Biology Reference
In-Depth Information
The cyclotides found in plants are mac-
rocyclic peptides, 28-37 amino acids long
and have a cyclic backbone (the name deriv-
ing from cyclopeptides: cyclotides), that is, a
continuous cycle of peptidic bonds (amide
bonds) forming the backbone. They contain
six cysteine residues (Cys) that form three
disulfide bridges that stabilize the molecule
(Craik et al ., 1999). Because of their well-
defined structure and potent biological
activities, these molecules can be regarded
as miniproteins (Felizmenio-Quimio et al .,
2001). Among these miniproteins, Kalata
B1, the circulins, cyclopsychotride and sev-
eral peptides from Viola arvensis can be
found. The physical properties of cyclotides,
which include resistance to thermal and
enzymatic breakdown (Colgrave and Craik,
2004; Ireland et al ., 2006), can be attributed
to the cyclic backbone and disulfide bridges
that, by means of the formation of a cystine
knot, provide the molecule with a great sta-
bility (Craik et al ., 2002).
Cyclotides are aligned according to the
six conserved Cys residues (named I to VI).
The backbone loops of the cyclotide corre-
sponding to the region located between the
Cys residues are numbered from 1 to 6. The
amino-acid chains responsible for the bio-
logical activities of cyclotides are located in
these loops. The outstanding stability of the
cystine knot structure makes it an excellent
framework on which to add a wide range of
biological activities (Craik, 2001). Most of
the cyclotides described so far in the plants
belonging to the Violaceae and Rubiaceae
families (Trabi and Craik, 2004; Trabi et al .,
2004; Simonsen et al ., 2005; Gruber et al. ,
2008) have sequences that allow classifying
them into two subgroups or main families
named 'bracelet' and Moëbius cyclotides.
As established by Craik et al . (2002), this
nomenclature is based upon the presence or
absence of a cis -proline residue in loop 5,
which introduces a twist into the circular
backbone of the peptide. Thus, those cyclo-
tides bearing such twist are named Moëbius
cyclotides (Rosengren et al ., 2003; Daly
et al. , 2006).
Even though cyclotides have been
found in roots, stems and leaves of
Oldenlandia affinis , these molecules are
mostly found in young leaves. Therefore,
the possibility that these compounds could
function in defence mechanisms against
plant pathogens was studied (Alexander,
2001). To this end, studies were carried out
on the basis of the ingestion of diets con-
taining 0.15% w/v of Kalata B1 by larvae of
Helicoverpa punctigera (Lepidoptera), a
moth that affects the harvests. Kalata B1
was able to inhibit the growth of these lar-
vae, hampering the development of the first
stage instar. It was not clear, however,
whether this alteration in the growth of lar-
vae was due to a toxic effect or to an anti-
alimentary action that led to death from
starvation (Jennings et al ., 2001).
It is known that small cysteine-rich
peptides present in plants have protease
or a-amylase inhibitory activities that can
delay the growth of insects by blocking
the digestion of proteins or starch.
Nevertheless, Kalata B1 and B2 are thought
to have a different mechanism of action,
because they did not have any effect on
the trypsins, chemotrypsins or a-amylase
isolated from the gut of Helicoverpa sp.
(Baillie Gerritsen, 2002). Furthermore,
Kalata peptides possess haemolytic activ-
ity. The latter observation raises the
hypothesis that the insecticide activity is
the result of damage to the gut membranes
of the insect (Jennings et al ., 2001). The
insecticide activity is a remarkable prop-
erty of cyclotides from both ecological and
environmental points of view, having tech-
nological and economical-commercial
applications. Trabi and Craik (2002) have
stated the importance of studying the
insecticide activity of cyclotides.
13.3 The Preparation and
Purification of Extracts
13.3.1
Extraction of the plant material
Hybanthus parviflorus ( syn. Ionidium gluti-
nosum Ventetat, Viola parvifolia Roemer
and Shultes; Ballard and Jorgensen, 1997),
family Violaceae, was collected, in part, at
the intersection of the Number 12 National
Road and the Feliciano stream, La Paz
Department, in the Province of Entre Ríos,
 
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