2.3. Extraction of Essential Oils. Thymus essentials oils
were obtained by hydrodistillation at atmospheric pressure
using leaves and small branches of aerial parts of the plants in a
clevenger-type glass apparatus for 3 h according to the method
recommended by the European Pharmacopoeia.10 The amount
of plant material used in these experiments was 30.0 ± 0.1 g,
and the plant/water ratio was kept constant in all cases. Each
experiment was run with three replications, and time was
measured after the falling of the first drop of distillate until
switching off heating and disassembling the apparatus.
Obtained yields were measured gravimetrically on a Mettler
Toledo AB265-S analytical balance.
Supercritical fluid extracts were obtained using 30.0 ± 0.1 g
of plant material and the SFE apparatus previously described by
Gomes et al.11 with minor modifications. Briefly, in the SFE
unit (in-house built equipment), the liquid CO2 (food grade) is
delivered from a standard cylinder, cooled (≈273 K), and
compressed with a high-pressure pump (air-driven piston
pump, model M71, Haskel) to an extraction pressure of
14 MPa. Compressed CO2 is heated up to 40 °C through a
stainless steel coil and enters the extraction cell containing the
plant material which is placed inside an oven with ventilation
and temperature control. This extractor was built from stainless
steel as well and has a capacity for 1 L of sample material.
Extraction time was equal to 3 h, and throughout the course of
the experiments, temperature and pressure variation was ±2 °C
and ±0.1 MPa, respectively. In this unit two different types of
SFE extracts were obtained using two independent separators
placed in series for the depressurization step. In this way, the
heaviest components were collected in the first separator
(working at 4 MPa) while the volatiles were collected in the
second separator (0.2 MPa). Both separators were cooled at
−15 °C using a thermostatic bath and an aqueous ethylene
glycol solution (50%) as thermal fluid to facilitate the retention
of the extracts. However, whenever the recovered quantities
were very small, it was necessary to use a solvent trap (hexane)
to recover the extract in the separator. After each experiment,
both tubing and valves were washed with hexane to collect the
extract that eventually had condensed there, and it was mixed
with the trapping solvent when used. Then, solvent was
distilled under vacuum to preconcentrate the extracted volatiles.
Finally, essential oils were stored in glass vials with Teflonsealed
caps and kept in the dark and cold (+4 °C) prior to
chemical analysis.