Arabian Journal of Chemistry (2017) xxx, xxxxxx

King Saud University

Arabian Journal of Chemistry

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ORIGINAL ARTICLE

Chemical composition, antibacterial and

antioxidant activities of essential oils from leaves
of three Melaleuca species of Pakistani flora
Saima Siddique a,*, Zahida Parveen b, Firdaus-e-Bareen a, Sania Mazhar c

a
College of Earth & Environmental Sciences, University of the Punjab, Lahore 54890, Pakistan
b
Applied Chemistry Research Centre, PCSIR Laboratories Complex, Lahore 54600, Pakistan
c
Food & Biotechnology Research Centre, PCSIR Laboratories Complex, Lahore 54600, Pakistan

Received 3 December 2016; accepted 30 January 2017

KEYWORDS Abstract This study presents essential oil composition of three Melaleuca species namely,
Melaleuca bracteata; Melaleuca bracteata F. Muell, Melaleuca fulgens R. Br. subsp. steedmanii and Melaleuca leucaden-
M. fulgens; dron (L.) L. collected from different regions of Pakistan. The chemical composition of essential oils
M. leucadendron; was analyzed by GC-FID and GCMS. Eugenol methyl ether was identified as a principal compo-
Essential oils; nent in M. bracteata (82.3%), M. fulgens (87.8%) and M. leucadendron (95.4%) oils. In vitro antibac-
Antibacterial activity; terial studies were done by agar well diffusion and microdilution method and the tested essential oils
Antioxidant potential exhibited bacteriostatic and bactericidal effects against the tested foodborne pathogens at 48 mg/ml.
Time kill assay showed significant bactericidal effect of oils for four weeks. The antioxidant potential
was assessed by free radical scavenging activity and reducing power assay. The oils showed strong
antioxidant activity with approximately 89.089.5% inhibition of 2,2-diphenyl-1-picrylhydrazyl
radical and ferric reducing power in the range of 1.94 0.0072.04 0.04% at 100 mg/ml.
2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction and Gould, 2003). However, the negative consumers perception of

Food preservation involves control of oxidative damage to the food
products and growth of foodborne pathogens. Different chemicals
are used to inhibit oxidation and microbial growth in foods (Russell
* Corresponding author.
E-mail address: saimesiddique@gmail.com (S. Siddique).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
chemical preservatives due to their harmful effects and increased
microbial resistance to the majority of currently used antibacterial
drugs (Shan et al., 2007), has directed the attention of scientists toward
the use of natural alternatives for the extension of products shelf-life.
Among the emerging natural preservatives, essential oils have been
widely investigated and have gained momentum in the recent years
due to the presence of antioxidants and their antibacterial, anti-
mutagenic and anti-carcinogenic properties (Gutierrez et al., 2009).
Myrtaceae is one of the most diverse and widespread plant families,
rich in essential oils. The main genera of Myrtaceae include Eucalyptus,
Eugenia, Leptospermum, Melaleuca, Myrtus, Pimenta, Plinia, Psidium,
Pseuocaryophyllus and Syzygium. The genus Melaleuca L. consists of
around 260 species and occurs predominantly in Australia but is also

http://dx.doi.org/10.1016/j.arabjc.2017.01.018
1878-5352 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Siddique, S. et al., Chemical composition, antibacterial and antioxidant activities of essential oils from leaves of three Melaleuca
species of Pakistani flora. Arabian Journal of Chemistry (2017), http://dx.doi.org/10.1016/j.arabjc.2017.01.018
2 S. Siddique et al.
domesticated in South-East Asia, the Southern United States and the
Caribbean (Tran et al., 2013). They are shrubs or trees, generally found
in open forests, woodlands or shrublands, particularly along the water-
courses and the edges of swamps. Melaleuca genus shows significant
phenotypic diversity in a variety of ecosystems. They can adapt to cli-
mate change through Wrights migrational adaptation, and can be
managed to attain sustainable benefits (Tran et al., 2013). The essential
oils from Melaleuca have demonstrated antibacterial (Hussein et al.,
2007), anti-inflammatory (Caldefie-Chezet et al., 2006), fungicidal
(Bagg et al., 2006), acaricidal (Iori et al., 2005), antioxidant (Pino
et al., 2010) and antiviral properties (Minami et al., 2003).
Studies on chemical composition of essential oils of various Mela-
leuca species from Benin, Brazil, Egypt, India, Thailand and Tunisia
have been previously reported. Eugenol methyl ether, 1,8-cineole,
terpinen-4-ol, terpinolene, a-terpinene, caryophyllene and caryophyl-
lene oxide were identified as major constituents in the essential oils
of most of the Melaleuca species (Lohakachornpan and
Rangsipanuratn, 2001; Farag et al., 2004; Kumar et al., 2005; Silva
et al., 2007; Chabir et al., 2011; Rini et al., 2012a, 2012b).
Melaleuca genus was introduced in the Punjab, Pakistan, long ago
(Parker, 1956). Literature survey revealed that essential oil composi-
tion of Melaleuca species from Pakistan has not yet been investigated.
The screening of these indigenous essential oil resources is needed for
their use in the industry. Three species of Melaleuca were selected for
identification of chemical constituents present in their oils and for
investigation of their biological properties. This research was also
aimed at identification of volatile constituents of the selected Mela-
leuca species and their evaluation for use in the food industry as alter-
nate to chemical preservatives.
2. Material and Methods
2.1. Plant material
Fresh leaves of M. bracteata F. Muell were collected from
Government College University Botanical Garden, Lahore
(Longitude 74.31E, Latitude 31.57N), M. fulgens R. Br.
subsp. steedmanii from Qarshi Botanical Garden, Hattar, Abot-
tabad (Longitude 72.85E, Latitude 33.85N) and M. leucaden-
dron (L.) L. from Jinnah Garden, Lahore (Longitude 74.26E,
Latitude 31.50N) in the Early Summer in 2014. Plant herbaria
were authenticated by Prof. Dr. A.N. Khalid at the Herbarium,
Department of Botany, University of Punjab, Lahore, Pakistan
and voucher specimens (BDSS # 4039, BDSS # 4040, BDSS #
4041) were also deposited in the same herbarium.
2.2. Chemicals
Homologous series of C8C25 n-alkanes used in this study were
obtained from Sigma Chemical Co. (St. Louis, MO, USA)
while 2,2-diphenyl-1-picrylhydrazyl (99.0%) was purchased
from ACE, Germany. Butylated hydroxytoluene (BHT,
99.0%) was obtained from ACROS-Organics, Belgium. Anhy-
drous sodium sulphate, ferrous chloride, potassium ferri-
cyanide, trichloroacetic acid, ethanol and methanol used in
this study were purchased from Merck (Darmstadt, Germany).
Culture media (Nutrient broth, Nutrient agar, Plate count
agar) were purchased from OXOID Ltd. Hampshire, UK.
2.3. Isolation of oils
Fresh leaves (4.30 kg, 0.97 kg and 1.25 kg) of M. bracteata,
M. fulgens and M. leucadendron respectively were subjected to
Please cite this article in press as: Siddique, S. et al., Chemical composition, antibacterial and antioxidant activities of essential oils from leaves of three Melaleuca
species of Pakistani flora. Arabian Journal of Chemistry (2017), http://dx.doi.org/10.1016/j.arabjc.2017.01.018
hydro-distillation for 3 h using Clevenger-type apparatus in
triplicate, according to the method recommended in the
European Pharmacopoeia (EDQM, 2005). The oils obtained
were dried over anhydrous sodium sulphate, filtered and stored
at
4 C further analyses. The essential oil contents (%) were
expressed as volume of essential oil vs. weight of fresh leaves
(v/w).
2.4. Chemical analysis of essential oils
2.4.1. GC-FID
GC analysis of the essential oils was carried out on
Shimadzu GC 2010 equipped with the flame ionization
detector (FID) and AOC-20i auto-sampler using a DB-5 MS
(30 m  0.25 mm id, 0.25 mm film thickness) capillary column.
The column oven temperature was programmed initially at
4090 C at the rate of 2 C/min and then raised to
90240 C at the rate of 3 C/min. The final temperature was
held constant for 5 min. Injector and detector temperatures
were maintained at 240 and 280 C, respectively. Essential oil
(0.5 ml) was injected in a split mode ratio of 1:5. Helium was
used as a carrier gas at the flow rate of 1 ml/min. Quantifica-
tion of constituents was carried out by integration of peak
areas without using the correction factors. The essential oils
samples were ran in triplicate.
2.4.2. GCMS
The identification of components was carried out on GCMS-
QP 2010 Plus, Shimadzu, Japan operating in electron ioniza-
tion mode at 70 eV. Mass units were monitored from 35 to
500 AMU. A DB-5 MS (30 m  0.25 mm id, 0.25 mm film
thickness) capillary column was used. Column conditions
and temperatures of injector and detector were the same as
in GC analysis.
Linear retention indices were calculated using a homolo-
gous series of n-alkanes (C8-C25) under the same
temperature-programmed conditions. The components were
identified by comparison with linear retention indices (RI)
from literature Adams (2001); mass spectra with those of NIST
mass spectral library (Mass spectral library, 2001) or co-
injection with standards.
2.5. Evaluation of antibacterial activities of essential oils
2.5.1. Tested Microorganisms
Seven bacterial strains from American Type Culture Collec-
tion (ATCC, Rockville) were selected for in vitro antibacterial
activity of the essential oils. Of the seven bacterial strains
Bacillus spizizenii (ATCC 6633) and Staphylococcus aureus
(ATCC 25923) were Gram positive while Enterobacter aeroge-
nes (ATCC 13048), Escherichia coli (ATCC 8739), Klebsiella
pneumoniae (ATCC 13882), Pseudomonas aeruginosa (ATCC
27853) and Salmonella enterica (ATCC 14028) were Gram neg-
ative strains. All the bacterial strains were sub-cultured at
35 C for 24 h on nutrient agar slants prior growing them in
nutrient broth overnight.
2.5.2. Agar well diffusion method
Antibacterial activity of the selected essential oils was checked
by agar well diffusion method (Zaika, 1988). Molten agar
Essential Oils from Leaves of Three Melaleuca Species
medium (20 ml) was inoculated with microbial suspension con-
taining the indicator strain having 106 cfu/ml concentration.
The inoculated medium was poured into Petri plates and
allowed to solidify. Wells were made on solidified agar with
a sterilized cork borer and 90 ll of the tested oil was added
to each. Ampicillin was used as a positive control. The plates
were incubated at 35 C for 24 h. The diameters of inhibition
zones were measured in millimeters and results were recorded
in triplicate.
2.5.3. Minimum Inhibitory Concentration (MIC) assay
Serial dilutions of 4 lg/ml, 8 lg/ml, 15 lg/ml, 65 lg/ml and
250 lg/ml were used in triplicate to determine MIC levels by
agar well method (Zaika, 1988). The lowest concentration of
oil inhibiting visible growth of each microbe after incubation
was taken as the MIC.
2.5.4. Minimal Bactericidal Concentration (MBC) assay
Minimum Bactericidal Concentration (MBC) was determined
by broth microdilution method (Rabe et al., 2002). Bacterial
load (106 cfu/ml) was poured in tubes containing respective
culture broth and oil with concentration of MIC. Broth tubes
with and without bacterial load were used as controls. The
tubes were incubated for 24 h at 35 C. After incubation,
100 ll from tubes having no visible growth was removed and
poured in plates along with agar to enumerate total viable
count. The lowest concentration with no visible growth after
24 h of incubation at 35 C was defined as the MBC, indicating
99.9% killing of the original inoculum.
2.5.5. Time kill assay
A time kill study was carried out with the MIC values found
previously by the agar well method to discern whether the
tested oils had bacteriostatic or bactericidal effect over a
period of time for use as a food preservative (White et al.,
1996). Microorganisms (bacterial suspension) with 106 cfu/ml
and oil having concentration equal to MIC were added
respectively in the tube of corresponding culture medium.
Broth tubes with and without microbial suspension were used
as controls. The cultures were incubated for one month at
35 C. An inoculant of 100 ll, removed after 2, 5, 8, 11, 14
and 30 d was poured in agar plates in triplicate to determine
the total reduction in viable counts. The mean number of the
colonies (cfu/ml) was counted and compared with that in the
control culture at the end of the incubation period. The test
tubes with turbidity after a certain incubation period
depicted bacteriostatic effect of the tested essential oil at the
applied concentration. To determine the bactericidal concen-
tration of essential oils against that particular strain, higher
concentrations (15 lg/ml, 65 lg/ml and 250 lg/ml) were
applied and lethal effect of essential oils was observed as
mentioned above.
2.6. Antioxidant activity
2.6.1. DPPH assay
The antioxidant activities of essential oils were evaluated by
measurement of their ability to scavenge 2,20 -diphenyl-1-picryl
hydrazyl (DPPH) stable radical. The assay was carried out
spectrophotometrically as described by Shimada et al. (1992).
Please cite this article in press as: Siddique, S. et al., Chemical composition, antibacterial and antioxidant activities of essential oils from leaves of three Melaleuca
species of Pakistani flora. Arabian Journal of Chemistry (2017), http://dx.doi.org/10.1016/j.arabjc.2017.01.018
3
Various solutions at different concentrations in the range of
20 to 100 lg/ml of essential oils were prepared in methanol. To
0.1 ml of each test concentration, 3 ml of methanolic solution
of DPPH (0.004%) were added. The resulting mixtures were
incubated in the dark for 30 min at room temperature and
absorbance was recorded as Asample at 517 nm using spec-
trophotometer (Cecil CE 7200). A blank experiment was also
carried out applying the same procedure to a solution without
essential oil, and absorbance was recorded as Ablank. Scaveng-
ing (%) of DPPH free radical by essential oils was calculated
as follows:
Scavenging % Ablank
Asample =Ablank  100
Antioxidant activity of essential oils or standard was
expressed as IC50 which is defined as the concentration of test
material required to cause a 50% decrease in initial DPPH
concentration. All determinations were performed in tripilcate.
Butylated hydroxytoluene (BHT) was used as a standard.
2.6.2. Total reduction ability by Fe3+-Fe2+ transformation
The total reduction ability of essential oils was determined by
the method of Oyaizu (1986). The capacity of essential oils to
reduce the ferric ion (Fe3+) to the ferrous ion (Fe2+) was eval-
uated by measuring the absorbance at 700 nm. To the different
concentrations of the essential oils 2.5 ml of phosphate buffer
(0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide (1%)
were added. The mixture was incubated at 50 C for 20 min.
Then 2.5 ml of trichloroacetic acid (10%) were added. The
mixtures were revolved at 3000 rpm for 10 min. The super-
natant (2.5 ml) was mixed with 2.5 ml of distilled water and
0.5 ml of ferric chloride. Absorbance was measured at
700 nm on UV spectrophotometer after allowing the solution
to stand for 30 min. Butylated hydroxytoluene (BHT) was
used as a standard.
2.7. Statistical analysis
The mean values, standard deviations were calculated using
MS Excel 2007. Data was analysed by using analysis of vari-
ance (ANOVA) and differences among the means were deter-
mined for significance at P < 0.05 using Duncans multiple
range test by SPSS (version 16.0).
3. Results & discussion
3.1. Essential oil yield
Hydrodistillation of fresh leaves of M. bracteata, M. fulgens
and M. leucadendron yielded 0.14 0.01%, 2.10 0.10%
and 0.42 0.03% of essential oils respectively. The essential
oil yield of M. bracteata and M. leucadendron was lower than
that reported by previous researchers (Zhong et al., 2009;
Oyedeji et al., 2014; Almarie et al., 2016). Essential oil yield
from M. fulgens leaves has not been reported previously.
3.2. Chemical composition
The chemical compositions of essential oils from Melaleuca
species is given in Table 1. The essential oils of M. bracteata,
M. fulgens and M. leucadendron were characterized by high
4 S. Siddique et al.

percentage of aromatic compounds (95.196.4%). Eugenol
methyl ether was identified as a principal component in M.
bracteata (82.3%), M. fulgens (87.8%) and M. leucadendron
(95.4%) oils. Melaleuca bracteata essential oil contained
methyl cinnamate (11.4%) in significant amounts along with
eugenol methyl ether. Our findings on M. bracteata essential
oil are in agreement with the previous reports (Aboutabl
et al., 1991; Ye et al., 2014; Almarie et al., 2016). Ye et al.
(2014) reported comparable methyl eugenol content (83.55%)
but a higher methyl cinnamate content (4.55%) in M. brac-
teata essential oil, extracted using petroleum ether. Zhong
et al. (2009) however, reported a higher concentration of euge-
nol methyl ether (>95%) in M. bracteata essential oil
extracted from branches and leaves.
Essential oil from M. fulgens and M. leucadendron leaves
showed qualitative and quantitative variability in chemical
composition when compared with the earlier reports.
Previously, 1,8-cineole was reported as the principal
component in the essential oil of M. leucadendron species
grown in Cuba, India, Indonesia, Ivory Coast and Egypt
(Farag et al., 2004; Kumar et al., 2005; Rini et al., 2012a,
2012b; Tia et al., 2013). Terpinolene (29.21%), a-terpinene
(22.55%), 2-c-carene (8.53%) and a-phellandrene (7.61%)
were reported as main components in the essential oil of
M. leucadendron grown in Thailand (Lohakachornpan and
Rangsipanuratn, 2001). Our results are similar to the Brazilian
M. leucadendron essential oil (Silva et al., 2007) wherein euge-
nol methyl ether (96.6%) was identified as the principal com-
ponent. However, Pakistan has a dry sub-tropical climate as
compared to Brazil having wet tropical climatic situation.
Contrasting the previous reports, 1,8-cineole (0.1%) and
terpinolene (0.1%) were found in traces in Pakistani variety
of M. leucadendron.
Lee et al. (2004a, 2004b) reported 1,8-cineole (77.5%) as a
major component in M. fulgens essential oil from Australia
along with an appreciable amount of limonene (6.14%),
a-pinene (3.21%), myrcene (1.43%) and methyl geranate
(1.0%). On the contrary, eugenol methyl ether (87.78%) was
found as the major component alongwith minor quantities
(0.10.2%) of limonene, a-pinene and 1,8-cineole in essential
oil from M. fulgens leaves in Pakistan. These differences in
essential oils composition might have arisen due to environ-
mental and climatic conditions, geographic variations and
genetic differences.

Table 1 Essential oil composition of the three Melaleuca species.

Compounds RIexp RIlit Area (%)
M. bracteata M. fulgens M. leucodendron
a-Pinene 930 932 0.2 0.1 0.1
a-Phellandrene 1002 1002 0.1 0.1
d-3-Carene 1004 1008 tr tr
p-Cymene 1026 1020 1.7 1.2 0.1
Limonene 1029 1024 0.2 0.2 0.1
1,8-Cineole 1028 1026 0.3 0.2 0.1
cis-b-Ocimene 1043 1032 tr 0.1
c-Terpinene 1055 1054 tr 0.1
Terpinolene 1083 1086 0.3 0.5 0.1
Linalool 1095 1095 1.0 1.0 0.4
Citronellal 1148 1148 tr tr
p-Menthan-3-ol 1167 1167 0.1
p-Cymen-8-ol 1183 1179 0.4 tr 0.1
a-Terpineol 1186 1186 1.0 0.9 0.7
Citronellol 1223 1223 0.4 tr 0.1
2-Isopropenyl-5-methylhex-4-enal 1198 0.1 tr
a-Cubebene 1345 1345 tr tr tr
3-Allyl-2-methoxyphenol 0.3 0.3 0.8
Nerol acetate 1365 1359 tr tr tr
a-Copaene 1374 1374 tr 0.1 tr
(E)-methyl cinnamate 1379 1376 11.4 5.8 0.8
Eugenol methyl ether 1402 1403 82.3 87.8 95.4
b-Caryophyllene 1417 1417 0.1 0.1
Germacrene D 1484 1484 0.2 0.6 0.4
Germacrene B 1559 1559 tr 0.2 0.1
Caryophyllene oxide 1582 1582 tr tr 0.1
Epiglobulol 1585 tr 0.1 tr
Total identified 99.6 99.4 98.7
Monoterpene hydrocarbons 0.8 1.1 0.3
Oxygenated monoterpenes 2.6 2.1 1.3
Sesquiterpene hydrocarbons 0.2 1.0 0.6
Oxygenated sesquiterpenes tr 0.1 0.1
Aromatic compounds 95.9 95.1 96.4
Others 0.1 tr
RIexp = Retention Indices relative to C9-C25 n-alkanes on the DB-5 column; RIlit = Retention Indices from literature (2); tr = trace < 0.05%.

Please cite this article in press as: Siddique, S. et al., Chemical composition, antibacterial and antioxidant activities of essential oils from leaves of three Melaleuca
species of Pakistani flora. Arabian Journal of Chemistry (2017), http://dx.doi.org/10.1016/j.arabjc.2017.01.018
Essential Oils from Leaves of Three Melaleuca Species 5

3.3. Antibacterial activity
The evaluated essential oils manifested good antibacterial
properties against all tested microbes but the level of bacterial
growth inhibition was found to be dependent on essential oils
concentration and the bacterial strain (Table 2).
Melaleuca bracteata exhibited excellent activity against
B. spizizenii with inhibition zones (IZ) of 12.244.0 mm
followed by M. fulgens (14.320.8 mm) and M. leucadendron
(13.216.3 mm). The tested essential oils showed moderate
activity against S. aureus with inhibition zones between
(11.013.2 mm). The evaluated essential oils showed good
activity against tested Gram negative bacterial strains with
zones of inhibition ranging from 11.0 to 18.9 mm at concentra-
tions of 4250 lg/ml. Melaleuca leucadendron essential oil
demonstrated appreciable activity against E. aerogenes
(IZ = 13.716.2 mm) while M. bracteata and M. fulgens
showed comparable zones of inhibition too i-e between 11.5
and 17.0 mm. Melaleuca bracteata showed moderate zones of
inhibition against K. pneumoniae (12.218.9 mm) while it ran-
ged from 14.5 to 17.8 mm for M. fulgens and M. leucadendron
essential oils. Melaleuca essential oils showed similar inhibitory
effect against E. coli, P. aeruginosa, and S. enterica as marked
by zones of inhibition ranging from 11.8 to 17.3 mm,
11.016.3 mm and 11.717.8 mm in the three species respec-
tively. Melaleuca fulgens exhibited a slightly larger zone of inhi-
bition as compared to M. bracteata and M. leucadenderon i-e.
13.817.2 mm and 10.816.3 mm for E. coli and P. aeruginosa
while M. bracteata was found more inhibitory to S. enterica
with inhibition zone 11.717.8 mm. The variation in the
antibacterial activities of Melaleuca essential oils with respect
to all species was statistically significant (P < 0.05).
The MIC and MBC values of Melaleuca essential oils ran-
ged from 4 to 8 lg/ml for the tested bacterial strains (Table 3).
There are a few reports on antibacterial activity of the selected
Melaleuca species for comparison. Higher MIC values were
reported against E. coli, P. aeruginosa and S. aureus (25
100 ll/ml) for M. leucadendron essential oil
(Lohakachornpan and Rangsipanuratn, 2001; Dehghan and
Bawazir, 2012). Antibacterial activity of M. fulgens has not
been reported previously. Our findings on antibacterial activity
of M. bracteata essential oils showed discrepancies with the
previously published data. Oyedeji et al. (2014) reported larger
zones of inhibition (10.315.3 mm) against S. aureus for differ-
ent varieties of M. bracteata from South Africa while smaller
IZ were observed against Gram negative bacteria viz. E. coli
(11.814.5 mm), P. aeruginosa (8.59.0 mm) and K. pneumo-
niae (11.514.5 mm). The MICs were found 0.621.25 lg/ml
for S. aureus and 0.621.25 lg/ml and 1.252.5 lg/ml for
K. pneumoniae and P. aeruginosa respectively.
The differences in the antibacterial activity of the selected
essential oils and subsequently the MICs and MBCs may result
from different chemical compositions and percentage content
of active constituents in essential oils. Factors such as the
choice of bacterial strains and their sensitivity, volume of
inoculum, cultivation conditions (incubation time, tempera-
ture, oxygen etc), concentration of test substance, the culture
medium, the solvents used to dilute the essential oils and differ-
ent methods used for in vitro antibacterial activity could also
be related to the variation in the experimental results.
The time kill assay was carried out to check the potency of
Melaleuca essential oils for use as food preservative. The study
was carried out to evaluate whether the tested essential oils
have bacteriostatic or bactericidal effects at 48 lg/ml for four

Table 2 Antimicrobial activity of essential oils by Agar well diffusion method.

Essential oil Conc. Zones of inhibition (mm) on tested microbial strains
(mg/ml)
B. spizizenii S. aureus E. aerogenes E. coli K. pneumoniae P. aeruginosa S. enterica
M. bracteata 250 44.0 3.5f 12.8 0.3a 15.7 0.6ab 16.7 0.3b 18.9 0.4bc 12.5 0.0a 17.8 0.8b
100 20.8 0.8c 12.3 0.3a 15.0 0.0ab 15.3 0.3ab 17.2 0.3b 12.0 0.0a 15.2 0.3ab
65 14.8 0.3ab 12.2 0.3a 14.3 0.6ab 14.8 0.3ab 16.3 0.3b 11.8 0.3a 13.7 0.3ab
15 13.2 0.3ab 11.7 0.3a 13.2 0.3ab 12.8 0.3a 14.0 0.0ab 11.7 0.3a 12.0 0.3a
8 12.7 0.3a 11.5 0.0a 13.0 0.0ab 12.5 0.0a 13.3 0.3ab 11.2 0.3a 11.8 0.3a
4 12.2 0.3a 11.3 0.3a 11.5 0.3a 11.8 0.3a 12.2 0.3a 11.0 0.0a 11.7 0.3a
M. fulgens 250 20.8 0.8c 12.7 0.6a 17.0 1.0b 17.2 0.3b 17.8 0.8b 16.3 0.5b 17.2 0.3b
100 19.2 0.3bc 12.5 0.0a 16.3 0.3b 16.3 0.3b 17.3 0.3b 15.7 0.3ab 16.3 0.3b
65 18.8 2.0bc 12.3 0.3a 16.0 0.5b 16.0 0.9b 17.2 1.0b 15.0 1.0ab 15.8 0.8ab
15 17.5 1.0b 11.7 0.3a 14.8 0.2ab 15.5 0.0ab 16.8 2.1b 11.3 0.6a 14.8 0.6ab
8 16.7 1.5b 11.2 0.3a 14.2 0.2ab 14.8 0.3ab 16.2 0.0b 11.0 0.0a 14.2 0.3ab
4 14.3 0.6ab 10.8 0.3a 13.2 0.3ab 13.8 0.8ab 14.5 0.5ab 10.8 0.3a 12.8 0.3a
M. leucodendron 250 16.3 0.6b 13.2 0.3ab 16.2 0.6b 17.3 0.8b 16.7 0.6b 12.2 0.3a 15.8 0.3ab
100 16.2 0.3b 12.5 0.0a 15.7 0.3ab 16.7 0.3b 16.5 0.0b 12.0 0.0a 15.7 0.3ab
65 16.0 0.0b 12.3 0.3a 15.5 2.6ab 15.3 0.8ab 16.3 0.6b 11.8 0.3a 15.5 0.5ab
15 15.0 0.0ab 12.3 0.3a 15.3 0.3ab 14.7 0.6ab 15.7 1.5ab 11.7 0.6a 14.5 0.0ab
8 13.8 0.3ab 11.8 0.3a 14.7 0.3ab 13.8 0.3ab 15.3 1.2ab 11.7 0.6a 13.5 0.0ab
4 13.2 0.3ab 11.0 0.0a 13.7 0.3ab 13.3 0.6ab 14.8 0.3ab 11.0 0.0a 11.8 0.3a
Ampicillin 1000 32.0 1.0d 16.3 0.5b 17.6 0.5b 39.3 0.5e 12.5 0.5a ND 33.3 0.5d
*
The diameter of the inhibition zones (mm), including the well diameter (6 mm), are given as mean SD of triplicate experiments. ND: Not
detected.
The values with the same lower case letters are not statistically significant at P = 0.05% according to Duncans Multiple Range Test.

Please cite this article in press as: Siddique, S. et al., Chemical composition, antibacterial and antioxidant activities of essential oils from leaves of three Melaleuca
species of Pakistani flora. Arabian Journal of Chemistry (2017), http://dx.doi.org/10.1016/j.arabjc.2017.01.018
6 S. Siddique et al.

Table 3 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) mg/ml of Melaleuca essential
oils after 24 h and four weeks.
Tested microbial strains M. bracteata M. fulgens M. leucodendron
MIC MBC, 24 h MBC, 4 w MIC MBC, 24 h MBC, 4 w MIC MBC, 24 h MBC, 4 w
B. spizizenii 4 4 8 4 4 250 4 4 65
S. aureus 8 8 8 8 8 8 8 8 8
E. aerogenes 4 4 65 4 4 250 4 4 15
E. coli 4 4 8 4 4 8 4 4 15
K. pneumoniae 4 4 8 4 4 8 4 4 8
P. aeruginosa 8 8 65 8 8 250 8 8 250
S. enterica 4 4 15 4 4 15 8 8 250
*
Abbreviation: w = week.

Table 4 Antioxidant activity of Melaleuca essential oils measured in term of DPPH radical scavenging capacity and Total Ferric
Reducing ability.
Test system Conc. (lg/ml) M. bracteata M. fulgens M. leucodendron BHT IC50 value, lg/ml
DPPH radical scavenging capacity (%) 20 35.3 0.4 34.9 1.6 34.1 1.1 30.8 0.6 37.3 0.9
40 54.3 1.0 54.1 0.5 52.6 0.9 52.4 1.0 M. bracteata
60 69.1 0.3 67.7 0.8 66.8 0.4 66.5 0.7 37.8 1.6
80 77.2 0.3 77.3 1.2 75.6 0.8 76.8 0.6 M. fulgens
100 89.2 0.4 89.0 0.6 89.5 0.8 85.8 0.8 39.1 0.3
M. leucodendron
41.5 0.50 (BHT)
Reducing power (absorbance at 700 nm) 20 1.20 0.02 1.25 0.03 1.22 0.08 1.16 0.18
40 1.38 0.01 1.44 0.05 1.40 0.05 1.36 0.03
60 1.60 0.01 1.68 0.05 1.63 0.03 1.57 0.05
80 1.88 0.02 1.92 0.03 1.89 0.03 1.82 0.07
100 2.02 0.02 2.11 0.04 2.06 0.05 1.95 0.04

weeks. Our data showed that the response of bacteria to the
tested essential oils varied among the strains and was concen-
tration and time dependent. Melaleuca bracteata essential oil
proved highly lethal to B. spizizenii with MBC value of
8 lg/ml whilst the highest bactericidal concentration
(250 lg/ml) was observed for M. fulgens essential oil against
B. spizizenii. Melaleuca leucodenderon essential oil showed
moderate bactericidal effects against B. spizizenii with MBC
of 65 lg/ml. Evaluated essential oils showed bactericidal effect
against S. aureus at 8 lg/ml for a month.
Klebsiella pneumoniae was found to be sensitive toward all
tested oils with lowest MBC i-e. 8 lg/ml among Gram negative
strains whilst P. aeruginosa was found to be most resistant
among the tested strains with MBC values of 250 lg/ml for
M. fulgens and M. leucadendron essential oils. Melaleuca
bracteata, however, showed relatively lower MBC value
against P. aeruginosa of 65 lg/ml.
Melaleuca leucadendron essential oil was resistant toward
S. enterica with MBC value of 250 lg/ml, but showed lower
MBC value against E. aerogenes and E. coli i-e 15 lg/ml.
The MBC values for M. bracteata ranged from 8 to 65 lg/ml
against E. aerogenes, E. coli and S. enterica. For M. fulgens
essential oil the MBC values were 8 lg/ml, 15 lg/ml and
250 lg/ml against E. coli, S. enterica and E. aerogenes
respectively.
The significant antibacterial activity of assayed essential
oils could be related to eugenol methyl ether; the major
compound with known antibacterial potential (Lawal et al.,
2014). The enhanced inhibitory effect of M. fulgens and
M. bracteata essential oils could be related to their higher
methyl cinnamate content which has also been reported to
possess antibacterial property (Stefanovic et al., 2015).
3.4. Essential oils antioxidant activities
DPPH assay and reducing power assay were used to assess
antioxidant potential of Melaleuca essential oils. The synthetic
antioxidant BHT was used as an equivalence parameter for the
antioxidant activity of the essential oils. The Melaleuca essen-
tial oils showed DPPH scavenging activity (89.089.5%). The
DPPH scavenging activity of the tested oils was found higher
than butylated hydroxytoluene (BHT) as is evident from lower
IC50 value of essential oils. In reducing power assay, Melaleuca
essential oils showed comparable ferric reducing power to
BHT at the tested concentrations of 20100 mg/ml (Table 4).
Previously, Hou et al. (2016) reported good ferric reducing
power (FRP) (2.37 0.01 mM Fe2+/g DW) and DPPH radi-
cal scavenging activity (86.0 0.3%) of M. bracteata ethano-
lic extract with eugenol methyl ether (86.86%) and trans-
cinnamic acid methyl ester (6.41%) as major components.
No data is available for comparison of antioxidant activity
of M. fulgens essential oil. Rini et al. (2012a, 2012b) reported
IC50 values of M. leucadendron essential oil ranging from
4240 to 9460 mg/ml in a DPPH scavenging assay. The IC50
value of evaluated M. leucadendron essential oil was quite
low (39.1 0.3 mg/ml) showing its strong antioxidant
potential.

Please cite this article in press as: Siddique, S. et al., Chemical composition, antibacterial and antioxidant activities of essential oils from leaves of three Melaleuca
species of Pakistani flora. Arabian Journal of Chemistry (2017), http://dx.doi.org/10.1016/j.arabjc.2017.01.018
Essential Oils from Leaves of Three Melaleuca Species
4. Conclusion
Our results show that the essential oils from leaves of Melaleuca
species are rich in eugenol methyl ether. The remarkable
antibacterial activity of Melaleuca essential oils based on
MIC, MBC, and kill-time study against common food-borne
pathogens and strong antioxidant activity suggests their possi-
ble use in the food industry as a potential new source of natural
antibacterial and antioxidant agents. However, in vivo studies
are recommended to determine the toxicity profile of essential
oils.
Authors contributions
Saima Siddique and Sania Mazhar designed and performed the
bioassays; Zahida Parveen collected the species and extracted
the essential oils; Saima Siddique and Firdaus-e-Bareen pre-
pared the manuscript and analyzed the data statistically.
Acknowledgement
The authors are thankful to Prof. Dr. A.N. Khalid in the
herbarium, University of the Punjab, Lahore for identification
of plants and the authentication of specimens. Authors are
also very thankful to Mr. Muhammad Akram (Senior Scien-
tific Officer), Medicinal Botanic Centre, Peshawar for his help
in the GC-MS analysis of essential oils.
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Please cite this article in press as: Siddique, S. et al., Chemical composition, antibacterial and antioxidant activities of essential oils from leaves of three Melaleuca
species of Pakistani flora. Arabian Journal of Chemistry (2017), http://dx.doi.org/10.1016/j.arabjc.2017.01.018