Supplementary material chemical composition and content of essential oil from aerial parts of Teucrium flavum L. subsp flavum growing spontaneously in Tunisia




Дата канвертавання27.04.2016
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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



aResearch Unit 13ES63, Applied Chemistry and Environment, Faculty of Sciences, 5000 Monastir, TUNISIA

bLaboratory of Botany and plant Ecology, Faculty of Sciences of Bizerta, 7021, Jarzouna, Bizerta, TUNISIA.

cIndustrial Technical Institute "Michele Giua", Via Montecassino  09134, Cagliari, ITALY

dDepartment 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

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.

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/71-T.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 % phenyl-methylpolysiloxane, 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 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.



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.


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