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Antioxidant and cytoprotective potentials of Parmeliaceae lichens and identification of active compounds
4. DISCUSSION
In the lack of taxonomically significant morphological
features especially at lower taxonomic level, molecular-
based techniques have provided a valuable additional tool
for species identification (29). The internal transcribed
spacer region of the nuclear ribosomal RNA (ITS rRNA)
is the most frequently sequenced fungal genetic marker,
including lichenized fungi, and this locus has been recently
accepted as universal DNA barcode marker for fungi (30).
The use of DNA barcoding as a major tool for
identification of fungal species largely depends on the
development of high-quality sequence databases that are
thoroughly curated by taxonomists. A reference DNA
sequence database generated from expertly identified
specimens of well-circumscribed taxa may provide an
effective alternative to phenotype-based identification of
species by using DNA barcoding (31). In a recent study, a
huge number of fungal ITS sequences especially type
species lodged in Genbank have been well-crated by
taxonomists (32). The National Institute of Health’s (NIH)
genetic sequence database, GenBank, currently provides
the largest number of ITS sequences from lichen-forming
fungi, especially Parmeliaceae representing a broad range
of taxonomic and geographic diversity
(http://www.ncbi.nlm.nih.gov/genbank/). This could
provide a valuable resource for species identification
through BLAST-based sequence similarity searches of
DNA sequence data obtained from unidentified samples.
Considering all these points, we selected the ITS
marker for our research in order to acquire correct species
identification and applied BLAST-based sequence
similarity searches approach to identify the 29 samples
under study.
Once a Genbank code had been assigned for each
lichen specimen, we proceeded to evaluate the antioxidant
activity through the ORAC assay. We obtained a varied
range of ORAC values for the different species, what may
be related to the differences in chemical composition
among lichen genus and species. Among in vitro
antioxidant tests, ORAC assay is classified as a hydrogen
atom transfer (HAT)-based assay and, therefore, such
donation of a hydrogen atom might be a plausible
mechanism for explaining the antiradical properties
exerted by the compounds present in the methanol extracts
(33). At this point, we selected the three species with the
highest ORAC values: Cetrelia braunsiana, Parmotrema
saccatilobum and Usnea ghattensis.
Their methanol extracts were tested on a cellular model
of neurons threatened by the hydrogen peroxide –induced
oxidative stress; the final aim was to assess the
cytoprotective properties of these lichens in nervous
system–like cells, since no previous studies had dealt with
the issue. Cell viability studies allowed the selection of the correlation between the main constituents and their
optimal concentration for each extract that was further
used in other experiments measuring some markers of
oxidative stress such as intracellular ROS levels, caspase-3
@Real Academia Nacional de Farmacia. Spain
activity and concentrations of the endogenous antioxidant
glutathione.
The incidence of exogenous H2O2 on intracellular ROS
levels was evaluated by DCFH-DA fluorimetric assay.
2´,7´-diclorodihidrofluorescein-diacetate (DCFH-DA) is a
non-fluorescent compound that is able to cross cell
membranes and is de-acylated by intracellular esterases,
turning into 2´,7´-diclorodihidrofluorescein (DCFH);
DCFH, in presence of ROS like hydrogen peroxide, reacts
with them and oxidizes to 2’,7’-diclorofluorescen (DCF),
which is already a high fluorescent compound.
Intracellular fluorescence of DCF allows to quantify
intracellular ROS formation and oxidative stress, since its
intensity is proportional to the amount of ROS produced.
Results obtained in this assay confirm that H2O2, under
established experimental conditions, exerts induction of
oxidative stress and increase intracellular ROS levels (34).
The decrease in ROS generation mediated by the
pretreatment with lichen extracts might suggest that
oxidative stress reduction as an important mechanism
underlying their cytoprotective action.
Consistence evidences demonstrate a direct
relationship between cellular oxidative stress and apoptosis
(35). Apoptosis is a complex process of programmed cell
death in which mitochondria play a crucial role, and
involves several molecular pathways including caspases
activation; inhibition of apoptosis arises as a plausible
mechanism for neuroprotection (36). Caspase-3 is a key
enzymatic mediator in external and internal apoptosis
pathways, and suppression of active caspase-3 contributes
to the cellular protection against oxidative stress (37).
Through a fluorimetric method, we demonstrated that
caspase-3 activity was enhanced after H2O2 treatment, but
that Cb and Ug were able to reverse such effect. Therefore,
the attenuation of neuronal apoptosis may also mediate
their neuroprotective effects.
Glutathione in its reduced state (GSH) is the major
endogenous antioxidant in the body due to the presence of
a reactive cysteine moiety. The ratio between the oxidized-
type (GSSG) and reduced-type glutathione levels is often
used as indicative of the cellular reducing power. As a
result of a decrease in GSH levels or because of changes in
GSH/GSSG ratio, cells experience greater oxidative
damage (38). The measurement of the concentrations of
GSH and GSSG in the different group of cells evidenced
that Ug favors a good antioxidant status within the
neurons; a pretreatment with this lichen extracts protected
cells from GSH depletion induced by oxidative stress, thus
promoting cell survival.
Results on chemical composition of the three species
obtained by a TLC method have been confirmed through
HPLC, and are in agreement with previous studies on the
phytochemistry of these species (39-41). A plausible
potential pharmacological activities might be established.
Hence, antioxidant activities exerted by methanol extracts
of Cetrelia braunsiana (Cb) and Parmotrema saccatilobum
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