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(5 cm diameter) with 3 g of sterilized wheat
bran, a plug of moistened cotton to preserve
humidity and held at 24.0 ± 1°C with 16:8
L:D photoperiod. The number of larvae that
successfully pupated, as well as the dura-
tion of the pupal stage (in days), were
recorded every 24 h for 30 days (end point
of the experiment) (Céspedes
et al.
, 2005).
of mortality obtained at each concentration of
the samples. Complete statistical analysis was
performed by means of the MicroCal Origin
6.0 statistical and graphs PC program.
15.5
Results and Discussion
15.5.1
Calceolaria talcana
15.4.8
Antioxidant activity
C. talcana
and
C. microphylla
were incor-
porated in our screening programme
designed to discover interesting biological
activities of plants from template regions.
Both species showed relevant insecticidal,
insect-growth-regulatory (IGR) and anti-
oxidant activities. On the basis of this
information, and the high resistance to
insect and pathogen attack exhibited by
these plants, we investigated the IGR acti-
vity of their
n
-hexane, ethyl acetate and
MeOH/aqueous extracts. Extracts of
C. tal-
cana
showed very satisfactory insecticidal
and IGR activities. Some bioassays were per-
formed at concentrations lower than 10 ppm.
In addition to these extracts, gedunin, and the
methanolic extracts from
Cedrela salvadoren-
sis
(Me-Ced),
Myrtillocactus geometrizans
(Me-Myrt) and
Yucca periculosa
(Me-Yuc),
were used as positive controls (Céspedes
et al.,
2000, 2005; Torres
et al.
, 2003).
A milled sample from
C. talcana
was
macerated with MeOH and further parti-
tioned with
n
-hexane, ethyl acetate and
water, respectively. These extracts were used
in a preliminary bioassay trial. Subsequently,
in order to obtain more satisfactory data for
the insecticidal activity, we used concentra-
tions as low as possible so that there was no
effect owing to the toxicity of the samples to
many targets that may not be specific to the
insect but general or broad spectrum for
plants, insects and humans.
The 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical was used in assays to measure the
antioxidant activity of the extracts from
C. tal-
cana
(Mensor
et al.
, 2001). A stock solution of
DPPH was prepared (0.03 g/l of DPPH in
methanol) from which 5 ml were mixed with
100 ml of each one of the extracts of
Calceolaria
(ethyl acetate, methanol,
n
-hexane and aque-
ous) in concentrations from 50 ppm to
300 ppm. The samples of
C. microphylla
did
not show significant antioxidant activity.
Each sample was stored in a dark room
for 30 min at room temperature, and then
the absorbance was measured at 517 nm. The
control corresponded to the measure of the
solvent in which is dissolved each extract
at 517 nm. As a negative control the same
mixture was used as described previously
but without including the extracts and, as a
positive control, quercetin, a-tocopherol
and an ethyl acetate extract from tecu-berry
fruit (
Aristotelia chilensis
; Céspedes,
et al.
,
2010) were used. Both samples' absorbance
was measured at 517 nm.
15.4.9
Statistical analyses
Data shown are average results obtained by
means of five replicates and are presented as
average ± standard errors of the mean (SEM).
Data were subjected to analysis of variance
(ANOVA) with significant differences between
means identified by generalized linear model
(GLM) procedures. Results are given in the
text at p <0.05. Differences between means
were established with a Student-Newman-
Keuls (SNK) test. The LD
50
values were calcu-
lated by PROBIT analysis based on percentage
15.5.2 Verbascoside and total
phenolics
The highest concentration of verbasco-
side was found in the ethyl acetate extract
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