Page 8 - Volume 4, Issue 2, July 2011

© Benaki Phytopathological Institute
Michaelakis et al. 36
to adults, while menthone, thymol and car-
vacrol against Culex species for larvicidal ac-
tivity (20, 23). These substances are known
active ingredients not only of Mentha species
essential oils but also of many other plants, in
which their concentrations vary (7).
Main objectives of this study were: a) to
evaluate the efficacy of essential oils derived
from three Mentha species, M. pulegium
(three populations), M. piperita and M. spi-
cata (spearmint) against Cx. pipiens larvae,
b) to isolate and evaluate essential oil major
components, c) to correlate larvicidal activi-
ty of isolated components with their precur-
sor oils and d) to study the structure activity
relationships for the isolated molecules.
Materials and Methods
Plant Materials
Three different species of the genus Men-
tha were used: M. pulegium (pennyroyal), M.
piperita (mint) and M. spicata (spearmint). All
plant materials were collected from various
natural habitats in Greece by members of
our team, between July and August 2007 in
the flowering stage. Specifically, in the case
of M. pulegium samples were collected from
three populations according to their geo-
graphical origin, the first one from Orestia-
da (North-East of Greece, named as PUL1),
the second from Karditsa (center of Greece,
PUL2) and the third fromHeraklion (Island of
Crete, PUL3). Samples of M. spicata (SP) and
M. piperita (PIP) were purchased from local
farmers from the region of Karditsa. All five
plant materials were air dried and stocked
for further use.
Chemicals
R-(+)-pulegone,terpinen-4-ol, β -farnesene,
α-pinene, β-pinene, mesityl oxide, meth-
yl vinyl ketone, menthol and menthone
were purchased from Aldrich (Steinheim,
Germany). Eucalyptol, β -myrcene, S -(-)-li-
monene, γ -terpinene, thymol, carvacrol and
β -caryophyllene were purchased from Sig-
ma (St. Louis, USA). Carvonewas bought from
Fluka (Steinheim, Germany). 3-methyl cyclo-
hexanone were bought from Jansen Chimi-
ca (Beerse, Belgium). Terpinolene, iso -men-
thone and piperitone were purchased from
Extra Synthese (Genay, France). Diethyl ether
(BHT free) was purchased from SDS (Cedex,
France). Pentane was bought from Lab-Scan
(Doublin, Ireland). Piperitenone was pre-
pared according to literature synthetic pro-
cedure (5), due to commercial unavailability.
Purity of the isolated product (97.3%) was es-
timated according to GC-MS analytical con-
ditions given bellow. Structural characteriza-
tion that was accomplished by mass spectral
analysis and NMR experiments was in agree-
ment to literature data (5). NMR spectra were
recorded on Brucer Avance DRX-500 instru-
ment. Potassium hydroxide, tetrahydrofuran
(THF), silica gel 60G and TLC plates (silica gel
60, F254) were purchased fromMerck (Darm-
stadt, Germany).
Isolation of the Essential Oils
Aerial parts from PUL1, PUL2, PUL3,
SP and PIP were powdered in an electrical
blender and 1 Kg of each sample submit-
ted to hydrodistillation for 4 h in a Cleveng-
er-type apparatus. The obtained essential
oils, named as EOpul1, EOpul2, EOpul3, EOsp
and EOpip respectively, were dried over an-
hydrous magnesium sulfate. After filtration
their volume were calculated and expressed
as ml of essential oil/100 g of dry material
(Table 1) and finally stored in labeled sterile
screw capped bottles at -22o C until use.
Gas chromatography – Mass spectrome-
try (GC-MS) analysis
The essential oils were analyzed using a
Hewlett Packard II 5890 gas chromatogra-
phy (GC) system, equipped with a FID de-
tector and HP-5ms capillary column (30 m
x 0.25 mm, film thickness 0.25 μm). Injector
and detector temperatures were set at 220o C
and 290 oC, respectively. GC oven tempera-
ture was programmed from 60oC to 240 oC at
a rate of 3o C /min and held isothermally for
10 min. Helium was the carrier gas at a flow
rate of 1 ml/min. Diluted samples (1/100 in
diethyl ether, mgl-1) of 1.0 μl were injected
manually and in the splitless mode. Quan-