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Antioxidant and cytoprotective potentials of Parmeliaceae lichens and identification of active compounds
strands were sequenced using the ABI Prism Dye minor variations. Cells were plated at a density of 5×104
Terminator Cycle Sequencing Ready Reaction Kit cells/well in 96-well plates overnight and then treated with
(Applied Biosystems, Foster City, California, USA) with different concentrations of lichens extracts (ranging from
the same primers used in the amplification step and with 0.5 to 250 µg/mL) for 24 h. Finally, MTT (2 mg/mL) was
following settings: initial denaturation at 94°C for 3 min; added and plates were incubated for 1 h at 37ºC. After
25 cycles at 96°C for 10 s, 50°C for 5 s, 60°C for 4 min.. removing medium, DMSO was added in order to dissolve
Sequencing reactions were electrophoresed on a 3730 the dark blue formazan crystals. Absorbance was then
DNA analyzer (Applied Biosystems) at the Unidad de measured at 550 nm using a microplate reader Digiscan
Genómica (Parque Científico de Madrid). 340 (ASYS HitechGmbH, Eugendorf, Austria).
Sequence fragments generated for this study were 2.6.c. Protection against H2O2- induced toxicity
assembled and edited using the program SeqMan v.7
(Lasergene R, DNASTAR, Madison, Wisconsin, USA). For evaluating a possible protective effect against
The sequences were blasted against GenBank database oxidative stress inductors, cells were exposed to 0.1 mM
following the methodology outlined in Sayers et al. (17) to H2O2 for 30 min after treatments with the extract (20),
approach the samples identity. The arbitrary = 97% and MTT assay was later conducted as previously
threshold value for assigning specimens to a closely described.
related congeneric species-group was selected as criterion
for assessing successful species identification. 2.6.d. Intracellular ROS production assay
2.4. Preparation of lichen extracts ROS production was assayed using the DCFH-DA
method (21), with some modifications. In brief, after
Methanol extract of the 29 lichen specimens were cellular treatments, 50 µL DCFH-DA/25 mL PBS–glucose
prepared by using 1 mL of metanol per mg of lichen was loaded for 30 min. After this time of incubation at
thallus. They were maintained for 1 hour in a sonicator 37ºC, cells were washed twice with PBS– glucose. ROS
(Branson) and then filtered. After evaporation of methanol generation was examined for 2 h (?exc 480 nm and ?em
at room temperature, dry extracts were kept at 4ºC in the 510 nm) in a Microplate Fluorescence Reader FLx800
fridge and later re-dissolved in methanol or PBS for the (Bio-Tek, Instrumentation, USA).
following experiments.
2.6.e. Determination of caspase-3 activity
2.5. Evaluation of antioxidant activity
Inhibition of H2O2-mediated apoptosis by extracts was
The Oxygen Radical Antioxidant Capacity (ORAC) assessed through the caspase-3 activity, by measuring the
assay was carried out as previously described (18). cleavage of a fluorogenic substrate (22). After treatments,
Dilutions of samples and Trolox (reference antioxidant, 100 µL of cell lysates were collected and added in a 96-
water soluble vitamin E analogue) were incubated in well plate. Well volume was completed with 50 µL
opaque 96-wells plates for 10 min at 37°C with reaction buffer (20 mM HEPES, 10% glycerol, 2 mM
fluorescein. After this period, the azocompound AAPH DTT, pH of 7.5) and 2 µL of caspase-3 substrate solution
(2,2´-azobis(2-methylpropionamidine) dihydrochloride) (1 mg/mL in DMSO) in the end. Substrate cleavage was
was added to the mixture. Fluorescence is read every 56 measured fluorometrically every hour using a FLx800
seconds for 98 minutes using a FLUOstar Optima (?exc 360/40 nm and ?em 480/20 nm).
fluorimeter (BMG Labtech) (?exc 485nm and ?em 520
nm). Area under the curve (AUC) was calculated for each 2.6.f. Glutathione levels
sample and compared with AUC of Trolox. ORAC values
are expressed as µmol Trolox equivalents (TE)/mg GSH and GSSG levels determination was performed
samples. according to the method described by Hissin and Hilf (23).
Reaction mixture for the determination of GSH contained
2.6. Cytoprotective activities 50 µL of the lichen sample, 150 µL of 0.1 M sodium
phosphate buffer (pH 8.0) and 20 µL of o-phthalaldehyde
2.6.a. Neurons cultures and treatments (1 mg/mL in methanol); instead, reaction mixture for
estimating of GSSG contained 50 µL of the sample and 3
Neurons from SH-SY5Y (human neuroblastoma) cell µL of N-ethylmaleimide and was maintained for 5 min in
line were maintained in DMEM supplemented with 10% darkness in this point before adding 150 µL of 0.1 N
FBS and 0.5% gentamicin in a humidified atmosphere NaOH (pH 12) and 20 µL of o-phthalaldehyde solution.
with 5% CO2 and 37ºC. H2O2 was used as oxidative Finally, both preparations were incubated for 15 min at
stress inductor, at a concentration of 0.1 mM for 30 min. room temperature in the dark, and fluorescence was
Cells were treated with lichen extracts at different measured at ?exc 528 nm and ?em 485 nm with a
concentrations for 24 h. Extracts and H2O2 were dissolved microplate fluorescence reader. Ratio GSH/GSSG was
in PBS for corresponding dilutions. obtained by dividing the nmols per mg of protein of GSH
and GSSG.
2.6.b. A ssessment of cell viability (MTT assay)
Mitochondrial integrity and activity, as cell viability
indicators, were determined by using MTT assay (19) with
@Real Academia Nacional de Farmacia. Spain 167
