Biology Reference
In-Depth Information
Argentina in October 1998. The plant was
identified by Dr Juan de Dios Muñoz,
keeping a voucher specimen - Muñoz 1514
(ERA) - in the Herbarium of the School of
Agricultural Sciences, National University
of Entre Ríos, Paraná City, Argentina.
Specimens were also collected in Cerro
Azul, Experimental Station of the National
Institute of Agricultural Technology (INTA),
J. Urdampilleta, L. N. Alem Department, in
the Province of Misiones, Argentina, on 14
April 2002. This vegetal material was identi-
fied by Dr Aníbal Amat, and a voucher speci-
men is kept in the Herbarium of the Pharmacy
Department of the School of Exact, Chemical
and Natural Sciences (MNEF 3980).
The method employed for obtaining and
purifying the extracts was described by
Claeson
et al
. (1998) with the aim of obtain-
ing peptide-enriched extracts. The vegetal
material was dried under sunlight and in a
forced air oven at a temperature below 40°C,
to preserve it and to avoid any enzymatic
degradation of the compounds present in it.
According to Claeson
et al.
(1998), there are
reports on plant peptides that contain sev-
eral disulfide bridges, rendering them stable
to heat and even to solution in boiling water.
The dried and ground aerial parts of
H. parv-
iflorus
(30.9 g) were extracted by maceration
with CH
2
Cl
2
(300 ml) for 1 h under continu-
ous shaking. This procedure was repeated
seven times, changing the solvent each time.
Unlike other lipophilic substances, such
as chlorophylls, lipids and other substances
of lower molecular weight (terpenoids and
phenylpropanoids), polypeptides are not sol-
uble in CH
2
Cl
2.
The insecticide activity was
assessed on the dried CH
2
Cl
2
extract. The
plant residue was then macerated in 50% v/v
ethanol in water. The latter solution is a bet-
ter solvent than pure water or the alcohol to
solubilize the polypeptides. Moreover, with
this solvent the extraction of most polysac-
charides and enzymes is avoided, and micro-
bial growth is inhibited as well.
column in order to remove the tannins that
bind to the polyamide with high affinity
and in an irreversible fashion. This method-
ology has proved to be efficient in removing
the tannins that were not desired for the
studies. The peptides are not therefore
retained in the column matrix. The column
was eluted with 2% AcOH with a subse-
quent rinsing with 50% EtOH/2% AcOH to
elute those peptides that are insoluble in
2% AcOH (Broussalis
et al
., 2001).
The remains of the extraction with 50%
v/v EtOH were macerated in 25% ace-
tonitrile (ACN)/0.1% trifluoroacetic acid
(TFA). The extraction of the remaining pep-
tides of the ethanolic extraction was assayed
with the same solvent mixture as that
employed in the high-performance liquid
chromatography (HPLC) fractionation. The
ACN extract obtained by this methodology
was eluted in a polyamide column in order
to eliminate any residual tannins. The same
mixture that was employed in the extrac-
tion step (25% ACN/0.1% TFA) was
employed as an elution solvent.
The next procedure, liquid-liquid par-
tition BuOH/H
2
O, was aimed at obtaining
the cyclotides. The partition between water
and BuOH of the tannin-free 50% EtOH
extracts and tannin-free ACN was performed,
taking into account the remarkable hydro-
phobic nature of the cyclotides and their
solubility in BuOH. A sample of the lyophi-
lized ethanolic extract (42.0 mg) was solubi-
lized in Milli Q water (10 ml) and partitioned
three times with 10 ml of n-BuOH. The
organic and aqueous phases were separated
by centrifugation (10,000 rpm, 20 min) and
subsequent settling. The butanolic phase
was dried under reduced pressure, at a tem-
perature lower than 40°C. This procedure
yielded a sample of 8.5 mg. The aqueous
phase was dried in a Speed Vac concentra-
tor with a refrigerated trap, obtaining 12.6 mg
of dried material. Both the butanolic and
aqueous fractions were subjected to HPLC
(Broussalis
et al
., 2001).
The purification process developed for
this work is simpler than the methodologies
previously described, such as gel filtration
on Sephadex G10 with subsequent extrac-
tion on solid phase (Claeson
et al
., 1998).
13.3.2
Extract purification
The ethanolic extract was acidified with 2%
AcOH and eluted through a polyamide
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