Biology Reference
In-Depth Information
The MeOH extracts were re-dissolved
with MeOH/H
2
O (6:4) and then these solu-
tions were deposited in a decantation fun-
nel. Twenty extractions were made, each
one with 150 ml of hexane. The hexane
phase was gathered and concentrated at
reduced pressure. The process was repeated
with ethyl acetate, subsequently leaving a
residue MeOH/water (Fig. 15.2).
of each component extracted per gram of
dried extract of
Calceolaria
.
15.4.4. Antifeedant test
against
Spodoptera frugiperda
The antifeedant activity of the extracts was
evaluated against
Spodoptera frugiperda
larvae according to the methodology used
by Valladares
et al.
(1997). Briefly, one larva
per Petri dish was deposited with two let-
tuce circles of 1 cm
2
, one sprayed with 10 ml
of extract dissolved in ethanol at a 100 ppm
concentration, and the other sprayed with
the same quantity of ethanol. Under the
lettuce, a round piece of paper towel mois-
tened with distilled water was placed to
reduce the dehydration of the plant material.
Ten repetitions were made for each extract
and ten repetitions for the
n-
hexane extract
in each one of the concentrations used. The
measurements were made for 24 h, estab-
lishing the percentage of consumed area
(estimated visually through the use of a grid),
and the antifeedant index rate was calcu-
lated, AI% = [(1−T/C) × 100], T being the
average food consumption treated with
extract and C the equivalent in the controls.
Once the fractions that had a higher AI%
were determined, a gradient was made in
the concentrations of these extracts and its
effect was measured for 24 h.
15.4.3
Determination of the total
phenolic content
The content of total phenolic compounds
was measured using a method described pre-
viously by Céspedes
et al.
(2010). An aliquot
(1 ml) of the proper diluted extract was added
to 1 ml of half diluted Folin-Ciocalteu rea-
gent (Sigma-Aldrich, Santiago, Chile). To
this was added 2 ml of 20% Na
2
CO
3
10 min
later. After 5 min the absorbance was meas-
ured at 730 nm with a UV2310 Techcomp
multichannel spectrophotometer. Add-
itionally, 10 ml of sample or standard (10-100
mM catechin) plus 150 ml of diluted Folin-
Ciocalteu reagent (1:4 reagent/water) was
placed in each well of a 96-well plate and
incubated at room temperature for 3 min.
Following the addition of 50 ml of sodium
carbonate (2:3 saturated sodium carbonate/
water) and a further incubation of 2 h at room
temperature, absorbance was read at 725 nm
with a BIOTEK Epoch microplate spectro-
photometer equipped with Gen5
™
microplate
data analysis software. Results are expressed
as micromoles of Cat E per gram using cate-
chin as the standard (Dominguez
et al.
, 2005).
All tests were conducted in triplicate.
Yield of verbascoside and total phenolic
compounds were expressed as the amount
15.4.5 Insecticidal bioassay
against
S. frugiperda
The
S. frugiperda
(J.E. Smith) larvae used
for this experiment were maintained under
Extraction of the aerial part (methanol)/concentrated
Methanol re-dissolved
MeOH/H
2
O (6:4)
Hexane partition
Ethyl acetate
partition
MeOH/H
2
O
residue
Fig. 15.2.
Method of obtaining extracts, partitions and fractions.
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