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Carlos Fernández-Moriano et al.

2.7. Phytochemical analysis lichens with known composition (Neofuscelia glabrans, N.
parviloba, Xanthoparmelia exemplaris) and analyzed in the
2.7.a. Thin Layer Chromatography (TLC) same conditions (27).

TLC analyses were performed based on Culberson 2.8. Statistical analysis
methods (24-26). Methanol extracts of lichen thallus were
charged on the silicagel plate and run with a mobile phase Results are expressed as means ± SD of at least three
composed of toluene/acetic acid (85:15). Once the plate independent experiments. Statistical differences were
had been saturated with the solvent within a glass cuvette, tested using one-way ANOVA followed by Tukey’s test
the plate was removed and dried at room temperature. It for multiple comparisons, using the Statgraphics Centurion
was then exposed and observed under UV light and XVI software. A value of p<0.05 was considered
revealed by spreading a sulfuric acid solution along the statistically significant.
plate, which was placed on a heater.
3. RESULTS
2.7.b. High Performance Liquid Chromatography (HPLC)
3.1. Identification of lichen species
HPLC analysis was performed in a SPECTRA-
PHYSICS SP 8800/8810LC coupled to a UV-vis detector, For the molecular identification of the lichen
measuring at a wavelength of 254nm. The column specimens, fragments of ITS rDNA sequences obtained
employed was a C18 Mediterranea Sea (150 x 4.6 mm, 5 were analyzed through BLAST function of NCBI
µm particle size; Teknokroma) and the mobile phase (National Center for Biotechnology Information) GenBank
consisted of A (acetonitrile / methanol (20:80 v/v)) and B database (04 March 2015), thus attributing to a certain
(4 % acetic acid solution). Elution was performed sequence a percentage of overlapping with the most
isocratically at a flow of 1 mL/min and at room similar by comparative analysis (see Table 2).
temperature. Reference chromatograms were used from

Table 2. Genbank code assigned to the 29 lichen species after comparison with the most similar appearing in BLAST.

Lichen specie Genbank Code

Bulbothrix meizospora EF 092102
Cetraria aculeata EU 409758
GU 994635
Cetraria canadensis AF 494383
Cetraria nigricans GU 994636
Cetrelia braunsiana AF 254630
Cetrelia cetrarioides GU 994638
Cetrelia olivatorum

Evernia prunastri DQ 923634
Flavoparmelia citrinescens GU 994641
HM 010928
Flavoparmelia euplecta HM 014233
Flavoparmelia haysonii DQ 084148
Flavoparmelia rutidota GQ 919258
EF 042917
Hipotrachyna execta EF 092128
Myelochroa entotheiochroa

Myelochroa irrugans

Parmelia saxatilis AF 391137
Parmotrema abessinicum HM 017025
Parmotrema austrosinense DQ 912360
AY 586580
Parmotrema perlatum DQ 912316
Parmotrema praeserediosum DQ 084184
AB 627399
Parmotrema reticulatum AF 297732
Parmotrema saccatilobum

Usnea arizonica

Usnea aurantiacoatra EF 179798
Usnea contexta AJ 249573
Usnea filipendula FR 799076
Usnea ghattensis EF 179806
Usnea sp. (Mot.) JF 794062
AF 262010
Xanthoparmelia coreana

168 @Real Academia Nacional de Farmacia. Spain