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associated with pod and stem blight, and
against
C. truncatum
growth, an antracnose-
producing fungus (Henning, 1987; Piolo
et al
., 2000). Otherwise the chalcones
showed high activity against
C. truncatum
(MIC = 6.25 mg/ml). Consequently, leaves and
twigs of
Z. punctata
contain antifungals
against fungi isolated from soybean plants
cultivated in different regions of Argentina.
Successive extractions of fruits, aerial
parts and exudates of
Z. punctata
(Svetaz
et al
., 2007) with non-polar solvents, under
reflux, allowed the separation of two main
fractions (petroleun ether and dichlorometh-
ane) with moderate antifungal activity against
the yeasts
Candida albicans, Saccharomyces
cerevisiae
and
Cryptococcus neoformance
(MIC: 62.5-250 mg/ml), and a strong anti-
fungal activity against the dermatophytes
Microsporum gypseum, Trichophyton rubrum
and
Trichophyton mentagrophytes
(MICs:
8-16 mg/ml), supporting the possible use
of this plant in pharmacological applica-
tions. Fractionation of the organic extracts
demonstrated that 2¢,4¢-dihydroxychalcone
and 2¢,4¢-dihydroxy-3¢-methoxychalcones are
the main compounds responsible for the
antifungal activity. Moreover, with the pur-
pose of investigating their possible use in
clinical applications, the active substances
were assayed on clinical isolates from immu-
nocompromised, infected patients.
The values of MIC
80
, MIC
50
and mini-
mum fungicidal concentration (MFC) of
both chalcones were analysed in an
extended panel of clinical isolates in a
scheme for three-dimensional steady flow
with a second-order accuracy for the most
sensitive fungi and also comprised a series
of targeted assays. The results suggested
that 2¢,4¢-dihydroxychalcone is a fungicide
and does not disrupt the fungal membrane
up to 4 × MFC or act on the cell wall.
Consequently, chalcones seem to have a dif-
ferent action mechanism than polyene and
azole existing drugs because both chalcones
are fungicidal and not fungistatic like azoles
(Ablordeppey
et al
., 1999), and 2¢,4¢-dihy-
droxychalcone did not disrupt membranes
as amphotericin B does (Carson
et al
., 2002).
Furthermore, 2¢,4¢-dihydroxychalcone seems
not to act by inhibition of the growth of
fungal cell walls (López
et al
., 2001).
Consequently,
Z. punctata
would be con-
sidered as a source of antifungals against
skin-infecting fungi.
12.4.3 Genotoxic and
anti-genotoxic activities
The possible genotoxic and anti-genotoxic
effect of an ethanolic extract of
Z. punctata
and 2¢,4¢-dihydroxychalcone was evaluated
(Zampini
et al
., 2008). Their toxicity was
assayed with the lethality test of
Artemia
salina
(Finney, 1971) in order to use sub-
lethal quantities to study cell damage in the
experiments of genotoxicity. The comet
assay (Moretti
et al
., 2002) was applied for
the analysis of DNA damage because it pro-
vides a direct determination of DNA single-
and double-strand breaks in individual
cells. This test was selected because it was
applied for the
in vivo
and
in vitro
assays
with several cell lines (CHO, V79, HepG2,
among others) (Valentin-Severin
et al
.,
2003). The HepG2 cells present an endo-
genous bioactivation capacity, retain many
of the morphological characteristics of liver
parenchymal cells, and contain phase I and
phase II drug-metabolizing enzymes (factor
S9) that play an essential role in the activa-
tion/detoxification of pro-mutagens/pro-
carcinogens, the latter being the major
advantage of HepG2 cells for their use in
mutagenicity/anti-mutagenicity studies
(Knasmüller
et al
., 1998). Controls of cell
viability were performed before the assays
of genotoxicity/anti-genotoxicity. The effect
of the co-treatment of HepG2 cells with a
direct genotoxic compound (4-nitroquino-
line-N-oxyde) and
Z. punctata
extract or
2¢,4¢-dihydroxychalcone decreased the DNA
cell damage. The pre-treatment of HepG2
cells with
Z. punctata
extract or 2¢,4¢-dihy-
droxychalcone and incubation with an indi-
rect mutagen (benzo[a]pyrene) significantly
decreased the DNA damage. Consequently,
the results suggest that the alcoholic extract
of
Z. punctata,
as well as one of its compo-
nents, is not genotoxic. Anti-genotoxic
activity was demonstrated in the chosen
experimental conditions, though in this
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