2016

NPC Natural Product Communications Vol. 11

No. 11
Detection and Identification of Antibacterial and Antioxidant 1705 - 1708
Components of Essential Oils by TLC-Biodetection and GC-MS
Ágnes M. Móricza,*, Györgyi Horváthb, Andrea Böszörményic and Péter G. Otta
a
Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Herman O. Str. 15,
H-1022 Budapest, Hungary
b
Györgyi Horváth, Department of Pharmacognosy, Faculty of Pharmacy, University of Pécs, Rókus u. 2.,
H-7624 Pécs, Hungary
c
Department of Pharmacognosy, Faculty of Pharmacy, Semmelweis University, Üllői Str. 26, 1085 Budapest,
Hungary9999

moricz.agnes@agrar.mta.hu

Received: January 31st, 2016; Accepted: March 27th, 2016

Dedicated to Prof. Dr. Wilhelm Fleischhacker on account of his 85th Birthday.

Components of cinnamon bark, rosemary, clove and thyme essential oils were screened for antioxidant and antibacterial activity utilizing thin-layer
chromatography (TLC) coupled with the DPPH• test and direct bioautography using Bacillus subtilis cells. The compounds in the active chromatographic
zones were identified by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) after their elution. Seven antibacterial
components were found: cinnamaldehyde and eugenol in cinnamon bark oil, 1,8-cineole, camphor, borneol and α-terpineol in rosemary oil, eugenol in clove
oil and thymol in thyme oil. Only two of them, thymol and eugenol displayed a free radical scavenging effect.

Keywords: Antibacterial and antioxidant activity, Essential oils, Thin-layer chromatography-direct bioautography, Thin-layer chromatography-DPPH• test,
Rosemary, Clove, Thyme, Cinnamon.

Essential oils (EOs), a mixture of volatile compounds (mainly Table 1: The percentage composition of the investigated EOs.
mono- and sesquiterpenoids, and phenylpropanoids) obtained by No. tR MS tR FID Compound Cinnamon Rosemary Clove Thyme
(min) (min) (Cn) (Rs) (Cl) (Th)
steam distillation, possess several types of biological activities, e.g.
1 13.2 15.6 Thymol - - - 46.3
analeptic, antimicrobial, anticancer, antinociceptive, antiphlogistic, 2 14.3 15.8 Eugenol 2.7 - 88.6 -
antioxidative, and sedative [1]. Their strong antimicrobial activity is 3 11.0 12.4 Borneol - 7.4 - 1.0
4 8.1 8.0 1,8-Cineole 2.1 45.6 - 9.8
mainly attributed to oxygenated terpenoids [2]. Because of their 5 12.9 14.9 trans- 74.0 - - -
pleasant odor or flavor, they are widely used in the food, fragrance, Cinnamaldehyde
and pharmaceutical industries. 6 11.4 12.6 α-Terpineol - 4.4 - -
7 10.5 11.5 Camphor - 23.2 - -
8 5.9 5.8 α-Pinene 0.5 4.0 - 0.9
Ceylon cinnamon bark oil (Cn) is obtained from the bark of the 9 6.3 6.2 Camphene - 2.4 - 0.9
shoots of Cinnamomum zeylanicum Nees (C. verum J.S. Presl.) and 10 6.9 6.7 β-Pinene - 4.0 - 1.4
11 7.7 7.2 α-Terpinene - - - 2.2
traditionally used for the symptomatic treatment of mild, spasmodic 12 8.0 7.45 Limonene 1.4 - - -
gastrointestinal complaints including bloating and flatulence [3]. 13 7.9 7.6 p-Cymene 1.2 4.5 - 22.1
14 8.6 8.5 γ-Terpinene - - - 0.3
Clove oil (Cl) can be isolated from the dried flower buds of 15 9.5 10.1 Linalool 3.8 - - 5.1
Syzygium aromaticum (L.) Merill et L. M. Perry. Its therapeutic 16 11.5 11.7 Terpinen-4-ol - - - 0.6
indication includes the symptomatic treatment of minor 27 13.0 13.1 Bornyl acetate - 2.3 - -
18 13.1 13.5 Anethol 2.3 - - -
inflammation in the mouth or the throat. Moreover, this is a 19 15.4 14.9 β-Caryophyllene - 1.2 8.6 2.5
traditional herbal product for the temporary relief of toothache due 20 16.0 15.5 α-Humulene - - 2.2 -
to a dental cavity [4]. Rosemary oil (Rs) is obtained from the 21 13.3 15.9 Carvacrol - - - 3.2
22 15.8 16.9 Cinnamyl 5.3 - - -
flowering aerial parts of Rosmarinus officinalis L. It can be used in acetate
the symptomatic relief of dyspepsia and mild spasmodic disorders 23 18.0 18.9 β-Caryophyllene - 0.2 0.5 -
oxide
of the gastrointestinal tract. In another indication, it is an adjuvant Total: 93.3 99.2 99.9 96.3
in the relief of minor muscular and articular pain and in minor
peripheral circulatory disorders [5]. Thyme oil (Th) can be distilled
combined with biological detection [7,8] is a high-throughput, easy-
from the fresh flowering aerial parts of Thymus vulgaris L. or T.
to-perform and relatively cheap method suitable for the detection of
zygis Loefl. ex L. or their mixture. Th with aromatic and spicy odor
antimicrobial activity of components with the desired effect in
is a traditionally used expectorant in cough associated with cold [6].
various matrices, such as EOs [9-11].
The conventional microbiological assays (e.g. disc diffusion, agar
absorption) are not appropriate for the antimicrobial testing of EOs Compared with other commonly used bio-tests its further advantage
due to their viscous, volatile, and non-water soluble characters. is to provide information about the bioactivity as well as
Therefore, it is highly important to develop standardized methods chromatographic property of the individual, separated compounds.
producing reliable results. Thin-layer chromatography (TLC) Therefore, it can be regarded as a bio-monitoring step underlying an
1706 Natural Product Communications Vol. 11 (11) 2016
Figure 2: Bioactive components in some EOs after chromatography with n-hexane–
isopropyl acetate 88:12 (v/v); A –TLC plate under UV 254, B and C – result with
vanillin–sulfuric acid reagent documented in visible light and under UV 365 nm,
respectively, D – TLC-DPPH• assay (visualization of antioxidant components as clear
zones), E – bioautograms using B. subtilis (bright zones indicate antibacterial effects);
Th- thyme oil (5 mg/mL); Cl- clove oil (10 mg/mL); Rs- rosemary oil (20 mg/mL); Cn-
cinnamon bark oil (10 mg/mL); 1- thymol (2 mg/mL); 2- eugenol (1 mg/mL); 3-
borneol (1 mg/mL); 4- 1,8-cineole (10 mg/mL); 5- trans-cinnamaldehyde (10 mg/mL);
6- α-terpineol; 7- camphor.
effect-directed isolation and/or identification system; the
identification needs further spectroscopic/spectrometric analyses
[9,12,13]. Direct bioautography [14,15] is the combination of TLC
and an antimicrobial test that occurs in situ in the adsorbent layer.
Móricz et al.
During investigation the developed layer is soaked in a cell
suspension and put into a humid chamber to create appropriate
conditions for biological functions on the surface of the plate. Such
a prepared bioautogram can be visualized e.g. by a tetrazolium vital
dye solution; the lack of the formation of a characteristic dark color
shows the presence of antimicrobial compounds.
The antioxidant (free radical scavenging) activity is in turn obtained
by the use of a chemical, a relatively stable free radical DPPH•. The
purple DPPH• is neutralized, thereby decolorized by electrons or
hydrogen atom transfers. As a result, the antioxidants in the
developed layer dipped into DPPH• solution appear as bright spots
against a purple background [16,17].
Currently, several research groups are focusing on the antibacterial
and/or antioxidant activities of EOs used in this study, but mostly
food-borne pathogens have been used in these investigations
[18,19]. Moreover, these experiments generally cannot provide
parallel information about the biological activities of the EOs and
their individual compounds.
In this study the aim was the detection and identification of
antioxidant and antibacterial compounds in cinnamon bark,
rosemary, clove and thyme EOs. For this purpose the TLC-DPPH•
test, TLC-Bacillus subtilis assay, gas chromatography-flame
ionization detector (FID), GC-mass spectrometry (MS) and solid-
phase microextraction (SPME)-GC-MS were utilized.
The chemical compositions of the EOs and the percentage of the
components were determined by GC-MS and GC-FID, respectively
(Table 1). The identification of the compounds was carried out by
comparing retention times and recorded spectra with the data of
authentic standards, and the NIST 02 library was also consulted.
trans-Cinnamaldehyde (73.2%) was the main component of the EO
of Cn, and 1,8-cineole (eucalyptol, 45.6%), eugenol (83.7%) and
thymol (49.9%) of Rs, Cl and Th, respectively. Apart from the main
components, some other high-abundance compounds were found in
each oil, such as p-cymene, camphor, borneol and β-caryophyllene.
The majority of the examined EO components were efficiently
separated by TLC (Figure 1A-C), except trans-cinnamaldehyde (5,
at RF 0.41) and eugenol (2, at RF 0.43). Apart from this co-
migration the developed TLC method provided an appropriate base
for further characterization of the individual components in situ in
the adsorbent layer with regard to their biological activity, namely
antioxidant and antibacterial.
The free radical scavengers were detected with the DPPH• test.
Clear chromatographic zones against a violet background appeared
in the tracks of three oils (Th, Cl and Cn) that indicate the presence
of a strong antioxidant (Figure 1D). These bio-effects corresponded
to two molecules: thymol (1) in Th and eugenol (2) in Cn and Cl.
Testing the eugenol and trans-cinnamaldehyde standards, it is
clearly observable that only eugenol showed antioxidant activity,
which was detected in the Cn oil (Table 1), where its presence
(together with trans-cinnamaldehyde) in the active spot was
confirmed by SPME-GC-MS after scraping off and eluting with
ethanol.
In Figure 1E the bright zones indicate inhibitory effects of EO
compounds against B. subtilis. The main components of Th and Cl,
the antioxidant thymol (1) and eugenol (2), and none of their minor
components, gave characteristic inhibition zones. In Cn there is also
one inhibition zone, but this chromatographic spot contains two
compounds, eugenol (2) and trans-cinnamaldehyde (5), which both
Antibacterial and antioxidant components of essential oils
exhibited an antibacterial effect. Four characteristic inhibition zones
were observed in Rs. Two of them corresponded to the investigated
standards: its main component 1,8-cineole (4) and borneol (3). The
compounds from the other two zones were eluted and identified by
SPME-GC-MS as α-terpineol (6) and camphor (7).
Planar chromatography hyphenations were successfully employed
for separation, detection and effect-directed isolation of antioxidant
and antibacterial components of Th, Cl, Rs and Cn. The
antibacterial effect determined with TLC-direct bioautography
using B. subtilis as test organism was attributed to the presence of
seven compounds that were identified by SPME-GC-MS. Two of
them showed adequate antioxidant activity in the TLC-DPPH• test
as well. Although the antioxidant activity of the main compounds in
the EOs used in our study is well-known, our study is the first to
prove their activities in a TLC-based system.
Experimental
Materials: For separation, 20 cm x 10 cm aluminum foil- or glass-
backed normal particle silica gel 60 F254 (both from Merck,
Darmstadt, Germany) plates were used. All solvents used were of
analytical grade from Reanal (Budapest, Hungary). Test substances
and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•)
were from Sigma–Aldrich (Budapest, Hungary) and dissolved in
ethanol. Dye reagent MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) was from Carl Roth GmbH
(Karlsruhe, Germany). EOs of clove (Syzygium aromaticum (L.)
Merr. et Perry), cinnamon bark (Cinnamomum zeylanicum Nees.),
thyme (Thymus vulgaris L.) and rosemary (Rosmarinus officinalis
L.) were obtained from Aromax Zrt. (Budapest, Hungary). Tryptone
(Microtrade, Budapest, Hungary), yeast extract (Scharlau,
Barcelona, Spain) and NaCl (Reanal) were purchased for cell media
preparation.
Chromatography: The sample was applied as an 8 mm band to the
layer with a Linomat IV (Camag, Muttenz, Switzerland) with an
8 mm distance from the bottom. TLC development was carried out
in unsaturated twin-trough chambers with isopropyl acetate – n-
hexane 88:12 (v/v) up to a migration distance of 85 mm. The
chromatoplates developed were dried by a cold air stream using a
hair-dryer (5 min). The chromatographic spots were visualized
using an UV lamp (λ = 254 and 365 nm) (Camag) and vanillin-
sulfuric acid reagent (40 mg vanillin + 10 mL ethanol + 200 L
concentrated sulfuric acid; the dipped plate was heated to 110oC for
5 min). For isolation of active compounds, 150 µL of EO was
sprayed onto the layer as 170 mm bands and after the development
the interesting zones were scraped off and eluted with 0.5 mL of
ethanol.
TLC-DPPH•-test: For visualization of the separated antioxidants
(free radical scavengers) the TLC plates were dipped into 0.02%
methanol DPPH• solution. Active compounds appeared as bright
yellow zones against a purple background.
TLC-direct bioautography: The bioassay was performed with the
Gram-positive soil bacterium Bacillus subtilis strain F1276 [20]. It
was grown in buffered (sodium phosphate, 10 mM, pH 7.2)
lysogeny broth (LB) without glucose (10 g/L tryptone, 5 g/L yeast
References
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Natural Product Communications Vol. 11 (11) 2016 1707
extract, 10 g/L NaCl) at 28.5C on an orbital shaker at 130 rpm to
reach late exponential phase (OD600 = 1.2).
The developed dried layers were immersed into the cell suspension
and after 2 h incubation in a humid chamber at 28C they were
dipped into an aqueous MTT solution (1 mg/mL) to visualize the
bioautogram. The yellow MTT is reduced to bluish MTT-formazan
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the darker background indicate the presence of antibacterial
components.
GC-FID: A Fisons GC 8000 gas chromatograph (Carlo Erba,
Milan, Italy), equipped with a flame ionization detector (FID) was
employed for the percentage evaluation of the EO samples. A Rt-β-
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GC-MS and SPME-GC-MS conditions: The EO compounds were
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autosampler. The injector temperature was 250°C and the split ratio
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headspace for 10 min at 100°C. Then, the fiber was transferred to
the injector port of the GC-MS and desorbed for 1 min at 250°C.
The SPME fiber was cleaned and conditioned in a Fibre Bakeout
Station (CTC Analytics) using pure nitrogen atmosphere at 250°C
for 15 min after desorption. The mass selective detector was
equipped with a quadrupole mass analyser and was operated in
electron ionization mode at 70 eV in full scan mode (41–500 amu at
3.2 scan/s). The data were evaluated using MSD ChemStation
D.02.00.275 software (Agilent).
Acknowledgments – This work was supported by NKFI 101271
and 104660 grants (previously Hungarian Scientific Research Fund,
OTKA K101271 and PD104660).
1708 Natural Product Communications Vol. 11 (11) 2016 Móricz et al.

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