ESEA RCH ARTI CLE
ScienceAsia 39 (2013): 472–476
Antibacterial activity of essential oils and their active
components from Thai spices against foodborne
Phanida Phanthonga, Pattamapan Lomarata, Mullika Traidej Chomnawangb,
aDepartment of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Sri Ayudhya Road, Rajathevi,
Bangkok 10400 Thailand
bDepartment of Microbiology, Faculty of Pharmacy, Mahidol University, Sri Ayudhya Road,
Rajathevi Bangkok 10400 Thailand
∗Corresponding author, e-mail: firstname.lastname@example.org
Received 15 Nov 2012
Accepted 25 Jun 2013
ABSTRACT: As the popularity of organic food especially fresh vegetables is increasing, it is a common practice to replace
chemical fertilizers by manure which leads to high bacterial contamination. Some essential oils such as Thymus vulgaris
(thyme) and Ocimum basilicum (basil) oils reduce spoilage ﬂora and foodborne pathogens when used in washing water.
This information prompted us to search for effective essential oils from Thai spices for vegetable washing products. Seven
out of nine essential oils; ﬁngerroot (Boesenbergia pandurata (Roxb.) Schltr.), galanga (Alpinia galanga (L.) Willd.), holy
basil (Ocimum tenuiﬂorum L.), makrut leaf, makrut peel (Citrus hystrix DC.), sweet basil (O. basilicum L.), and turmeric
(Curcuma longa L.) oils showed antibacterial activity. The active components were identiﬁed by thin layer chromatography
(TLC) bioautography, preparative TLC, and gas chromatography-mass spectrometry. The results indicated that the active
components were major components of the oils. The essential oils exhibited higher potency than their active components
suggesting that the whole essential oils were more suitable than the pure compounds for product development.
KEYWORDS: TLC bioautography, GC-MS, active compounds, citral, biological guided separation
Diarrhoea, a disease which causes high morbidity and
mortality, caused at least two million deaths world-
wide in year 2000. Besides, each year 30% of people
in industrialized countries suffer from this foodborne
disease1. The bacteria which are foodborne pathogens
including Escherichia coli,Salmonella Enteritidis,
Salmonella Typhimurium, Salmonella Typhi, Shigella
ﬂexneri,Bacillus cereus, and Staphylococcus aureus,
often contaminate food particularly fresh vegetable2.
At present, the popularity of organic foods, especially
fresh vegetables is increasing and it is a common
practice to use manure instead of chemical fertilizers
which leads to high bacterial contamination. Hence to
control bacterial contamination, an effective and safe
vegetable washing product is needed. Currently, veg-
etable washing products available in the Thai market
are used to remove pesticides, therefore it is necessary
to have the vegetable washing products with antibac-
terial agent in order to decrease the contamination of
the foodborne pathogens.
Some essential oils such as thyme and basil oils
are effective against spoilage ﬂora and foodborne
pathogens when used in washing water3,4. This
information prompted us to search for the active com-
pounds of essential oils from Thai spices. Previous
studies indicated that some essential oils of Thai
spices such as lemongrass, holy basil, turmeric, and
galanga oils had antibacterial properties5–7. However,
an extensive study to identify the active components
has only been done on lemongrass oil, in which citral
was identiﬁed as the active compound5,8.
The aims of this study were to determine the
efﬁcacy of nine commercially available essential oils
of Thai spices as antimicrobial against foodborne
pathogens (E. coli,S. Enteritidis, S. Typhimurium,
S. Typhi, S. ﬂexneri,B. cereus, and S. aureus),
and to identify the active components of each oil.
Biological guided separation was used along with
gas chromatography-mass spectrometry (GC-MS) to
identify active components of the tested oils. Since
ScienceAsia 39 (2013) 473
the essential oils selected for this study were obtained
from edible plants and were volatile, it is likely that
they will be safe and easy to remove. With proper
research and development, essential oil products may
be an alternative for cleaning fresh vegetables to
eliminate microbial contamination.
MATERIALS AND METHODS
Nine essential oils of Thai spices; black pepper fruit
(Piper nigrum L.), ﬁngerroot rhizome (Boesenbergia
pandurata (Roxb.) Schltr.), galanga rhizome (Alpinia
galanga (L.) Willd.), holy basil leaf (Ocimum tenui-
ﬂorum L.), lemongrass stem (Cymbopogon citratus
Stapf), makrut leaf and peel (Citrus hystrix DC.),
sweet basil leaf (O. basilicum L.), and turmeric rhi-
zome (Curcuma longa L.) oils were obtained from
Thai-China ﬂavour & fragrances industry Co., Ltd.
The major compounds of essential oils; (+)-camphor,
(−)-trans-caryophyllene, 1,8-cineole, citral, citronel-
lal, citronellol, eugenol, geraniol, α-humulene, D-lim-
onene, (±)-linalool, methyl chavicol, (−)-α-terpineol,
and terpinen-4-ol were obtained from Sigma-Aldrich
Chemical Co. Inc. (St. Louis, MO). Camphene was
obtained from Carl Roth Co. (Schoemperlenstr, Karl-
sruhe). Methyl cinnamate and methyl eugenol were
obtained from Tokyo Chemical Industry (Tokyo).
Bacterial strains used in this study, E. coli (ATCC
25922), S. Enteritidis (DMST 15676), S. Typhimu-
rium (ATCC 13311), B. cereus (ATCC 11778), S. au-
reus (ATCC 25923), S. ﬂexneri, and S. Typhi were
inoculated and maintained on tryptic soy agar (TSA).
All bacteria were obtained from the Microbiology
Laboratory Culture Collection, Department of Micro-
biology, Faculty of Pharmacy, Mahidol University,
Identiﬁcation of the components in the essential oil
The components of the essential oils were identiﬁed
by GC-MS on GCMS-QP 2010 GC-MS (Shimadzu)
using a DB-5MS bonded phase fused silica capil-
lary column (30 m ×0.25 mm i.d., 0.25 µm ﬁlm
thickness; J&W Scientiﬁc, Folsom, CA). Puriﬁed
helium was used as carrier gas at constant ﬂow rate
of 0.68 ml/min. The oven temperature program for
essential oil was modiﬁed from method of previous
studies9–12. The injector temperature and ion source
temperature were 200 °C and 250 °C, respectively.
Electron impact mass spectra were obtained at 70 eV
by operating in the full scan acquisition mode in
the range of m/z 40–400. The identiﬁcation of the
active compounds were performed by comparing the
obtained mass spectra with those from the Wiley and
NIST spectral library and their retention time and
mass spectral with those of the reference compounds.
Determination of minimum inhibitory
concentration (MIC) and minimum bactericidal
The broth macrodilution method was used to deter-
mine the MICs of oils and reference compounds.
Stock solutions of oils were initially dissolved in
tween 80 and 95% ethanol then a series of two-
fold dilutions of each oil (ranging 200 µg/ml to
1.5625 µg/ml) was prepared in tryptic soy broth
(TSB). Freshly grown bacterial suspensions in TSB
were adjusted to approximately 106CFU/ml. For MIC
determination, a set of 10 assay tubes in duplicate was
employed per sample. The ﬁrst test tube contained
1.8 ml of the seeded broth and the remaining tubes
had 1 ml each. The solution of the test agent (0.2 ml)
was added to the ﬁrst tube to bring the volume to
2 ml. One ml of this was transferred to the next tube
and the process was repeated giving a two-fold serial
dilution. The tubes were incubated at 37 °C for 24 h.
The ﬁrst assay tube with no apparent growth of the
microorganism containing the lowest dilution of the
test essential oil represented the MIC. The bactericidal
effect was assessed by MBC determination. Samples
were removed from tubes that showed no turbidity
and were dropped onto TSA plate. After incubation
at 37 °C for 24 h the minimum concentration without
visible growth was reported as MBC13 .
Determination of the active components by thin
layer chromatography (TLC) bioautography
The TLC bioautographic method was used to detect
the active components. After application of oils and
reference standards on three silica gel G60 F254 alu-
minium plates (A, B, C), the plates were developed us-
ing toluene-ethyl acetate (93:7) as the solvent. Plate A
was visualized under UV light at 254 nm and sprayed
with anisaldehyde sulphuric acid. Then, it was heated
at 110 °C for 5 min to detect the separated components
and used as reference chromatogram. Plate B was
prepared for the isolation of active compounds of
The TLC bioautography method was modiﬁed
from the method of Chomnawang et al14 . The TLC
plate (C) was developed as described above. The
TLC plate (C) was placed on the top of agar base
474 ScienceAsia 39 (2013)
and inocula of bacterial strains in TSA media (agar
seed) was distributed over TLC plate (C). The plate
(C) was incubated at 37 °C for 24 h. The active
compounds were identiﬁed by the spots within the
inhibition zones. Rf of the corresponding spots were
compared with Rf of reference standard on plate A.
Plate B was compared with plates A and C. The areas
on the TLC plate which corresponded to the inhibition
zones were scraped from the plate and the substances
were eluted from the adsorbent with hexane. Eluted
samples were ﬁltered with ﬁlter paper (Whatman
No 1), concentrated by removing the solvent under
vacuum. Then, the active components were conﬁrmed
by gas chromatography-mass spectrometry (GC-MS).
RESULTS AND DISCUSSION
GC analysis of the components of the essential
oils from Thai spices revealed that the major com-
ponents of black pepper, ﬁngerroot, galanga, holy
basil, lemongrass, makrut leaf and peel, sweet basil,
and turmeric oils, were δ-3-carene, trans-β-ocimene,
1,8-cineole, eugenol, E-citral, β-citronellal, limonene,
methyl chavicol, and ar-tumerone, respectively
(Table 1). The antibacterial testing against foodborne
pathogens showed that seven essential oils of Thai
spices exhibited the activity with MIC lower than
125 µg/ml (Table 1). In this study, the cut off limit was
set up as recommended by UNIDO for commercially
potential agents, which were 125 µg/ml and 10 µg/ml
for crude extract and pure compound13, respectively.
Since lemongrass oil has been well known to contain
citral as active component, it was used as a positive
control in this study. As a result of the activity
against the tested bacteria, none of the tested oils
were comparable to lemongrass oil. However, among
seven essential oils, holy basil and sweet basil oils
showed the widest spectrum (6 strains) followed by
makrut leaf and ﬁngerroot oils (4 strains), galanga,
makrut peel, and turmeric oils (1 strain), in that order
(Table 1). The holy basil, makrut leaf, and sweet
basil oils also showed the highest potency (25 µg/ml).
The results suggest that besides lemongrass oil, sweet
basil oil showed the highest potential for commercial
products followed by holy basil and makrut leaf oils.
Some of our results were in agreement with previous
Preliminary identiﬁcation by TLC autobiography
found positive bands on TLC chromatogram of each
oil (Table 1). However, the identiﬁcation by TLC
was limited. The large amount of the oil required to
produce antibacterial activity resulted in overlapping
of the adjacent components and prevented a precise
identiﬁcation. Hence the identiﬁcation was conﬁrmed
by GC-MS. Most of the active components were
found to be the major constituents of essential oils.
The major active components of ﬁngerroot, galanga,
holy basil, makrut leaf, makrut peel, sweet basil, and
turmeric oils were 1,8-cineole/camphor, 1,8-cineole,
eugenol, citronellal, terpinen-4-ol/α-terpineol, methyl
chavicol, and ar-tumerone, respectively. All other
active components are shown in Table 1. The at-
tempt to isolate active components by preparative
TLC was not successful due to the instability of each
component. Hence all the commercially available
active compounds were used and subjected to an-
tibacterial testing and all of the compounds showed
the MICs over 50 µg/ml which is over the cut-off
limit recommended by UNIDO13. Thus the oils are
recommended for product development rather than the
Some of the antibacterial components found
in Thai spices were reported to be active including
trans-α-bergamotene, camphor, trans-caryophyllene,
1,8-cineole, citronellal, eugenol, α-humulene,
(±)-linalool, methyl eugenol, methyl chavicol, neral,
α-terpineol, and terpinen-4-ol 15–21 . In this study,
new active components against foodborne pathogen,
citronellol, ar-curcumene, β-farnesene, cis-farnesol,
trans-farnesol, and turmerone, were found. None of
these active compounds showed a potency comparable
to that of citral (5 µg/ml). They exhibited the activity
with MICs over the UNIDO cut off limit (10 µg/ml)13 .
Based on the results, the oils were more appropriate
than active components for product development.
The potential role of essential oil as food preser-
vatives has been recognized but the concentration nec-
essary to inhibit foodborne pathogens is high which
leads to the undesirable organoleptic effect22 . Only
few articles reported on the use of essential oil in
the washing solution for decontamination of fresh
vegetable3,4,15 . Based on the antibacterial activity
and the ﬂavour of the essential oils, lemongrass, sweet
basil, holy basil, and makrut leaf oils were suggested
to be used in washing solution for fresh vegetables.
In this study, sweet basil oil contained methyl
chavicol (estragol) as active components against
S. ﬂexneri,S. Typhi, and S. aureus which was in agree-
ment with previous study15. Methyl chavicol has been
reported to exhibit weaker activity against Shigella
spp. than carvacrol and thymol which is probably
due to the substitution of phenolic hydroxyl group
with methyl group15. However, Thai sweet basil oil
which contained 90% methyl chavicol showed the
inhibitory activity against E. coli with the MIC of
25 µg/ml. Hence it was considered to be potential for
the vegetable washing products.
ScienceAsia 39 (2013) 475
Table 1 The constituents, minimum inhibitory concentration (MIC), maximum bactericidal concentration (MBC), and
active components of essential oils from Thai spices identiﬁed by TLC-bioautography and GC-MS.
Essential oil Constituents MIC MBC Bacterial Active Identiﬁcation by TLC and GC-MS
(µg/ml) (µg/ml) strainsaband
Black pepper δ-3-carene (37%), >200 >200 All 7 bacteria ND ND
Fingerroot trans-β-ocimene (27%), 100 200 B. cereus 3 geraniol, α-terpineol, 1,8-cineole, camphor,
camphor (24%), neral, and unidentiﬁedb
1,8-cineole (17%), 100 100 S. Typhi 5 geraniol, α-terpineol, (±)-linalool,
geraniol (11%), 1,8-cineole, camphor, neral, and unidentiﬁedb
camphene (8%), 100 >200 S. ﬂexneri 3 geraniol, α-terpineol, (±)-linalool, and unidentiﬁedb
cis-ocimene (3%), 100 >200 E. coli 2 geraniol, α-terpineol, and unidentiﬁedb
Galanga 1,8-cineole (34%) 50 50 B. cereus 6 1,8-cineole,tau-muurolol, and unidentiﬁedb
Holy basil eugenol (42%), 25 50 B. cereus 2 eugenol, methyl eugenol, and unidentiﬁedb
caryophyllene (26%), 50 >200 S. aureus 3 eugenol, methyl eugenol, and unidentiﬁedb
methyl eugenol (15%), 50 100 S. Typhi 1 eugenol, methyl eugenol
(−)-β-elememe (12%) 100 >200 S. Typhimurium 2 eugenol, methyl eugenol, and unidentiﬁedb
100 200 S. ﬂexneri 2 eugenol, methyl eugenol, and unidentiﬁedb
100 200 E. coli 2 eugenol, methyl eugenol, and unidentiﬁedb
Makrut leaf β-citronellal (78%), 25 25 B. cereus 5 citronellol, cis-farnesol, trans-farnesol, citronellal,
citronellyl acetate (6%), and unidentiﬁedb
β-citronellol (5%) 50 50 S. Typhi 4 citronellol, cis-farnesol, citronellal, and unidentiﬁedb
50 >200 S. Typhimurium 1 unidentiﬁedb
50 >200 S. ﬂexneri 5 citronellol, cis-farnesol, citronellal, and unidentiﬁedb
Makrut peel limonene (46%), 100 100 S. Typhi 3 α-terpineol, terpinen-4-ol, and unidentiﬁedb
Sweet basil methyl chavicol (90%), 50 100 B. cereus 2 2 unidentiﬁedb
α-bergamotene (3%) 100 >200 S. aureus 4 methyl chavicol and 3 unidentiﬁedb
50 50 S. Typhi 3 methyl chavicol and 2 unidentiﬁedb
50 >200 S. Typhimurium 1 unidentiﬁedb
25 50 S. ﬂexneri 3 methyl chavicol and 2 unidentiﬁedb
25 50 E. coli 2 2 unidentiﬁedb
Turmeric Ar-tumerone (45%), 50 >200 S. Typhi 2 tumerone, Ar-curcumene, and trans-caryophyllene
Lemongrass E-citral (53%), 25 25 S. aureus 1 citral
Z-citral (38%), 12.5 12.5 B. cereus 1 citral
geraniol (4%) 100 100 S. Typhi 1 citral
*Positive control 100 100 S. Typhimurium 1 citral
50 50 S. ﬂexneri 1 citral
100 100 S. Enteritidis 1 citral
50 50 E. coli 1 citral
aThe bacterial strains not present in this table exhibited MIC and MBC of oils higher than 200 µg/ml.
bLack of reference compound.
ND: Not determined.
Holy basil, one of the active oils contained
eugenol (42%) and methyl eugenol (15%) which was
found to be active against S. Typhimurium, S. Ty-
phi, S. ﬂexneri,E. coli,S. aureus, and B. cereus.
The results from this study supported previous report
which indicated that eugenol was active. Eugenol
exerted the activity not only at the membrane but also
inhibited the production of amylase and protease by
organisms21,23. However, the unpleasant ﬂavour of
eugenol may not be accepted by consumers. Another
potential oil, makrut leaf oil contained different active
compounds. The major active components were cit-
ronellal (78%) and citronellol (5%). The activities of
citronellal and citronellol were due to aldehyde and
alcohol functional groups, respectively22 .
In conclusion, four essential oils of Thai spices,
lemongrass, holy basil, makrut leaf, and sweet basil
oils showed the inhibitory activity against foodborne
pathogens with the MICs 25–100 µg/ml. The major
active components were identiﬁed along with other
476 ScienceAsia 39 (2013)
components using TLC-bioautography and GC-MS
techniques. Since most of the components are less
active than the whole oils, it is recommended to
use the whole oil for vegetable washing solution.
This study also identiﬁed the new antibacterial agents
against foodborne pathogens including citronellol, ar-
curcumene, and turmerone.
Acknowledgements:The authors are grateful to the Co-
ordinating Centre for Research and Development to increase
value of the Plants Indigenous to Thailand, Mahidol Univer-
sity and the Thailand Research Fund for ﬁnancial support.
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