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SUPPLEMENTARY MATERIAL
Chemical composition and content of essential oil from aerial parts of
Teucrium flavum L. subsp. flavum growing spontaneously in Tunisia
Saoussen Hammamia*, Ridha El Moknib, Khaled Faidia, Danilo
Falconieric,d, Alessandra Pirasd, Silvia Proceddad , Zine Mighria and
Mohamed Hédi El Aounib
a
Research Unit 13ES63, Applied Chemistry and Environment, Faculty of Sciences,
5000 Monastir, TUNISIA
b
Laboratory of Botany and plant Ecology, Faculty of Sciences of Bizerta, 7021,
Jarzouna, Bizerta, TUNISIA.
c
Industrial Technical Institute "Michele Giua", Via Montecassino 09134, Cagliari,
ITALY
d
Department of Chemical and Geological Science, University of Cagliari, Cittadella
Universitaria di Monserrato, S.P. Monserrato-Sestu km 0,700 - 09042, Monserrato
(CA), ITALY
*
Corresponding author: saoussenhammami@voila.fr
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Abstract
The objectives of this study were to chemically characterize and evaluate the
antioxidant potential of the essential oil of Teucrium flavum L. subsp. flavum growing
spontaneously in Tunisia. The volatile oil was extracted by hydrodistillation of the
aerial parts in a Clevenger type apparatus. 40 constituents were identified via GC and
GC-MS analysis. β-caryophyllene (32.5 %) and α-humulene (17.8 %) were the most
abundant components. The evaluation of free radical scavenging activity using stable
DPPH free radical showed that the volatile oil exhibit a moderate antioxidant activity
and reduce DPPH to 50% at EC50 value of 1230 µg.mL-1.
Key words: Teucrium flavum L. subsp. flavum, Lamiaceae, essential oil, aerial parts,
GC/MS, β-caryophyllene, α-humulene, antioxidant effects.
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Experimental
Plant material
Teucrium flavum species was collected during the flowering stage from North of
Tunisia (Mogods region) and identified by Ridha El Mokni, a botanist in the
Laboratory of Botany and Plant Ecology, Faculty of Sciences of Bizerta, Jarzouna,
Bizerta-Tunisia, where Voucher specimens have been deposited [LAM/71T.fl.fl/010].
Hydrodistillation
Hydrodistillation (HD) was performed for 3 h in a circulatory Clevenger-type
apparatus. The sample thus obtained, was dried over anhydrous sodium sulfate
yielding 0.1% of yellow oil (w/w) with a pleasant smell.
GC-FID and GC-MS analysis
Analysis of the volatile extracts were carried out by gas chromatography (GC) and by
gas chromatography-mass spectrometry (GC-MS).
Analytical GC was carried out in a gas chromatograph (Agilent, Model 7890A, Palo
Alto, CA), equipped with a flame ionization detector (FID), an autosampler (Agilent,
Model 7683B), Agilent HP5 fused silica column (5 % phenyl-methylpolysiloxane), 30
m × 0.25 mm i.d., film thickness 0.25 m, and a Agilent ChemStation software
system. Oven temperature was settled at 60 ºC, raising at 3 ºC min-1 to 246 ºC and
then held 20 min at 246 ºC; injector temperature: 250 ºC; carrier gas: helium at 1.0
mL min-1; splitting ratio 1:10; detectors temperature: 300 ºC.
GC-MS analysis was carried out using a gas chromatograph (Agilent, Model 6890N,
Palo Alto, CA, USA) equipped with a split-splitless injector, an Agilent model 7683
autosampler and an Agilent HP5-MS fused silica column (5 % phenylmethylpolysiloxane, 30 m × 0.25 mm i.d., film thickness 0.25 µm).
The GC
conditions included programmed heating from 60 °C to 246 °C at 3 °C min-1,
followed by 20 min under isothermal conditions. The injector was maintained at 250
°C. Helium was the carrier gas, at 1.0 mL min-1. Samples were run diluted in hexane
with a dilution ratio of 1:100 and (1 µL) were injected in the split mode (1:20). The
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GC was fitted with a quadrupole mass spectrometer with an Agilent model 5973
detector. The MS conditions were as follows: ionization energy, 70 eV; electronic
impact ion source temperature, 200 °C; quadrupole temperature, 150 °C; scan rate, 3.2
scan s-1; mass range, 30 ÷ 480 u. The software that was used to handle and analyse the
mass spectra and chromatograms was an Agilent MSD ChemStation E.01.00.237. The
linear retention indices (RIs) for all of the compounds were determined by injection of
a hexane solution containing the homologous series of C8-C26 n-alkanes (Vand Den
Dool & Kratz 1963). The identification of the essential oil constituents was
accomplished by comparison of their retention indices and their mass spectra with the
literature data and the mass spectra databases, including HPCH2205 (Adams 2007)
and W8N05ST (Wiley ver. 8.0 & NIST, ver. 5.0). The table 1 show the
chromatographic results, expressed as area percentages (GC) calculated without any
response factor.
Antioxidant activity
DPPH radical scavenging assay: 2,2’-diphenyl-1-picrylhydrazyl (DPPH) free radical
assay was carried out to measure the free radical scavenging activity as reported
previously (Yu et al 2008). A volume of 1.0 mL of each ethanol solution from B.
officinalis prepared at different concentrations was mixed with an equal volume of
ethanolic solution of DPPH (0.1mM). The disappearance of the DPPH was measured
after 30 min of incubation at room temperature. The inhibition percentage of the
DPPH radical by the essential oil was calculated according to the formula of Yen and
Duh (Tian et al 2012).
% RSA=[(Acontrol-Asample)/Acontrol] x 100
Where Acontrol is absorbance of the control sample (t=0h)
Asample is the absorbance of a tested sample at the end of the reaction (t=1h).
The essential oil concentration providing 50% inhibition (IC50) was calculated from
the graph plotting percentage of radical scavenging activity (% RSA) against T.
flavum essential oil concentration.
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Adams
RP.
2007.
Identification
of
essential
oil
components
by
gas
chromatography/mass spectroscopy. 4th Ed. Carol stream. Illinois: Allured Publishing
Corporation.
Vand Den Dool H, Kratz PD. 1963. A generalization of the retention index system
including linear temperature programmed gaz-liquid partition chromatography. J.
Chromatogr. 11: 463-471.