Fumigant Toxicity of Nutmeg Seed Essential Oil ( Myristica fragrans Houtt.) ... 129
spice can be toxic when ingested in large quanti-
ties (1–3 nutmegs) (Forrest and Heacock 1972 ).
The utility of nutmeg as a spice has been
known since ancient times in Indonesia. Nutmeg
has aromatic, stimulant, narcotic, carminative,
astringent, aphrodisiac, hypolipidaemic, anti-
thrombotic, antiplatelet aggregation, antifungal,
antidysenteric, and anti-inflammatory activities.
The spice is used as a remedy for stomach ache,
rheumatism, and vomiting during pregnancy
(Olajide et al. 1999 ; Sonavane et al. 2002 ). This
chapter describes a laboratory study to assess the
potential fumigant effects of essential oils ex-
tracted from the fruits of M. fragrans on adults of
Callosobruchus maculatus.
Material and Methods
Cowpea ( Vigna unguiculata) Blackeye, a variety
susceptible to C. maculatus (Baker et al. 1989 ),
was stored in a freezer at 18 °C for a week and
subsequently dried in a stove at 60 °C for about
a week to guarantee the absence of viable insects
without having to use chemicals. The beans were
stored in airtight plastic containers at room tem-
perature before use. Only visually uninfested
beans were used for the experiments.
C. maculatus was collected in Urmia on local
cowpea. The beetles were reared on cowpea in
laboratory for a year (≈ 12 generations) prior to
the experiments. The rearing was done in a cli-
mate chamber at 30 ± 1 °C with a 12-h photope-
riod and 50–80 % relative humidity (RH) For the
tests, newly emerged (1–1.5 h) insects were used.
For repellence tests, female beetles were used
that had been kept for 1 h with a surplus of newly
emerged males and were assumed to have mated.
In the experiments, the day of death of the
adults was determined as the day the antennae
and legs did not move upon gentle disturbance
with forceps. Unhatched eggs recognized by the
color of the egg. In the cowpea variety that pro-
duced big yellowish seeds, the tunnel that is dug
by the developing larva could be seen a few days
before emergence of the beetle as a bluish spot
AQ2AQ3AQ4under the seed coat. From the size and the clear-
ness of this spot, the stage of development could
be estimated, but further investigation was avoid-
ed to keep the beans intact. If no beetles emerged
from the beans for 5 days, the larvae were con-
sidered dead.Plant Materials
Plants were collected and dried in Urmia (North-
west Iran). The plant samples were stored in plas-
tic bags (1 L volumes) in the dark at 4 °C. Shortly
before use, after warming up to room tempera-
ture, the dry plant material was isolated.Isolation and Extraction of Essential Oil
The essential oil of nutmeg seeds was isolated
with a yield of 6.85 % w/w. The essential oils
were extracted by hydrodistillation of dried plant
material (100 g of each sample in 500 ml of dis-
tilled water) using a modified Clevenger-type
apparatus for 4 h. The oils were dried over anhy-
drous sodium sulfate and stored in sealed glass
vials at 4–5 °C prior to analysis. Yields were av-
eraged over four experiments and calculated ac-
cording to dry weight of the plant materials.Gas Chromatography Mass
Spectrometry (GC/MS) Analysis
Measurements were performed using a QP-
5050A (Shimadzu) gas chromatograph coupled
to a VG autospect mass spectrometer at 70 eV,
40–550 amu with a fused silica capillary column
(DB-5MS, 30 m × 0.25 mm) using helium as a
carrier gas and with temperature programming
from 60 °C/5 min to 300 °C/2 min (10 °C/min) for
essential oils (Adams, 1995 ). The MS was oper-
ated using an interface temperature of 240 °C,
and an electron impact ionization of 70 eV with
a scan mass range of 40–350 m/z (sampling rate
of 1.0 scan/s).