Taking into account the results of this study , it can be concluded that Cymbopogon
citratus and Syzygium aromaticum oils are good candidate s for the biopreservation of foods.

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